The Anticancer Research Of Antisense Oligonucleotides Targeting Survivin

Survivin, an important member of the inhibitor of apoptosis (IAP) family, is implicated in both control of apoptosis and regulation of cell division. It is over-expressed in almost human neoplasmas, but is undetectable in most terminally differentiated tissues. In most cancers, expression of survivin is correlated with reduced apoptotic index, poor prognosis and increased risk of recurrence. These features of survivin make it promising target for cancer therapy. In previous work, an ASODN (SV-10) against survivin from 12 antisense sequences was obtained, which exhibited preliminary inhibition in HepG2 cell. In the present study, we mainly evaluated the antitumor effects of antisense oligonucleotides against survivin in vitro and in vivo, probed correlative molecular mechanism and provide evidence in screening new drugs for anti-cancer research.Firstly, the inhibitory effect of SV-10 against survivin on proliferation of twelve human tumor cells was estimated by MTS assay in vitro. The results shown that SV-10 inhibited the cells growth of 12 cancer cell lines, and the IC₅₀ (was defined as the dose of SV-10 for getting 50% inhibitory rate in cells) of SV-10 on twelve human cancer cells proliferation ranged between 0.122-0.477μmol/L. These data suggested that SV-10 had wide antitumor activities in vitro. To investigate the specific activity of SV-10, we employed HepG2 cells to observe the effects of SV-10 on cells growth and survivin expression (both mRNA and protein) by MTS assay, RT-PCR and Western blot analysis, repectively. Results showed that SV-10 compounds inhibited HepG2 cells growth and reduction expression of survivin mRNA and protein, in a dose-dependent manner, and the effects of control oligonucleotides Sen and Mis on cells were comparatively lower. Result from caspase-3 protease activation assays suggest that inhibition of survivin greatly decreased cells growth and induced cells apoptosis. Moreover, HepG2 was tansfected with SV-10 and then treated with 5-fluorouracil (5-FU), then cell proliferation was evaluated by MTS assay. The results suggested that the synergism was generated in conbined treatment, and SV-10 also increased chemosensitization. On the basis of studies in vitro, we try to investigate the pharmacological activity of SV-10 in animal models on tumor growth. In this study, we used an orthotopic transplant metastatic model of HCC in nude mice to evaluate anti-tumor activity of SV-10, and ODNs reagents were delivered by intravenous injection. Interestingly, this systemic treatment also resulted in significant inhibition in tumor growth. Tumor growth in mice treated with SV-10 (50 and 75 mg/kg per day) was significantly inhibited (45.31% and 60.94%, respectively) compared with saline-injected group (P<0.01), in a dose-dependent manner. Furthermore, SV-10 also reduced survivin expression in tumor xenografts by using RT-PCR, Western blot and immunohistochemical staining, and induced tumor cell apoptosis with the use of in situ TUNEL (i.e., terminal deocynucleotidyl transferase-mediated deoxyuridine triphosphatedigoxigenin nick end labeling) staining. Tumor proliferative activity was decreased significantly in SV-10-treated groups by immunohitochemical analysis of Ki67 expression, as compared with Sen- and saline-injected groups. Moreover, the level of serum alpha-fetoprotein. in SV-10-treated groups was also decreased in a dose-dependent manner.In summary, our results demonstrate that the SV-10 targeting survivin can specifically down-regulate the expression level of survivin and cause apoptotic cell death in vitro and in vivo. It is important that we have for the first time demonstrated that systemic administration of ASODN resulted in a significant tumor growth inhibition in orthotopic transplant metastatic model of HCC in nude mice, which suggests that the use of SV-10 deserves further investigation as a novel approach to increase clinical effectiveness in cancer treatment.