The Role Of Of Involucrin In Arsenite-induced Human Keratinocytes Transformation

ObjectiveInvolucrin is protein precursor of crosslinked membrane. It is not only the marker of early differentiation in epidermal keratinocyte cells, but also the marker of terminal differentiation in squamous epithelial cell. Studies have shown that Involucrin shows the high expression level in multiple tumors, and it plays an important role in cancer diagnosis. However, it remains unclear that how it changes and the mechanism of its changes in different stages of the tumor. In this study, we based at immortalized human keratinocytes for the research. The expression of Involucrin was detected in different stages of arsenic-induced neoplastic transformation in human keratinocytes, and with the key point of JNK/c-Jun signaling pathways, we studied the molecular mechanism of these changes.Methods1.Neoplastic transformation of Ha Ca T cells induced by long-term low-dose sodium arsenite: After cells were cultured in fresh medium for 24 h, Ha Ca T human immortalized keratinocytes were continuously exposed to 1.0 μmol/L sodium arsenite for 35 passages(about 15 weeks).2.Growth kinetics of cells: 14, 21, 28 and 35 passage cells during logarithmic phase in control and exposure groups were selected, and then inoculated with the same number into 24-well plate. After that, counted with blood cell counters continuously for 6 days, and calculated the doubling time finally.3.Anchorage-independent growth: Colony formation was analyzed in 21,28,35 passage cells treated by 1.0 μmol/L Na As O2 continuously and passage matched control cells in soft agar. Cells were collected and supplied with 0.7% low melting point agarose culture media, and then was overlaid onto 0.7% agar medium with a density of 2×103,then added culture media on the frozen face. After 3 weeks of incubation, colonies were manually counted.4.Flow cytometry to detect ROS levels: Intracellular reactive oxygen species levels were detected by a cell-permeating fluorescent probe DCFH-DA. 0, 1, 7, 14, 21, 28 and 35 passages cells in arsenite-treated and control groups were collected and loaded with the probe. The expressions of fluorescent were measured by flow cytometry.5.Western blot: Ha Ca T cells in the two groups(as in the above passages) were collected in logarithmic growth phase. Extracted and detected total protein, then proteins were separated by SDS-PAGE and transferred to NC membranes. With specific antibody incubated overnight at 4℃and combining secondary antibodies. The expression of protein and the levels of phosphorylation were detected by chemiluminescence.6.The inhibitor of JNK(SP600125): Ha Ca T human immortalized keratinocytes were continuously exposed to 1.0 μmol/L sodium arsenite for 35 passages. Ha Ca T cells treated with 20 of μmol/L the inhibitors of JNK(SP600125) for 30 min, then cells were cultured in fresh medium for 24 h containing for two and five days. Then tested the related protein phosphorylation levels and the degree of cells malignant transformation.Results1.Neoplastic transformation of Ha Ca T cells chronic induced by low-dose of sodium arsenite: A marked increase in the secretion of MMP-9 activity in the arsenite-treated(1.0μmol/L Na As O2) cells was observed in comparison to the passage-matched untreated control(0.0 μmol/L Na As O2) cells at 28 and 35 passages. And compared with the control group, the proliferation rate and doubling time in exposure group was much faster at 21(64.37±15.92 h) and 28(64.04±12.84 h) passages with a significant statistical difference at passage 35(54.00±2.35 h)(P<0.05). Furthermore, the colony number in 35 passage cells treated by Na As O2(107±11) was obviously higher than the passage-matched untreated control cells(P<0.05), and the colonies were much larger.2.The levels of Involucrin and JNK / c-Jun pathway during the long-time low-dose sodium arsenite induced neoplastic transformation in human keratinocytes: There were no obvious regularity changes of intracellular ROS levels before 14 passages of arsenite exposure, except for passage 1. Surprisingly, after 14 passages, with the increased passages of exposure to arsenite, the ROS levels were decreased gradually, even the level at passage35 was significant lower compared to passage 0(P<0.05). Western blot results showed that the expression levels of Involucrin, c-Jun and p-JNK were gradually increased in the later-stage of arsenite-induced transformation, while the expression levels of Jun B, Jun D,JNK did not change significantly. The results suggested that the production of reactive oxygen species induced by Na As O2 activated JNK and c-Jun, and this process might be involved in the rising expression of Involucrin.3.The levels of Involucrin and JNK / c-Jun pathway in tranformed Ha Ca T cells induced by the inhibitor SP600125: Ha Ca T human immortalized keratinocytes were continuously exposed to 1.0 μmol/L sodium arsenite for 35 passages. After adding SP600125 to the low-dose of sodium arsenitein chronic induced neoplastic transformation in human keratinocytes two days and five days. Our results provided that JNK pathway was successfully blocked, and the levels of JNK and p-JNK were declined. Meanwhile the levels of c-Jun and Involucrin were also decreased. The results suggested that the expression of Involucrin may be regulated by JNK /c-Jun pathway in neoplastic transformation of Ha Ca T cells.ConclusionIn the process of Ha Ca T cells transformation induced by low-dose sodium arsenite,the expression of Involucrin is gradually build up, and is regulated by JNK/c-Jun signaling pathway. The pathway can reduce the expression of Involucrin in the malignant transformation by inhibting JNK activity, and in order to reduce malignant degree of Ha Ca T cells.