Combination Of PTD4-apoptin Protein With Dacarbazine Showed Synergistic Antitumor Effect On B16-F1 Melanoma In Vitro And In Vivo

Bcl-2 is an antiapoptotic protein that is generally expressed in melanoma and high level of Bcl-2 expression has been associated with resistance to chemotherapy in melanoma. Apoptin is a small molecule protein derived from chicken anemia virus (CAV), which can selectively induce cell death in tumor cells and transformed cells, but not in normal cells. Apoptin induces cell death independently of the tumor suppressor p53 and can be stimulated by Bcl-2. Recently, it has been identified that CDK2 phosphorylates apoptin's C-terminal Thr-108, which is crucially required for apoptin-induced cell death.PTD4-apoptin protein harbors penetrating membrane capacity and tumor-selective cell death activity. Dacarbazine (DTIC) is mainstay of treatment for malignant melanoma. In this study, we investigated the cytotoxic effect of PTD4-apoptin protein alone and combined with dacarbazine on mouse B16-F1 and human A875 and SK-MEL-5 melanoma cells in vitro and by means of an mouse B16-F1 melanoma model in vivo, and preliminarily investigated the mechanism of the combination.B16-F1, A875 and SK-MEL-5 cells were cultured in PTD4-apoptin protein or/and DTIC in vitro. Cell viability was analyzed by MTT assay and the nature of drug interaction was analyzed by the coefficient of drug interaction (CDI). B16-F1 cells were incubated with PTD4-apoptin protein or/and DTIC, cell morphological changes, flow cytometric analysis and the expression of Bcl-2 and CDK2 were analyzed. For in-vivo therapeutic experiments, C57BL/6 mice bearing B16-F1 melanoma were treated with PTD4-apoptin protein or/and DTIC in different strategies. The inhibitory effect on the tumor growth was observed and the apoptosis were investigated. The experiments for determining possible side effects by PTD4-apoptin and/or dacarbazine were carried out in tumor-free C57BL/6 mice. The blood parameters were measured and compared with normal values.In vitro, PTD4-apoptin protein alone could inhibit the growth of B16-F1, A875 and SK-MEL-5 cells in a dose-dependent manner, and it combined with DTIC produced synergistic inhibitory effect on the growth of the three cell lines (CDI<1). The DAPI staining and the Annexin-V/PI staining assay indicated that PTD4-apoptin protein combined with dacarbazine achieved the most prominent apoptotic-inducing effect in B16-F1 melanoma-derived tumor cells. The level of CDK2 and Bcl-2 expression in B16-F1 cells increased after incubation with 2-drug combination.In vivo, PTD4-apoptin protein could penetrate into the tumors via the epidermal tissue of the mice and effectively inhibited the growth of tumor in C57BL/6 mice. The inhibitory effect were enhanced in "half dose" or "full dose" combination treatment group, and the apoptotic cells in combined treatment group were more than that in either single treatment group. Analyzing the bone marrow suppression of dacarbazine and/or PTD4-apoptin protein in C57BL/6 tumor-free mice, the results showed that the dacarbazine treatment alone revealed bone-marrow suppression. PTD4-apoptin protein alone and "half-dose" drug combination both had no detectable bone marrow suppression. In contrast, "full dose" drug combination had, but less in comparison to the treatment of dacarbazine alone. One of the potential mechanisms may be that the apoptosis-induced activity of apoptin was enhanced by high CDK-2 and Bcl-2 expression and the high Bcl-2 expression exerting resistance to chemotherapy in melanoma was avoided by changing the properties of Bcl-2 from an anti-apoptotic to a proapoptotic molecule.In conclusion, PTD4-apoptin protein combined with the chemotherapy drug dacarbazine showed synergistic inhibitory effects on the growth of mouse melanoma B16-F1 cells in vitro and in vivo, and of human melanoma A875 and SK-MEL-5 cells in vitro. As important, PTD4-apoptin was shown to enhance the anti-tumor activity induced by dacarbazine without side effects on bone marrow suppression. One of the potential mechanisms may be related with Bcl-2 and CDK2. Our preclinical observations indicate that a combinatorial therapy based on PTD4-apoptin protein and dacarbazine is a promising novel anti-cancer strategy.