Original Contribution
Arsenic-induced malignant transformation of human keratinocytes: Involvement of Nrf2

https://doi.org/10.1016/j.freeradbiomed.2008.05.020Get rights and content

Abstract

Arsenic is a well-known human skin carcinogen but the underlying mechanisms of carcinogenesis are unclear. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism, and emerging data suggest that constitutive activation of Nrf2 contributes to malignant phenotype. In the present study when an immortalized, nontumorigenic human keratinocyte cell line (HaCaT) was continuously exposed to an environmentally relevant level of inorganic arsenite (100 nM) for 28 weeks, malignant transformation occurred as evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice. To investigate the mechanisms involved, a broad array of biomarkers for transformation were assessed in these arsenic-transformed cells (termed As-TM). In addition to increased secretion of matrix metalloproteinase-9 (MMP-9), a set of markers for squamous differentiation and skin keratinization, including keratin-1, keratin-10, involucrin, and loricrin, were significantly elevated in As-TM cells. Furthermore, As-TM cells showed increased intracellular glutathione and elevated expression of Nrf2 and its target genes, as well as generalized apoptotic resistance. In contrast to increased basal Nrf2 activity in As-TM cells, a diminished Nrf2-mediated antioxidant response induced by acute exposure to high doses of arsenite or tert-butyl hydroxyquinone occurred. The findings that multiple biomarkers for malignant transformation observed in As-TM cells, including MMP-9 and cytokeratins, are potentially regulated by Nrf2 suggest that constitutive Nrf2 activation may be involved in arsenic carcinogenesis of skin. The weakened Nrf2 activation in response to oxidative stressors observed in As-TM cells, coupled with acquired apoptotic resistance, would potentially have increased the likelihood of transmittable oxidative DNA damage and fixation of mutational/DNA damage events.

Introduction

Inorganic arsenic is a well-recognized, multisite human carcinogen and exposure is associated with an increased risk for dermal malignancies [1]. Arsenic is also carcinogenic in rodent models, producing liver, lung, ovary, and adrenal tumors after transplacental exposure [2] and skin tumors in combination with ultraviolet (UV) irradiation or phorbol esters in mice [3], [4]. However, arsenic alone does not appear to induce skin cancer in these mouse models [2], [3], [4], suggesting that events associated with arsenic-induced dermal carcinogenesis may be distinct from other target tissues. Accumulating evidence suggests that oxidative stress occurs in response to arsenic exposure [5], [6] and may be one factor in dermal arsenic carcinogenesis. Indeed, evidence of arsenic-induced oxidative DNA damage has been observed in cells [7], [8], [9], [10], [11], [12], in rodents [13], and in humans [14].

Transcription factor NF-E2-related factor 2 (Nrf2), a cap ‘n’ collar basic leucine zipper protein, regulates critically important cellular defense transcriptional programs that maintain cellular redox homeostasis and serve to limit oxidative damage and inflammation [15]. Nrf2 controls expression of a variety of genes encoding for antioxidative and phase 2 drug-metabolizing enzymes through antioxidant response elements (ARE) [15]. Nrf2-controlled genes encode for various enzymes, including detoxification enzymes like glutathione S-transferases (GST), NAD(P)H: quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1) and antioxidant enzymes like the γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM). Nrf2 also controls expression of several transporters including the multidrug resistance protein-1 and cysteine-glutamate-exchange transporter, among many other proteins. Supporting the importance of Nrf2 in cellular defense is the finding that Nrf2-deficient mice show a deficiency in this coordinated gene regulatory program and have a higher susceptibility to both oxidative damage and chemical carcinogenesis [16], [17]. In apparent contrast to extensive data showing that low Nrf2 activity predisposes cells to chemical carcinogenesis, emerging evidence suggests that constitutive activation of Nrf2 may contribute to a malignant phenotype [18]. Indeed, increased expression and activity of Nrf2 have been observed in various tumor cells [19], [20]. These inconsistencies suggest that Nrf2 may play paradoxical roles in different stages of tumorigenesis.

Given the potential importance of oxidative DNA damage in dermal carcinogenesis, as well as the critical roles of Nrf2 in the defense against oxidative damage, here we study whether Nrf2 and its target genes are involved in arsenic-induced skin carcinogenesis using a human dermal cell model system in which we find that arsenic induces malignant transformation of human keratinocytes.

Section snippets

Cell culture

The HaCaT cell is a spontaneously immortalized human epithelial cell line developed by Boukamp et al. [21]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U penicillin/ml, and 100 μg streptomycin/ml. Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere with an oxygen concentration of 19.9%. For chronic arsenic exposure, cells were maintained continuously in medium containing 100 nM sodium arsenite (NaAsO2, Sigma,

Low level, chronic arsenic treatment of HaCaT cells induces malignant transformation

To achieve oncogenic transformation, HaCaT cells were continuously exposed to a low level (100 nM) of inorganic arsenite. The arsenic concentration used in this study is comparable to human blood arsenic levels found in chronic arsenosis patients in Inner Mongolia, China, where arsenic-induced skin lesions and cancers are common [22]. After 28 weeks of continuous arsenic exposure, the arsenic-treated cells exhibited unique morphological alterations with the frequent occurrence of giant

Discussion

Low-level, chronic arsenic exposure can induce malignant transformation in various human and rodent cells [28], [29], [30], [31], [32]. In the present study when HaCaT cells were continuously exposed to environmentally relevant levels of arsenic for a protracted period, malignant transformation occurred as evidenced by the formation of highly aggressive SCC, a common form of skin cancer in arsenic-exposed humans [1], after inoculation of nude mice. Importantly, by studying this cell model

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This research was also funded in part with Federal Funds from the National Cancer Institute under contract No. NO1-CO-124000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the United

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