Inhibition of bcl-2 Enhances the Efficacy of Chemotherapy in Renal Cell Carcinoma

Abstract

Objectives: Renal cell cancer (RCC) is highly resistant to chemotherapy. Increased expression of the antiapoptotic gene bcl-2 in tumors is known to be associated with poor responses to systemic treatment of cancer. Down-regulation of bcl-2 expression using antisense oligonucleotides (asON) has been shown to increase chemosensitivity in clinical phase I–III studies with various cancers. However, no studies on the efficacy of this approach in RCC have been reported so far. This study aimed to evaluate whether bcl-2 asON could enhance efficacy of chemotherapy in human RCC. Material and Methods: Expression of bcl-2 mRNA and protein was analyzed in different RCC cell lines by RT-PCR and Western blot. Cells with high or low bcl-2 mRNA and protein expression were treated with different concentrations of bcl-2 asON in combination with cisplatin. AsON-induced down-regulation of bcl-2 mRNA and protein was documented by RT-PCR and Western blot. Treatment effects on cell viability were analyzed by colorimetric tetrazolium (MTT) assay. Immunohistochemical staining of M30-positive cells was performed for quantification of apoptotic cells. Results: Transfection of high bcl-2 expressing cells with bcl-2 asON alone induced no reduction of cell viability at a concentration range from 100–1000 nM. In combination therapy, pretreatment with asON significantly enhanced MTT reduction after cisplatin treatment. IC 50 concentrations of cisplatin were 1 μg/ml with and 2.7 μg/ml without prior incubation. The marked reduction of cell viability correlated with an 8-fold increase of apoptotic cells after combination treatment. Only a minor increase of cisplatin effectivity was noted after asON preincubation of cells with lower bcl-2 expression. Conclusions: The combination of cisplatin and bcl-2 antisense ON exerts significantly greater effects on cell viability and apoptosis than either agent used alone on human RCC cells. These data indicate that inhibition of bcl-2 expression may be an attractive therapeutic strategy in RCC tumors with high bcl-2 expression.