Georg T Wondrak

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Books

• Wondrak, G. T. (2016). ‘Skin Stress Response Pathways: Environmental Factors and Molecular Opportunities’. SPRINGER (Biomedicine; book and e-book: ISBN 978-3-319-43155-0). doi:10.1007/978-3-319-43157-4
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  Editor, ‘Skin Stress Response Pathways: Environmental Factors and Molecular Opportunities’, 2016, Springer, 457 p.; ISBN 978-3-319-43155-0; http://www.springer.com/us/book/9783319431550; hardcopy and ebook; [including a book chapter: 'The Aryl Hydrocarbon Receptor (AhR) as an Environmental Stress Sensor and Regulator of Skin Barrier Function: Molecular Mechanisms and Therapeutic Opportunities' Justiniano R. and Wondrak GT, in: Skin Stress Response Pathways: Environmental Factors and Molecular Opportunities, 2016, Springer, 457 p.; ISBN 978-3-319-43155-0; pp. 325-359-309, 2016]
• Wondrak, G. T. (2015). ‘Stress Response Pathways in Cancer: From Molecular Targets to Novel Therapeutics’. SPRINGER (Biomedicine; book and e-book: ISBN 978-94-017-9420-6).
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  Editor, ‘Stress Response Pathways in Cancer: From Molecular Targets to Novel Therapeutics’, 2015, Springer, 446 p.; ISBN 978-94-017-9420-6; http://www.springer.com/biomed/cancer/book/978-94-017-9420-6; hardcopy and ebook; published online: November 11, 2014. Hardcopy: 2-1-2015 [including a book chapter: 'Melanomagenic Gene Alterations Viewed from a Redox Perspective: Molecular Mechanisms and Therapeutic Opportunities.' Wondrak GT, in: ‘Stress Response Pathways in Cancer'; ISBN 978-94-017-9420-6; pp. 285-309, 2015]

Journals/Publications

• Jandova, J., & Wondrak, G. T. (2020). Genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth. Redox biology, 39, 101838.
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  Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human malignant melanoma cells and patient tumors and substantiated a novel role of GLO1 as a molecular determinant of invasion and metastasis in melanoma. Here, employing NanoString™ gene expression profiling (nCounter™ 'PanCancer Progression Panel'), we report that CRISPR/Cas 9-based GLO1 deletion from human A375 malignant melanoma cells alters glucose metabolism and redox homeostasis, observable together with acceleration of tumorigenesis. Nanostring™ analysis identified TXNIP (encoding thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis, displaying the most pronounced expression change in response to GLO1 elimination, confirmed by RT-qPCR and immunoblot analysis. TXNIP was also upregulated in CRISPR/Cas9-engineered DU145 prostate carcinoma cells lacking GLO1, and treatment with MG or a pharmacological GLO1 inhibitor (TLSC702) mimicked GLO1_KO status, suggesting that GLO1 controls TXNIP expression through regulation of MG. GLO1_KO status was characterized by (i) altered oxidative stress response gene expression, (ii) attenuation of glucose uptake and metabolism with downregulation of gene expression (GLUT1, GFAT1, GFAT2, LDHA) and depletion of related key metabolites (glucose-6-phosphate, UDP-N-acetylglucosamine), and (iii) immune checkpoint modulation (PDL1). While confirming our earlier finding that GLO1 deletion limits invasion and metastasis with modulation of EMT-related genes (e.g. TGFBI, MMP9, ANGPTL4, TLR4, SERPINF1), we observed that GLO1_KO melanoma cells displayed a shortened population doubling time, cell cycle alteration with increased M-phase population, and enhanced anchorage-independent growth, a phenotype supported by expression analysis (CXCL8, CD24, IL1A, CDKN1A). Concordantly, an accelerated growth rate of GLO1_KO tumors, accompanied by TXNIP overexpression and metabolic reprogramming, was observable in a SCID mouse melanoma xenograft model, demonstrating that A375 melanoma tumor growth and metastasis can be dysregulated in opposing ways as a consequence of GLO1 elimination.
• Jandova, J., Perer, J., Hua, A., Snell, J. A., & Wondrak, G. T. (2020). Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo. Cancers, 12(6).
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  Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by ), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with deletion (A375-_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven construct (A375-_R). After confirming target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-_KO versus A375-_WT), pronounced modulation of epithelial--mesenchymal transition (EMT)-related genes [upregulated: , , ; (downregulated): and (up to tenfold; < 0.05)] was observed-all of which are consistent with EMT suppression as a result of deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-_R cells. Differential expression of as a function of status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-_KO cells was strongly attenuated as compared to A375-_WT cells. Taken together, these prototype data provide evidence in support of a novel function of in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of as a novel melanoma target.
• Justiniano, R., de Faria Lopes, L., Perer, J., Hua, A., Park, S. L., Jandova, J., Baptista, M. S., & Wondrak, G. T. (2020). The Endogenous Tryptophan-derived Photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo. Photochemistry and photobiology, 97(1), 180-191.
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  UV-chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol  cm ), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC-25, HaCaT-ras II-4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm ) and FICZ (≥10 nm), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg-sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 "high-risk" mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV-induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.
• Perer, J., Jandova, J., Fimbres, J., Jennings, E. Q., Galligan, J. J., Hua, A., & Wondrak, G. T. (2020). The sunless tanning agent dihydroxyacetone induces stress response gene expression and signaling in cultured human keratinocytes and reconstructed epidermis. Redox biology, 36, 101594.
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  Sunless (chemical) tanning is widely regarded as a safe alternative to solar UV-induced skin tanning known to be associated with epidermal genotoxic stress, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous amino-carbonyl ('glycation') reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined the cutaneous effects of acute DHA-exposure on cultured human HaCaT keratinocytes and epidermal reconstructs, profiled by gene expression array analysis and immunodetection. In keratinocytes, DHA-exposure performed at low millimolar concentrations did not impair viability while causing a pronounced cellular stress response as obvious from rapid activation of phospho-protein signal transduction [p-p38, p-Hsp27(S15/S78), p-eIF2α] and gene expression changes (HSPA6, HMOX1, CRYAB, CCL3), not observable upon exposure to the non-ketose, tanning-inactive DHA-control glycerol. Formation of advanced glycation end products (AGEs) from posttranslational protein-adduction was confirmed by quantitative mass spectrometric detection of N-ε-(carboxyethyl)-l-lysine (CEL) and N-carboxyethyl-l-arginine, and skin cells with CRISPR-Cas9-based elimination of the carbonyl stress response gene GLO1 (encoding glyoxalase 1) displayed hypersensitivity to DHA-cytotoxicity. In human epidermal reconstructs a topical use-relevant DHA-dose regimen elicited a comparable stress response as revealed by gene expression array (HSPA1A, HSPA6, HSPD1, IL6, DDIT3, EGR1) and immunohistochemical analysis (CEL, HO-1, p-Hsp27-S78). In DHA-treated SKH-1 hairless mouse skin IHC-detection revealed epidermal occurrence of CEL- and p-Hsp27-epitopes. For comparison, stress response gene expression array analysis was performed in epidermis exposed to a supra-erythemal dose of solar simulated UV (2 MEDs), identifying genes equally or differentially sensitive to either one of these cutaneous stimuli [DHA ('sunless tanning') versus solar UV ('sun-induced tanning')]. Given the worldwide use of chemical tanners in consumer products, these prototype data documenting a DHA-induced specific cutaneous stress response deserve further molecular exploration in living human skin.
• Schmidlin, C. J., Rojo de la Vega, M., Perer, J., Zhang, D. D., & Wondrak, G. T. (2020). Activation of NRF2 by topical apocarotenoid treatment mitigates radiation-induced dermatitis. Redox biology, 37, 101714.
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  Radiation therapy is a frontline treatment option for cancer patients; however, the effects of radiotherapy on non-tumor tissue (e.g. radiation-induced dermatitis) often worsen patient quality of life. Previous studies have implicated the importance of redox balance in preventing dermatitis, specifically in reference to modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Due to the cytoprotective functions of transcriptional target genes of NRF2, we investigated how modulation of NRF2 expression could affect DNA damage, oxidative stress, and cell viability in response to radiotherapy. Specifically, it was noted that NRF2 knockdown sensitized human skin keratinocytes to ionizing radiation; likewise, genetic ablation of NRF2 in vivo increased radiosensitivity of murine epidermis. Oppositely, pharmacological induction of NRF2 via the apocarotenoid bixin lowered markers of DNA damage and oxidative stress, while preserving viability in irradiated keratinocytes. Mechanistic studies indicated that topical pretreatment using bixin as an NRF2 activator antagonized initial DNA damage by raising cellular glutathione levels. Additionally, topical application of bixin prevented radiation-induced dermatitis, epidermal thickening, and oxidative stress in the skin of SKH1 mice. Overall, these data indicate that NRF2 is critical for mitigating the harmful skin toxicities associated with ionizing radiation, and that topical upregulation of NRF2 via bixin could prevent radiation-induced dermatitis.
• Blohm-Mangone, K., Burkett, N. B., Tahsin, S., Myrdal, P. B., Aodah, A., Ho, B., Janda, J., McComas, M., Saboda, K., Roe, D. J., Dong, Z., Bode, A. M., Petricoin, E. F., Calvert, V. S., Curiel-Lewandrowski, C., Alberts, D. S., Wondrak, G. T., & Dickinson, S. E. (2018). Pharmacological TLR4 Antagonism Using Topical Resatorvid Blocks Solar UV-Induced Skin Tumorigenesis in SKH-1 Mice. Cancer Prevention Research (Philadelphia, Pa.), 11(5), 265-278.
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  An urgent need exists for the development of more efficacious molecular strategies targeting nonmelanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) has been implicated in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. We have previously shown in keratinocyte cell culture and SKH-1 mouse epidermis that topical application of the specific TLR4 antagonist resatorvid (TAK-242) blocks acute UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators and MAP kinase phosphorylation. We therefore explored TLR4 as a novel target for chemoprevention of UV-induced NMSC. We selected the clinical TLR4 antagonist resatorvid based upon target specificity, potency, and physicochemical properties. Here, we confirm using permeability assays that topical resatorvid can be effectively delivered to skin, and using studies that topical resatorvid can block UV-induced AP-1 activation in mouse epidermis. We also report that in a UV-induced skin tumorigenesis model, topical resatorvid displays potent photochemopreventive activity, significantly suppressing tumor area and multiplicity. Tumors harvested from resatorvid-treated mice display reduced activity of UV-associated signaling pathways and a corresponding increase in apoptosis compared with tumors from control animals. Further mechanistic insight on resatorvid-based photochemoprevention was obtained from unsupervised hierarchical clustering analysis of protein readouts via reverse-phase protein microarray revealing a significant attenuation of key UV-induced proteomic changes by resatorvid in chronically treated high-risk SKH-1 skin prior to tumorigenesis. Taken together, our data identify TLR4 as a novel molecular target for topical photochemoprevention of NMSC. .
• Jung, A. M., Dennis, L. K., Jacobs, E. T., & Wondrak, G. T. (2018). Sun sensitivity and sun protective behaviors during sun exposure among indoor office workers in the American Midwest. Photodermatology, photoimmunology & photomedicine, 34(6), 393-399.
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  Sun sensitivity, a confounder between sun exposure and sun protection, is often overlooked. We examined how sun exposure and sun protection behaviors among indoor workers varied by sun sensitivity.
