Study Of Sodium Arsenite-induced Neoplastic Transformation In Human Skin HaCaT Cells And Mechanism Of Action

Epidemiological studies from many countries and observed that chronic exposure to arsenic caused primarily skin cancer, but also lung cancer, urinary bladder cancer and liver cancer. However, inorganic arsenic fails to induce tumors in most laboratory animals. To explore the mechanisms of arsenic carcinogenesis, and to investigate the molecular mechanism of arsenic-induced skin cancer from cellular origin aspect may provide us some novel clues for explaining further development and progression of arsenic-induced skin cancer and for finding the valuable biomarkers of early diagnosis, prevention and treatment. The skin is a major target of arsenic exposure and one of the most sensitive tissues to chronic arsenic exposure. Thus, human skin keratinocytes are widely used for study of the arsenic toxicity. The human spontaneously immortalized skin keratinocytes (HaCaT) was originally derived from normal human adult skin, which maintains full epidermal differentiation capacity and is immortalized but nontumorigenic. It is an ideal model for the study of arsenic carcinogenicity.Here, we repeatedly exposed the HaCaT cell line to an environmentally relevant level of arsenic (0.05ppm, about0.4u M) in vitro for about4months, determined the acquired cancer phenotype. Through a continuous exposure of HaCaT to non-toxic doses of sodium arsenite(0.05ppm) for several months,HaCaT cells became apparently tumorigenic in nude mice, pathological studiesof xenogratf tumor sections showed a highly undifferentiated, pleomorphicnature of tumors from arsenic-treated HaCaT cells, and the tumor testified assquamous cell carcinomas (SCC), a type of cancer that induced by arsenic inskin.The present results suggest that the malignant population from chronic,low-level arsenic-treated HaCaT cells may develop from a rare population,which "the existence" of cancer stem cells (CSCs) with the capacity ofself-renewal and potential differentiation. CD44v6may be a biomarker ofarsenic-induced neoplastic transformation in human skin cells, and that arsenicpromotes malignant transformation in HaCaT cells through its activation ofNF-kB and inactivation of P53stimulated expression of CD44v6.The changes of gene expression profile between the arsenic-treated cellsand passage-matched control cells was obtained through oligo microarraydetection and qPCR respectively. Bioinformaties analysis suggested that theimbalance between cell proliferation, immune system process, cell developmentand cell adhesion, etc. may play an important role in arsenic-induced neoplastictransformation of HaCaT cells. From the pathway comparison(Base on KEGGand Biocarta database), the differentially expressed genes involved in severalsignaling pathways that play an important role in cancer and cancer stem cells,such as Wnt, MAPK, Jak-STAT, TGF-beta, NOTCH, PI-3K/AKT and NF-kBsignaling pathway. Alteration of gene expression patterns and especiallyup-regulation of AKT2, MYC, DMD2, CCDN3, MAP3K14anddown-regulation of JAK1, PTEN, ATM, STAT5A may account for the malignanteffect of inorganic arsenic. Part one Study of sodium arsenite-induced neoplastic transformation in human skin HaCaT cellsTo explore the mechanisms of arsenic carcinogenesis, we have investigated the growth property and induced malignant transformation in spontaneously immortalized human skin keratinocytes (HaCaT), that were continuously exposed to non-toxic doses of arsenite (0.05ppm, about0.4μM) for approximately4months. This research was intended to provide a foundation for further work in analysis of cancer stem cells specific molecular marker and gene expression profile during arsenic-induced HaCaT cells malignant transformation.The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with10%fetal bovine serum (FBS), and antibiotics (100U/ml penicillin and100μg/ml streptomycin). For chronic exposure, cells were maintained continuously in a medium containing0.05ppm of NaAsO2for30passages (about4months). Morphological change was observed under light microscope or transmission electron microscope. We assessed colony forming efficiency (CFE) in soft agar every5passages (3weeks) to ascertain acquired malignant phenotype. Meanwhile, the proliferative ability of arsenic-treated HaCaT cells and passage-matched control cells were detected by MTT analysis, cell cycle was measured by flow cytometry (FCM). Once the soft agar colony assay suggested that arsenic-induced carcinogenic conversion had occurred, arsenic-treated and control cells were injected subcutaneously on the left axillary fossa of male Balb/c nude mice, animals were observed for tumor formation over a6-month period, and tumor tissue was examined by immunohistochemistry.Through a continuous exposure of HaCaT to non-toxic doses of arsenite(0.05ppm) over25passages, HaCaT cells became apparently tumorigenic in nude mice, and the tumor testified as squamous cell carcinomas (SCC), a type of cancer that induced by arsenic in skin. In addition, we have observed the following changes caused by long term, low-level exposure to arsenite in HaCaT cells:(1) higher colony forming efficiency on soft agar;(2) higher cell density at confluence and exhibited morphological alterations with the frequent occurrence of giant multinuclear cells;(3) Higher capacity of proliferation; and (4) more population in S, G2/M phase.These results suggested that HaCaT cells treated by chronic, low-level arsenite have neoplastic transformation. The