Sorafenib treatment of clear-cell renal cell carcinoma (CCRCC) and colorectal carcinoma (CRC) cells: Differential effects on gene expression and cell death pathways

Abstract

15612 Background: The von Hippel Lindau gene (VHL) is often mutated in CCRCC and leads to loss of VHL protein (pVHL) expression. Sorafenib is a TKI with clinical activity in metastatic CCRCC. Studies to define mechanisms governing anti-tumor activity of this agent in CCRCC or CRC cell lines that express wild-type pVHL were conducted. Methods: We evaluated CAKI-1 (CCRCC) and HCT- 116/p53 +/+ (CRC) cell lines as model systems expressing wild-type pVHL. Cells were treated at 37°C in an atmosphere of normoxia (21% O2) or hypoxia (1% O2), 5% CO2 and the remainder N2 in the absence (control) or presence of sorafenib (2.5–20 μM) for 24–96 hours. Expression of target angiogenesis, apoptotic and anti-apoptotic genes was determined by real-time RT- PCR. Fluorescence microscopy following staining with Hoechst 33342 plus propidium iodide was used to analyze cell death by apoptosis and/or necrosis. Caspase-3 activity was measured using the target substrate DEVD-AFC. Results: In CAKI-1 and HCT-116 cells, exposure to 1% O2 relative to 21% O2, led to increased expression (2 to 6-fold) of angiogenesis (VEGF) and anti-apoptosis (TNFAIP3 & MCF2) genes. However, in an atmosphere of 1% O2 relative to 21% O2, a decreased (>2-fold) and increased (>3-fold) expression of the apoptotic (TNFRSF25) gene was observed in CAKI-1 cells and HCT-116 cells. Sorafenib treatment (7.5 μM) of CAKI-1 cells in 1% O2 led to a >3–4-fold decrease in expression of the VEGF and TNFAIP3 and a 3-fold increase TNFRSF25 genes. Following treatment with 10 μM sorafenib for 48h, cell death was >80% by necrosis in CAKI-1 cells and >95% by apoptosis in HCT-116 cells. Apoptotic cell death in the HCT-116 was also confirmed by increased caspase-3 activity in cell extracts following sorafenib treatment. Apoptotic cell death or necrotic cell death induced by sorafenib was unaffected by normoxia or hypoxia. Conclusions: In contrast to CCRCC cells, hypoxia led to upregulation of the apoptotic gene TNFRSF25 in the CRC cells. Anti- proliferative effects of sorafenib were primarily by necrosis in CCRCC cells and by apoptosis in CRC cells. No significant financial relationships to disclose.