Regulatory Effects of Apatinib in Combination with Piperine on MDM-2 Gene Expression, Glutathione Peroxidase Activity and Nitric Oxide level as Mechanisms of Cytotoxicity in Colorectal Cancer Cells

Purpose: Apatinib has been utilized in colon cancer therapies but its efficiency and molecular mechanism are not fully understood. Chemotherapy in combination with non-toxic compounds can be an effective treatment strategy for cancer. Consequently, this study was carried out to evaluate the effects of apatinib and piperine on colorectal cancer (CRC) cell line and their potential anti-cancerous mechanisms in vitro. Methods: The effects of apatinib and piperine on HCT-116 CRC cells were detected by assessing cell viability using MTT assay. The potential cytotoxic mechanisms of apatinib and piperine were investigated by evaluating MDM-2 gene expression ratio using real-time PCR assay. Moreover, the glutathione peroxidase (GPX) activity and nitric oxide (NO) levels were assessed by colorimetric assays. Results: The proliferation rate of CRC cells decreased by increasing the concentrations of piperine or apatinib. When HCT-116 cells were treated with different concentrations of apatinib in combination with piperine, the synergistic effects were observed (combination index 1). In HCT-116 cells treated with apatinib and piperine at the concentrations of 0.5×IC50 and 0.2×IC50, the MDM-2 gene expression was downregulated and NO levels increased compared to the untreated control cells and related single treatments. In addition, GPX activity significantly decreased in combination treatment at 0.5×IC50 concentration of both agents versus single treatments. Conclusion: Apatinib in combination with piperine could significantly inhibit the growth of CRC cells. These cytotoxic effects were induced by regulation of MDM-2 gene expression and inhibition of antioxidant marker.