| Objective:Inorganic arsenic has long been recognized as a human carcinogen. However, the exact molecular and cellular mechanisms involved in arsenic toxicity are rather unrevealed given that the complicated metabolism of arsenic in the human body. To date, considerable progress has been made to address arsenic toxicity, among the various hypotheses of arsenic carcinogenic mechanism, induction of oxidative stress and epigenetic dysregulation have been steadily gaining attention in recent studies. The Keap1-Nrf2/ARE signaling pathway represents a critical cellular defense in response to oxidative stress, however,emerging data suggest that constitutive activation of Nrf2 is associated with cancer development and chemotherapy resistance. The reasons why Nrf2 continuous accumulates in cancer cells remain to be fully understood. The aim of the present study was to elucidate dynamic changes of Nrf2 and its target antioxidant genes in different stages of arsenic-induced neoplastic transformation in human keratinocytes, and also to explore the molecular mechanisms responsible for those variances.Methods:1. To establish the malignant transformed Ha Ca T cell model induced by low level Na As O2: Ha Ca T cells were maintained continuously in DMEM medium containing 0.0 or1.0μM Na As O2 for 35 passages(about 18weeks). Cell transformation was identified by the changes of cell morphology, the ability of cell proliferation, MMP-9 secretion, and soft agar colony formation. 2. Detection of ROS, MDA and Keap1-Nrf2/ARE signaling pathway in different stages(0, 1, 7, 14, 21, 28, and 35 passages) of low level Na As O2 induced neoplastic transformation in Ha Ca T cells: The ROS level was determined by ?ow cytometer, the changes of MDA and SOD were detected by biochemical techniques, theexpression levels of Nrf2, HO-1, Keap1, and Bach1 were analyzed by Western-blot. 3.Determination of DNA methylation status of Keap1 gene promoter region in the later stage(14, 28, and 35 passages) of low level Na As O2 induced neoplastic transformation in Ha Ca T cells: Bisulfite sequencing PCR(BSP) was used to detect the methylation status in Keap1 gene promoter region. 4. Changes after 5-Aza-2’-deoxycytidine(5-Aza-d C)treatment: Both normal Ha Ca T and transformed Ha Ca T cells(T-Ha Ca T) were treated with or without 5-Aza-d C(10μM) for five days with medium changed daily. Then detected methylation status in Keap1 gene promoter region by BSP, analyzed the changes of Keap1,Nrf2, and HO-1 protein levels by Western-blot, and then determined the ability of anchorage-independent growth by colony formation assay.Results:1. After long-term exposure to 1.0μM Na As O2, the proliferation rate and doubling time in 1.0μM Na As O2 group was much faster at 21 and 28 passages with a significant statistical difference at passage 35(p<0.05). Besides, a marked increase in the secretion of active MMP-9 in 1.0μM Na As O2 group was observed in comparison to the passage-matched untreated control(0.0μM Na As O2) group at 28 and 35 passages. In addition, after exposure to 1.0μM Na As O2 for 35 passages, the Ha Ca T cells formed significantly higher colonies in soft agar than passage control cells. 2. Detection of ROS,MDA and Keap1-Nrf2/ARE signaling pathway in different stages(0, 1, 7, 14, 21, 28, and35 passages) of low level Na As O2 induced neoplastic transformation in Ha Ca T cells, the results showed that: the ROS level was increased at passage 1 after exposure to 1.0μM Na As O2, no obvious regularity changes of ROS level was found between 1 to 14 passages of arsenite exposure. Surprisingly, after 14 passages, with the increased passages of exposure to arsenite, the ROS level decreased gradually and gave a significant lower at passage35 compared to passage 0(p<0.05). The MDA level was increased at passage 1,then decreased gradually, but no obvious changes were found compared to passage control cells. The Nrf2 and its target enzymes(HO-1, SOD) were increased at passage 1 after arsenite exposure, then decreased after passage 7. However, after passage 14, with increased passages of exposure to arsenite, an up-expression of Nrf2 in nuclear fractions as well as in whole cell lysates was observed. In addition, high expression of HO-1 and SOD activity also occurred in the later stage of arsenite exposure. No such changes, however,were observed in passage control cells. In contrast, a continuous decrease in Keap1 expression both in cytosolic fractions and whole cell lysates was observed after 14 passages of arsenite exposure. For Bach1 expression, a slight elevated protein level in cytosolic fractions was found after arsenite exposure, but no significant changes in whole cell lysates of arsenite-treated cells compared to passage control cells. 3. Determination of DNA methylation status of Keap gene promoter region in the later stage of arsenite-induced Ha Ca T cell transformation, the results showed that: Dramatic increase in methylated Cp G dinucleotides in an upstream region(-433 to-1bp) of the Keap1 TSS was revealed in Ha Ca T cells at passage 14 after exposed to arsenite compared with passage control cells, the methylation rate was 51.1±6.5%. In addition, with increased passages of arsenite exposure, the sites of methylation in Keap1 promoter region also increased accordingly, the methylation rate was 77.1±6.5% and 90.6±4.3%, respectively, which was significantly higher than passage control cells(7.1 ± 2.8% and 6.3 ± 3.8%).Interestingly, in a Cp G island region that is downstream of the Keap1 TSS(+1to +159 bp),all of Ha Ca T cells exhibited complete absence of methylation both in arsenite-treated group and passage control group. 4. After treated with 5-Aza-d C, obvious demethylation of keap1 gene was found in T-Ha Ca T cells, the methylation rate(9.7 ± 2.4%) was significantly lower than untreated T-Ha Ca T cells(93.1±2.4%), p<0.05. As a result of the treatment, the protein level of Keap1 was significant restored both in cytosolic fractions and whole cell lysates(p<0.05). On the other hand, the expression levels of Nrf2 and HO-1protein in 5-Aza-d C treated T-Ha Ca T cells showed substantial reduction compared to T-Ha Ca T cells. In addition, restoration of Keap1 expression by 5-Aza-d C treatment also reduced the anchorage-independent growth capacity of the arsenite-transformed cells as determined by the colony formation in soft agar.Conclusions:1. Ha Ca T cells could be transformed by chronic exposure to low level of sodium arsenite.2. The imbalance of oxidation-antioxidation system is involved in arsenite-induced human keratinocyte transformation. Continuous reduction of ROS and up-regulation of Nrf2 mediated antioxidant levels occur in the later-stage of arsenite-induced Ha Ca T cell transformation.3. Hypermethylation of Keap1 gene promoter region inactivates its function is responsible for continuous up-regulation of Nrf2 mediated antioxidant levels in thein Arsenic-induced Neoplastic Transformation in Human Keratinocytes Abstract later-stage of arsenite-induced Ha Ca T cell transformation.4. Reduce methylation level of Keap1 gene promoter region could restore its function, decrease Nrf2 mediated antioxidant levels and colony formation of the arsenite-transformed cells.5. The induction of oxidative stress in combination with epigenetic dysregulation may jointly contribute to the carcinogenesis induced by low level, long-term arsenite exposure. |
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