Dual-Inhibition of mTOR and Bcl-2 Enhances the Anti-tumor Effect of Everolimus against Renal Cell Carcinoma In Vitro and In Vivo

Abstract

Renal cell carcinoma (RCC) is the predominant type of kidney cancer. Mammalian target of rapamycin (mTOR) inhibitor everolimus is currently used as a second-line therapy for sorafenib or sunitinib-refractory metastatic RCC patients. The clinical limitation confronted during everolimus therapy is the onset of drug resistance that decreases the efficacy of the drug. Elevated level of anti-apoptotic Bcl-2 protein is proposed to be an emerging feedback loop for the acquired drug-resistance in various cancer types. In this study, the Bcl-2 inhibitor ABT-737 was used in combination with everolimus to enhance its anti-tumor effectiveness in everolimus-resistant RCC cell lines. Everolimus and ABT-737 combination synergistically led to a decrease in the proliferation of primary site A-498 and metastatic site Caki-1 RCC cell lines, which was accompanied by a reduction in protein levels of cell cycle and mTOR pathway proteins. In both RCC cell lines, everolimus-ABT-737 combination not only induced apoptosis, caspase and PARP-1 cleavage but also a decrease in Bcl-2 protein levels in parallel with a concomitant increase in Bim and Noxa levels. In order to confirm our in vitro findings, we have generated everolimus-resistant RenCa cell line (RenCa^res) to establish a RCC mouse xenograft model. Animals co-treated with everolimus and ABT-737 exhibited a complete suppression of tumor growth without any notable toxicity. This study thus proposes the everolimus-ABT-737 combination as a novel therapeutic strategy for the treatment of RCC to overcome the current clinical problem of everolimus resistance.

Keywords: Bcl-2; Everolimus-ABT 737 combination; Renal Cell Carcinoma; mTOR.

Figure 1

Effect of everolimus and ABT-737 co-treatment on the proliferation of RCC cell lines. (A) Western blot analysis was performed to determine the basal levels of anti-apoptotic Bcl-2 proteins in A-498, Caki-2, Caki-1 and ACHN cells. β-Actin was used as loading control. A-498 and Caki-1 cells in 96-well plates were treated with increasing concentrations of everolimus (B and D) or ABT-737 (C and E) or DMSO (as control) for 24, 48 and 72 hours. The effect of monotherapies on cell proliferation was analyzed by WST-1 assay and the proliferation rate for control cells was considered as 100% at 24 hours. (F) A-498 cells were exposed to 1 µM everolimus and/or 5µM ABT-737, and (G) Caki-1 cells were subjected to 1 µM everolimus and/or 10 µM ABT-737 treatment for 24 to 72 hours. WST-1 assay was performed to analyze the effect of the combination therapy on A-498 and Caki-1 cells. Absorbance values obtained for control cells treated with DMSO at the end of 24 hours was set to 100%. Graphs show the mean ± S.D. of three independent experiments, each performed in triplicate. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Figure 2

Inhibitory effect of everolimus and ABT-737 combination on cell cycle and mTORC1 complex. (A) A-498 cells were treated either with 1 µM everolimus and/or 5 µM ABT-737 and Caki-1 cells were exposed to 1 µM everolimus and/or 10 µM ABT-737. After 24 hours of treatment, protein extracts of A-498 and Caki-1 cells were used to determine the protein levels of CDK4, Cyclin D1, Cyclin D3, CDK2, Cyclin E1, and p27^Kip1 by Western blot analysis. β-Actin was used as loading control. (B) Effect of the combination treatment on mTOR pathway was investigated by the determination of protein levels of p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K, p-S6 (Ser235/236), p-S6 (Ser240/244), S6, p-4E-BP1 (Ser65), and 4E-BP1 in A-498 cells treated with 1 µM everolimus and/or 5 µM ABT-737, and Caki-1 cells exposed to 1 µM everolimus and/or 10 µM ABT-737 for 24 hours. β-Actin was used as loading control.

