Research On Antitumor Properties Of Anti-tumor Peptide Of Tumstatin For Human Ovarian Cancer

Ovarian cancer is malignant tumor of ovary, one of the most common malignant tumors of female genital system. Due to the lack of early diagnose methods, it often be diagnosed too late. Its mobility is tending to become the leading course of gynecologic malignant tumors. Though the prognosis of patients suffered from ovarian cancer is much better than before, the 5-year survival rate is only 25~30%. Today, to decrease the side effect, improve life quality, prolong survival time, new treatments for ovarian cancer are urgently needed. With the rapid development of molecular biology, biotherapy for cancer is come into clinic use. Biotherapy becomes the lively research field of neoplasm.The organization of BM is dependent on the assembly of type IV collagen (Col IV) networks . There are six distinct gene products, a1~a6, which encode for Col IV . Each of the six chains of Col IV consists of three domains: a 7S domain at the N-terminal, a collagenous domain in the middle portion of the molecule, and a noncollagenous domain (NC1) positioned at the C-terminal. The distribution of a3 chain of type IV collagen is restricted and localized to glomerular BM, several BM of the cochlea, ocular BM of the anterior lens capsule, Descemet's membrane, ovarian and testicular BM, alveolar capillary BM, and the capillary BM of several organs. Tumstatin, an angiogenesis inhibitor, is the bioactive NC1 domain (28 kDa) of Col IVa3. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis. Interestingly the peptide containing amino acids 185~203 of tumstatin inhibit the activation of polymorphonuclear leukocytes and suppresses the tumor growth, such as in a mouse melanoma model, associated with inhibition of tumor cell proliferation and a decrease in cell invasive properties. This study project aimed at finding potential activities of tumstatin (185~203) against ovarian and providing a potential way for the clinical treatment of ovarian carcinoma.Firstly, we synthesized the nucleotide sequence encoding 185～203 amino acids (19 peptide) of tumstatin according to the sequence of tunstatin cDNA searched from the Genebank, and constituted the recombinant plasmid expressed 19 peptide- pTYB2 which allow the fusion of target gene to the intein. The recombinant plasmid was confirmed by restriction digestion and sequencing. Then, this recombinant plasmid was transformed into E.coli ER2566 strain. Transformants were efficiently induced by IPTG, and then expressed. We used a self-cleavable affinity tag to complete one-step purification of the soluble 19 peptide by DTT reduction and chitin affinity chromatography. 19 peptide was identified by the western-blot with the rabbit anti-human tumstatin (185~203) serum.Secondly, we investigated the basic activities of 19 peptide in vitro. The MTS assay was used to assess cytostatic activity induced by 19 peptide. Flow cytometry and Hoechst 33258 stain were adopted to evaluate the cell apoptosis after 19peptide treatment. Cell invasion assay were employed to observe the cell migration and invasion ability. Semiquantitative RT-PCR was applied to analyzing the expression of MTI-MMP, TIMP-2 and MMP-2 in SKOV3 cells incubated with 19 peptide. Western blot was used to the influences of 19 peptide on SKOV3 cells. The MTS assay indicated that the proliferation of SKOV3, HO8910 and OVCAR3 cells was inhibited significantly by the 19 peptide in vitro. The 50% inhibition concentration (IC50) ranged from 26.92 to 33.09μg/ml. The 19 peptide led to a large decrease in SKOV3 cell migration after 24 h. Semiquantitative RT-PCR analysis confirmed the decreased expression of MTI-MMP in SKOV3 cells incubated with 19 peptide. No alterations of TIMP-2 or MMP-2 were observed. Thus, 19 peptide inhibited the migration of SKOV3 cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP. Moveover, Hoechst 33258 showed that 19 peptide significantly induced SKOV3 cells apoptosis and this phenomenon was quantified by flow cytometry. According to the results of western blot, treatment with 19 peptide increased expression of bax and activated of caspase -9 or -3.Finally, the subcutaneous xenotransplanted SKOV3 tumor model in nude mice was established and has the characteristics of human epithelium ovarian cancer. 24 nude mice of ovarian carcinoma model were freely divided into 4 groups: positive control group (treated with saline), 19 peptide group(tail vein treated with 5mg/kg of 19peptide), paclitaxel group (tail vein treated with 10mg/kg of paclitaxel) and combination group (simultaneously treated with paclitaxel and 19peptide). The treatments were performed every other day and amounted to seven times. The weight of body and diameter of tumor were measured everyday. Five days after the treatments were over, firstly, we executed the nude mice and detached the tumors completely and calculated the inhibition rate of tumor growth by weighing the tumors; secondly, we got pathological sections of the tumors and stained by tunel assay; at last, we analyzed the expression level of protein relating to apoptosis by immunohistochemistry. The results showed that the tumor growth of those treated with drugs was all inhibited. The inhibition rate of tumor growth from the combination treated group was better than that from the single 19 peptide or paclitaxel treated group. Tunel assay showed that 319 peptide could obviously induce apoptosis of SKOV3 cells in vivo. The results of immunohistochemistry showed that the expression levels of caspase 3 protein in treated groups were higher than those of control groups and that of the combination treated group was higher that single drug treated groups. From the above the 19 peptide from tumstatin (185~203) could potentially be used as a remedy or supplementary measure for antitumor therapy of ovarian carcinoma.In this study, anti-tumor 19 peptide of tumstatin was highly expression in E.coli and successfully purified by chitin affinity chromatography. Subsequently, we demonstrated that the purified 19 peptide inhibited proliferation and migration of SKOV3 cell and induced apoptosis in vitro, and had a good antitumor activity in subcutaneous xenotransplanted tumor model of human ovarian carcinoma in nude mice. These data provide support for antitumor 19 peptide of tumstatin as a potential treatment or supplementary measure of ovarian carcinoma.