• Rojo de la Vega, M., Zhang, D. D., & Wondrak, G. T. (2018). Topical Bixin Confers NRF2-Dependent Protection Against Photodamage and Hair Graying in Mouse Skin. Frontiers in Pharmacology, 9, 287.
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  Environmental exposure to solar ultraviolet (UV) radiation causes acute photodamage, premature aging, and skin cancer, attributable to UV-induced genotoxic, oxidative, and inflammatory stress. The transcription factor NRF2 [nuclear factor erythroid 2 (E2)-related factor 2] is the master regulator of the cellular antioxidant response protecting skin against various environmental stressors including UV radiation and electrophilic pollutants. NRF2 in epidermal keratinocytes can be activated using natural chemopreventive compounds such as the apocarotenoid bixin, an FDA-approved food additive and cosmetic ingredient from the seeds of the achiote tree (). Here, we tested the feasibility of topical use of bixin for NRF2-dependent skin photoprotection in two genetically modified mouse models [SKH1 and C57BL/6J ( versus )]. First, we observed that a bixin formulation optimized for topical NRF2 activation suppresses acute UV-induced photodamage in but not SKH1 mice, a photoprotective effect indicated by reduced epidermal hyperproliferation and oxidative DNA damage. Secondly, it was demonstrated that topical bixin suppresses PUVA (psoralen + UVA)-induced hair graying in but not C57BL/6J mice. Collectively, this research provides the first evidence that topical application of bixin can protect against UV-induced photodamage and PUVA-induced loss of hair pigmentation through NRF2 activation. Topical NRF2 activation using bixin may represent a novel strategy for human skin photoprotection, potentially complementing conventional sunscreen-based approaches.
• Teves, J. M., Bhargava, V., Kirwan, K. R., Corenblum, M. J., Justiniano, R., Wondrak, G. T., Anandhan, A., Flores, A. J., Schipper, D. A., Khalpey, Z., Sligh, J. E., Curiel-Lewandrowski, C., Sherman, S. J., & Madhavan, L. (2018). Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy. Frontiers in Neuroscience, 11, 737.
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  The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.
• Dickinson, S. E., & Wondrak, G. T. (2017). TLR4-directed Molecular Strategies Targeting Skin Photodamage and Carcinogenesis. Current medicinal chemistry.
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  Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and inflammatory dysregulation is a key mechanism underlying detrimental effects of acute and chronic UV exposure. The health and economic burden of skin cancer treatment is substantial, creating an increasingly urgent need for the development of improved molecular strategies for photoprotection and photochemoprevention. The role of Toll-like receptor 4 (TLR4) as a key regulator of skin anti-microbial defense, wound healing, and cutaneous tumorigenic inflammation has now been recognized, and recently published evidence suggests that TLR4 represents a novel molecular target for skin photoprotection and cancer photochemoprevention. Specifically, it has been shown that pharmacological and genetic antagonism of TLR4 suppresses UV-induced inflammatory signaling involving the attenuation of cutaneous NF-κB and AP-1 stress signaling observable in vitro and in vivo. A number of TLR4-directed small molecule pharmacological antagonists [including eritoran, (+)-naloxone, ST2825, and resatorvid] have now been identified and are at various stages of preclinical and clinical development for the modulation of dysregulated TLR4-dependent inflammatory signaling.
• Justiniano, R., Perer, J., Hua, A., Fazel, M., Krajisnik, A., Cabello, C. M., & Wondrak, G. T. (2017). A Topical Zinc Ionophore Blocks Tumorigenic Progression in UV-exposed SKH-1 High-risk Mouse Skin. Photochemistry and photobiology, 93(6), 1472-1482.
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  Nonmelanoma skin cancer (NMSC) is the most common malignancy in the United States representing a considerable public health burden. Pharmacological suppression of skin photocarcinogenesis has shown promise in preclinical and clinical studies, but more efficacious photochemopreventive agents are needed. Here, we tested feasibility of harnessing pharmacological disruption of intracellular zinc homeostasis for photochemoprevention in vitro and in vivo. Employing the zinc ionophore and FDA-approved microbicidal agent zinc pyrithione (ZnPT), used worldwide in over-the-counter (OTC) topical consumer products, we first demonstrated feasibility of achieving ZnPT-based intracellular Zn2+ overload in cultured malignant keratinocytes (HaCaT-ras II-4; SCC-25) employing membrane-permeable fluorescent probes. Zinc overload was accompanied by induction of intracellular oxidative stress, associated with mitochondrial superoxide release as substantiated by MitoSOX Red™ fluorescence microscopy. ZnPT-induced cell death observable in malignant keratinocytes was preceded by induction of metal (MT2A), proteotoxic (HSPA6, HSPA1A, DDIT3, HMOX1) and genotoxic stress response (GADD45A, XRCC2) gene expression at the mRNA and protein levels. Comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions. In a photocarcinogenesis model (UV-exposed SKH-1 high-risk mouse skin), topical ZnPT administration post-UV caused epidermal zinc overload and stress response gene expression with pronounced blockade of tumorigenesis. Taken together, these data suggest feasibility of repurposing a topical OTC drug for zinc-directed photochemoprevention of solar UV-induced NMSC.
• Justiniano, R., Williams, J. D., Perer, J., Hua, A., Lesson, J., Park, S. L., & Wondrak, G. T. (2017). The B6 -vitamer Pyridoxal is a Sensitizer of UVA-induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis. Photochemistry and photobiology, 93(4), 990-998.
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  UVA-driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3-hydroxypyridine-derived chromophores including B6 -vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA-induced photooxidative stress in human skin cells. Here, we report that the B6 -vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA-induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ-H2AX (Ser139)], comet analysis indicated the formation of Fpg-sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA-driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B6 -vitamers.
• Rojo de la Vega, M., Krajisnik, A., Zhang, D. D., & Wondrak, G. T. (2017). Targeting NRF2 for Improved Skin Barrier Function and Photoprotection: Focus on the Achiote-Derived Apocarotenoid Bixin. Nutrients, 9(12).
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  The transcription factor NRF2 (nuclear factor-E2-related factor 2) orchestrates major cellular defense mechanisms including phase-II detoxification, inflammatory signaling, DNA repair, and antioxidant response. Recent studies strongly suggest a protective role of NRF2-mediated gene expression in the suppression of cutaneous photodamage induced by solar UV (ultraviolet) radiation. The apocarotenoid bixin, a Food and Drug Administration (FDA)-approved natural food colorant (referred to as 'annatto') originates from the seeds of the achiote tree native to tropical America, consumed by humans since ancient times. Use of achiote preparations for skin protection against environmental insult and for enhanced wound healing has long been documented. We have recently reported that (i) bixin is a potent canonical activator of the NRF2-dependent cytoprotective response in human skin keratinocytes; that (ii) systemic administration of bixin activates NRF2 with protective effects against solar UV-induced skin damage; and that (iii) bixin-induced suppression of photodamage is observable in Nrf2+/+ but not in Nrf2-/- SKH-1 mice confirming the NRF2-dependence of bixin-induced antioxidant and anti-inflammatory effects. In addition, bixin displays molecular activities as sacrificial antioxidant, excited state quencher, PPAR (peroxisome proliferator-activated receptor) α/γ agonist, and TLR (Toll-like receptor) 4/NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) antagonist, all of which might be relevant to the enhancement of skin barrier function and environmental stress protection. Potential skin photoprotection and photochemoprevention benefits provided by topical application or dietary consumption of this ethno-pharmacologically validated phytochemical originating from the Americas deserves further preclinical and clinical examination.
• Janda, J., Burkett, N. B., Blohm-Mangone, K., Huang, V., Curiel-Lewandrowski, C., Alberts, D. S., Petricoin, E. F., Calvert, V. S., Einspahr, J., Dong, Z., Bode, A. M., Wondrak, G. T., & Dickinson, S. E. (2016). Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochemistry and Photobiology, 92(6), 816-825.
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  Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of nonmelanoma skin cancer (NMSC) are urgently needed. Toll-like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression, and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV-induced NF-κB and AP-1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis, also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA-based genetic TLR4 inhibition blocks UV-induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK-242), a molecularly targeted clinical TLR4 antagonist, blocks UV-induced NF-κB and MAP kinase/AP-1 activity and cytokine expression (Il-6, Il-8, and Il-10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV-induced cutaneous signaling, and future experiments will explore the potential of TLR4-directed strategies for prevention of NMSC.
• Tao, S., Rojo de la Vega, M., Quijada, H., Wondrak, G. T., Wang, T., Garcia, J. G., & Zhang, D. D. (2016). Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner. Scientific Reports, 6, 18760.
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  Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage, and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2(+/+) but not in Nrf2(-/-) mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment.
• Davis, A. L., Qiao, S., Lesson, J. L., Rojo de la Vega, M., Park, S. L., Seanez, C. M., Gokhale, V., Cabello, C. M., & Wondrak, G. T. (2015). The quinone methide aurin is a heat shock response inducer that causes proteotoxic stress and Noxa-dependent apoptosis in malignant melanoma cells. The Journal of Biological Chemistry, 290(3), 1623-38.
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  Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.
• Long, M., Rojo de la Vega, M., Wen, Q., Bharara, M., Jiang, T., Zhang, R., Zhou, S., Wong, P. K., Wondrak, G. T., Zheng, H., & Zhang, D. D. (2015). An essential role of NRF2 in diabetic wound healing. Diabetes.
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  The high mortality and disability of diabetic non-healing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens we show that perilesional skin tissues from diabetic patients are under more severe oxidative stress and display higher activation of the NRF2-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In an STZ-induced diabetes mouse model, Nrf2(-/-) mice have delayed wound closure rates compared to Nrf2(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low TGF-β1 and high MMP9 expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in HaCaT cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers.
• Long, M., Tao, S., Rojo de la Vega, M., Jiang, T., Wen, Q., Park, S. L., Zhang, D. D., & Wondrak, G. T. (2015). Nrf2-dependent suppression of azoxymethane/dextran sulfate sodium-induced colon carcinogenesis by the cinnamon-derived dietary factor cinnamaldehyde. Cancer Prevention Research (Philadelphia, Pa.), 8(5), 444-54.
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  The progressive nature of colorectal cancer and poor prognosis associated with the metastatic phase of the disease create an urgent need for the development of more efficacious strategies targeting colorectal carcinogenesis. Cumulative evidence suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defence, represents a promising molecular target for colorectal cancer chemoprevention. Recently, we have identified cinnamon, the ground bark of Cinnamomum aromaticum (cassia cinnamon) and Cinnamomum verum (Ceylon cinnamon), as a rich dietary source of the Nrf2 inducer cinnamaldehyde (CA) eliciting the Nrf2-regulated antioxidant response in human epithelial colon cells, conferring cytoprotection against electrophilic and genotoxic insult. Here, we have explored the molecular mechanism underlying CA-induced Nrf2 activation in colorectal epithelial cells and have examined the chemopreventive potential of CA in a murine colorectal cancer model comparing Nrf2(+/+) with Nrf2(-/-) mice. In HCT116 cells, CA caused a Keap1-C151-dependent increase in Nrf2 protein half-life via blockage of ubiquitination with upregulation of cytoprotective Nrf2 target genes and elevation of cellular glutathione. After optimizing colorectal Nrf2 activation and target gene expression by dietary CA-supplementation regimens, we demonstrated that CA suppresses AOM/DSS-induced inflammatory colon carcinogenesis with modulation of molecular markers of colorectal carcinogenesis. Dietary suppression of colorectal cancer using CA supplementation was achieved in Nrf2(+/+) but not in Nrf2(-/-) mice confirming the Nrf2 dependence of CA-induced chemopreventive effects. Taken together, our data suggest feasibility of colorectal cancer suppression by dietary CA, an FDA-approved food additive derived from the third most consumed spice in the world.
