Study On The Apoptosis Of Colorectal Cancer Stem Cells Induced By20(S)-Ginsenoside Rg3

Colorectal cancer is the second most common cause of cancer-related death in the world,which relapsed and produced drug resistance easily. A increasing evidence about that there area small subset of cells as well as the normal stem cells exist in the tumor tissues, which iscapable of self-renewal、driving tumor growth、differentiating to form all of the lineages andstay longer in G0found within a cancer, also be resistant to the chemotherapy called cancerstem cell, which is the root of tumor formation, development and deterioration.Ginsenoside Rg3is part of ginseng as well as one of the main effective components offive plus families Panax ginseng plant ginseng c. a. Mey and with two kinds of configurations:type S and type R. Existing studies show that Rg3(R) can inhibit tumor angiogenesis, reducetumor microvascular density, induce tumor cells apoptosis and so on. But rarely in the research of ginsenoside Rg3(S), especially the research on the role of cancer stem cells.Therefore,based on the preliminary work, a immune magnetic bead was used, and the surface markesEpCAM and CD44, screening the EpCAM~+CD44~+HCT-116cells, then cultured in serumfree medium, formated tumor in soft agar、differented in serum、identified stem cellsspecificity gene with RT-PCR. Cultured EpCAM~+CD44~+HCT-116cells and normal cellswith Rg3(S) in different concentrateions and then identified the inhibition of the Rg3(S) andselected the useful concentrateions with CCK-8Kit. Respectively, made use of AO/EB、AnnexinV–FITC/PI double staining、spectrophotometric assay of Caspase、methods of DNALadder, At last, with the method of Mitochondrial membrane potential method detectedapoptosis gene with RT-PCR and preliminary discussed the apoptosis mechanism.Results show that EpCAM~+CD44~+HCT-116cells which were clutrued in serum-freemedium formed tumor microspheres gradually, the clone formed as well as in the soft agarmedium, which then adhered to grow in serum medium. Result of RT-PCR showed that thereexisted SOX2, Nanog and Cldn-6as the specific genes of stem cells.20-Rg3(S) of400μmol/L gave us a good inhibition against to EpCAM~+CD44~+HCT-116cells for24h. Therefore, theeffective concentration choosed100μmol、200μmol、400μmol and and with the20-Rg3(S) of100μmol、200μmol、400μmol/L had no effection on the3T3cells compared with DDP (P<0.05). It showed a relationship between the cell apoptosis and dose of Rg3(S); Afterdealed with20-Rg3(S), the EpCAM~+CD44~+HCT-116cells showed a high activity ofCaspase-3and Caspase-9, which was significantly higher than the control (P<0.01); Also,there was a reduction of mitochon drial membrane potential. Correspondingly, result showeda high expression of Bax and low expression of Bcl-2.20-Rg3(S) can inhibit the proliferation of EpCAM~+CD44~+HCT-116cells, induceapoptosis in a certain concentration. The possible classic apoptosis pathways:（1）reduce mitochondrial membrane potential and release the Cyto, then activate the precursor of Caspase-9, followed by cascade amplification to activate the upstream Caspase-9anddownstream Caspase-3respectively. At last, destroy the dimer of Bax and Bcl-2to raise the expression of Bax and lower the expression of Bcl-2which promote the cellsoccur a irreversible apoptosis；（2）Turn on the way of CD95which is activated bythe TNF, the activated Caspase-8destroies the dimer of Bax and Bcl-2to raise the expression of Bax and lower the expression of Bcl-2which promotes the cells occur airreversible apoptosis；In combination with the corresponding change of detection indexes suggests, the apoptosis mechanism of the EpCAM~+CD44~+HCT-116cells could take the way of aboved.Therefore, Rg3(S) can inhibit the proliferation of colon cancer stem cells, and induceapoptosis through a certain way that play a role in anti-tumor area.