The Effect Of Anti-CD44 A3D8 On Proliferation And Apoptosis Of Human Ovarian Stem/progenitor Cells And Its Preliminary Mechanism

Objective:To explore the effect of anti-CD44 monoclonal antibody A3D8 on the cell proliferation,apoptosis and cycle in ovarian cancer sphere-information cells from ascites and solid tumor, and research the preliminary mechanisms on apoptosis. So it can provide a new method for curing ovarian cancer.Methods:We cultured ovarian cancer sphere-information cells from ascites and solid tumor by serum-free medium. This method can screen tumor stem cells.1. To determine the cell proliferation of A3D8 (0.2,2,6μg/ml and cisplatin on ovarian cancer sphere-information cells from ascites and solid tumor by MTS.2. To observation the morphology changes of apoptosis cells by acridine orange/ethidium(AO/EB) doubly staining in Fluorescence microscope.3. After treated with A3D8 2μg/ml at 24,36 hours,the effect of A3D8 on cell cycle of ovarian cancer sphere-information cells from ascites and solid tumor was assayed by FCM4. The effect of A3D8 2μg/mL on apoptosis of ovarian cancer ascetic sphere-information cells was assayed by FCM.5. RT-PCR was applied for P21,P27,Bcl-2,cyclinD1,cyclinA,CDK2,CDK4 mRNA level in ovarian cancer sphere-information cells from ascites and solid tumor by A3D8.6. After treated with A3D8 2μg/ml,western blot was applied for CDK2 and cyclinA protein expression level in ovarian cancer initial cells and ascitic sphere cells.Results:1. A3D8 could inhibit ovarian cancer ascitic sphere cells and initial cells proliferation in time and dose-dependent2. After treated with different dosages of A3D8, the lived cells were dyed green; early stage apoptosis cells were orange, and late stage apoptosic cells were red,chromatin condensation, nuclear margination, fragmented chromatin and apoptosis bodies. The percentage of the apoptosis cells added with the increasing dose of A3D8.3. The ovarian cancer sphere-information cells from ascites and solid tumor were arrested in cell cycle S phase by A3D8 2μg/ml. A3D8 can increase the percentage of S phase cells, decreasing the percentage of G0/G1 phase cells.4. A3D8 could induce the ovarian ascitic sphere-information cells apoptosis. The apoptosis rate of the combined treated cells are significantly higher than those cells treated only by cisplatin or A3D8.5. A3D8 could not effect the mRNAlevel of P21,P27,Bcl-2,cyclinD1,cyclin A,CDK2,CDK4.There was no statistical significance compared with control group.6. A3D8 (2μg/ml) could remarkably inhibit the CDK2 and cyclinA protein expression.Conclusion:A3D8 could inhibit ovarian cancer cells proliferation, arrest the cell cycle in S phase and induce cells apoptosis.The mechanisms may be that A3D8 caused cells injured, CDK/cyclin complexes firsted experienced the damage. It induced the cell cycle arrested for repaired the injured cells.when the cells could not repair, the cells entered apoptosis at last.