Figure 3

Induction of apoptosis by the combination regimen of everolimus and ABT-737. Flow cytometry analysis was performed with A-498 cells (A) and Caki-1 (C) exposed to everolimus and/or ABT-737 at indicated concentrations for 48 hours to analyze the effect of combinatorial drug treatment on cell death. The percentage of early apoptotic (bottom right quarter) and late apoptotic (top right) cells was presented in the histogram images and the graphical representation of percentages for Annexin V-positive cells (early and late apoptosis) was shown. Graphs represent percentage analysis of Annexin V-positive cells (n = 3). Each data point in bar graph represents the mean ± S.D. of three independent experiments, each performed in triplicate. N.S. (not significant); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. The effect of drug treatments on apoptotic proteins including caspase 9 and 3, and PARP were analyzed in protein extracts of (B) A-498 or (D) Caki-1 cells exposed to everolimus and/or ABT-737 for 24 hours.

Figure 4

Effect of everolimus-ABT-737 combination on the expression levels of Bcl-2 family proteins. Total cell lysates of A-498 cells treated with 1 µM everolimus and/or 5 µM ABT-737, and of Caki-1 cells exposed to 1 µM everolimus and/or 10 µM ABT-737 for 24 hours were used to determine the expression of (A) anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1), and (B) p53, Bim, Puma, Noxa, and Bax proteins. As loading control, β-actin was used.

Figure 5

Effect of everolimus and/or ABT-737 on RenCa and RenCa^res cells. (A) Everolimus response of parental RenCa cells was characterized by investigating the effect of increasing everolimus concentration on cell viability at 24, 48, and 72 hours. (B) RenCa cells were subjected to increasing concentrations of ABT-737 at indicated time points. (C) RenCa^res cells were subjected to increasing doses of everolimus for 24 to 72 hours to test acquired drug resistance by performing cell viability assay. (D) The response of RenCa^res cells to ABT-737 monotherapy was investigated by cell viability assay upon the treatment of cells with various ABT-737 concentrations for indicated time points. Cell viability of untreated parental and resistant control cells was considered as 100% for 24 hours and graphs show the mean ± S.D of three independent experiments, each performed in triplicate. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) The effect of the acquired everolimus resistance on mTOR pathway was determined in cell lysates of RenCa and RenCa^res cells by investigating the protein levels of p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K, p-S6 (Ser235/236), p-S6 (Ser240/244), S6, p-4E-BP1 (Ser65), 4E-BP1. (F) Western blot analysis of Bcl-2, Bcl-xL, and Mcl-1 was performed with proteins isolated from RenCa and RenCa^res cells. β-Actin was used as loading control. (G) RenCa^res cells exposed to 20 µM ABT-737 were used for Annexin-V/PI staining to determine the effect of ABT-737 monotherapy for 72 hours. The graph shows the percentage of Annexin V-positive cells. Each data value represents the mean ± S.D. of three independent experiments, each performed in triplicate. *, P ≤ 0.05.

Figure 6

Anti-tumor effect of everolimus and ABT-737 combination on RenCa^res xenograft mice model. BALB/c mice were randomized into four groups (n = 8) and administered every other day with the vehicle control, 2 mg/kg everolimus, 75 mg/kg ABT-737 or the combination of single drug doses. (A) Representative pictures of tumors isolated from each group after 21 days of treatment were shown. (B) Average weight of tumors from each cohort subjected to everolimus and/or ABT-737 treatment or control was shown. Scatter blot represents the mean ± S.D. The significant analysis of tumor weights of control groups compared to treatment groups was performed by two-tailed Student's t-test. *, P ≤ 0.05; ****, P ≤ 0.0001. (C) Images are the representatives for hematoxylin and eosin staining of tumor tissue sections. (D) The graph shows the change of mice body weight at the days of injection. Each data point in the graph represents the mean ± S.D. of body weights of eight mice in each group.