| Objective::To investigate the inhibition of the proliferation and the molecular mechanism of Raddeanin A on human gastric carcinoma cells in vitro.Method:Effects of different concentrations of Raddeanin A on proliferation of high (MKN-28), moderately(SGC-7901), low(BGC-823) differentiated human gastric cancer cell line were investigated with MTT assay; induced apoptosis were detected of Raddeanin A on human high, moderately, low differentiated gastric cancer cell line with Annexin V/PI double staining and flow cytometer; Inhibition of the invasion of Raddeanin A on human low differentiated gastric cancer cell BGC-823with transwell assay; wounding heal assay were used to measure the effect of migration ability of Raddeanin A on human low differentiated gastric carcinoma cell BGC-823; cell adhesion assays were used to measure the effect of adherence ability of Raddeanin A on human low differentiated gastric cancer cell BGC-823; RT-PCR method were used to measure the changing of the mRNA of Bax, Bcl-2, Bcl-xl, Survivin of Raddeanin A on human high, moderately, low differentiated gastric cancer cell and reversion inducing cysteine rich proteinwith Kazal motifs (RECK), matrix metalloproteinases-2(MMP-2), MMP-9,MMP-14of Raddeanin A on human low differentiated gastric cancer cell BGC-823; Western Bolt assay were used to measure the changing of the protein activity of Bax, Bcl-2, caspase-3, caspase-8, caspase-9, poly-ADP ribose polymerase (PARP) of Raddeanin A on human high, moderately, low differentiated gastric carcinoma cell and the protein activity of RECK, MMP-9, MMP-14, RhoC, E-cadherin (E-cad) and the STAT3signaling pathway of Raddeanin A on human low differentiated gastric carcinoma cell BGC-823.Result:After the three differentiated human cancer cells were treated by Raddeanin A on12h,compared with control group, Raddeanin A inhibited proliferation of human carcinoma cancer cell (P<0.01). The inhibition effects increased with the dose. Annexin V/PI double staining showed that Raddeanin A could induce human gastric cancer cell apoptosis. At the16μM, the inhibition rate of Raddeanin A on MKN-28, SGC-7901and BGC-823gastric cancer cells reached to95.47%,34.01%and34.01%, respectively, and the apoptosis-inducing effect rose with the dose; RT-PCR and Western Bolt assay demonstrated that Raddeanin A increased the activity of Bax and reduce the activity of Bcl-2、Bcl-x1、Survivin, also Raddeanin A significantly activated caspase-3、caspase-8、caspase-9、PARP. The expressions showed the concentration dependent. After the human low differentiated cancer cells BGC-823were treated by Raddeanin A on24h, compared with control group, Raddeanin A inhibited significantly the abilities of the invasion of the BGC-823cell. At the8μM and16μM, transwell assay showed that the microscopic shows the number of invasive cells to the bottom of the chamber was significantly lower than the control group. The inhibition rates reached to43.66%±4.37%and 82.48%±1.37%, respectively, and the invasion effect rose with the dose; wounding heal assay demonstrated that Raddeanin A could inhibit the migration ability of BGC-823cell significantly, and the inhibition rates present the concentration dependence; cell adhesion assays proved that the adhesion inhibition rate of Raddeanin A on BGC-823showed the concentration and time dependence; RT-PCR and Western Bolt assay demonstrated that Raddeanin A increased the activity of RECK, E-cad and reduce the activity of MMP-2, MMP-9, MMP-14, Rhoc, also the expressions showed the concentration dependent. Furthermore, we detected STAT3signaling pathway which was closely associated with cell proliferation, apoptosis, angiogenesis, invasion and metastasis. The result showed that the expression of phosphorylated STAT3(p-STAT3), which was the active form of STAT3, decreased with the dose increased.Conclusion:Raddeanin A could inhibit proliferation of human cancer cell and induce it’s apoptosis; The molecular mechanism of apoptosis of human cancer cell lines induced by Raddeanin A was through activation of the caspase-8、caspase-3、caspase-9and PARP cascades and increase the activity of Bax and reduce the activity of Bcl-2、Bcl-xL、Survivin; Raddeanin A could inhibit the abilities of the invasion, migration and adherence of the BGC-823cell; the molecular mechanism could be that it increased the activity of RECK, E-cad and reduce the activity of MMP-2, MMP-9, MMP-14, Rhoc. On the one hand, Raddeanin A has the negative regulation in STAT3signaling pathway attribute to inhibiting the p-STAT3expression which was the active form of STAT3in STAT3signaling pathway. |
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