The Effects And Mechanisms Of Monoclonal Antibody Against CD44(A3D8) On Proliferation Of Sphere-forming Cells From Ovarian Cancer

Objective:To explore the effects and mechanisms of monoclonal antibody against CD44(A3D8) on proliferation of sphere-forming cells from ovarian cancer.Methods:1. To evaluate the cell proliferation of A3D8(0.2、2、6μg/ml) and DDP on sphere-forming cells of ovarian cancer by MTS.2. To observe the morphology changes of apoptosis cells by acredine orange/ethidium(AO/EB) doubly staining in Fluorescence microscope.3. The apoptosis-inducing function of A3D8on sphere-forming cells of ovarian cancer was assayed by FCM.4. The effect of A3D8on mitochondrial membrane potential of sphere-forming cells of ovarian cancer was assayed by FCM.5. Expression of Caspase-3and Bcl-2proteins after treatment with A3D8was evaluated by western blot assay.Results:1. A3D8could inhibit the proliferation of sphere-forming cells of ovarian cancer; the inhibition was in dose-and time-dependant manner; compared with treatment with DDP alone, the inhibition rate caused by A3D8combined with DDP was increased significantly.2. After treated with different dosages of A3D8, the living cells were dyed in 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 treated with A3D8were upregulated along with the increasing dose and time of A3D8, the apoptosis rate caused by A3D8combined with DDP was increased significantly.3. A3D8could induce apoptosis of sphere-forming cells of ovarian cancer, the apoptosis rate caused by A3D8combined with DDP was increased significantly. 4. A3D8can remarkably decrease mitochondrial membrane potential of sphere-forming cells of ovarian cancer after24hours, compared with treatment with DDP alone,the rate of descent caused by A3D8combined with DDP was increased significantly.5. A3D8could promote the protein expression of activated Caspase-3fragment and could decrease the protein expression of apoptosis inhibitor Bcl-2after24hours.Conclusion:A3D8could inhibit the proliferation of sphere-forming cells of ovarian cancer, and induce sphere-forming cells of ovarian cancer apoptosis. The mechanism of its anticancer effect may be as follows:it decrease mitochondrial membrane potential, injury tumor cell mitochondria, increase the protein expression of activated Caspase-3fragment, decrease protein expression of apoptosis inhibitor Bcl-2and induce the apotosis of sphere-forming cells of ovarian cancer, thus made tumor cell proliferation is restrained in order to achieve the purpose of suppressing tumor growth.