Nutlin-3 enhances sorafenib efficacy in renal cell carcinoma

Abstract

The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi-specific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53, and increase in the levels of p53 pro-apoptotic effector PUMA, Bax, and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.

Figure 1. Apoptotic effects of sorafenib and nutlin-3 in RCC

Caki-1 and caki-2 cells were incubated with 0-50 μM of sorafenib (panel A) and 0-30 μM of nutlin-3 (panel B) for 72 h and MTT assay was performed to asses cell death. Caki-2 cells were incubated with 0-20 μM of sorafenib alone or in combination with 20 μM of nutlin-3 for 72 h to evaluate synergistic effect (panel C). Examination of phosphatidyl serine (PS) exposure at the cell surface by confocal microscopy with Alexa Fluor 488-conjugated annexin V. Caki-1 and caki-2 (1 × 10⁵) were treated with 20 μM sorafenib and 20 μM nutlin-3, alone or in combination for 4 h to differentiate apoptosis from necrosis. After drug treatment, cells were washed with wash buffer followed by incubation with Alexa Fluor 488-conjugated annexin V at room temperature for 15 min. Cells were again washed with wash buffer and slides were mounted with Vectashield DAPI mounting medium and observed under a fluorescence microscope (Olympus) using the standard filter sets for DAPI and Alexa Fluor 488 (panel D).

Figure 2. In situ analysis of activation of caspase-3 by sorafenib and nutlin-3

Caki-1 and caki-2 (1 × 10⁵) RCC cells were treated with 20 μM sorafenib and 20 μM nutlin-3, alone or in combination for 12 h. The activation of caspase-3 in these cells was examined by staining with 5 μM CaspACE™ FITC-VAD-FMK in situ marker according to the manufacturer's instructions. The slides were mounted with Vectashield DAPI mounting medium and observed under a f LSM510 Meta confocal system equipped with Axio Observer Z1 microscope (Zeiss, Germany) using the standard filter sets for DAPI and FITC (panel A), and cleaved caspase was demonstrated by Western blot analysis. GAPDH was used as a loading control (panel B).

Figure 3. Effects of co-treatment of sorafenib and nutlin-3 on p53 mediated apoptotic pathway in caki-2 cells

Caki-2 cells were treated with 20 μM sorafenib and 20 μM nutlin-3, alone or in combination for 24 h at 37 °C. The protein lysates (30 μg) were analyzed by Western blotting for the protein expression of p53, p-p53, p21, PUMA, Bcl-2 and Bax. GAPDH was used as a loading control (panel A). Caki-2 cells were treated with sorafenib (0-30 μM) for 24 h at 37 °C. The protein lysates (30 μg) were analyzed by Western blotting for the protein expression of p53 and p-p53 (panel B).

Figure 4. Effects of co-treatment of sorafenib and nutlin-3 on VEGFR-2 and ERK in caki-2 cells

Caki-2 cells were treated with 20 μM sorafenib and 20 μM nutlin-3, alone or in combination for 24 h at 37 °C. The protein lysates (30 μg) were analyzed by Western blotting for the protein expression of VEGFR-2, p-VEGFR-2, ERK and pERK. GAPDH was used as a loading control (panel A). Caki-2 cells were treated with 20 μM sorafenib and 20 μM nutlin-3, alone or in combination for 1, 6 and 24 h at 37 °C. The protein lysates (30 μg) were analyzed by Western blotting for the protein expression of ERK and pERK. GAPDH was used as a loading control (panel B).

Figure 5. Co-treatment of sorafenib and nutlin-3 inhibits wound healing

Caki-1 and Caki-2 cells at confluence were injured by a scratch with a 10 μL pipette tip. Wounded cells were allowed to heal for 24 h in the presence or absence of sorafenib (20 μM), nutlin-3 (20 μM) and combination of both of sorafenib (20 μM) and nutlin-3 (20 μM). Bars represents migration distance in wound healing assay was quantitated using NIH Image J program (panel A). Caki-1 and caki-2 cells were grown in monolayers were treated with sorafenib (20 μM), nutlin-3 (20 μM) and in combination for 24 h. Phase contrast images were taken on Nikon TMS-F for cell morphology (panel B). The cells were fixed, permeabilized and stained with rhodamine-conjugated phalloidin to detect F-actin. Cells were imaged using a confocal system (panel C).

Figure 6. Mechanisms of action of synergistic combination of sorafenib and nutlin-3 in RCC

Our studies revealed that sorafenib and nutlin-3 induced synergistic inhibition of cell-survival and enhance apoptosis in RCC. For the first time, we show that sorafenib increases the phosphorylation of p53 in a concentration dependent manner which indicates that the pro-apoptotic effects of sorafenib, a known multi-specific tyrosine kinase inhibitor, are mediated by p53 in RCC. Treatment of nutlin-3 will enhance the levels of p53 in cytoplasm which in turn will lead to activation of p53 by sorafenib. The other mechanisms include known inhibition of VEGFR2 and ERK by sorafenib, which also potentiated by the presence of nutlin-3. The enhanced expression of PUMA, Bax and decrease in Bcl-2 levels leads to increased caspase-3 activation following cytochrome c release from mitochondria to initiate apoptosis.