• Park, S. L., Justiniano, R., Williams, J. D., Cabello, C. M., Qiao, S., & Wondrak, G. T. (2015). The Tryptophan-Derived Endogenous Aryl Hydrocarbon Receptor Ligand 6-Formylindolo[3,2-b]Carbazole Is a Nanomolar UVA Photosensitizer in Epidermal Keratinocytes. The Journal of Investigative Dermatology, 135(6), 1649-58.
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  Endogenous UVA chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high-affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar-simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, endoplasmic reticulum stress, and oxidative stress response gene expression observed only upon FICZ/UVA cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin.
• Tao, S., Park, S. L., de la Vega, M. R., Zhang, D. D., & Wondrak, G. T. (2015). Systemic administration of the apocarotenoid bixin protects skin against solar UV-induced damage through activation of NRF2. Free Radical Biology & Medicine, 89, 690-700.
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  Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and an urgent need exists for improved molecular photoprotective strategies different from (or synergistic with) photon absorption. Recent studies suggest a photoprotective role of cutaneous gene expression orchestrated by the transcription factor NRF2 (nuclear factor-E2-related factor 2). Here we have explored the molecular mechanism underlying carotenoid-based systemic skin photoprotection in SKH-1 mice and provide genetic evidence that photoprotection achieved by the FDA-approved apocarotenoid and food additive bixin depends on NRF2 activation. Bixin activates NRF2 through the critical Cys-151 sensor residue in KEAP1, orchestrating a broad cytoprotective response in cultured human keratinocytes as revealed by antioxidant gene expression array analysis. Following dose optimization studies for cutaneous NRF2 activation by systemic administration of bixin, feasibility of bixin-based suppression of acute cutaneous photodamage from solar UV exposure was investigated in Nrf2(+/+) versus Nrf2(-/-) SKH-1 mice. Systemic administration of bixin suppressed skin photodamage, attenuating epidermal oxidative DNA damage and inflammatory responses in Nrf2(+/+) but not in Nrf2(-/-) mice, confirming the NRF2-dependence of bixin-based cytoprotection. Taken together, these data demonstrate feasibility of achieving NRF2-dependent cutaneous photoprotection by systemic administration of the apocarotenoid bixin, a natural food additive consumed worldwide.
• Shen, T., Chen, X., Harder, B., Long, M., Wang, X., Lou, H., Wondrak, G. T., Ren, D., & Zhang, D. D. (2014). Plant extracts of the family Lauraceae: a potential resource for chemopreventive agents that activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element pathway. Planta medica, 80(5), 426-34.
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  Cells and tissues counteract insults from exogenous or endogenous carcinogens through the expression of genes encoding antioxidants and phase II detoxifying enzymes regulated by antioxidant response element promoter regions. Nuclear factor-erythroid 2-related factor 2 plays a key role in regulating the antioxidant response elements-target gene expression. Hence, the Nrf2/ARE pathway represents a vital cellular defense mechanism against damage caused by oxidative stress and xenobiotics, and is recognized as a potential molecular target for discovering chemopreventive agents. Using a stable antioxidant response element luciferase reporter cell line derived from human breast cancer MDA-MB-231 cells combined with a 96-well high-throughput screening system, we have identified a series of plant extracts from the family Lauraceae that harbor Nrf2-inducing effects. These extracts, including Litsea garrettii (ZK-08), Cinnamomum chartophyllum (ZK-02), C. mollifolium (ZK-04), C. camphora var. linaloolifera (ZK-05), and C. burmannii (ZK-10), promoted nuclear translocation of Nrf2, enhanced protein expression of Nrf2 and its target genes, and augmented intracellular glutathione levels. Cytoprotective activity of these extracts against two electrophilic toxicants, sodium arsenite and H2O2, was investigated. Treatment of human bronchial epithelial cells with extracts of ZK-02, ZK-05, and ZK-10 significantly improved cell survival in response to sodium arsenite and H2O2, while ZK-08 showed a protective effect against only H2O2. Importantly, their protective effects against insults from both sodium arsenite and H2O2 were Nrf2-dependent. Therefore, our data provide evidence that the selected plants from the family Lauraceae are potential sources for chemopreventive agents targeting the Nrf2/ARE pathway.
• Williams, J. D., Bermudez, Y., Park, S. L., Stratton, S. P., Uchida, K., Hurst, C. A., & Wondrak, G. T. (2014). Malondialdehyde-derived epitopes in human skin result from acute exposure to solar UV and occur in nonmelanoma skin cancer tissue. Journal of Photochemistry and Photobiology. B, Biology, 132, 56-65.
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  Cutaneous exposure to solar ultraviolet radiation (UVR) is a causative factor in photoaging and photocarcinogenesis. In human skin, oxidative stress is widely considered a key mechanism underlying the detrimental effects of acute and chronic UVR exposure. The lipid peroxidation product malondialdehyde (MDA) accumulates in tissue under conditions of increased oxidative stress, and the occurrence of MDA-derived protein epitopes, including dihydropyridine-lysine (DHP), has recently been substantiated in human skin. Here we demonstrate for the first time that acute exposure to sub-apoptogenic doses of solar simulated UV light (SSL) causes the formation of free MDA and protein-bound MDA-derived epitopes in cultured human HaCaT keratinocytes and healthy human skin. Immunohistochemical staining revealed that acute exposure to SSL is sufficient to cause an almost twenty-fold increase in general MDA- and specific DHP-epitope content in human skin. When compared to dose-matched solar simulated UVA, complete SSL was more efficient generating both free MDA and MDA-derived epitopes. Subsequent tissue microarray (TMA) analysis revealed the prevalence of MDA- and DHP-epitopes in nonmelanoma skin cancer (NMSC). In squamous cell carcinoma tissue, both MDA- and DHP-epitopes were increased more than threefold as compared to adjacent normal tissue. Taken together, these date demonstrate the occurrence of MDA-derived epitopes in both solar UVR-exposed healthy human skin and NMSC TMA tissue; however, the potential utility of these epitopes as novel biomarkers of cutaneous photodamage and a functional role in the process of skin photocarcinogenesis remain to be explored.
• Lamore, S. D., & Wondrak, G. T. (2013). UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts. Journal of Photochemistry and Photobiology B: Biology, 123, 1-12.
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  PMID: 23603447;PMCID: PMC3710731;Abstract: Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display 'UVA-mimetic' effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. © 2013 Elsevier B.V. All rights reserved.
• Lamore, S. D., & Wondrak, G. T. (2013). UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts. Journal of photochemistry and photobiology. B, Biology, 123, 1-12.
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  Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display 'UVA-mimetic' effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts.
• Qiao, S., Tao, S., Rojo, M., Park, S. L., Vonderfecht, A. A., Jacobs, S. L., Zhang, D. D., & Wondrak, G. T. (2013). The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- And chemotherapy-induced cell death. Autophagy, 9(12), 2087-2102.
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  Abstract: Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A , E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells. © 2013 Landes Bioscience.
• Tao, S., Justiniano, R., Zhang, D. D., & Wondrak, G. T. (2013). The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV. Redox biology, 1, 532-41.
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  Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photocarcinogenesis and photoaging, and an urgent need exists for improved strategies for skin photoprotection. The redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defense against environmental electrophilic insult, has recently emerged as an important determinant of cutaneous damage from solar UV, and the concept of pharmacological activation of Nrf2 has attracted considerable attention as a novel approach to skin photoprotection. In this study, we examined feasibility of using tanshinones, a novel class of phenanthrenequinone-based cytoprotective Nrf2 inducers derived from the medicinal plant Salvia miltiorrhiza, for protection of cultured human skin cells and reconstructed human skin against solar simulated UV. Using a dual luciferase reporter assay in human Hs27 dermal fibroblasts pronounced transcriptional activation of Nrf2 by four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A) and cryptotanshinone (CT)] was detected. In fibroblasts, the more potent tanshinones T-I and DHT caused a significant increase in Nrf2 protein half-life via blockage of ubiquitination, ultimately resulting in upregulated expression of cytoprotective Nrf2 target genes (GCLC, NQO1) with the elevation of cellular glutathione levels. Similar tanshinone-induced changes were also observed in HaCaT keratinocytes. T-I and DHT pretreatment caused significant suppression of skin cell death induced by solar simulated UV and riboflavin-sensitized UVA. Moreover, feasibility of tanshinone-based cutaneous photoprotection was tested employing a human skin reconstruct exposed to solar simulated UV (80 mJ/cm(2) UVB; 1.53 J/cm(2) UVA). The occurrence of markers of epidermal solar insult (cleaved procaspase 3, pycnotic nuclei, eosinophilic cytoplasm, acellular cavities) was significantly attenuated in DHT-treated reconstructs that displayed increased immunohistochemical staining for Nrf2 and γ-GCS together with the elevation of total glutathione levels. Taken together, our data suggest the feasibility of achieving tanshinone-based cutaneous Nrf2-activation and photoprotection.
• Tao, S., Zheng, Y., Lau, A., Jaramillo, M. C., Chau, B. T., Lantz, R. C., Wong, P. K., Wondrak, G. T., & Zhang, D. D. (2013). Tanshinone I activates the Nrf2-dependent antioxidant response and protects against As(III)-induced lung inflammation in vitro and in vivo. Antioxidants & redox signaling, 19(14), 1647-61.
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  The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators.
• Whitman, S. A., Long, M., Wondrak, G. T., Zheng, H., & Zhang, D. D. (2013). Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy. Experimental Cell Research, 319(17), 2673-2683.
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  PMID: 23896025;PMCID: PMC3809009;Abstract: The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy. Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1α, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1α protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment. We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy. © 2013 Elsevier Inc.
• Whitman, S. A., Long, M., Wondrak, G. T., Zheng, H., & Zhang, D. D. (2013). Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy. Experimental cell research, 319(17), 2673-83.
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  The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy. Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1α, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1α protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment. We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy.
• Wondrak, G., Davis, A. L., Cabello, C. M., Qiao, S., Azimian, S., & Wondrak, G. T. (2013). Phenotypic identification of the redox dye methylene blue as an antagonist of heat shock response gene expression in metastatic melanoma cells. International journal of molecular sciences, 14(2).
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  Repurposing approved and abandoned non-oncological drugs is an alternative developmental strategy for the identification of anticancer therapeutics that has recently attracted considerable attention. Due to the essential role of the cellular heat shock response in cytoprotection through the maintenance of proteostasis and suppression of apoptosis, small molecule heat shock response antagonists can be harnessed for targeted induction of cytotoxic effects in cancer cells. Guided by gene expression array analysis and a phenotypic screen interrogating a collection of 3,7-diamino-phenothiazinium derivatives, we have identified the redox-drug methylene blue (MB), used clinically for the infusional treatment of methemoglobinemia, as a negative modulator of heat shock response gene expression in human metastatic melanoma cells. MB-treatment blocked thermal (43 °C) and pharmacological (celastrol, geldanamycin) induction of heat shock response gene expression, suppressing Hsp70 (HSPA1A) and Hsp27 (HSPB1) upregulation at the mRNA and protein level. MB sensitized melanoma cells to the apoptogenic activity of geldanamycin, an Hsp90 antagonist known to induce the counter-regulatory upregulation of Hsp70 expression underlying cancer cell resistance to geldanamycin chemotherapy. Similarly, MB-cotreatment sensitized melanoma cells to other chemotherapeutics (etoposide, doxorubicin). Taken together, these data suggest feasibility of repurposing the non-oncological redox drug MB as a therapeutic heat shock response antagonist for cancer cell chemosensitization.
