15-deoxy-Δ12, 14-prostaglandin J2 enhances anticancer activities independently of VHL status in renal cell carcinomas

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Clear cell RCC (ccRCC) accounts for the majority of RCC, which have mutations or epigenetic silencing of the von Hippel–Lindau (VHL) gene. VHL-positive Caki-2 cells are killed by an endogenous anticancer substance, 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2). The MTT reduction assay reflecting mitochondrial succinate dehydrogenase activity was employed for assessment of cell viability. We confirmed anticancer activities of camptothecin (topoisomerase I inhibitor), etoposide (topoisomerase II inhibitor), doxorubicin (topoisomerase II inhibitor) in VHL-positive Caki-2 cells. Combination of topoisomerase inhibitors with 15d-PGJ2 exhibited the synergistic effect in VHL-positive Caki-2 cells. However, 15d-PGJ2 did not increase cytotoxicities of topoisomerase inhibitors on VHL-negative 786-O cells. In addition, the 15d-PGJ2-enhanced antitumor activity of topoisomerase inhibitors was detected in neither VHL-positive nor VHL-negative RCC4 cells. Our finding indicated that 15d-PGJ2 enhanced the antitumor activity of topoisomerase inhibitors independently of VHL.


Introduction
Renal cell carcinomas (RCCs) account for approximately 2% of adult carcinomas and arise from renal tubular epithelial cells that encompasses 85% of all primary renal neoplasms. RCCs are classified into several types such as clear cell RCC (ccRCC) accounting for the majority of RCC [1], papillary RCC and chromophobe RCC. The common genes involved in the pathogenesis of ccRCC include von Hippel-Lindau (VHL) [2]. VHL can be altered and transmitted in an autosomal dominant fashion (VHL disease) or in a sporadic manner. Despite extensive evaluation of many different treatment modalities, advanced metastatic RCC remains highly resistant to radiotherapy and chemotherapy [3].

Reagents
15d-PGJ 2 (ab141717) was obtained from Abcam (Tokyo, Japan). Camptothecin (CPT), doxorubicin (DOX), etoposide (VP-16) and RPMI-1640 were purchased from FUJIFILM Wako Pure Chemical Corporation, Ltd. (Osaka, Japan). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye (MTT) was purchased from Dojindo Laboratories (Kumamoto, Japan). The protein concentration was measured using the bicinchoninic acid (BCA) protein assay reagent obtained from Takara (Shiga, Japan). The principle of the assay is based on monovalent copper ions interact with a BCA reagent to form a violet reactive complex, which shows a strong absorbance at 562 nm. The peptide bonds in the protein reduce copper ions from Cu 2+ to Cu + . The quantity of reduced Cu 2+ is proportional to the amount of protein. The sample solution was added the BCA reagent and incubated at 37°C for 30 min. The colorimetric variations were analyzed by spectrophotometer (iMark Microplate Reader, Bio Rad Laboratories, Hercules, CA, USA) at 562 nm. The experiments were analyzed in triplicate.

Cell viability analysis
MTT reduction assay reflecting mitochondrial succinate dehydrogenase activity was employed. The cells were seeded on a 96-well tissue culture plate at 10,000 cells/cm 2 and incubated for 24 h prior to drug exposure. The cells were incubated with 15d-PGJ 2 and doxorubicin at the indicated concentrations. After 20 h or 24 h, the cells were incubated with MTT solution (0.1 mg/ml in phosphate-buffered saline) for an additional 3 h at 37°C. The MTT solution was then aspirated off. To dissolve the formazan crystals formed in viable cells, 100 μl 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).

Statistical analysis
Data are given as means ± SE (n = numbers of observations). We performed two experiments at least on different days, and confirmed their reproducibility. We analyzed observations obtained on the same day, and presented the typical experimental results among independent ones on different days to minimize experimental errors. Data were statistically analyzed with the Student's t-test for comparison with the control group. Data on various drugs were statistically analyzed by twoway ANOVA followed by Dunnett's test for comparison between the groups.

