Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

Abstract

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.

Keywords: Bcl-2; Telmisartan; apoptosis; caspase-3; human cancer cells.

© 2014 by the Society for Experimental Biology and Medicine.

Figures

Figure 1

Telmisartan reduced cell viability in a concentration-dependent manner. Half-maximal inhibition of RCC growth was observed for concentrations of telmisartan ranging from 25 to 400 µM. ***P < 0.001

Figure 2

Cisplatin reduced cell viability in a concentration-dependent manner. Half-maximal inhibition of 786 RCC cell growth was observed for concentrations of telmisartan ranging from 25 to 400 µM. ***P < 0.001

Figure 3

Apoptosis induced by telmisartan and cisplatin in 786 RCC cells was detected using flow cytometry. Levels of early apoptosis (EA) (a) and late apoptosis (LA) (b) were detected for cells treated with 100 µM and 200 µM telmisartan for 4 h, 24 h, and 48 h. Levels of EA (c) and LA (d) were also detected for cells treated with 50 µM cisplatin for 4 h, 24 h, and 48 h. Apoptosis induced by telmisartan and cisplatin in HEK cells was detected using flow cytometry. Levels of EA (e) and LA (f) were detected for cells treated with 200 µM telmisartan for 24 h and 48 h. Levels of EA (g) and LA (h) were detected for cells treated with 50 µM cisplatin for 24 h and 48 h. ***P < 0.001; **P < 0.01; *P < 0.05

Figure 4

Effects of telmisartan and cisplatin on early and late apoptosis as detected using flow cytometry. Treatment of 786 RCC cells with 100 µM and 200 µM telmisartan induced early- or late-stage apoptosis after 24 h (e, f) and 48 h (h, i), but not at the 4 h timepoint (b, c). In contrast, treatment of normal HEK cells with 200 µM telmisartan did not induce apoptosis after 24 h (q), yet did after 48 h (s). The lower right quadrants represent the Annexin V-FITC-positive/PI-negative cells in the early stages of apoptosis, while the top right quadrants include Annexin V-FITC-positive/PI-positive cells in the late stages of apoptosis. Treatment with 50 µM cisplatin induced either early or late apoptosis in 786 RCC cells at the 4 h (k), 24 h (m), and 48 h (o) timepoints, and a greater number of late stage cells were observed. Treatment with 50 µM cisplatin also induced early and late apoptosis in HEK cells 24 h (u) and 48 h (x) after treatment, with the latter being more predominant. (A color version of this figure is available in the online journal.)

Figure 4

Effects of telmisartan and cisplatin on early and late apoptosis as detected using flow cytometry. Treatment of 786 RCC cells with 100 µM and 200 µM telmisartan induced early- or late-stage apoptosis after 24 h (e, f) and 48 h (h, i), but not at the 4 h timepoint (b, c). In contrast, treatment of normal HEK cells with 200 µM telmisartan did not induce apoptosis after 24 h (q), yet did after 48 h (s). The lower right quadrants represent the Annexin V-FITC-positive/PI-negative cells in the early stages of apoptosis, while the top right quadrants include Annexin V-FITC-positive/PI-positive cells in the late stages of apoptosis. Treatment with 50 µM cisplatin induced either early or late apoptosis in 786 RCC cells at the 4 h (k), 24 h (m), and 48 h (o) timepoints, and a greater number of late stage cells were observed. Treatment with 50 µM cisplatin also induced early and late apoptosis in HEK cells 24 h (u) and 48 h (x) after treatment, with the latter being more predominant. (A color version of this figure is available in the online journal.)

Figure 4

Effects of telmisartan and cisplatin on early and late apoptosis as detected using flow cytometry. Treatment of 786 RCC cells with 100 µM and 200 µM telmisartan induced early- or late-stage apoptosis after 24 h (e, f) and 48 h (h, i), but not at the 4 h timepoint (b, c). In contrast, treatment of normal HEK cells with 200 µM telmisartan did not induce apoptosis after 24 h (q), yet did after 48 h (s). The lower right quadrants represent the Annexin V-FITC-positive/PI-negative cells in the early stages of apoptosis, while the top right quadrants include Annexin V-FITC-positive/PI-positive cells in the late stages of apoptosis. Treatment with 50 µM cisplatin induced either early or late apoptosis in 786 RCC cells at the 4 h (k), 24 h (m), and 48 h (o) timepoints, and a greater number of late stage cells were observed. Treatment with 50 µM cisplatin also induced early and late apoptosis in HEK cells 24 h (u) and 48 h (x) after treatment, with the latter being more predominant. (A color version of this figure is available in the online journal.)

Figure 5

Detection of caspase-3 and Bcl-2 in 786 RCC cells. Cells were stained with DAPI (blue) and anti-Bcl-2 and anti-caspase-3 antibodies (green) to detect caspase-3 activation and Bcl-2 expression as described in the ‘Materials and methods’ section. Untreated 786 cells (a, e), and HEK control cells (i) are shown. Following exposure of 786 RCC cells to 100 µM and 200 µM telmisartan for 24 h and 48 h, positive staining for active caspase-3 (b, c) and negative staining for Bcl-2 were observed (f, g). In contrast, positive staining for caspase-3 was observed at the 24 h and 48 h timepoints following treatment with 50 µM cisplatin (d), while Bcl-2 expression remained negative (h). B.1 and C.1 show the staining of positive markers for caspase-3 without an overlay of DAPI staining. Exposure of HEK cells to dose of telmisartan (200 µM) for 24 h and 48 h did not activate caspase-3 (j). In panel (k), positive staining for caspase-3 is shown for HEK cells treated with 50 µM cisplatin for 24 h and 48 h. (A color version of this figure is available in the online journal.)

Figure 5

Detection of caspase-3 and Bcl-2 in 786 RCC cells. Cells were stained with DAPI (blue) and anti-Bcl-2 and anti-caspase-3 antibodies (green) to detect caspase-3 activation and Bcl-2 expression as described in the ‘Materials and methods’ section. Untreated 786 cells (a, e), and HEK control cells (i) are shown. Following exposure of 786 RCC cells to 100 µM and 200 µM telmisartan for 24 h and 48 h, positive staining for active caspase-3 (b, c) and negative staining for Bcl-2 were observed (f, g). In contrast, positive staining for caspase-3 was observed at the 24 h and 48 h timepoints following treatment with 50 µM cisplatin (d), while Bcl-2 expression remained negative (h). B.1 and C.1 show the staining of positive markers for caspase-3 without an overlay of DAPI staining. Exposure of HEK cells to dose of telmisartan (200 µM) for 24 h and 48 h did not activate caspase-3 (j). In panel (k), positive staining for caspase-3 is shown for HEK cells treated with 50 µM cisplatin for 24 h and 48 h. (A color version of this figure is available in the online journal.)