• Lamore, S. D., & Wondrak, G. T. (2012). Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 11(1), 163-72.
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  Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and α-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, α-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.
• Qiao, S., Lamore, S. D., Cabello, C. M., Lesson, J. L., Muñoz-Rodriguez, J. L., & Wondrak, G. T. (2012). Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes. Biochemical Pharmacology, 83(9), 1229-1240.
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  PMID: 22321511;PMCID: PMC3299892;Abstract: Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma. © 2012 Elsevier Inc.
• Qiao, S., Lamore, S. D., Cabello, C. M., Lesson, J. L., Muñoz-Rodriguez, J. L., & Wondrak, G. T. (2012). Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes. Biochemical pharmacology, 83(9), 1229-40.
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  Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B', HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.
• Wondrak, G. T., & Jacobson, E. L. (2012). Vitamin B6: beyond coenzyme functions. Sub-cellular biochemistry, 56, 291-300.
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  Endogenous reactive intermediates such as photoexcited states of tissue chromophores, reactive oxygen species (ROS), reactive carbonyl species (RCS), and transition metal ions are mediators of tissue damage involved in initiation and progression of human pathologies including tumorigenesis, atherosclerosis, diabetes, and neurodegenerative disease. A large body of evidence now suggests that B6 vitamers antagonize the harmful activity of endogenous reactive intermediates fulfilling a very different role than that established as a cofactor for numerous enzymes. In this chapter, the structural basis of vitamin B6 activity as a potent antioxidant, metal chelator, carbonyl scavenger, and photosensitizer is presented and the physiological relevance is discussed.
• Wondrak, G., Cabello, C. M., Lamore, S. D., Bair, W. B., Qiao, S., Azimian, S., Lesson, J. L., & Wondrak, G. T. (2012). The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis. Investigational new drugs, 30(4).
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  Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.
• Wondrak, G., Qiao, S., Cabello, C. M., Lamore, S. D., Lesson, J. L., & Wondrak, G. T. (2012). D-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis. Apoptosis : an international journal on programmed cell death, 17(10).
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  D-Penicillamine (3,3-dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2α, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.
• Wondrak, G., Cabello, C. M., Lamore, S. D., Bair, W. B., Davis, A. L., Azimian, S. M., & Wondrak, G. T. (2011). DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma. Free radical research, 45(3).
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  Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B', p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.
• Wondrak, G., Lamore, S. D., & Wondrak, G. T. (2011). Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 24(5).
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  Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309-322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm™) exposed to topical ZnPT (0.1-2% in Vanicream™), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm™ confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug.
• Zheng, H., Whitman, S. A., Wu, W., Wondrak, G. T., Wong, P. K., Fang, D., & Zhang, D. D. (2011). Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy. Diabetes, 60(11), 3055-66.
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  To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.
• Lamore, S. D., Azimian, S., Horn, D., Anglin, B. L., Uchida, K., Cabello, C. M., & Wondrak, G. T. (2010). The malondialdehyde-derived fluorophore DHP-lysine is a potent sensitizer of UVA-induced photooxidative stress in human skin cells. Journal of Photochemistry and Photobiology B: Biology, 101(3), 251-264.
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  PMID: 20724175;PMCID: PMC2963670;Abstract: Light-driven electron and energy transfer involving non-DNA skin chromophores as endogenous photosensitizers induces oxidative stress in UVA-exposed human skin, a process relevant to photoaging and photocarcinogenesis. Malondialdehyde is an electrophilic dicarbonyl-species derived from membrane lipid peroxidation. Here, we present experimental evidence suggesting that the malondialdehyde-derived protein epitope dihydropyridine (DHP)-lysine is a potent endogenous UVA-photosensitizer of human skin cells. Immunohistochemical analysis revealed the abundant occurrence of malondialdehyde-derived and DHP-lysine epitopes in human skin. Using the chemically protected dihydropyridine-derivative (2S)-Boc-2-amino-6-(3,5-diformyl-4-methyl-4H-pyridin-1-yl)-hexanoic acid-t-butylester as a model of peptide-bound DHP-lysine, photodynamic inhibition of proliferation and induction of cell death were observed in human skin Hs27 fibroblasts as well as primary and HaCaT keratinocytes exposed to the combined action of UVA and DHP-lysine. DHP-lysine photosensitization induced intracellular oxidative stress, p38 MAPkinase activation, and upregulation of heme oxygenase-1 expression. Consistent with UVA-driven ROS formation from DHP-lysine, formation of superoxide, hydrogen peroxide, and singlet oxygen was detected in chemical assays, but little protection was achieved using SOD or catalase during cellular photosensitization. In contrast, inclusion of NaN3 completely abolished DHP-photosensitization. Taken together, these data demonstrate photodynamic activity of DHP-lysine and support the hypothesis that malondialdehyde-derived protein-epitopes may function as endogenous sensitizers of UVA-induced oxidative stress in human skin. © 2010 Elsevier B.V.
• Lamore, S. D., Cabello, C. M., & Wondrak, G. T. (2010). HMGB1-Directed drug discovery targeting cutaneous inflammatory dysregulation. Current Drug Metabolism, 11(3), 250-265.
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  PMID: 20406187;Abstract: Extracellular cytokine function of the non-histone nuclear protein high-mobility group box 1 (HMGB1) has recently been recognized as an important drug target for novel anti-inflammatory therapeutics. Accumulating evidence supports the mechanistic involvement of the alarmin HMGB1 in skin response to microbial infection and ultraviolet-induced solar damage. Moreover, HMGB1 modulation of inflammatory signaling and tissue remodeling is now emerging as a causative factor in wound repair, autoimmune dysregulation, and skin carcinogenesis, representing cutaneous pathologies that affect large patient populations with unmet therapeutic needs. Recent structure-based drug discovery efforts have aimed at increasing the number of small molecule- and biologics-based prototype therapeutics targeting HMGB1. Small molecule drugs that may provide therapeutic benefit through HMGB1-directed mechanisms involve HMGB1 inhibitory ligands, Toll-like receptor antagonists, RAGE antagonists, α7 nicotinic acetylcholine receptor agonists, G2A antagonists, serine protease inhibitors, and α-dicarbonyl-based soft electrophiles. Using some of these agents, pharmacological modulation of HMGB1-associated cutaneous pathology has been achieved with an acceptable toxicity profile, and preclinical proof-of-concept experimentation has demonstrated feasibility of developing HMGB1-modulators into novel systemic and topical therapeutics that target cutaneous inflammatory dysregulation. © 2010 Bentham Science Publishers Ltd.
• Lamore, S. D., Cabello, C. M., & Wondrak, G. T. (2010). The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells. Cell Stress and Chaperones, 15(3), 309-322.
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  PMID: 19809895;PMCID: PMC2866994;Abstract: The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of γ-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin. © 2009 Cell Stress Society International.
• Lamore, S. D., Qiao, S., Horn, D., & Wondrak, G. T. (2010). Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts. Photochemistry and Photobiology, 86(6), 1307-1317.
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  PMID: 20946361;PMCID: PMC3001288;Abstract: Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference- gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin. © 2010 The Authors. Journal Compilation. The American Society of Photobiology.
• Wondrak, G. T., Villeneuve, N. F., Lamore, S. D., Bause, A. S., Jiang, T., & Zhang, D. D. (2010). The cinnamon-derived dietary factor cinnamic aldehyde activates the Nrf2-dependent antioxidant response in human epithelial colon cells. Molecules, 15(5), 3338-3355.
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  PMID: 20657484;PMCID: PMC3101712;Abstract: Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and γ-glutamyl-cysteine synthetase (γ-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis. © 2010 by the authors.
• Wondrak, G., Bair, W. B., Cabello, C. M., Uchida, K., Bause, A. S., & Wondrak, G. T. (2010). GLO1 overexpression in human malignant melanoma. Melanoma research, 20(2).
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  Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. On the basis of the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative reverse transcription polymerase chain reaction analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase I protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using two-dimensional proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target adduction by the glycolytic byproduct methylglyoxal in malignant melanoma.
• Bause, A. S., Lamore, S. D., & Wondrak, G. T. (2009). More than skin deep: The human skin tissue equivalent as an advanced drug discovery tool. Frontiers in Drug Design and Discovery, 4(1), 135-161.
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  Abstract: Advanced human tissue equivalents are an emerging drug discovery tool that aims at minimizing costly candidate attrition in later stages of preclinical and clinical development. 3-D cellular tissue models amenable to screening in 96-well plate format and drug monitoring in real time provide more accurate compound screening in a physiologically relevant context, not achievable in traditional two dimensional, single cell type-based assays. Currently, equivalents that represent the healthy or pathological physiology of human tissues including skin, vasculature, lung, prostate, and various malignant tumors are used for rapid activity screening, toxicity profiling, and pharmacokinetic characterization of compound libraries. Human skin equivalents with intact stratum corneum reconstructed in vitro from matrix-embedded dermal fibroblasts and epidermal primary keratinocytes closely resemble the complex architecture and functional complexity of skin. Using human skin equivalents, delivery of novel topical agents for photoprotection has been studied, and toxicity and activity profiling of skin care products and FDA-approved drugs including sunscreens has been performed. Importantly, human skin equivalents are better predictors of drug activity than available animal models as mouse and guinea pig skin do not reflect the architecture of human skin in terms of physico-optical properties such as light reflectance and scattering, epidermal thickness, cellularity, and biochemical composition. Recently, cellular composition of advanced commercial skin reconstructs has been further optimized by incorporation of melanogenic melanocytes and immunomodulatory dendritic (Langerhans) cells in order to assess drug modulation of cutaneous pigmentation, inflammation, photo-immunosuppression, and photo-carcinogenesis. Continuous progress in skin equivalent engineering will ensure the expanding role of skin equivalents in disease model-based assays for rapid identification and development of novel cutaneous therapeutics. © 2009 Bentham Science Publishers.
• Cabello, C. M., B., W., Bause, A. S., & Wondrak, G. T. (2009). Antimelanoma activity of the redox dye DCPIP (2,6-dichlorophenolindophenol) is antagonized by NQO1. Biochemical Pharmacology, 78(4), 344-354.
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  PMID: 19394313;PMCID: PMC2742658;Abstract: Altered redox homeostasis involved in the control of cancer cell survival and proliferative signaling represents a chemical vulnerability that can be targeted by prooxidant redox intervention. Here, we demonstrate that the redox dye 2,6-dichlorophenolindophenol (DCPIP) may serve as a prooxidant chemotherapeutic targeting human melanoma cells in vitro and in vivo. DCPIP-apoptogenicity observed in the human melanoma cell lines A375 and G361 was inversely correlated with NAD(P)H:quinone oxidoreductase (NQO1) expression levels. In A375 cells displaying low NQO1 activity, DCPIP induced apoptosis with procaspase-3 and PARP cleavage, whereas G361 cells expressing high levels of enzymatically active NQO1 were resistant to DCPIP-cytotoxicity. Genetic (siRNA) or pharmacological (dicoumarol) antagonism of NQO1 strongly sensitized G361 cells to DCPIP apoptogenic activity. DCPIP-cytotoxicity was associated with the induction of oxidative stress and rapid depletion of glutathione in A375 and NQO1-modulated G361 cells. Expression array analysis revealed a DCPIP-induced stress response in A375 cells with massive upregulation of genes encoding Hsp70B' (HSPA6), Hsp70 (HSPA1A), heme oxygenase-1 (HMOX1), and early growth response protein 1 (EGR1) further confirmed by immunodetection. Systemic administration of DCPIP displayed significant antimelanoma activity in the A375 murine xenograft model. These findings suggest feasibility of targeting tumors that display low NQO1 enzymatic activity using DCPIP. © 2009 Elsevier Inc. All rights reserved.
