Investigation On The Apoptosis Effects And Mechanism Of Sitibinin In Cervical Cancer And Gastric Cancer

Silymarin, an extract of the milk thistle seeds, is mixture of flavonolignansconsisting of among others of Silibinin, isosilibinin, silicristin, and silidianin.Silibinin itself is mixture of two diastereomers silibinin A and silybinin B inapproximately equimolar ratio. Both in vitro and animal research suggest thatsilibinin, as a ‘narural hepatoprotective drug’, has hepatoprotective (antihepatotoxic)properties that protect liver cells against toxins. Animal experiments showed thatSilibinin has no obivousely toxin side effect. Recent studies have showed strongerefficacy of Silibinin and more progress has been made towards antitumor role andmolecular mechanism. In vitro studies have also showed Silibinin processesinhibitory role against male hormone-dependent and hormone-nondependent cancerincluding prostate cancer, skin cancer, urinary bladder cancer, lung cancer, coloncancer, breast cancer, ovarian cancer, kindney cancer, hepatic cancer, cervical cancer,and tongure cancer.The purpose of this study was to investigate the function and molecular mechanismsof Silibinin in cell growth in cervical cancer and gastric cancer. First, we had cervicalcancer (HeLa) as a model. Flow cytometry results showed Silibinin arrested HeLacells in G2/M phase. After24h treatment with200μM silibinin, percentages of G2/Mphase cells increased from5.64%(control) to22.67%. Using immunoblot analysis, wealso observed that Silibinin decreased the expression of CDK2and CDK1(Cdc2)levels in HeLa cell lines in a dose-dependent manner, which might be the possiblemolecular mechanism of Silibinin efficacy in growth inhibition in HeLa cells. In thestudies assessing Silibinin effect on cyclin levels, no changes were observed in CyclinA and Cyclin B1protein levels. Morphological analysis using DAPI staining revealedthe presence of nuclei with chromatin condensation and the formation of apoptoticbodies in cells cultured with Silibinin. In addition, flow cytometric analyses revealedenhanced apoptosis of HeLa cells treated with Silibinin in a concentration-dependentand time-dependent manner. Western-blot analysis revealed Silibinin significantly cleaved the PARP and reduced pro-caspase3/8/9in dose-dependent manner.Furthermore，our data also demonstrated that Silibinin significantly downregulated theratio of Bcl-2/Bax that was accompanied by a subsequent evident release ofcytochrome c from mitochondria to cytoplasm and activation of caspase-9. Meanwhile,the expression of Fas and Fas L were upregulated in a dose-dependent manner andcaspase8was activated. All these findings implicated that the Silibinin-drivenapoptosis was conducted in both the mitochondrial pathway and the deathreceptor-mediated pathway.Silibinin, a flavonoid compound, has shown to be of chemopreventive potentialagainst many cancers. However, its efficacy against gastric cancer has not been wellelucidated. Here, we assessed the activity of Silibinin on apoptosis and cell-cyclearrest in human gastric cells culture system using SGC-7901, BGC-823, HGC-27asthe model. We observed that SGC-7901, BGC-823, HGC-27have different sensitivityto Silibinin. In SGC-7901cells, Silibinin showed a G2phase arrest upregulating theexpression p53, p21protein, downregulating the expression CDK1, and decreasing theactivity of CDK1-Cyclin B1complex. Silibinin induced G2phase arrest in thecell-cycle progression of SGC-7901cell line. Surprisingly, our data demonstrated thatSilibinin treatment did not show any increase in the activities of Caspase3,8,9. Thesefindings clearly demonstrated Silibinin treatment resulted in apoptotic death ofSGC-7901cells, which was independent of caspase activation. In BGC-823cells, theeffects of Silibinin are the most insensitive. Silibinin slightly downregulated CDK1and slightly activated Caspase3,9. However, Silibinin could significantlydownregulate the expression of CDK1and Cyclin B1in HGC-27cells. Furthermore,Silibinin could activite Caspase3,8,9, downregulate the Bcl-2, and upregulate theBax. These data demonstrated that Silibinin arrested G2/M phase and inducedapoptosis in both the mitochondrial pathway and the death receptor-mediated pathwayin HGC-27cells.Palliative chemotherapy may play a very important role in the treatment of patientswith advanced gastric cancer. However, most patients who are sensitive to initial chemotherapy will eventually fail to react to further cytotoxic chemotherapeuticagents. Therefore, phytotherapeutic agents with high anticancer efficacy and leasttoxicity to normal tissues are suggested as possible candidates to be investigated fortheir synergistic efficacy in combination with anticancer drugs. Herein we assessedwhether Silibinin could synergize the therapeutic efficacy of Paclitaxel, or doxorubicin,or camptothecine in human gastric cancer cell line SGC-7901. The results showed thatSilibinin strongly synergized human gastric cancer SGC-7901cells toPaclitaxel-induced growth inbition, G2/M cell cycle arrest, and apoptosis. Together,we gained a deeper insight into synergetic efficacy of Silibinin in combination withPaclitaxel in human gastric cancer cells.