The Molecular Mechanism Of Antitumor Drug Induced Tumor Cell Aging

Objective: Explore the effect of grugs like curcumol and cis-platinuminducing aging in human liver cancer cell line HepG2，the human ovariancarcinoma cell line SKOV3，the human ovarian cancer cisplatin-resistant celllines SKOV-3/DDP and human nasopharyngeal carcinoma cell line CNE2,anddiscuss its possible mechanism. in order to provides the theory basis foranti-tumor treatment of inducing tumor cells senecence. Methods:Culturedhuman hepatoma cells HepG2, human ovarian cancer SKOV3,cisplatin-resistant human ovarian cancer SKOV3-DDP and humannasopharyngeal carcinoma cells CNE2, with Eshu different concentrations ofalcohol culture medium were treated HepG2, SKOV3, SKOV3-DDP and CNE2cells to an equal volume of ethanol&culture medium as a control group; usingdifferent concentrations of cis-platinum-containing culture medium were treatedHepG2, SKOV3and CNE2cells to an equal amount of deionized water&culture medium as a control group. respectively;1. Using tetrazolium blue(MTT) on HepG2, SKOV3, SKOV3-DDP curcumol observed inhibition assayand CNE2cell proliferation; inhibitory effect of cisplatin on HepG2, SKOV3and empathy CNE2cell proliferation was observed；2. Proliferation theSenescence-associated β-galactosidase (SA-β-gal) activity after the judgehandling curcumol HepG2, SKOV3, SKOV3-DDP, morphological changes CNE2cell senescence, and cisplatin HepG2, SKOV3, CNE2cell senescencemorphology histological changes；3. Curcuma were analyzed by flow cytometryafter alcohol treatment HepG2, SKOV3, SKOV3-DDP, CNE2cell cycledistribution, and post-cisplatin treatment HepG2, SKOV3, CNE2cell cycledistribution;4. Using Sybe Green real-time fluorescence dye method, aftertreatment with real-time PCR detected curcumol HepG2, SKOV3, SKOV3-DDP,the expression level of the cell cycle CNE2cell-related genes, and cisplatinHepG2, SKOV3, CNE2cells the expression levels of cell cycle-related genes;5.test the result of QRT-PCRby testing the protein product related to the aginggenes using the way of western blot. Results:1. MTT assay curcumol found onthe proliferation of HepG2cells significantly inhibited, and within a certainrange (2.5~10mg·L-1) in a dose-and time-dependent; cisplatin on theproliferation of cells with CNE2significant inhibitory effect, and within acertain range (0.4~4.0mg·L-1) in a dose-and time-dependent;2. SA-β-galstaining showed curcumol processing HepG2, SKOV3, SKOV3-DDP, CNE2cells, and cisplatin HepG2, SKOV3, after CNE2cells, significantly reduced thenumber of cells becomes large, flat, blue cytoplasm stained;3. were detected byflow cytometry curcumol treated HepG2, SKOV3, SKOV3-DDP, CNE2cells,HepG2cisplatin treatment, SKOV3, CNE2cells24h,48h, cell cycle arrest, theresults show that drug treatment most of the group of cells was inhibited in theG1phase of the cell cycle, with negative group (0h) comparing the differencewas significant (P <0.05or0.01);4. conduct aging-related genes Sybe Greenreal-time quantitative PCR screening test found that some aging-relatedgene-specific expression occurs, such as p53，p63，p16Ink4a, p27Kip1，PTEN，pRb，TBX3，Cdc25C，Gadd45a，IGF1R，PIK3CA，BMI-1，B2M，MORC3，MYC and SPARC expression increased，and Cyclin A2，Cyclin B1，Cyclin E1， CDK6，ATM，E2F1，ETS1，ETS2，MDM2，FN1，IGFBP3，RBL2，SERPINB2，SIRT1expression reduced;5. Verify part of aging gene expression by Westernblot, found that the expression of tumor suppressor gene PTEN, AKT, p53, p21,such as increased cell cycle regulatory proteins p16, p27, CyclinA2etc.Downregulated. Conclusion: In vitro anticancer drugs-curcumol cisplatin onthe human hepatoma cells HepG2, human ovarian cancer SKOV3,cisplatin-resistant human ovarian cancer SKOV3-DDP, who has a pro-agingCNE2nasopharyngeal carcinoma cells action, the mechanism involved in p16and p27-mediated p53-pRB and PTEN signaling regulates aging network.