• Wondrak, G. T. (2009). Redox-directed cancer therapeutics: Molecular mechanisms and opportunities. Antioxidants and Redox Signaling, 11(12), 3013-3069.
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  PMID: 19496700;PMCID: PMC2824519;Abstract: Redox dysregulation originating from metabolic alterations and dependence on mitogenic and survival signaling through reactive oxygen species represents a specific vulnerability of malignant cells that can be selectively targeted by redox chemotherapeutics. This review will present an update on drug discovery, target identification, and mechanisms of action of experimental redox chemotherapeutics with a focus on pro-and antioxidant redox modulators now in advanced phases of preclinal and clinical development. Recent research indicates that numerous oncogenes and tumor suppressor genes exert their functions in part through redox mechanisms amenable to pharmacological intervention by redox chemotherapeutics. The pleiotropic action of many redox chemotherapeutics that involves simultaneous modulation of multiple redox sensitive targets can overcome cancer cell drug resistance originating from redundancy of oncogenic signaling and rapid mutation. Moreover, some redox chemotherapeutics may function according to the concept of synthetic lethality (i.e., drug cytotoxicity is confined to cancer cells that display loss of function mutations in tumor suppressor genes or upregulation of oncogene expression). The impressive number of ongoing clinical trials that examine therapeutic performance of novel redox drugs in cancer patients demonstrates that redox chemotherapy has made the crucial transition from bench to bedside. Antioxid. Redox Signal. 11, 3013-3069. © 2009 Mary Ann Liebert, Inc.
• Wondrak, G., Cabello, C. M., Bair, W. B., Lamore, S. D., Ley, S., Bause, A. S., Azimian, S., & Wondrak, G. T. (2009). The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth. Free radical biology & medicine, 46(2).
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  Redox dysregulation in cancer cells represents a chemical vulnerability that can be targeted by pro-oxidant redox intervention. Dietary constituents that contain an electrophilic Michael acceptor pharmacophore may therefore display promising chemopreventive and chemotherapeutic anti-cancer activity. Here, we demonstrate that the cinnamon-derived dietary Michael acceptor trans-cinnamic aldehyde (CA) impairs melanoma cell proliferation and tumor growth. Feasibility of therapeutic intervention using high doses of CA (120 mg/kg, po, daily, 10 days) was demonstrated in a human A375 melanoma SCID mouse xenograft model. Low-micromolar concentrations (IC(50)< 10 microM) of CA, but not closely related CA derivatives devoid of Michael acceptor activity, suppressed proliferation of human metastatic melanoma cell lines (A375, G361, LOX) with G1 cell-cycle arrest, elevated intracellular ROS, and impaired invasiveness. Expression array analysis revealed that CA induced an oxidative stress response in A375 cells, up-regulating heme oxygenase 1, sulfiredoxin 1 homolog, thioredoxin reductase 1, and other genes, including the cell-cycle regulator and stress-responsive tumor suppressor gene cyclin-dependent kinase inhibitor 1A, a key mediator of G1-phase arrest. CA, but not Michael-inactive derivatives, inhibited NF-kappaB transcriptional activity and TNFalpha-induced IL-8 production in A375 cells. These findings support a previously unrecognized role of CA as a dietary Michael acceptor with potential anti-cancer activity.
• Wondrak, G., Cabello, C. M., Bair, W. B., Ley, S., Lamore, S. D., Azimian, S., & Wondrak, G. T. (2009). The experimental chemotherapeutic N6-furfuryladenosine (kinetin-riboside) induces rapid ATP depletion, genotoxic stress, and CDKN1A(p21) upregulation in human cancer cell lines. Biochemical pharmacology, 77(7).
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  Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N(6)-furfuryladenosine (FAdo, kinetin-riboside) displays anti-proliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo exposure in MiaPaCa-2 pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N(6)-furfuryladenine (FA), displayed potent anti-proliferative activity that was also observed in human primary fibroblasts and keratinocytes. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60-180min of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, and GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase (ADK) suppressed FAdo cytotoxicity and also prevented ATP depletion and p21 upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative factors involved in FAdo cytotoxicity.
• Wang, X. J., Sun, Z., Villeneuve, N. F., Zhang, S., Zhao, F., Li, Y., Chen, W., Yi, X., Zheng, W., Wondrak, G. T., Wong, P. K., & Zhang, D. D. (2008). Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis, 29(6), 1235-43.
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  Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a 'good' transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2-small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.
• Wondrak, G. T., Cabello, C. M., Villeneuve, N. F., Zhang, S., Ley, S., Yanjie, L. i., Sun, Z., & Zhang, D. D. (2008). Cinnamoyl-based Nrf2-activators targeting human skin cell photo-oxidative stress. Free Radical Biology and Medicine, 45(4), 385-395.
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  PMID: 18482591;PMCID: PMC3710742;Abstract: Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress. © 2008 Elsevier Inc. All rights reserved.
• Wondrak, G., & Wondrak, G. T. (2008). Reactivity-based drug discovery using vitamin B(6)-derived pharmacophores. Mini reviews in medicinal chemistry, 8(5).
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  Endogenous reactive intermediates including photoexcited states of tissue chromophores, reactive oxygen species (ROS), reactive carbonyl species (RCS), transition metal ions, and Schiff bases have been implicated in the initiation and progression of diverse human pathologies including tumorigenesis, atherosclerosis, diabetes, and neurodegenerative disease. In contrast to structure-based approaches that target macromolecules by selective ligands, reactivity-based drug discovery uses chemical reagents as therapeutics that target reactive chemical species involved in human pathology. Reactivity-based design of prototype agents that effectively antagonize, modulate, and potentially even reverse the chemistry underlying tissue damage from oxidative and carbonyl stress therefore holds great promise in delivering significant therapeutic benefit. Apart from its established role as an essential cofactor for numerous enzymes, a large body of evidence suggests that B(6)-vitamers contain reactive pharmacophores that mediate therapeutically useful non-vitamin drug actions as potent antioxidants, metal chelators, carbonyl scavengers, Schiff base forming agents, and photosensitizers. Based on the fascinating chemical versatility of B(6)-derived pharmacophores, B(6)-vitamers are therefore promising lead compounds for reactivity-based drug design.
• Cabello, C. M., III, W. B., & Wondrak, G. T. (2007). Experimental therapeutics: Targeting the redox Achilles heel of cancer. Current Opinion in Investigational Drugs, 8(12), 1022-1037.
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  PMID: 18058573;Abstract: Reactive oxygen species (ROS) have recently emerged as promising targets for anticancer drug discovery. Constitutively elevated levels of cellular oxidative stress and dependence on mitogenic and anti-apoptotic ROS signaling represent a specific vulnerability of malignant cells that can be selectively targeted by novel pro- and antioxidant redox chemotherapeutics. This review discusses small-molecule anticancer redox drugs currently in various phases of preclinical and clinical development that are characterized by their unique mechanism of action, including small-molecule superoxide dismutase and catalase mimetics, bioreductively activated pro-oxidant redox catalysts, metal-based pro-oxidants, hypoxia-selective free radical precursors, and specific antagonists of the cancer cell antioxidant glutathione or thioredoxin redox systems. Based on ongoing redox biomarker discovery and validation, future redox phenotyping and genotyping may guide the selection of novel redox chemotherapeutics that efficiently target the redox Achilles heel of the individual tumor. © The Thomson Corporation.
• Wondrak, G. T. (2007). Let the sun shine in: Mechanisms and potential for therapeutics in skin photodamage. Current Opinion in Investigational Drugs, 8(5), 390-400.
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  PMID: 17520868;Abstract: Photoaging and photocarcinogenesis are the two Janus faces of skin photodamage. Reactivity-based design of prototype agents that antagonize, modulate and reverse the chemistry of skin photodamage holds promise in delivering unprecedented therapeutic benefit. In contrast to structure-based approaches that use selective ligands to target macromolecules, reactivity-based drug discovery uses chemical reagents as therapeutics to target reactive chemical species as key mediators of skin photo-oxidative stress. The following classes of reactivity-based agents for skin photoprotection can be distinguished based on their mechanism of action: direct antagonists of photo-oxidative stress (sunscreens, quenchers of photo-excited states, antioxidants, redox modulators and glycation inhibitors) and skin photo-adaptation inducers (nuclear factor erythroid 2-related factor 2 [Nrf2] activators, heat-shock response inducers and metallothionein inducers). © The Thomson Corporation.
• Wondrak, G. T. (2007). NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis. Free Radical Biology and Medicine, 43(2), 178-190.
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  PMID: 17603928;PMCID: PMC2705808;Abstract: Altered redox signaling and regulation in cancer cells represent a chemical vulnerability that can be targeted by selective chemotherapeutic intervention. Here, we demonstrate that 3,7-diaminophenothiazinium-based redox cyclers (PRC) induce selective cancer cell apoptosis by NAD(P)H:quinone oxidoreductase (NQO1)-dependent bioreductive generation of cellular oxidative stress. Using PRC lead compounds including toluidine blue against human metastatic G361 melanoma cells, apoptosis occurred with phosphatidylserine externalization, loss of mitochondrial transmembrane potential, cytochrome c release, caspase-3 activation, and massive ROS production. Consistent with reductive activation and subsequent redox cycling as the mechanism of PRC cytotoxicity, coincubation with catalase achieved cell protection, whereas reductive antioxidants enhanced PRC cytotoxicity. Unexpectedly, human A375 melanoma cells were resistant to PRC-induced apoptosis, and PRC-sensitive G361 cells were protected by preincubation with the NQO1 inhibitor dicoumarol. Indeed, NQO1 specific enzymatic activity was 9-fold higher in G361 than in A375 cells. The critical role of NQO1 in PRC bioactivation and cytotoxicity was confirmed, when NQO1-transfected breast cancer cells (MCF7-DT15) stably overexpressing active NQO1 displayed strongly enhanced PRC sensitivity as compared to vector control-transfected cells with baseline NQO1 activity. Based on the known overexpression of NQO1 in various tumors these findings suggest the feasibility of developing PRC lead compounds into tumor-selective bioreductive chemotherapeutics. © 2007 Elsevier Inc. All rights reserved.
• Wondrak, G. T., Jacobson, M. K., & Jacobson, E. L. (2006). Antimelanoma activity of apoptogenic carbonyl scavengers. Journal of Pharmacology and Experimental Therapeutics, 316(2), 805-814.
