Enhanced inhibition of bladder cancer cell growth by simultaneous knockdown of antiapoptotic Bcl-xL and survivin in combination with chemotherapy

Abstract

The overexpression of antiapoptotic genes, such as Bcl-xL and survivin, contributes to the increased survival of tumor cells and to the development of treatment resistances. In the bladder cancer cell lines EJ28 and J82, the siRNA-mediated knockdown of survivin reduces cell proliferation and the inhibition of Bcl-xL sensitizes these cells towards subsequent chemotherapy with mitomycin C and cisplatin. Therefore, the aim of this study was to analyze if the simultaneous knockdown of Bcl-xL and survivin might represent a more powerful treatment option for bladder cancer than the single inhibition of one of these target genes. At 96 h after transfection, reduction in cell viability was stronger after simultaneous inhibition of Bcl-xL and survivin (decrease of 40%-48%) in comparison to the single target treatments (decrease of 29% at best). Furthermore, simultaneous knockdown of Bcl-xL and survivin considerably increased the efficacy of subsequent chemotherapy. For example, cellular viability of EJ28 cells decreased to 6% in consequence of Bcl-xL and survivin inhibition plus cisplatin treatment whereas single target siRNA plus chemotherapy treatments mediated reductions down to 15%-36% only. In conclusion, the combination of simultaneous siRNA-mediated knockdown of antiapoptotic Bcl-xL and survivin-a multitarget molecular-based therapy-and conventional chemotherapy shows great potential for improving bladder cancer treatment.

Figure 1

Effects of siRNA transfection on the expression of Bcl-xL and survivin. (a) Relative mRNA expression levels of Bcl-xL and survivin in EJ28 and J82 bladder cancer cells, 48 h after transfection. Expression values are normalized to the reference gene TBP and are shown relative to the control siRNA “ns-si” (=100%). Values represent averages of two independent experiments with their mean deviation; (b) Bcl-xL and survivin protein content detected by Western Blotting 48 h after transfection. Bcl-xL and survivin levels are shown normalized to the reference protein β-actin and relative to the ns-si control.

Figure 2

Reduction in cell counts and induction of apoptosis after single and combined siRNA-mediated inhibition of Bcl-xL and survivin in EJ28 and J82 bladder cancer cells. Analyses were performed 48 h after transfection. (a) Cell counts are shown relative to the control siRNA “ns-si” (=100%). Values shown are averages of two independent experiments with their mean deviation; (b) Percentage of apoptotic cells presented as sum of early and late apoptotic cells. Values shown are representatives of two independent experiments.

Figure 3

Induction of polyploidy after knockdown of survivin in single target and target combination treatments. DNA content of EJ28 cells, 48 h after single and combined siRNA-mediated inhibition of Bcl-xL and survivin, is shown. Arrows point at polyploid cells with DNA content of 8N. Representative images of two independent experiments are shown.

Figure 4

Reductions in viability of EJ28 cells after siRNA-mediated inhibition of Bcl-xL and survivin, with or without subsequent chemotherapy (CT). EJ28 cells were transfected with a total of 40 nM siRNAs for four hours. Twenty-four hours after transfection start, cells were treated with 2.1 μg/mL cisplatin (CDDP) for 24 h or with 0.9 μg/mL mitomycin C (MMC) for two hours. Cell viability was examined 96 h after transfection. Values shown are relative to the control siRNA “ns-si” (=100%, for all siRNA treatments) or relative to untreated cells (only for CDDP and MMC single treatments) and are averages of a fourfold determination. Error bars represent the 95% confidence interval. An unpaired Student’s t-test was used to compare the differences in cell viability between target-specific siRNA and ns-si treated cells (*p ≤ 0.05) as well as between target-specific siRNA + CT and ns-si + CT treated cells (^#p ≤ 0.05).

Figure 5

Induction of apoptosis and reduction in cell counts after combined siRNA plus chemotherapy (CT) treatments in EJ28 bladder cancer cells. Cells were transfected with the respective siRNAs for four hours. “CT only” cells were treated with serum-free OptiMEM medium during transfection. Twenty-four hours after transfection start, cells were treated with 2.1 μg/mL cisplatin for 24 h (a,c) or with 0.9 μg/mL mitomycin C for 2 h (b,d). Rate of apoptosis—presented as sum of early and late apoptotic cells—(a,b), as well as cell counts (c,d) were determined 48 h and 72 h after transfection start.

Figure 6

Apoptosis rate after treatment of J82 bladder cancer cells with siRNAs and chemotherapy (CT). Cells were transfected with the respective siRNAs for four hours. “CT only” cells were treated with serum-free OptiMEM medium during transfection. Twenty-four hours after transfection start, cells were treated with 1.2 μg/mL cisplatin for 24 h or with 1.0 μg/mL mitomycin C for two hours. Apoptosis rate—presented as sum of early and late apoptotic cells—was determined 72 h after transfection start. Values shown are averages of two independent experiments. Error bars represent the mean deviation.

Figure 7

Analyses of cell viability, cytotoxicity and caspase-3/7 activity after treatment of EJ28 cells with siRNAs and subsequent chemotherapy (CT). Cells were transfected with the respective siRNAs for four hours. “CT only” cells were treated with serum-free OptiMEM medium during transfection. Twenty-four hours after transfection start, cells were treated with 0.9 μg/mL mitomycin C for two hours. ApoTox-Glo triplex assay was performed 72 h after transfection. Values shown are mean values of duplicates and are relative to the control siRNA “ns-si” plus CT treatment (=100%).