Effects of 15d-PGJ 2 on the anti-cancerous agents in 786-O cells
To ascertain whether topoisomerase inhibitors and 15d-PGJ 2 synergistically exhibit the pharmacological effects on the VHL-negative ccRCC as well as the VHL-positive ccRCC, Caki-2 cells, 786-O cells were used as the VHL-negative ccRCC. In 786-O cells, CPT (Fig. 1A), VP-16 ( Fig. 1B) or DOX (Fig. 1C) induced cell death in a concentration-dependent manner. As shown in Fig. 1D, 15d-PGJ 2 also induced cell death in a concentration-dependent manner. We evaluated the synergism of 0.05 μM CPT, 5 μM VP-16 or 10 μM DOX with 20 μM 15d-PGJ 2 by MTTreducing activity (Fig. 1E). Although 15d-PGJ 2 decreased the MTT-reducing activity and the cell number ( Fig. 1F) significantly, it did not increase anticancer activities of the above three drugs significantly. At 10 μM, CPT, VP-16 and DOX degenerated morphologies slightly, moderately and severely, respectively. However, these degenerative morphologies were not deteriorated by 15d-PGJ 2 (Fig. 1F). Although various concentrations of drugs were tested, we have not yet succeeded in detecting synergistic effect of CPT, VP-16 and DOX with 15d-PGJ 2 . Thus, the synergism of topoisomerase inhibitors and 15d-PGJ 2 was not detected in the VHL-negative ccRCC, suggesting that VHL might be involved in the combinational effect of topoisomerase inhibitors and 15d-PGJ 2 .

Effects of 15d-PGJ 2 on the anti-cancerous agents in RCC4 (-) cells
To confirm the result obtained from the VHL-negative 786-O cells, RCC4 (-) was used as another VHL-negative RCC. In RCC4 (-) cells, CPT ( Fig. 2A), VP-16 (Fig. 2B) or DOX (Fig. 2C) induced cell death in a concentration-dependent manner. As shown in Fig. 2D, 15d-PGJ 2 induced cell death in a concentration-dependent manner. We evaluated the synergism of 0.05 μM CPT, 20 μM VP-16 or 1 μM DOX with 20 μM 15d-PGJ 2 by the MTT-reducing activity (Fig. 2E) and the morphological criteria (Fig. 2F). CPT and VP-16 decreased the MTT-reducing activity to around 90% of control, whereas 15d-PGJ 2 and DOX did it to around 70% of control. 15d-PGJ 2 did not increase the anticancer activities of the above three drugs. Morphologies of RCC4(-) were similar to those of RCC4(+). Although cell densities appeared to be reduced by the four drugs, morphologies of RCC4(+) were not deteriorated by them significantly. Although various concentrations of drugs were tested, we have not yet detected synergistic effect of CPT, VP-16 and DOX with 15d-PGJ 2 . Thus, 15d-PGJ 2 did not increase the anti-cancerous activities of topoisomerase inhibitors in the two VHL-negative RCCs.

Effects of 15d-PGJ 2 on the anti-cancerous agents in RCC4 (+) cells
To confirm the plausible involvement of VHL in the combinational effect of topoisomerase inhibitors and 15d-PGJ 2 , RCC4(+) and RCC4(-) were evaluated as another VHL-positive and VHL-negative RCCs, respectively. In RCC4(+) cells, CPT (Fig. 3A), VP-16 (Fig. 3B) or DOX (Fig. 3C) induced cell death in a concentration-dependent manner. As shown in Fig. 3D, 15d-PGJ 2 induced cell death in a concentration-dependent manner. We evaluated the synergism of 0.05 μM CPT, 20 μM VP-16 or 1 μM DOX with 20 μM 15d-PGJ 2 by the MTT-reducing activity    . 3E) and the morphological criteria (Fig. 3F). Although these four anti-cancer agents did not alter the morphology of RCC4(+) clearly, they exhibited cytotoxicities slightly, but significantly. 15d-PGJ 2 increased the anticancer activity of CPT additively, whereas it did not those of the two topoisomerase II inhibitors. In spite of testing various concentrations of drugs, we have not yet succeeded in detecting synergistic effect of CPT, VP-16 and DOX with 15d-PGJ 2 . Contrary to the result obtained from the VHL-positive Caki-2 cells, 15d-PGJ 2 did not enhanced the anti-cancer activity of topoisomerase inhibitors synergistically in the VHL-positive RCC4(+) cells.

