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

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, non-tumorigenic human keratinocyte cell line (HaCaT) was continuously exposed to 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, 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 dose 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 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.

Loss of Notch1-dependent p21Waf1/Cip1expression influences the Notch1 outcome in tumorigenesis

“…Cells were maintained as monolayers in a humidified atmosphere containing 5% CO 2 at 37 °C, and culture medium was replaced every 2 d. The HaCaT cell is a spontaneously immortalized human epithelial cell line developed by Boukamp et al 57 For generating arsenic-resistant cells, cells were maintained continuously in medium containing 2.5 μM of As 2 O 3 similarly as described previously. 12 Cells were seeded at 3 × 10 6 cells per 10-mm diameter/dish and maintained in 2.5 μM As 2 O 3 . At passage 23, cells started growing and reached confluency.…”

“…It is well established that when human keratinocyte cell line (HaCaT) is chronically exposed to arsenic trioxide, malignant transformation occurs as evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice. 12 To determine a possible functional link between Notch1 downmodulation and arsenic trioxide-induced malignant transformation we analyzed the Notch1 expression in HaCaTtransformed cells after acute re-exposure to As 2 O 3 (see "Materials and Methods"). Surprisingly, arsenic trioxide treatment in HaCaT-transformed cells (HaCaT-R) did not result in Notch1 inhibition, even when cells were treated with the highest dose, 10 μM (Fig.…”

MiRNA expression changes in arsenic-induced skin cancer in vitro and in vivo.

“…Immortalized cells are needed for chronic exposure studies because it is necessary to maintain these cells are maintained for an extended time required for transformation. Immortalized human keratinocytes (HaCaT) are an in vitro model for normal human keratinocytes that can be used to study arsenic-induced skin cancer (39,64). Pi et al (2008) showed that continuous exposure of HaCaT cells for 28 weeks to a low level (100 nM) of sodium arsenite transformed these cells and resulted in an aggressive SCC phenotype upon inoculation of nude mice (39).…”

“…Immortalized human keratinocytes (HaCaT) are an in vitro model for normal human keratinocytes that can be used to study arsenic-induced skin cancer (39,64). Pi et al (2008) showed that continuous exposure of HaCaT cells for 28 weeks to a low level (100 nM) of sodium arsenite transformed these cells and resulted in an aggressive SCC phenotype upon inoculation of nude mice (39). The concentration of 100 nM sodium arsenite was selected based on an epidemiological study of a population in China that consumed well water containing high concentrations of arsenic.…”

“…However, the various stages starting from the early stages of the transformation process in this model have not been studied yet. Furthermore, there has been no further evaluation for the model as an SCC model as suggested by Pi et al (2008).…”