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  PMID: 16210394;Abstract: Therapeutic induction of apoptosis is an important goal of anticancer drug design. Cellular carbonyl stress mediated by endogenous reactive carbonyl species (RCS) such as glyoxal and methylglyoxal (MG) affects proliferative signaling and metastasis of human tumor cells. Recent research suggests that RCS produced constitutively during increased tumor cell glycolysis may be antiapoptotic survival factors and thus represent a novel molecular target for anticancer intervention. Here, we demonstrate the tumor cell-specific apoptogenicity of carbonyl scavengers, which act by covalently trapping RCS, against human (A375, G361, and LOX) and murine (B16) melanoma cell lines. A structure-activity relationship study identified nucleophilic carbonyl scavenger pharmacophores as the functional determinants of apoptogenic antimelanoma activity of structurally diverse agents such as 3,3-dimethyl-D-cysteine and aminoguanidine. Previous work has demonstrated that covalent adduction of protein-arginine residues in the mitochondrial permeability transition (MPT) pore and heat shock protein 27 by intracellular MG produced in tumor cell glycolysis inhibits mitochondrial apoptosis and enhances cancer cell survival. Indeed, in various melanoma cell lines, carbonyl scavenger-induced apoptosis was antagonized by pretreatment with the membrane-permeable RCS phenylglyoxal (PG). Carbonyl scavenger-induced apoptosis was associated with early loss of mitochondrial transmembrane potential, and cyclosporin A antagonized the effects of carbonyl scavengers, suggesting a causative role of MPT pore opening in carbonyl scavenger apoptogenicity. Consistent with RCS inhibition of mitochondrial apoptosis in melanoma cells, staurosporine-induced apoptosis also was suppressed by PG pretreatment. Our results suggest that carbonyl scavengers acting as direct molecular antagonists of RCS are promising apoptogenic prototype agents for antimelanoma drug design. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.
• Wondrak, G. T., Jacobson, M. K., & Jacobson, E. L. (2006). Endogenous UVA-photosensitizers: Mediators of skin photodamage and novel targets for skin photoprotection. Photochemical and Photobiological Sciences, 5(2), 215-237.
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  PMID: 16465308;Abstract: Endogenous chromophores in human skin serve as photosensitizers involved in skin photocarcinogenesis and photoaging. Absorption of solar photons, particularly in the UVA region, induces the formation of photoexcited states of skin photosensitizers with subsequent generation of reactive oxygen species (ROS), organic free radicals and other toxic photoproducts that mediate skin photooxidative stress. The complexity of endogenous skin photosensitizers with regard to molecular structure, pathways of formation, mechanisms of action, and the diversity of relevant skin targets has hampered progress in this area of photobiology and most likely contributed to an underestimation of the importance of endogenous sensitizers in skin photodamage. Recently, UVA-fluorophores in extracellular matrix proteins formed posttranslationally as a consequence of enzymatic maturation or spontaneous chemical damage during chronological and actinic aging have been identified as an abundant source of light-driven ROS formation in skin upstream of photooxidative cellular stress. Importantly, sensitized skin cell photodamage by this bystander mechanism occurs after photoexcitation of sensitizers contained in skin structural proteins without direct cellular photon absorption thereby enhancing the potency and range of phototoxic UVA action in deeper layers of skin. The causative role of photoexcited states in skin photodamage suggests that direct molecular antagonism of photosensitization reactions using physical quenchers of photoexcited states offers a novel chemopreventive opportunity for skin photoprotection. © The Royal Society of Chemistry and Owner Societies 2006.
• Jacobson, E. L., Kim, H., Kim, M., Wondrak, G. T., & Jacobson, M. K. (2005). Developing topical prodrugs for skin cancer prevention. Fundamentals of Cancer Prevention, 215-237.
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  Abstract: The skin plays multiple roles in protection from environmental insults yet skin damage, particularly that derived from sunlight, constitutes a major public health problem. End stage skin damage in the form of non-melanoma skin cancers (NMSC) are the most frequent malignancies in the United States with more than 1,000,000 cases diagnosed annually (Karagas et al. 1999). Melanoma skin cancer is the most rapidly increasing cancer. Actinic keratosis (AK), skin lesions that can progress to NMSC are far more prevalent than skin cancers. The occurrence of DNA damage and cellular responses to DNA damage are major determinants of skin damage including skin cancer (Ames 2001; Ullrich 2002). A compelling body of evidence now indicates that there are multiple targets for reducing skin damage and that several key micronutrients are candidates for skin damage prevention. However, a major challenge for the development of prevention strategies for skin damage is the difficulty of delivering micronutrients to skin. Delivery to skin via the blood circulation of nutrients taken orally is inherently inefficient since this delivery is distal to other organs, particularly the liver, which removes many agents by first pass metabolism. In addition the major cell targets for prevention of skin cancer are located in the epidermis, which is non-vascular. Described here are strategies to limit skin damage and thus skin cancer by targeting multiple mechanisms that include preventing DNA damage, enhancing DNA repair, preventing immune suppression, and preventing migration of transformed cells from epidermis to dermis. Further, an approach for delivery of key protective agents to skin cells using prodrugs specifically tailored for topical delivery is described. Finally, this approach is illustrated using niacin as a model micronutrient demonstrating that topical delivery of this polar compound to skin cells via prodrugs is feasible and that targeted delivery provides prevention benefit for skin. © 2008 Springer-Verlag Berlin Heidelberg.
• Wondrak, G. T., Jacobson, M. K., & Jacobson, E. L. (2005). Identification of quenchers of photoexcited states as novel agents for skin photoprotection. Journal of Pharmacology and Experimental Therapeutics, 312(2), 482-491.
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  PMID: 15475591;Abstract: Photooxidative stress is a key mechanism in UVA-induced skin photodamage. Photoexcited states of endogenous UVA chromophores such as porphyrins, melanin precursors, and crosslink-fluorophores of skin collagen exert skin photodamage by direct reaction with substrate molecules (type I photosensitization) or molecular oxygen (type II), leading to formation of reactive oxygen species. Based on our previous research on the role of photoexcited states of endogenous skin chromophores as sensitizers of photooxidative stress, we describe here the identification of a novel class of chemopreventive agents for topical skin photoprotection: quenchers of photoexcited states (QPES). QPES compounds antagonize the harmful excited state chemistry of endogenous sensitizers by physical quenching, facilitating the harmless return of the sensitizer excited state to the electronic ground state by energy dissipation. To identify QPES compounds suitable for development, we designed a primary screening assay based on QPES suppression of photosensitized plasmid cleavage using conditions that exclude antioxidants. This screen is followed with a screen to test for nonsacrificial quenching of dye-sensitized singlet oxygen (1O 2) formation by electron paramagnetic resonance detection of 2,2,6,6-tetramethyl-piperidine-1-oxyl, a stable free radical indicative of 1O2 formation. These initial screens identified a pyrrolidine pharmacophore with pronounced QPES activity, and L-proline and other noncytotoxic proline derivatives containing this pharmacophore were then screened for efficacy in cellular models of sensitized photodamage. These compounds showed QPES protection against dye-sensitized and psoralen-UVA-induced apoptosis and suppression of proliferation in cultured human skin keratinocytes and fibroblasts. Furthermore, QPES photoprotection of reconstructed full thickness human skin exposed to solar simulated light has been demonstrated.
• Wondrak, G. T., Roberts, M. J., Jacobson, M. K., & Jacobson, E. L. (2004). 3-Hydroxypyridine chromophores are endogenous sensitizers of photooxidative stress in human skin cells. Journal of Biological Chemistry, 279(29), 30009-30020.
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  PMID: 15133022;Abstract: Photocarcinogenesis and photoaging are established consequences of chronic exposure of human skin to solar irradiation. Accumulating evidence supports a causative involvement of UVA irradiation in skin photo-damage. UVA photodamage has been attributed to photosensitization by endogenous skin chromophores leading to the formation of reactive oxygen species and organic free radicals as key mediators of cellular photooxidative stress. In this study, 3-hydroxypyridine derivatives contained in human skin have been identified as a novel class of potential endogenous photosensitizers. A structure-activity relationship study of skin cell photosensitization by endogenous pyridinium derivatives (pyridinoline, desmosine, pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5′-phosphate) and various synthetic hydroxypyridine isomers identified 3-hydroxypyridine and N-alkyl-3-hydroxypyridinium cation as minimum phototoxic chromophores sufficient to effect skin cell sensitization toward UVB and UVA, respectively. Photosensitization of cultured human skin keratinocytes (HaCaT) and fibroblasts (CF3) by endogenous and synthetic 3-hydroxypyridine derivatives led to a dose-dependent inhibition of proliferation, cell cycle arrest in G2/M, and induction of apoptosis, all of which were reversible by thiol antioxidant intervention. Enhancement of UVA-induced intracellular peroxide formation and p38 mitogen-activated protein kinase-dependent stress signaling suggest a photooxidative mechanism of skin cell photosensitization by 3-hydroxypyridine derivatives. 3-Hydroxypyridine derivatives were potent photosensitizers of macromolecular damage, effecting protein (RNase A) photocross-linking and peptide (melittin) photooxidation with incorporation of molecular oxygen. Based on these results, we conclude that 3-hydroxypyridine derivatives comprising a wide range of skin biomolecules, such as enzymatic collagen cross-links, B6 vitamers, and probably advanced glycation end products in chronologically aged skin constitute a novel class of UVA photosensitizers, capable of skin photooxidative damage.
• Roberts, M. J., Wondrak, G. T., Laurean, D. C., Jacobson, M. K., & Jacobson, E. (2003). DNA damage by carbonyl stress in human skin cells. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 522(1-2), 45-56.
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  PMID: 12517411;Abstract: Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of Nε-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress. © 2002 Elsevier Science B.V. All rights reserved.
• Wondrak, G. T., Roberts, M. J., Cervantes-Laurean, D., Jacobson, M. K., & Jacobson, E. L. (2003). Proteins of the extracellular matrix are sensitizers of photo-oxidative stress in human skin cells. Journal of Investigative Dermatology, 121(3), 578-586.
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  PMID: 12925218;Abstract: Sensitized production of reactive oxygen species after photo-excitation of endogenous chromophores is thought to contribute to skin photo-oxidative stress. Here we present experimental evidence in support of a potential role of extracellular matrix proteins as skin photosensitizers. Human and bovine type I collagen and elastin sensitized of hydrogen peroxide generation upon irradiation with solar simulated light or ultraviolet A. Induction of intracellular oxidative stress by extracellular matrix-protein sensitization was demonstrated by flow cytometric analysis of fibroblasts preloaded with the intracellular redox dye dihydrorhodamine 123 and exposed to pre-irradiated type I collagen. Pre-irradiated collagen and elastin induced pronounced inhibition of proliferation in cultured keratinocytes and fibroblasts, which was reversed by antioxidant or catalase treatment and reproduced by exposure to concentrations of H2O2 formed during extracellular matrix-protein irradiation. In fibroblasts, chromosomal DNA damage as a consequence of collagen-sensitized H2O2 formation was demonstrated using a single cell electrophoresis assay. The enzymatic cross-links pyridinoline and desmosine were examined as candidate sensitizer chromophores contained in collagen and elastin, respectively. Pyridinoline, but not desmosine, sensitized light-driven H2O2 production and inhibition of fibroblast proliferation. Our results support the hypothesis that extracellular matrix proteins play a functional role in skin photoaging and carcinogenesis by sensitization of photo-oxidative damage.
• Wondrak, G. T., Cervantes-Laurean, D., Roberts, M. J., Qasem, J. G., Kim, M., Jacobson, E. L., & Jacobson, M. K. (2002). Identification of α-dicarbonyl scavengers for cellular protection against carbonyl stress. Biochemical Pharmacology, 63(3), 361-373.