Effects of 15d-PGJ 2 on the anti-cancerous agents in ACHN cells
To confirm the plausible involvement of VHL in the combinational effect of topoisomerase inhibitors and 15d-PGJ 2 , ACHN cells were evaluated as another VHL-positive RCCs. In ACHN cells, CPT (Fig. 4A), VP-16 (Fig. 4B) or DOX (Fig. 4C) induced cell death in a concentrationdependent manner. As shown in Fig. 4D, 15d-PGJ 2 induced cell death in a concentration-dependent manner. We evaluated the synergism of 0.5 μM CPT, 50 μM VP-16 or 0.5 μM DOX with 30 μM 15d-PGJ 2 by the MTT-reducing activity (Fig. 4E) and the morphological criteria (Fig. 4F). Although these four anti-cancer agents did not alter the morphology of ACHN clearly, they exhibited cytotoxicities slightly, but significantly. 15d-PGJ 2 did not increase the anticancer activities of the three topoisomerase inhibitors. In spite of testing various concentrations of drugs, we have not yet succeeded in detecting synergistic effect of CPT, VP-16 and DOX with 15d-PGJ 2 . Contrary to the result obtained from the VHL-positive Caki-2 cells, 15d-PGJ 2 did not enhanced the anticancer activity of topoisomerase inhibitors synergistically in the VHLpositive ACHN cells.
VHL is a tumor suppressor protein and localized in the nucleus or cytoplasm. VHL forms a protein complex, which determines ubiquitindependent proteolysis of large cellular proteins. When normal oxygen levels are present, the complex binds to, and targets, α subunits of hypoxia-inducible factors (HIF) 1 and 2 for ubiquitin-mediated degradation of protein [22]. Caki-2 cell line has been established from a primary tumor of the kidney. Although it has been primarily defined as the ccRCC cell line, it expresses wild-type pVHL. However, a low expression of HIF-1α and no expression of HIF-2α is detected in Caki-2 cell line [23]. 786-O has many characteristics of ccRCC and is defective in VHL expression, as it harbors mutated VHL [24]. In 786-O cells, cytotoxicities of topoisomerase I and II inhibitors were detected. Although 15d-PGJ 2 induced cell death in 786-O cells, it did not potentate the anti-tumor activity of topoisomerase inhibitors. Another cell line is RCC4, a VHL mutant derived from a primary tumor widely used as a model for VHL-dependent mechanisms, with a commercially available counterpart cell line with restored wild-type gene [22]. Cytotoxicities of CPT, VP-16 and DOX were detected in the two RCC4(-) and RCC4(+) cells. However, 15d-PGJ 2 enhanced the anti-tumor activity of these topoisomerase inhibitors in neither RCC4(-) nor RCC4(+) cells. Thus, the pharmacological synergism of 15d-PGJ 2 and topoisomerase inhibitors were not depend on the state of VHL.
Previously, we have reported that the PI3K/Akt signaling played an important role in the cytoprotection and proliferation of RCCs [13]. 15d-PGJ 2 markedly decreased the phosphorylation of Akt. The Akt inhibitor showed cytotoxicity with a low IC 50 value, suggesting that 15d-PGJ 2 exerted cytotoxicity via the inactivation of Akt. The PI3K inhibitor mimicked the anti-tumor activity of 15d-PGJ 2 . However, we could not detect the synergistic effect between DOX and PI3K inhibitor. In addition, the PI3K inhibitor did not enhanced cytotoxicities of another topoisomerase II inhibitor, etoposide, and a topoisomerase inhibitor I, camptothecin. Neither PPARγ nor PI3K was involved in the 15d-PGJ 2enhanced chemosensitivity of Caki-2 cells to topoisomerase inhibitors. Further studies are required to identify targets for 15d-PGJ 2 , which reduces the chemoresistance of topoisomerase inhibitors.

Conclusion
In the present study, we ascertained whether VHL was involved in the synergy between topoisomerase inhibitors and 15d-PGJ 2 or not. We demonstrated that 15d-PGJ 2 enhanced anticancer activities independently of VHL status in renal cell carcinomas.