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Abstract
Caveolin-1 (CAV1) is an integral membrane protein that may function as a scaffold for plasma membrane proteins and acts as a tumor suppressor protein. One causative factor of chemotherapy-resistant cancers is P-plycoprotein (P-gp), the product of the multidrug resistance-1 gene (MDR1), which is localized in the caveolar structure. Currently, the interactive roles of CAV1 and MDR1 expression in the death of cancer cells remain controversial. In this study, we investigated the effects of indomethacin on the cell viability and the expression levels of MDR1 mRNA and protein in a CAV1-siRNA-mediated gene knockdown hepatoma cell line (SK-Hep1). Cell viability was significantly decreased in CAV1-siRNA-transfected cells compared with that of control-siRNA-transfected cells. Furthermore, the viability of cells pretreated with CAV1 siRNA was markedly decreased by treatment with indomethacin (400μM for 24 h). However, the protein and mRNA levels of MDR1 were unchanged in CAV1-siRNA-transfected cells. These results suggest that CAV1 plays an important role as a major survival enzyme in cancer cells, and indomethacin can sensitively induce cell death under conditions of reduced CAV1 expression, independent of MDR1 expression.
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Fig. 1.
Indomethacin-induced cell death. Cell viability was determined by cell counting after treatment with different concentrations of indomethacin (0, 100, 500, and 1,000μM) for 24 h.
Fig. 2.
Viability of CAV1-siRNA-transfected cells. Cell viability was determined by cell counting after treatment with indomethacin (400μM) for 24 h. Cell viability was decreased significantly in CAV1-siRNA-transfected SK-Hep1 cells, and was markedly decreased by indomethacin treatment compared with that in each control.
Fig. 3.
Effects of indomethacin treatment on the expression of CAV1 and MDR1 mRNAs in CAV1-siRNA-transfected cells. Cells were first transfected for 24 h with control siRNA or CAV1 siRNA. To complete the knockdown of CAV1 gene expression, a second transfection was performed for 24 h. Forty-eight hours after the first transfection, the cells were treated with indomethacin (400μ M) for 24 h. DMSO was used as the vehicle. Expression levels of CAV1 (B) and MDR1 (C) mRNAs were determined by RT-PCR (A) and normalized against that for 18S rRNA.
Fig. 4.
Effects of indomethacin treatment on the expression of CAV1 and MDR1 proteins in CAV1-siRNA-transfected cells. Crude protein was extracted from the cells. DMSO was used as the vehicle. Expression levels of CAV1 (B) and MDR1 (C) proteins were determined by Western-blot analysis (A) and normalized against those for β-actin.
Fig. 5.
Effects of indomethacin treatment on the expression of CAV1 and MDR1 mRNAs in SK-Hep1 cells. Cells were treated with different concentrations of indomethacin (0, 100, 500, and 1,000μM) for 31 h. Expression levels of CAV1 (B) and MDR1 (C) mRNAs were determined by RT-PCR (A) and normalized against those for 18S rRNA.
Fig. 6.
Effects of indomethacin treatment on the expression of CAV1 and MDR1 proteins in SK-Hep1 cells. Crude protein was extracted from cells that had been treated with different concentrations of indomethacin (0, 100, 500, and 1,000μM) for 31 h. Expression levels of CAV1 and MDR1 proteins were determined by Westernblot analysis and normalized against those for β-actin.
Table 1.
Nucleotide sequences of primers used in RT-PCR analysis
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