Abstract 3536: Rapamycin in combination with gemcitabine reduced viability, growth and metastasis of the murine osteosarcoma cell line LM8 in vitro and in vivo
Authors: Ando Takashi , Ichikawa Jiro , Sato Nobutaka , Ohba Tetsuro , Koyama Kensuke , Hagino Tetsuo , Haro Hirotaka 
Published: July 2016

Abstract
Introduction
Rapamycin is an mTOR inhibitor with anti-tumor activity in several human cancers. However, the efficacy of this compound in osteosarcoma has not been fully elucidated. It acts downstream in the PI3K pathway and impedes cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription. Here, we assessed the anti-tumor activity of rapamycin. We had previously reported that gemcitabine reduced viability, growth and metastasis of osteosarcoma cell lines. In this study, we investigated whether gemcitabine could enhance the anti-tumor effects of rapamycin in the murine osteosarcoma cell line LM8 in vitro and in vivo.
Results
In the LM8 cell line, rapamycin synergized with gemcitabine to reduce cell proliferation as determined by the WST assay and to induce apoptosis as analyzed by flow cytometry using the annexin V and 7AAD staining method. Rapamycin at doses &gt;10 μM showed significant cytotoxicity. By combining with gemcitabine, the amount of rapamycin required for apoptosis was reduced to 1 μM. Autophagy was evaluated with the Cyto-ID® Autophagy detection kit. Not only rapamycin but also gemcitabine induced autophagy in the LM8 cell line. However, no synergistic effects were observed. Cell mobility was evaluated by migration assay using a trans-well system (LM8 cells in the upper layer and conditioned medium in the lower chamber). Rapamycin synergized with gemcitabine to reduce LM8 cell mobility.
In C3H mice inoculated subcutaneously with LM8, we observed frequent metastasis to the lung. Treatment of the mice with rapamycin reduced the size of the primary tumor; elevated levels of apoptosis were observed. Moreover, rapamycin in combination with gemcitabine had additive effects on the primary tumor and almost no metastatic lesions in the lung were seen. Consequently, combination therapy prolonged the overall survival of tumor-bearing mice.
Conclusions
Rapamycin synergized with gemcitabine to reduce cell proliferation, and to induce apoptosis, autophagy and cell mobility of the LM8 murine osteosarcoma cell line in vitro. Rapamycin in combination with gemcitabine exhibited synergistic antitumor effects. The combination prolonged overall survival and reduced the number of lung metastases in osteosarcoma-bearing mice. Rapamycin has been used to treat osteosarcoma in clinical trials and gemcitabine combined with docetaxel chemotherapy has shown good clinical results. Rapamycin combined with gemcitabine chemotherapy might be a novel and promising therapeutic approach to the treatment of osteosarcoma.
Citation Format: Takashi Ando, Jiro Ichikawa, Nobutaka Sato, Tetsuro Ohba, Kensuke Koyama, Tetsuo Hagino, Hirotaka Haro. Rapamycin in combination with gemcitabine reduced viability, growth and metastasis of the murine osteosarcoma cell line LM8 in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3536.