A Study Of The Effects And Mechanism Of Monoclonal Antibody A3D8on Biological Behaviors Of Three Types Of Human Ovarian Cancer CSC-LCs

Ovarian cancer (OVCA) is considered as the most lethal malignancy of the female reproductive system all over the world. Being lack of early symptoms and specific tumor marker makes the early detection of OVCA more difficult. Besides, OVCA has a feature of direct invasion and implantation in the abdominal cavity, which renders radical cure nearly impossible. Conventional therapies, such as surgery and chemotherapy, can reduce the size of the ovarian tumor but ignorance of the survival of chemo-resistant ovarian cancer stem cell (CSC) following treatment. CSC are defined as cells within a tumor that possess the capacity to self-renew, multi-differentiate into the heterogeneous cancer cells, expand, promote tumor growth as well as metastasize. Recently, the studies on the isolation and identification of CSC or CSC-like cells (CSC-LCs) in OVCA were reported in succession. In further researches, it was found that CD44of human ovarian CSC was upregulated compared to normal OVCA cells. Compelling evidence reveals that CD44is involved in the occurring and development of cancers. If CD44is ligated by anti-CD44mAb, the growth of ovarian CSC will be affected, at least to some extent, which maybe provide some novel ideas of developing new therapeutic strategies directed specifically against CSC.In order to provide experimental basis for improved targeted therapies of OVCA and other correlative clinical drug applications, the present study aims to explore the effects and molecular mechanism of A3D8, one of anti-CD44monoclonal antibodies, on the biological behaviors, such as cell proliferation, apoptosis and tumorigenicity, of three kinds of human ovarian CSC-LCs. In our earlier experiments, three kinds of CSC-LCs (S-CSC-LCs, T-CSC-LCs and A-CSC-LCs) were isolated from different materials (human ovarian cell line SKOV-3, solid tumor tissue or ascites of epithelial OVCA patients) by "suspension spheres culture" with serum-free SGM (DMEM/F12culture medium supplemented with several factors). In the thesis, we evaluated the role of A3D8on ovarian CSC-LCs in vitro or in vivo by using MTS assay, soft agar test, cell cycle analysis assay, microscopy with AO/EB double fluorescent staining, normal and magnified Giemsa staining, Annexin V-FITC/PI kit assay by FCM, Rhl23AψM detection kit assay by FCM, real-time RT-PCR, western blot analysis and animal test in vivo. After CD44ligation with A3D8, cell proliferation was notably attenuate, cellular CFE was decreased, the percentage of cells in G0/G1phase decreased with increasing percentage of S phase, cell cycle progression arrested in S phase, cell apoptosis significantly enhanced, micronuclei and typical morphological change of apoptosis observed by microscopy. The effect of A3D8was enhanced in a dose-and time-manner and the effect of DDP (a kind of chemotherapeutic drugs for OVCA) on cell proliferation and apoptosis is enhanced by being combined with A3D8. Furthermore, we found that the mRNA and protein expression level of p21and caspase-3in CSC-LCs treated by A3D8was upregulated while CDK2, cyclinA and bcl-2downregulated. In conclusion, CD44ligation with A3D8inhibits cell growth of CSC-LCs via S-arrest by upregulation of p21, which is an important regulator of cell cycle, can negatively regulate CDK2activity, decrease the formation of CDK2/cyclinA and stop cell cycle progression in response to regulatory signals. At the same time, A3D8induces apoptosis by disturbing cellular AψM and downregulating of bcl-2in human ovarian CSC-LCs. Results also showed that A3D8could decrease CSC-LCs’ tumorigenicity in NOD/SCID mice.A3D8, as an anti-CD44mAb, maybe be applied to a novel therapy with mAbs targeting CSC of OVCA after surgery by inhibiting cell growth, inducing apoptosis of CSC to decrease the enrichment in situ and implantation in the abdominal cavity of ovarian CSC. The clinical application of DDP combined with A3D8will enhance the toxic effect of DDP on cell proliferation and apoptosis of ovarian CSC and improve tumor therapeutic efficacy. The present study may provide power experimental evidences for the making of targeting-strategy against OVCA and the application of clinical drug-therapy of OVCA.