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  PMID: 11853687;Abstract: Tissue deterioration and aging have long been associated with the accumulation of chemically induced protein and DNA damage. Reactive oxygen species (ROS) and reactive carbonyl species (RCS), especially α-dicarbonyl compounds, are key mediators of damage caused by oxidative stress, glycation, and UV-irradiation. The toxic effects of ROS are counteracted in vivo by antioxidants and antioxidant enzymes, and the deleterious effects of one RCS, methylglyoxal, are counteracted by a ubiquitous glyoxalase system. Carbonyl stress as a result of toxic effects of various mono-dicarbonyls (e.g. 4-hydroxynonenal) and α-dicarbonyls (e.g. glyoxal and deoxyosones) cannot be directly antagonized by antioxidants, and only a small number of biological carbonyl scavengers like glutathione (GSH) have been identified to date. We have developed a new screening method for the identification of carbonyl scavengers using a rapid glycation system that proceeds independent of oxygen and therefore, excludes identification of inhibitory compounds acting as antioxidants. Using this screening assay adapted to 96-well microtiter plates, we have identified the cysteine derivative 3,3-dimethyl-D-cysteine as a potent inhibitor of non-oxidative advanced glycation. Comparative kinetic analyses demonstrated the superior α-oxoaldehyde-scavenging activity of D-penicillamine over that of aminoguanidine. D-Penicillamine traps α-oxoaldehydes by forming a 2-acylthiazolidine derivative as shown by structure elucidation of reaction products between D-penicillamine and methylglyoxal or phenylglyoxal. We demonstrated that upon co-incubation, D-penicillamine protects human skin keratinocytes and fibroblasts (CF3 cells) against glyoxal- and methylglyoxal-induced carbonyl toxicity. Our research qualifies α-amino-β-mercapto-β,β-dimethyl-ethane as a promising pharmacophore for the development of related α-dicarbonyl scavengers as therapeutic agents to protect cells against carbonyl stress. © 2002 Published by Elsevier Science Inc.
• Wondrak, G. T., Jacobson, E. L., & Jacobson, M. K. (2002). Photosensitization of DNA damage by glycated proteins. Photochemical and Photobiological Sciences, 1(5), 355-363.
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  PMID: 12653475;Abstract: Photosensitized DNA damage in skin is thought to be an important mechanism of UV phototoxicity. Here we demonstrate that proteins modified by advanced glycation endproducts (AGE-proteins) are photosensitizers of DNA damage and show that multiple mechanisms are involved in AGE-sensitization. AGE-chromophores accumulate on long-lived skin proteins such as collagen and elastin as a consequence of glycation, the spontaneous amino-carbonyl reaction of protein-bound lysine and arginine residues with reactive carbonyl species. AGE-proteins accumulate in both the nucleus and the cytoplasm of mammalian cells. To test the hypothesis that protein-bound AGEs in close proximity to DNA are potent UV-photosensitizers, a simple plasmid DNA cleavage assay was established. Irradiation of supercoiled ΦX 174 DNA with solar simulated light in the presence of AGE-modified bovine serum albumin or AGE-modified RNAse A induced DNA single strand breaks. The sensitization potency of the glycated protein correlated with increased AGE-modification and the unmodified protein displayed no photosensitizing activity. AGE-sensitized formation of reactive oxygen species was not fully responsible for the observed DNA damage and other mechanisms such as direct electron transfer interaction between photoexcited AGE and DNA are likely to be involved. Glycated proteins in skin may equally function as potent photosensitizers of DNA damage with implications for photoaging and photocarcinogenesis.
• Wondrak, G. T., Roberts, M. J., Jacobson, M. K., & Jacobson, E. L. (2002). Photosensitized growth inhibition of cultured human skin cells: Mechanism and suppression of oxidative stress from solar irradiation of glycated proteins. Journal of Investigative Dermatology, 119(2), 489-498.
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  PMID: 12190875;Abstract: Chronic exposure to sunlight plays a role in skin aging and carcinogenesis. The molecular mechanisms of photodamage by ultraviolet A, the sunlight's major ultraviolet constituent, are poorly understood. Here we provide evidence that advanced glycation end products on proteins are sensitizers of photo-oxidative stress in skin cells. Glycation is a process of protein damage by reducing sugars and other reactive carbonyl species leading to the formation of advanced glycation end products, which accumulate on long-lived proteins such as dermal elastin and collagen during skin aging. Growth inhibition as a result of advanced glycation end product photosensitization of ultraviolet A and solar-simulated light was demonstrated in human keratinocytes and fibroblasts. Using advanced glycation end product bovine serum albumin and advanced glycation end product collagen as model photosensitizers, ultraviolet A- induced formation of H2O2 was identified as the key mediator of skin cell growth inhibition as evidenced by complete protection by catalase treatment and equivalent growth inhibition of unirradiated cells treated with pre-irradiated advanced glycation end product protein. D-penicillamine protected against advanced glycation end product-photosensitized growth inhibition even when added following irradiation, suggesting the feasibility of therapeutic approaches for protection against skin ultraviolet A damage. Photosensitized growth inhibition increased with the degree of advanced glycation end product modification paralleled by the amount of H2O2 formed upon solar-simulated light irradiation of the protein. Photosensitization was not observed using bovine serum albumin modified with the major advanced glycation end product, Nε-carboxymethyl-L-lysine, ruling out effects of cellular advanced glycation end product receptor (RAGE) stimulation. In contrast to bovine serum albumin, unglycated collagen showed photosensitization in CF3 fibroblasts and generation of H2O2 upon solar-simulated light irradiation. This study supports the hypothesis that advanced glycation end product-modified proteins are endogenous sensitizers of photo-oxidative cell damage in human skin by ultraviolet A-induced generation of reactive oxygen species contributing to photoaging and photocarcinogenesis.
• Jacobson, E. L., Giacomoni, P. U., Roberts, M. J., Wondrak, G. T., & Jacobson, M. K. (2001). Chapter 34 Metabolic effects of solar radiation and enhancers of energy metabolism. Comprehensive Series in Photosciences, 3(C), 677-690.
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  Abstract: Ionizing- and ultraviolet radiation provoke cell damage or death in a sequel that begins with DNA damage followed by NAD consumption. Several investigators have shown that interruption of the Embden-Meyerhof pathway and the depletion of cellular ATP occur subsequently. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteosome that degrades damaged nuclear proteins including histones. Identifying ways to optimize the function of PARPs is likely to be very important in minimizing the deleterious effects of solar radiation on skin. © 2001 Elsevier B.V. All rights reserved.
• Jacobson, E. L., Giacomoni, P. U., Roberts, M. J., Wondrak, G. T., & Jacobson, M. K. (2001). Optimizing the energy status of skin cells during solar radiation. Journal of Photochemistry and Photobiology B: Biology, 63(1-3), 141-147.
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  PMID: 11684461;Abstract: Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin. © 2001 Elsevier Science B.V. All rights reserved.
• Wondrak, G. T., Cervantes-Laurean, D., Jacobson, E. L., & Jacobson, M. K. (2000). Histone carbonylation in vivo and in vitro. Biochemical Journal, 351(3), 769-777.
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  PMID: 11042133;PMCID: PMC1221418;Abstract: Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N'-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.
• Wondrak, G. T., Varadarajan, S., Butterfield, D. A., & Jacobson, M. K. (2000). Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1. Free Radical Biology and Medicine, 29(6), 557-567.
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  PMID: 11025199;Abstract: Glycation, the nonenzymatic reaction between protein amino groups and reducing sugars, induces protein damage that has been linked to several pathological conditions, especially diabetes, and general aging. Here we describe the direct identification of a protein-bound free radical formed during early glycation of histone H1 in vitro. Earlier EPR analysis of thermal browning reactions between free amino acids and reducing sugars has implicated the sugar fragmentation product glycolaldehyde in the generation of a 1,4-disubstituted pyrazinium free radical cation. In order to evaluate the potential formation of this radical in vivo, the early glycation of BSA, lysozyme, and histone H1 by several sugars (D-glucose, D-ribose, ADP-ribose, glycolaldehyde) under conditions of physiological pH and temperature was examined by EPR. The pyrazinium free radical cation was identified on histone H1 glycated by glycolaldehyde (g = 2.00539, a(N) = 8.01 [2N], a(H) = 5.26 [4H], a(H) = 2.72 [4H]), or ADP-ribose. Reaction of glycolaldehyde with poly- L-lysine produced an identical signal, whereas reaction with BSA or lysozyme produced only a minor unresolved singlet signal. In the absence of oxygen the signal was stable over several days. Our results raise the possibility that pyrazinium radicals may form during glycation of histone H1 in vivo. (C) 2000 Elsevier Science Inc.
• Tressl, R., Wondrak, G. T., Garbe, L., Krüger, R., & Rewicki, D. (1998). Pentoses and Hexoses as Sources of New Melanoidin-like Maillard Polymers. Journal of Agricultural and Food Chemistry, 46(5), 1765-1776.
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  Abstract: N-Substituted pyrroles (1), 2-furaldehyde (2), and N-substituted 2-formylpyrroles (3), formed in pentose (hexose) Maillard systems, were identified as components of extraordinary polycondensation activity. The polycondensation was studied in model experiments with N-methylpyrrole (1a)/N-methyl-2-formylpyrrole (3a), N-(2-methoxycarbonylethyl)pyrrole (1b)/N-(2-methoxycarbonylethyl)-2-formylpyrrole (3b), N-methyl-2-formylpyrrole (3a), N-methylpyrrole (1a)/2-furaldehyde (2), and N-(2-methoxycarbonylethyl)pyrrole (1b)/2-furaldehyde (2), respectively. MALDI-TOF-MS spectra indicated regular oligomers of up to 15-30 methine-bridged N-methyl(or N-2-methoxycarbonylethyl)-pyrroles. With participation of aldehyde 2, furan rings instead of pyrrole rings were incorporated. The oligomers 5-11 were isolated and identified by MS and NMR techniques. A complementary experiment with N-methyl-2-[13C]formylpyrrole ([13CHO]-3a)/N-methylpyrrole (1a) was performed. The relevance of the new (type II) melanoidin-like oligomers/polymers in Maillard reactions is discussed and, in conclusion, a corresponding structure for native melanoidins is proposed. The oligomers 5, 6, 8, and 9 were tested for antioxidative activity in an iron(III) thiocyanate assay.
• Tressll, R., Wondrak, G., Kersten, E., Kriiger, R. P., & Rewicki, D. (1998). Identification of Maillard Type Polymers with Antioxidative Activity. ACS Symposium Series, 702, 209-220.
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  Abstract: In a series of 13C-isotopic labelling experiments, Maillard reaction pathways of intact or fragmented sugars to N-alkylpyrroles and furan compounds were elucidated. The polymerizing activity of these compounds has been investigated. Complex mixtures of polymers and individual oligomers were characterized by FAB-/MALDI-TOF-MS and 1H/13C-NMR spectroscopy. The antioxidative activity of individual compounds was assessed by the Fe(III)-thiocyanate method and the DPPH-reduction assay.
• Wondrak, G. T., Tressl, R., & Rewicki, D. (1997). Maillard Reaction of Free and Nucleic Acid-Bound 2-Deoxy-D-ribose and D-Ribose with ω-Amino Acids. Journal of Agricultural and Food Chemistry, 45(2), 321-327.
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  Abstract: The Maillard reaction of free and nucleic acid-bound 2-deoxy-D-ribose and D-ribose with ω-amino acids (4-aminobutyric acid, 6-aminocaproic acid) was investigated under both stringent and mild conditions. Without (with) amines 2-deoxy-D-ribose (D-ribose) displays the strongest browning activity, and DNA is much more reactive than RNA. From stringent reaction between 2-deoxy-D-ribose (or DNA) and methyl 4-aminobutyrate, methyl 4-[2-[(oxopyrrolidinyl)methyl]-1-pyrrolyl]-butyrate (12) was identified by GC/MS and NMR as a new 2-deoxy-D-ribose specific key compound trapped by pyrrolidone formation. Levulinic acid-related N-substituted lactames 13-15 were identified as predominant products from DNA with amino acids, whereas RNA paralleled the reaction with D-ribose. α-Angelica lactone (2), a significant degradation product of DNA, and thiols leads under mild conditions to new addition products (e.g., 17 with glutathione). Probable reaction pathways considering activating effects of the polyphosphate backbone of nucleic acids are discussed.
