Study On Cell Circles Of A2780 By HDACIs Trichostatin A

Study on cell circles of A2780 by HDACIs trichostatin AObjectiveHistone deacetylase inhibitors(HDACIs) can inhibit the proliferation of many kinds of tumor cells and induce the apoptosis and differentiation of them.. They have exclusive cytotoxic effect on tumor cells and can cooperate with many chemotherapeutic agents without serious cytotoxic effect on normal hemopoietic cells. Recently, the clinical trials about HDACIs are on theâ… orâ…¡phase. Recetly, it was founded that TSA has the special function of HDACIs. TSA can reversible inhibites histone deacetylase activity of mammal through Zn²⁺ chelation to induce the cell circls blockage and apoptosis and differentiation of tumor cells. So far, there're no reports about the effects and mechanisms of TSA on cell circl human ovarian cancer cell line A2780. Cell cyclin dely kinase inhibitor (CDKI) P21^WAF/CIPI is depress or no express in tumor cells and correlate to cell circl. In this experiment, we took A2780 cells as targets and examined the cell circl and express of P21^WAF/CIPI. At the same time, we analyzed the effect and mechanism of TSA on cell circl human ovarian cancer cell line A2780.Methods1. Cell cultureThe human ovarian cancer cell line A2780 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100u/ml penicilin and hpytomycin. Cultures were grown at 37℃in a humid 95% air: 5% CO₂ chamber and periodically tested to ensure they remained free of Mycoplasma infection during the course of the experiments.2. Detect cell circl by flow cytometryCulture A2780 cells in six shadow mast and cell denisity is 1×10⁵/ml. That be treated by TSA 0,50,100,200,400nM for 24 hours. 100nM TSA treat the A2780 cells 6,12,24,36,48 hours. Collect cells by pancreatin and centrifuge in 1000rpm and 5 min. 75% alcohol fix 24 hours. Then centrifuge in 1000rpm and 5 min. PI 200μl (100μg/ml), RNaseA 10μl(10μg/ml), PBS 290μl dyeing 30 min away from light. Be tested by flow cytometry in 4 hours. Repeat three times.3. Test P21^WAF/CIPI mRNA with RT-PCRCells denisty is 1×10⁷/ml. Be treated 0,50,100,200,400nM TSA in 24 hours. Another is treated 6,12,24,36,48 hours by TSA. Test express of P21^WAF/CIPI mRNA with RT-PCR.. Repeat three times.ResultsAfter be treated 12 hours by TSA, the express of P21^WAF/CIPI mRNA was increased in A2780 cells. That is correlation with times and maximun in 24 hours. The effects begin decrease in 48 hours (P<0.05). Cells in G2/M is increase and S-phase is decrease that is correlated to times. G2/M phase is maximun and S-phase minimun in 36 hours. (P<0.05). Different levels TSA treat A2780 cells in 24 hours. The express of P21^WAF/CIPImRNA increase in 100nM(P<0.05). That is correlation with levels. The cells of G2/M phase increase and S phase decrease. That is correlated to levels.ConclusionsTrichostatin A effect the activity of cyclin-dependent kinase through increase the express of P21^WAF/CIPImRNA. TSA make A2780 cells blockage G2/M phase and inhibit cell proliferation of A2780 cells. The minimun level of activitve TSA is 100nM and the minimum time is 12 hours. The effect rely on times and levels.