Long-term Low-dose Cadmium Exposure Leads To The Enhancement Of L-02Proliferation Via Down-regulating Caspase-8by DNA Hypermethylation

ObjectiveTo establish and identify a transformed cell model of low dosechronic cadmium in L-02cell (human normal hepatocyte). Discussing thecarcinogenic mechanism of low dose chronic cadmium by analyzing theaberration of morphology, proliferation, apoptosis ratio and methylationstatus of transformed cell.Methods1) Establish and identify the transformed cell model. MTT assay formeasurement the culture concentration of cadmium, morphology andWesternblot for ensuring the culture length of cadmium; Trypan bluestaining method and Cell clone formation method were used for countingRelative growth rate, Doubling time and Colony-forming efficiency; Theinvasiveness and migration capability of transformed cell was tested byTranswell; Cell cycle, ROS level and cell apoptosis rate were detected byFCM.2) Experiment of transformed cells. The cells were divided into fourgroups, the control group (L-02cells), CDT-L-02group，5-aza group，CDT-L-02+5-aza group. Trypan blue staining method were used forcounting Relative growth rate；cell apoptosis rate were detected by FCM；The expression of DNMTs, apoptosis and cycle related proteinand PCNA were detected by Westernblot; HpaII/MspI enzymeelectrophoresis and MSP analysis to detect methylation status of genomicDNA and caspase-8gene promoter.3) Experiment of tumor cells. The cells were divided into fourgroups, the control group (L-02cells), CDT-L-02group, HepG-2group，HepG-2+5-aza group. To assay the cell viability of5-aza treated HepG-2by Trypan blue staining method; Methylation status of caspase-8genepromoter of5-aza treated HepG-2was detected by MSP analysis; Theexpression of DNMT1and Caspase-8protein of5-aza treated HepG-2were detected by Westernblot.Results1) After L-02cells were cultured with1μM CdCl2for2weeks, themacroaxis of the cells became longer; for10weeks, the shape of cellstransformed from polygon to long spindle, and compared with normalL-02(Control group) the cadmium transformed L-02(CDT-L-02group)expressed significantly and stably up-regulation DNMT1associated withdown-regulation Caspase-8.Compared with normal L-02, the transformedcells showed significantly increased proliferation, colony-formingefficiency and invasiveness and migration capability, however,demonstrated significantly reduced ROS level, doubling time andapoptosis rate.2) Methylation inhibitor5-aza treated transformed cells (CDT-L-02+5-aza group) demonstrated reduced proliferation and increasedapoptosis rate compared with transformed cells;5-aza restored the expression of DNMTs, caspase-8and other apoptosis and cycle relatedprotein significantly compared with normal L-02;5-aza changed theaberration of methylation status (of genomic DNA and caspase-8genepromoter) of transformed cells.3) After48hours treatment of5-aza HepG-2cells showedsignificantly reduced cell viability; compared with normal L-02, HepG-2cells demonstrated higher methlation status of caspase-8gene promoter,but the treatment of5-aza down-regulated the methylation status.Conclusion1.Low dose chronic cadmium promoted L-02cells demonstratingincreased proliferation and reduced apoptosis rate, and altered the shapeof L-02cells, leading to the transform of L-02cells.2.Correlated mechanism of cadmium carcinogenic is that cadmiumaltered the methylation status of genomic DNA and caspase-8genepromoter. The methylation of caspase-8may be concerned with the lowapoptosis ratio and malignant proliferation of tumor cell HepG-2.