Despite the development of various chemotherapeutic strategies, RCC remains a challenging tumor entity

Despite the development of various chemotherapeutic strategies, RCC remains a challenging tumor entity. independently of PPAR. Keywords:etoposide; 15-deoxy-12,14-prostaglandin J2; renal cell carcinoma == Introduction == Renal cell carcinoma (RCC) accounts for ~2% of all cancer cases and is characterized by diverse clinical manifestations, few early warning signs and a resistance to radiotherapy and chemotherapy (1). Clear cell PF-562271 RCC accounts for the majority of RCC cases (2) and one-third of the patients present with metastases at initial diagnosis. Due to the resistance of RCC to radiotherapy and chemotherapy, the PF-562271 5-year survival rate for patients with metastatic RCC is <10% (3). The responsiveness of RCC to treatment with conventional anticancer agents, such as 5-fluorouracil (5-FU) and cisplatin (CDDP), was reported to be lower compared to other types of cancer (4,5). Despite the development of various chemotherapeutic strategies, RCC remains a challenging tumor entity. A few patients were reported to exhibit complete or partial response to frequently used chemotherapeutic agents, such as gemcitabine, 5-FU, capecitabine and vinblastine (6). As RCC is known to be immunogenic, several clinical trials investigated the potency of cytokines, mainly interleukin 2 and/or interferon- (7,8). Targeted therapies, including monoclonal antibodies and small-molecule inhibitors, have significantly modified the treatment of cancer over the last 10 years through inhibiting tyrosine kinase activity or vascular endothelial growth factor receptors (9). However, despite these novel therapies, the clinical outcome of patients with metastatic RCC remains poor (6). Thus, there is a pressing need to establish alternative therapeutic modalities against RCC. In our previous study, we reported that the topoisomerase-I inhibitor camptothecin exhibited toxicity synergistically with a peroxisome proliferator-activated receptor- (PPAR) agonist (10). 15-Deoxy-12,14-prostaglandin J2(15d-PGJ2) is an endogenous carcinostatic agent, whose nuclear receptor is a PPAR. PPAR activation was shown to induce growth inhibition in human RCC Smad5 cells (11). Furthermore, 15d-PGJ2was also implicated in antiproliferation independently of PPAR (12). The antitumor activity of 15d-PGJ2was also found to be associated with the inhibition of topoisomerase-II (13). We previously identified novel binding sites for 15d-PGJ2on the cell surface (14). With regard to targets for 15d-PGJ2in the plasma membrane, molecular chaperones, glycolytic enzymes and cytoskeletal components, such as -actin, were also identified (15). PPAR agonists were shown to enhance 5-FU-, CDDP- PF-562271 or topoisomerase-II inhibitor-induced apoptosis in cancer cell types other than RCC (1619). The aim of the present study was to evaluate the therapeutic efficacy of the combination treatment with 15d-PGJ2and the topoisomerase-II inhibitor etoposide (VP-16) in RCC. == Materials and methods == == Cell lines and cell culture == The Caki-2 human RCC cell line was obtained from Summit Pharmaceuticals International (Tokyo, Japan). The Caki-2 cells were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 mg/l penicillin G and 50 mg/l streptomycin (Invitrogen, Tokyo, Japan), at 37C in a 5% CO2atmosphere. == Reagents == 15d-PGJ2was obtained from Cayman Chemicals (Ann Arbor, MI; Cabru, Milan, Italy). Etoposide (VP-16) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GW9662 was obtained from Sigma-Aldrich (St. Louis, MO, USA); and MTT was purchased from Dojindo Laboratories (Kumamoto, Japan). == Cell viability analysis == To evaluate the effects of 15d-PGJ2and VP-16, alone or in combination, PF-562271 on the growth of Caki-2 cells, cell viability was determined by the MTT assay. The cells were seeded on a 96-well tissue culture plate at 10,000 cells/cm2and incubated for 24 h prior to drug exposure. The cells were incubated with 15d-PGJ2and VP-16 at increasing concentrations (0, 10, 20, 30, 40 and 50 M of 15d-PGJ2; and 0, 10, 20, 30, 40, 50, 60 and 70 M of VP-16) for 24 h. After 24 h, the cells were incubated with MTT solution (0.1 mg/ml in phosphate-buffered saline) for an additional 3 h at 37C. The MTT solution was then aspirated off. To dissolve the formazan crystals formed in viable cells, 100 l dimethyl sulfoxide was added to each well. Absorbance was measured at 570 nm using a spectrophotometer (iMark Microplate Reader, Bio-Rad Laboratories, Hercules, CA, USA). == Detection of chromatin condensation (fluorescence microscopy) == For nuclear staining, the cells were treated with 15d-PGJ2and VP-16 for 24.