Study On The Mechanism Of Flavokawain A-induced Apoptosis In Gastric Cancer SGC-7901Cells

Flavokawain A, a plant extract from the dry roots of piper methysticum, is a type of physiologically active component, known as a novel anticarcinogenic drug, and its interaction mechanisms were still unclear. In this paper, we determined the effects of Flavokawain A on the apoptosis and inhibition of SGC-7901, as well as the initial investigation of interaction mechanisms.1. Effects of Flavokawain A on proliferation and apoptosis of SGC-7901cells Gastric cancer cells SGC-7901were incubated with different concentrations of Flavokawain A (0.781,1.56,3.13,6.25and12.55g/ml) for24h,48h and72h, and MTT assay was applied to obtain IC50for the determination of cell survival and growth conditions. The morphological changes of SGC-7901cells were observed with an inverted microscope. Cell apoptosis was determined by flow cytometry with Annexin V-FITC/PI. The quantitative determination of cell cycle distribution relied on flow cytometry. Flavokawain A inhibited the proliferation of SGC-7901cells in a time-dependent and dose-dependent manner. According to the experiment results, IC50value of SGC-7901cells were8.55,5.42and3.33μg/ml after treated with Flavokawain A for24,48and72h,, respectively. Apoptosis results indicated an increase in both early and late apoptosis rates. After treated with6.25μg/ml Flavokawain A for48h, the percentage of Annexin-V+/PI+and Annexin-V+/PI-cells were5.90%and5.44%, respectively; while, after treated with12.5μg/ml Flavokawain A for48h, the percentages of Annexin-V+/PI+and Annexin+/PI-cells were14.37%and20.70%.2. Effects of Flavokawain A on the expression of caspase-9and caspase-3mRN A SGC-7901cells were incubated in the medium with suitable concentrations of Flavokawain A in, and total RNAs were extracted with Trizol reagents. The mRNA expressions of caspase-9and caspase-3were measured by real-time fluorescent quantitative PCR, and results showed that they were significantly increased, and the incensement performed positive correlation with the extending of time.3. Effects of Flavokawain A on the expression of caspase-9and caspase-3protein To determine whether or not caspase-9and caspase-3protein were also up-regulated in SGC-7901cells, the expressions of the proteins in SGC-7901cells were determined by western blot analysis. Statistical results suggested that caspase-9protein was significantly increased in SGC-7901cells after treated with Flavokawain A for72hours, and caspase-3was actived.4. Flavokawain A cell uptake kinetics reasearch SGC-7901cells were incubated for15,30,60,120and240min in the medium with suitable concentrations of Flavokawain A in, and samples were collected for the investigation of cell uptake using HPLC. Accroding to the results obtained, Flavokawain A could be uptaken at the highest amout1-2h after administration.5. ConclusionFlavokawain A could significantly inhibit the proliferation of SGC-7901cells in a concentration and time-depent manner, and cause severe damage to its cell morphology. Study on cell cycles also indicated that Flavokawain A blocked cells at the G2/M phase. And apoptosis experiments showed an increasing rate of apoptosis induced by Flavokawain A. Finally, expressions of caspase-9and caspase-3were determined by Western Blot and real-time fluorescent quantitative PCR, which suggested the possible mechanism might be chondriosome-induced cell apoptosis.