Synergistic Activity of Paclitaxel, Sorafenib, and Radiation Therapy in advanced Renal Cell Carcinoma and Breast Cancer

Abstract

Advanced cancer has been shown to be associated with a higher percentage of epigenetic changes than with genetic mutations. Preclinical models have shown that the combination of paclitaxel, sorafenib, and radiation therapy (RT) plays a crucial role in renal cell carcinoma (RCC) and breast cancer. This study aimed to investigate the involvement of mitochondrial cytochrome c-dependent apoptosis in the mechanism of action of a combination of paclitaxel, sorafenib, and RT in RCC and breast cancer. RCC and breast cancer cell lines were exposed to paclitaxel and sorafenib alone or combined in the presence of radiation, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The synergistic anticancer effects of the combination therapy on cell cycle and intracellular signaling pathways were estimated using flow cytometry and immunoblot analysis. RCC and breast cancer cell line xenograft models were used to examine the antitumor activity in vivo. Our results suggest that paclitaxel, sorafenib, and RT synergistically decreased the viability of RCC and breast cancer cells and significantly induced their apoptosis, as shown by caspase-3 cleavage. Paclitaxel, sorafenib, and radiation cotreatment reduced antiapoptotic factor levels in these cells and, thereby, significantly reduced the tumor volume of RCC and breast cancer cell xenografts. The current study suggests that paclitaxel, sorafenib, and radiation cotreatment was more effective than cotreatment with paclitaxel or sorafenib and radiation. These findings may offer a new therapeutic approach to RCC and breast cancer.

Figure 1

Cotreatment with paclitaxel, sorafenib, and RT suppressed cancer cell proliferation. Effects of various combinations of paclitaxel, sorafenib, and RT on renal cell carcinoma (RCC, Caki-1; A and B) and breast cancer (MB-231; C and D) cell using cell proliferation and viability assays. Points indicate mean (%) of value observed in the solvent-treated control. All experiments were repeated at least three times. The data represent the mean ± SD. Experiments were repeated at least three times with similar results. *P < .05 vs. control, ** P < .01 vs. control, *** P < .005 vs. control.

Figure 2

RT, paclitaxel, and sorafenib combination was the most induced cancer cell cycle arrest and apoptosis. Cell cycle analysis of the indicated cell lines following exposure to various combinations of paclitaxel, sorafenib, and RT. Paclitaxel, sorafenib, and RT combination potently induced cell cycle arrest and apoptosis of RCC (Caki-1; A) and breast cancer (MB-231; B) cells. Cells were exposed to the indicated inhibitors.

Figure 3

RT, paclitaxel, and sorafenib combination significantly induced apoptosis mediated by induction of cytochrome c release. Immunofluorescence assay: cotreatment with paclitaxel, sorafenib, and RT showed the most significant induction of cytochrome c release into cytosol, indicating apoptosis of RCC (A) and breast cancer (B) cells.

Figure 4

RT, paclitaxel, and sorafenib combination induced highest tumor shrinkage of cancer cell xenografts in vivo. Combination of paclitaxel, sorafenib, and RT induced more potent inhibition of tumor progression than paclitaxel or sorafenib with RT or RT only did in RCC (Caki-1; A) and breast cancer (MDA-MB-231; D) cell xenografts (n = 10 mice/group). Athymic nude mice with established tumors were treated with the indicated drugs or RT. Data represent the mean tumor volumes. The compounds had no significant effect on body weight in RCC (Caki-1; B) and breast cancer (MDA-MB-231, E) cell xenografted mice. Cotreatment with paclitaxel, sorafenib, and RT showed the highest reduction of tumor weights of the dissected weight in RCC (Caki-1, C) and breast cancer (MDA-MB-231, F) cell xenografts. *P < .05, **P < .01, and ***P < .005 vs. control.

Figure 5

RT, paclitaxel, and sorafenib combination affected antiapoptosis-related proteins in tumor tissues derived from RCC and breast cancer cells. Immunohistochemical analysis of Bcl-2 proteins in tumor tissues following the indicated treatments. Each assay was performed in triplicate, and representative images are displayed. ^⁎P < .05, ^⁎⁎P < .01, and ^⁎⁎⁎P < .005 vs. control. MetaMorph 4.6 image-analysis software was used to quantify the immunostained target protein.