Inhibition of PPARα induces cell cycle arrest and apoptosis, and synergizes with glycolysis inhibition in kidney cancer cells

Abstract

Renal cell carcinoma (RCC) is the sixth most common cancer in the US. While RCC is highly metastatic, there are few therapeutics options available for patients with metastatic RCC, and progression-free survival of patients even with the newest targeted therapeutics is only up to two years. Thus, novel therapeutic targets for this disease are desperately needed. Based on our previous metabolomics studies showing alteration of peroxisome proliferator-activated receptor α (PPARα) related events in both RCC patient and xenograft mice materials, this pathway was further examined in the current study in the setting of RCC. PPARα is a nuclear receptor protein that functions as a transcription factor for genes including those encoding enzymes involved in energy metabolism; while PPARα has been reported to regulate tumor growth in several cancers, it has not been evaluated in RCC. A specific PPARα antagonist, GW6471, induced both apoptosis and cell cycle arrest at G0/G1 in VHL(+) and VHL(-) RCC cell lines (786-O and Caki-1) associated with attenuation of the cell cycle regulatory proteins c-Myc, Cyclin D1, and CDK4; this data was confirmed as specific to PPARα antagonism by siRNA methods. Interestingly, when glycolysis was blocked by several methods, the cytotoxicity of GW6471 was synergistically increased, suggesting a switch to fatty acid oxidation from glycolysis and providing an entirely novel therapeutic approach for RCC.

Figure 1. PPARα protein level was higher in grade 4 RCC tissues than grade 1 RCC tissues.

RCC tumor tissues of different Fuhrman grades were prepared for immunohistochemistry as described in Materials and Methods and probed with PPARα antibody. The photomicrographs shown are representative of at least three patients for each group. Bar = 50 µm.

Figure 2. PPARα antagonist inhibited RCC and NHK cell viability.

RCC cells (Caki-1 and 786-O) were treated with DMSO, WY14,643 (WY), or GW6471 (GW) at the indicated doses from 12.5 to 100 µM for 72 hours and a cell viability assay was performed as described in Materials & Methods. The data shown are representative of at least three repeats. *p<0.05 compared to DMSO. Error bars indicate standard deviation.

Figure 3. PPARα antagonist arrested cell cycle at G0/G1 phase and attenuated cell cycle related proteins.

RCC cells (Caki-1 and 786-O) were treated with DMSO (Cont) or GW6471 (GW) 25 µM for 24 hours and cell cycle analysis was performed as described in Materials and Methods. The data shown are representative of at least three repeats.

Figure 4. PPARα antagonist induced apoptosis in RCC cells.

A. RCC cells (Caki-1 and 786-O) were treated with DMSO or GW6471 (GW) at the indicated doses for 24 hours and an annexin V-based apoptosis assay was performed as described in Materials and Methods. B. RCC cells (Caki-1 and 786-O) were treated with DMSO or GW6471 (GW) at the indicated doses for 24 hours and immunoblotting was performed as described in Materials and Methods. ß-actin was immunoblotted as a loading control. The data shown are representative of at least three repeats.

Figure 5. Levels of cell cycle and apoptosis relevant proteins were altered by PPARα Antagonist.

RCC cells (Caki-1 and 786-O) were treated with DMSO (Cont) or GW6471 (GW) 25 µM for 24 hours and immunoblotting was performed as described in Materials and Methods. The pictures shown are representative of at least three patients for each group.

Figure 6. Downregulation of PPARα by siRNA transfection confirmed the antagonist’s on-target effects in Caki-1 cells.

Caki-1 cells were transfected with control siRNA or PPARα siRNA at 100 nM for 72 hours and processed for immunoblotting, cell cycle analysis, and apoptosis assay as described in Materials and Methods. A. PPARα protein level was attenuated in the cells transfected with PPARα siRNA. B. PPARα siRNA transfection arrested cell cycle at G0/G1 phase. C. CDK4, cyclin D1, and c-Myc protein levels were attenuated in the cells transfected with PPARα siRNA. D. PPARα siRNA transfection induced apoptosis. The data shown are each representative of at least three repeats.

Figure 7. Glucose depletion synergized cytotoxicity of PPARα antagonist but not agonist.

A. RCC cells (Caki-1 and 786-O) were treated with DMSO (Cont), GW6471 (GW) 25 µM, and/or 2-DG (5 mM) for 72 hours and cell viability assay was performed as described in Materials and Methods. B. RCC cells (Caki-1 and 786-O) were treated with DMSO (Cont), GW6471 (GW) 25 µM, low glucose media (Lo Glu), or GW6471 in low glucose for 72 hours and cell viability was assessed by an MTT assay as described in Materials and Methods. The data shown are representative of at least three repeats. **Synergistic effect compared to each treatment separately. Error bars indicate standard deviation.