ZOL inhibited the mevalonate pathway, thereby suppressing the translocation of prenylated Ras and RhoA proteins to membrane fractions. An in vivo study showed the inhibitory potential of ZOL on tumor growth in mice without changes

Renal cell carcinoma (RCC) has been shown to be resistant to chemotherapy and radiotherapy. In order to examine the potential of zoledronate (ZOL), a bisphosphonate, as an anticancer agent, we investigated the effects of ZOL on RCC cells and the involvement of the mevalonate pathway in antiproliferative effects, as well as the effects of ZOL administration on mice inoculated with RCC. ACHN cells were used and cell viability was measured via intra cellular reductase activity. Chromatin condensation was detected by Hoechst 33342 staining. Proteins were detected by western blot analysis. Tumor volume was measured bidimensionally in mice inoculated with ACHN cells after vehicle or ZOL subcutaneous administration. ZOL exhibited antiproliferative effects with an IC50 value of 2.29±0.53 μM in ACHN cells and chromatin condensation was observed when treated with ZOL. Farnesol (FOH) and geranylgeraniol (GGOH), precursors of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, exhibited potency to rescue cells treated with ZOL. Additionally, Ras and RhoA proteins located in the membrane fraction decreased when treated with ZOL and recovered by FOH or GGOH treatment, suggesting that ZOL inhibited the mevalonate pathway, thereby suppressing the translocation of prenylated Ras and RhoA proteins to membrane fractions. An in vivo study showed the inhibitory potential of ZOL on tumor growth in mice without changes in body weight. Our study showed that ZOL could be useful as an anticancer agent for the treatment of RCC, and the mevalonate pathway could be an efficient target for novel therapeutic agents against RCC. Introduction Renal cell carcinoma (RCC) can be resistant to chemotherapy and radiotherapy, making nephrectomy the most effective treatment (1). Although nephrectomy can be expected to cure patients without metastatic involvement, the 5-year survival rate of those with metastasis is <10%. In addition, recent advances in drug development have allowed us to use novel agents for RCC targeting the cancer-specific pathway, such as bevacizumab, sorafenib and sunitinib (2-4). Although they prolonged overall survival in patients with advanced RCC, the effect was still insufficient; thus, it is necessary to discover novel chemotherapy for RCC. There have been studies by us and other groups, showing the possibility of other targets to treat RCC (5-7). Bisphosphonates have been widely used for the treatment of bone disease with potent antiresorptive activity, especially nitrogen-containing bisphosphonates, including aldendronate (ALN), risedronate (RISE), and zoledronate (ZOL), which have been characterized to have strong inhibitory effects on the mevalonate pathway, thereby inhibiting the prenylation of Ras and Rho inducing cell apoptosis (8,9). ZOL is the most potent nitrogen-containing bisphosphonate and it is widely used for bone diseases caused by metastasis of breast, prostate, bladder, and renal cancer (10,11). In addition, ZOL has been shown to have cytotoxic effects on cancer cells, including multiple myeloma and pancreatic, colon, prostate, and breast cancer (12-18), which anticipate direct effects on cancer cells (19). In addition, ZOL has been suggested to have cytotoxic effects mediated by caspase-dependent apoptotic cell death in RCC (20,21); however, limited information is available in regard to the mevalonate pathway. ZOL potency in vivo is also unknown in RCC. The aims of this study were to investigate the cytotoxic effects of ZOL in ACHN cells, derived from RCC, and to clarify the importance of the mevalonate pathway and the potency of ZOL in mice inoculated with ACHN cells. Materials and methods Materials. Alendronate (ALN) and Risedronate (RISE) were purchased from LKT Laboratories (St. Paul, MN), and zoledronate (ZOL) was purchased from Toronto Research Involvement of the mevalonate pathway in the antiproliferative effect of zoledronate on ACHN renal cell carcinoma cells MEGUMI FUJITA1, MAKIKO TOHI1, KYOKO SAWADA1, YASUHIRO YAMAMOTO2, TSUTOMU NAKAMURA2, TATSUROU YAGAMI2, MOTOHIRO YAMAMORI1 and NOBORU OKAMURA1 1Department of Clinical Pharmacy, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo 663-8179; 2Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan Received November 26, 2011; Accepted January 9, 2012 DOI: 10.3892/or.2012.1683 Correspondence to: Dr Noboru Okamura, Department of Clinical Pharmacy, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien-kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan E-mail: [email protected]