transformed cells provided us basis for further study.Part two The expression and significance of the cancer stem cell markers in arsenic-induced neoplastic transformation in human skin cellsTo explore the effects of long-term, low concentration arsenic on cancer stem cell markers function in HaCaT cells. After treatment of Oppm,0.05ppm sodium arsenite for0passage,5passages,10passages,15passages,20passages,25passages and30passages. Western blotting and immunofluorescence staining were used to observe the expression of CD44v6, CD133, NF-κB and p53protein in arsenic-treated HaCaT cells and passage-matched control cells at different time points.The research results show that the protein level of cancer stem cell marker, CD44v6was up-regulated after chronic, low level sodium arsenite administration while CD133protein level has no obvious change. CD44v6protein level increased steadily duration of arsenic treatment, and has significance difference from passage-matched control cells at25passages (P＜0.05). After treatment of0.05ppm sodium arsenite for15passages, CD44v6expression in arsenic-treated cells increased sharply, CD44v6immunoreactivity positive cells localized primarily in the membrane of some HaCaT cells, CD44v6positive ratio of CD44v6immunoreactivity positive cells is about8%at25and30passages of arsenic-treated cells. Moreover, the extent of CD44v6immunoreactivity in arsenic-treated cells was found to positively correlate with the cloning efficiency in soft agar (r=0.949, P=0.01). Arsenic exposure promoted abnormal expression of CD44v6possibly involved in its activation of NF-κB and inactivation of P53.The present results suggested that the malignant population from chronic, low-level arsenic-treated HaCaT cells may develop from a rare population, which "the existence" of cancer stem cells (CSCs) with the capacity of self-renewal and potential differentiation. CD44v6may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin cells through its activation of NF-κB and the inactivation of P53. Part three Arsenic promotes malignant transformation in human epidermal keratinocytes through alteration of gene expression patternsTo get more insight into the carcinogenic mechanism of arsenic action, we performed genome-wide expression profiling of arsenic-treated and untreated HaCaT cells using oligo microarrays (Agilent Whole Human Genome Oligo Microarray) that contain41,000genes. Data analysis included hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and pathway analysis. Eleven genes that involved in cancer were selected for further evaluation by quantitative RT-PCR (qPCR).Gene expression profile analysis revealed that2,871were differentially expressed between malignant HaCaT cells and normal HaCaT cells. Of these,1,415were significantly up-regulated and1,456were statistically significantly down-regulated in malignant HaCaT cells. Up-regulated genes included AKT2(v-akt murine thymoma viral oncogene homolog2/protein kinase Bβ), MYC(v-myc myelocytomatosis viral oncogene homolog), MDM2(Mdm2p53binding protein homolog, murine double mimute2), CCND3(cyclin D3), MAP3K14(mitogen-activated protein kinase kinase kinase14), etc. Down-regulated genes had Jakl (Janus kinase1), MMP9(matrix metallopeptidase9), PTEN (Phosphatase and tensin homolog deleted on chromosome ten), ATM (Ataxia telangiectasia mutated), STAT5A(Signal transducer and activator of transcription5A), ROCK2(Rho-associated, coiled-coil containing protein kinase2),etc. The differentially expressed genes with functions that include cell proliferation, immune system process, cell development and cell adhesion, and involved in several signaling pathways that play an important role in cancer and cancer stem cells, such as Wnt, MAPK, Jak-STAT, TGF-beta, NOTCH, PI-3K/AKT and NF-κB signaling pathway. Alteration of gene expression patterns and especially up-regulation of AKT2, MYC, DMD2, CCDN3, MAP3K14and down-regulation of JAK1, PTEN, ATM, STAT5A may account for the malignant effect of inorganic arsenic.Conclusion:Low level, chronic arsenic exposure promotes malignant transformation in human epidermal keratinocytes, which is mediated through alteration of gene expression profiles directly. Information gained from our work is valuable for the direction of future experiments involving arsenic toxicity and skin cancer formation.Conclusions1. Low level, chronic arsenic exposure promotes malignant transformation in human epidermal keratinocytes (HaCaT).2. The malignant population from chronic, low-level arsenic-treated HaCaT cells may develop from a rare population, which "the existence" of cancer stem cells (CSCs) with the capacity of self-renewal and potential differentiation. CD44v6may be a biomarker of arsenic-induced neoplastic transformation in human skin cells.3. Arsenic promotes malignant transformation in HaCaT cells possibly involved in its activation of NF-κB and inactivation of P53.4. The profile of gene expression between the arsenic-induced malignant transformation HaCaT cells and control cells were analyzed. Differential genes during transformation were involved in the regulation of cell proliferation, immune system process, cell development and cell adhesion. Wnt, MAPK, Jak-STAT, TGF-beta, NOTCH, PI-3K/AKT and NF-κB signaling pathway play an important role during arsenic-induced malignant transformation of HaCaT cells. Alteration of gene expression patterns and especially up-regulation of AKT2, MYC, DMD2, CCDN3, MAP3K14and down-regulation of JAK1, PTEN, ATM, STAT5A may account for the malignant effect of inorganic arsenic.