Osteosarcoma is one of the primary malignant bone tumors that often occur around the ages of 15 to 20. Despite advances in combined chemotherapy and surgical resection, 30% of osteosarcoma patients still die from tumor or metastasis. Therefore, new treatment strategy is being required. We previously reported that telomerase-specific, replication-competent oncolytic adenovirus (Telomelysin, OBP-301), showed the in vitro and in vivo antitumor effects in human bone and soft tissue sarcoma cells; however OBP-301 could not efficiently prevent bone destruction in an orthotopic xenograft tumor model. Recently, third-generation bisphosphonates, zoledronic acid (ZOL), is widely used to inhibit bone destruction in metastatic bone tumors in the clinical settings. Moreover, ZOL has been shown to induce the antitumor effect synergistically in combination with chemotherapeutic agents in human sarcoma cells. In this study, we investigated the in vitro and in vivo antitumor effects of combination therapy with ZOL and OBP-301 in human osteosarcoma cells. Four human osteosarcoma cell lines, HOS, SaOS-2, U2OS and MNNG/HOS were used. We used XTT assay to examine the antitumor effect of ZOL and OBP-301 individually and combinatory on days 2,3,5. ZOL was treated at concentration of 0 to 10µM, and OBP-301 was infected at multiplicity of infections (MOI) of 0 to 100 plaque forming units (PFU)/cell. Combination index was calculated with the CalcuSyn software (BioSoft) and combined antitumor effect between ZOL and OBP-301 was analyzed. XTT assay revealed that combination treatment of ZOL and OBP-301 showed synergistic and additive effects. To analyze the molecular mechanism in the synergistic effect of ZOL and OBP-301, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Treatment with ZOL induced apoptosis, which was confirmed by the cleavage of PARP, and OBP-301infection induced both apoptosis and autophagy, which is confirmed by conversion of LC3- I to LC3- II and p62 downregulation. Combination treatment showed increased apoptotic cell death in all human osteosarcoma cells. Moreover, in vivo antitumor effect of combination therapy was investigated in an orthotopic xenograft tumor model, in which MNNG/HOS cells (2×106 cells per site) were inoculated into the left tibias of female athymic nude mice. ZOL and OBP-301 were injected subcutaneously and intratumorally, respectively, for three times every week. Tumor volume was measured once a week and bone destruction status was analyzed using 3D-CT imaging system at final assessment. Combination treatment showed increased antitumor effect and efficiently prevented bone destruction compared to monotherapy in an orthotopic xenograft MNNG/HOS tumor model. These results suggest that combination treatment of ZOL and OBP-301 is a promising antitumor strategy for improvement of clinical outcome in patients with osteosarcomas.

Citation Format: Yasuaki Yamakawa, Joe Hasei, Hiroshi Tazawa, Toshinori Omori, Shuhei Osaki, Tsuyoshi Sasaki, Aki Yoshida, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Combination therapy of telomerase-specific oncolytic adenovirus and zoledronic acid in human osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2014-725