Abstract 5396: Synergistic effect of cucurbitacin B and low-dose methotrexate for treatment of human osteosarcoma

Abstract

Abstract Human osteosarcoma (OS) is well known for its metastasis and high recurrence rate even after aggressive treatment. Although current chemotherapeutic regimens have greatly improved the 5-year overall survival rate of the OS patients from 20 % to 70 %, only a limited number of drugs are available to treat OS patients, which makes the development of better treatment protocols difficult. In this research, we tested cucurbitacin B as a potential chemotherapeutic agent for the treatment of human OS. Cucurbitacin B is a plant-derived tetracyclic triterpenoid known to have an antiproliferative effect on several human cancers. In our research, cucurbitacin B showed an antiproliferative effect on 6 human OS cell lines in vitro (ED50 = ∼5×10^-8 M), and the response was dose- and time-dependent. These cells underwent marked morphological changes, multinucleation, G2/M cell cycle arrest, and subsequent cell death. This antiproliferative effect of cucurbitacin B as tested on MG-63 and SAOS-2 human OS cell lines was mainly achieved by the down-regulation of mTOR (mammalian target of rapamycin) pathway through decreased activation of ERK (extraceullar signal-related kinase) independent of Akt phosphorylation. This led to the subsequent inhibition of the downstream targets such as ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Interestingly, when MG-63 and SAOS-2 cells were exposed to both cucurbitacin B and methotrexate (MTX) in combination in vitro, their growth was significantly and synergistically inhibited (combination index (CI) < 0.9). For this reason, we used MG-63 xenografts in athymic nude mice to test if cucurbitacin B can augment the therapeutic effect of MTX in vivo. Whereas low-dose cucurbitacin B (0.5 mg/kg body weight) or low-dose MTX (150 mg/kg body weight) alone failed to decrease the tumor size, the combination of the two compounds at these concentrations significantly inhibited the tumor growth by 70 %. Strikingly, similar synergism was observed even when the MTX was lowered to 50 mg/kg body weight. No significant toxicity was found in combination groups compared to monotherapeutic regimen in terms of body weight loss, bone-marrow clonogenic growth, or a variety of whole-blood and serum parameters. In conclusion, cucurbitacin B showed a promising antiproliferative activity against human OS. We also identified for the first time that mTOR pathway was down-regulated through the inhibition of ERK by cucurbitacin B in human OS cells. Moreover, strong synergistic effects of cucurbitacin B with low-dose methotrexate were observed both in vitro and in vivo. Considering that many current chemotherapeutic regimens require a high-dose of methotrexate (2,400 mg/kg body weight) including leucovorin rescue, the use of cucurbitacin B with low-dose methotrexate provides a promising clinical option for the treatment of human OS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5396.