• Wondrak, G., Pier, T., & Tressl, R. (1995). Light from Maillard reaction: photon counting, emission spectrum, photography and visual perception.. Journal of bioluminescence and chemiluminescence, 10(5), 277-284.
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  PMID: 8533609;Abstract: Several authors have reported on high-sensitivity measurement of oxygen-dependent low-level chemiluminescence (CL) from Maillard reactions (MR), i.e. nonenzymatic amino-carbonyl reactions between reducing sugars and amino acids (also referred to as nonenzymatic browning). Here we report for the first time, that light from Maillard reactions can be seen by the human eye and also can be photographed. In parallel with visual perception and photography CL was monitored by means of a CL-detection programme of a liquid scintillation counter (LSC, single photon rate counting). CL emission spectrum was recorded by a monochromator-microchannel plate photomultiplier arrangement. CL intensity from reaction of 6-aminocaproic acid with D-ribose (200 mg each) in 5 mL H2O at pH 11 at 95 degrees C was high enough for visual perception after adaptation to absolute darkness. Reaction in dimethylsulphoxide (DMSO) exhibited strongly enhanced CL (10 mg each in 5 mL were sufficient for visual detection) and could be photographed (15 minutes' exposure, ASA 6400); all characteristics of Maillard specific CL (O2-dependence, no CL from nonreducing sugars, inhibition by sulphur compounds) remained. Visual detection of CL and measurement by LSC were in full concordance. The CL emission spectrum showed two broad peaks at around 500 nm and 695 nm. Fluorescence emission of the brown reaction mixture matched the blue-green part of the CL emission spectrum. Emission of visible light during Maillard reactions may partly originate from oxygen-dependent generation of excited states and energy transfer to simultaneously formed fluorescent products of the browning reaction.
• Tressl, R., Wondrak, G., Kersten, E., & Rewicki, D. (1994). Structure and potential cross-linking reactivity of a new pentose-specific Maillard product. Journal of Agricultural and Food Chemistry, 42(12), 2692-2697.
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  Abstract: The Maillard reaction of model compounds for peptide-bound lysine (4-aminobutyric acid, 6-aminocaproic acid, Nα-acetyl-L-lysine) with reducing sugars (D-ribose, D-xylose, D-arabinose, D-glucose, D-fructose, D-glyceraldehyde) was investigated under both stringent and mild conditions. With pentoses the corresponding ω-(dimethylmaleimido)carboxylic acids 1-3 were identified as substantial, hitherto unknown components by GC/MS and NMR. Their structure was confirmed by synthesis. A pathway to compounds 1-3 was derived from the results of 4-aminobutyric acid/[1-13C]-D-arabinose isotopic labeling experiments. The new N-(dimethylmaleoyl)-ω-aminocarboxylic acid derivatives are shown to react under very mild conditions (pH 7, 32°C) with thiols (methyl 3-mercaptopropionate, Nα-acetyl-L-cysteine methyl ester) to form the addition products 4-6. This reaction demonstrates that compounds of type 1-3 can be considered as potential protein cross-linkers in Maillard reactions. © 1994 American Chemical Society.

Presentations

• Dickinson, S. E., & Wondrak, G. T. (2021, September). Topical Prevention of Skin Photocarcinogenesis: Targeting Toll-like Receptor 4 (TLR4) and Beyond. Translational Advances in Cancer Prevention Agent Development (TACPAD) Virtual Workshop on Immunomodulatory Agents. Virtual: Division of Cancer Prevention, National Cancer Institute.
• Wondrak, G. T. (2018, May 12-15). invited speaker & session chair: 2018 ASP: ‘Novel Molecular Strategies in Skin Photoprotection’, oral presentation. 2018 Biennual Meeting, American Society for Photobiology, Tampa , FL.
• Wondrak, G. T. (2018, May 12-15). invited speaker: 'From AGEs to RAGE to TLR4: Pharmacological Modulation of Skin Stress Response Pathways for Photoprotection and Photochemoprevention'; oral presentation. Ventana/Roche, headquarters, Tucson, AZVentana.
• Wondrak, G. T. (2018, OCT 28 – 30). invited speaker: 'Molecular actors in multiple roles: Photodynamic and receptor-directed activities of endogenous skin photosensitizers' ; oral presentation. THE 13TH INTERNATIONAL SKIN CARCINOGENESIS CONFERENCE (ISCC) OCTOBER 28 – 30, 2018, The University of Texas at AustinUniversity of Texas at Austin.
• Wondrak, G. T. (2018, Oct 09). invited speaker: 'Redox-directed drug discovery targeting skin photodamage and carcinogenesis' ; oral presentation. UAB Comprehensive Cancer Center, Birmingham, ALUniversity of Alabama at Birmingham.
• Wondrak, G. T. (2018, Sep 6-9). invited speaker, session chair, & organizer: 'The skin exposome: Pharmacological modulation of cellular stress response pathways for skin photoprotection and photochemoprevention'; oral presentation. 8th International Conference on Oxidative Stress in Skin Medicine and Biology; The University of Athens, Greece & OCC (Oxygen Club of California). Andros, Greece: OCC (Oxygen Club of California) & University of Athens, Greece.
• Wondrak, G. T. (2017, April 6-7). invited speaker & session chair: 2017 ASP Presidential Evening Symposia: ‘Photo-excited States: From Tissue Damage to Photomedicine’, oral presentation. 2017 Presidential Evening Symposia, American Society for Photobiology, San Diego , CA.
• Wondrak, G. T. (2017, April 7). invited speaker: 'Harnessing the Endogenous Tryptophan-derived Photosensitizer and AhR-ligand 6-formylindolo[3,2-b]carbazole (FICZ) for Experimental Photodynamic Intervention’, oral presentation. 2017 Annual Meeting, American Society for Laser Medicine and Surgery [ASLMS], San Diego, CA.
• Wondrak, G. T. (2017, February 12-15). invited speaker: 'Glycation Damage and Excited States’, oral presentation. THE BANBURY CENTER, COLD SPRING HARBOR LABORATORY, NY - 'Chemiexcitation in Human Disease'.
• Wondrak, G. T. (2017, September 5). invited keynote speaker: ‘From photo-excited states to molecular interventions: Targeting skin photo-oxidative stress’, oral presentation. 2017 European Society for Photobiology [ESP], Pisa , Italy.
• Wondrak, G. T., & Dickinson, S. (2017, April 26-29). session chair - 'skin pharmacology' and presenter: 'A novel strategy for topical photochemoprevention: Pharmacological TLR4 antagonism blocks non-melanoma skin cancer'. Society for Investigative Dermatology (SID) Annual Meeting, April 26-29, 2017, Portland, OR; published abstract: Journal of Investigative Dermatology, May 2017, Volume 137, Issue 5, Supplement 1, Page S131.
• Wondrak, G. T., Steinfass, T., Hua, A., Park, S. L., & Justiniano, R. (2017, April 26-29). talk: 'Repurposing the clinical anti-malarial quinacrine for chaperone-mediated autophagy (CMA)-directed anti-melanoma intervention'. Society for Investigative Dermatology (SID) Annual Meeting, April 26-29, 2017, Portland OR; published abstract: Journal of Investigative Dermatology, May 2017, Volume 137, Issue 5, Supplement 1, Page S124.
• Wondrak, G. T. (2016, August 4-5). talk: Repurposing the clinical antimalarial mefloquine for the therapeutic induction of lethal ER stress targeting BRAF-kinase inhibitor-resistant malignant melanoma. 2016 SKAGGS Biomedical Research Symposium, Idaho State University. Pocatello, Idaho.
• Wondrak, G. T. (2016, June 3). invited speaker: Therapeutic induction of oxidative stress targeting malignant melanoma. Free Radical and Radiation Biology Graduate Program; translational Radiation Oncology , The University of Iowa. Iowa City, Iowa.
• Wondrak, G. T. (2016, May 11-15). talk: Repurposing a clinical antimalarial for the therapeutic induction of lethal ER stress targeting BRAF-kinase inhibitor-resistant malignant melanoma. Society for Investigative Dermatology (SID) Annual Meeting, May 11-14, 2016, Scottsdale, AZ; published abstract: Journal of Investigative Dermatology, Volume 136, Issue 5, Supplement 1, May 2016, Page S98.
• Wondrak, G. T. (2016, May 11-15). talk: The topical OTC antimicrobial zinc pyrithione (ZnPT) suppresses tumorigenic progression in a SKH-1 mouse model of solar UV-induced skin carcinogenesis. Society for Investigative Dermatology (SID) Annual Meeting, May 11-14, 2016, Scottsdale, AZ; published abstract: Journal of Investigative Dermatology, Volume 136, Issue 5, Supplement 1, May 2016, Page S97.
• Wondrak, G. T. (2016, May 21-26). invited speaker & session chair: ‘Frontiers in Skin Photodamage and Sun Protection', oral presentation. American Society for Photobiology, biannual meeting, Tampa, FL.
• Wondrak, G. T. (2016, September 21-23). invited speaker: 'Targeting BRAF-kinase inhibitor-resistant malignant melanoma'. The 12th International Skin Carcinogenesis Conference, The Hormel Institute, University of Minnesota. Austin, Minnesota.
• Wondrak, G. T. (2015, 12). speakers-collaborators; Wondrak/Zhang: 'Harnessing NRF2 for Cytoprotection: From the Inside to the Outside',. Collaborative Cancer Grand Rounds, UA Cancer Center, December 4, 2015, Tucson, AZ..
• Wondrak, G. T. (2015, May 6-9). talk: Identification of glycolysis-derived α-dicarbonyl metabolites as the smallest known endogenous UVA-photosensitizers in human skin cells and reconstructed epidermis. Society for Investigative Dermatology (SID) Annual Meeting, May 6-9, 2015, Atlanta, GA; published abstract: Journal of Investigative Dermatology (2015), 135: 609..
• Wondrak, G. T. (2015, November). oral presentation: The Clinical Antimalarial Mefloquine Induces Lethal ER and Oxidative Stress Targeting BRAF-Kinase Inhibitor-Resistant Malignant Melanoma Cells.. Society for Redox Biology and Medicine, Annual Meeting, November 18-21 2015, Boston, MA;.
• Wondrak, G. T. (2015, September 2-4). invited speaker, ‘Redox-directed interventions targeting skin photodamage', oral presentation,. Society for Free Radical Research-Europe (SFRR-E/SNFS) Conference, September 2-4, 2015, Stuttgart, Germany..

Poster Presentations

• Dickinson, S. E., Wondrak, G. T., Curiel, C. N., Khawam, M., Vaishampayan, P., & Kirschnerova V, V. (2021, April). TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes.. AACR Annual Meeting. Virtual.

Others

• Janda, J., Blohm-Mangone, K., Burkett, N., Einspahr, J. G., Alberts, D. S., Dong, Z., Bode, A. M., Curiel, C. N., Wondrak, G. T., & Dickinson, S. E. (2016, May). TLR4 as a novel molecular target for non-melanoma skin cancer prevention.. Soc. Invest. Derm. #85,.
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  Abstract for poster presented at a conference.