The angiotensin II type 1 receptor antagonist telmisartan inhibits cell proliferation and tumor growth of esophageal adenocarcinoma via the AMPKα/mTOR pathway in vitro and in vivo

Abstract

Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB). This drug inhibits cancer cell proliferation, but the underlying mechanisms in various cancers, including esophageal cancer, remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human esophageal cancer cell proliferation in vitro and in vivo. We assessed the effects of telmisartan on human esophageal adenocarcinoma (EAC) cells using the cell lines OE19, OE33, and SKGT-4. Telmisartan inhibited the proliferation of these three cell lines via blockade of the G0 to G1 cell cycle transition. This blockade was accompanied by a strong decrease in cyclin D1, cyclin E, and other cell cycle-related proteins. Notably, the AMP-activated protein kinase (AMPK) pathway, a fuel sensor signaling pathway, was enhanced by telmisartan. Compound C, which inhibits the two catalytic subunits of AMPK, enhanced the expression of cyclin E, leading to G0/G1 arrest in human EAC cells. In addition, telmisartan reduced the phosphorylation of epidermal growth factor receptor (p-EGFR) and ERBB2 in vitro. In our in vivo study, intraperitoneal injection of telmisartan led to a 73.2% reduction in tumor growth in mice bearing xenografts derived from OE19 cells. Furthermore, miRNA expression was significantly altered by telmisartan in vitro and in vivo. In conclusion, telmisartan suppressed human EAC cell proliferation and tumor growth by inducing cell cycle arrest via the AMPK/mTOR pathway.

Keywords: AMPKα; angiotensin II type 1 receptor blocker; cell cycle arrest; esophageal adenocarcinoma; telmisartan.

Figure 1. The effects of the ARBs telmisartan, irbesartan, losartan, and valsartan on the proliferation of EAC cell lines in vitro

Telmisartan suppresses the proliferation of EAC cells. OE19, OE33, and SKGT-4 cells were seeded in 96-well plates. After 24 h, ARB (telmisartan, irbesartan, losartan, and valsartan; 1, 10, and 100 μM) or vehicle was added to the culture medium; 24 h later, the cells were evaluated with CCK-8 assays. Cell viability was assayed daily from 0 to 48 h. The viability of the ARB-treated cells was significantly different from that of the control cells (**, P < 0.01).

Figure 2. The antiproliferative effects of telmisartan in EAC cells are mediated via cell cycle arrest

A. Cell cycle analysis of OE19, SKGT-4, and OE33 cells treated with 100 μM telmisartan at 48 h (*, P < 0.05). B. Western blot analysis of PPAR-γ in EAC cells. C. Western blot analysis of cyclin D1, Cdk4, Cdk6, Cdk2, cyclin E, and E2F2 in OE19 and SKGT-4 cells treated with 100 μM telmisartan. D. Western blot analysis of p21Cip1 and p27Kip in OE19 and SKGT-4 cells at 24 and 48 h after the addition of 100 μM telmisartan.

Figure 3. Telmisartan induces cell cycle arrest via activation of the AMPK pathway and suppression of mTOR signaling

A. Three EAC cell lines were treated with 100 μM telmisartan for the indicated times, and the activation status of AMPKα was assessed by western blotting. B. OE19 cells were treated with 100 μM telmisartan, and the activation status of the LKB1/AMPK/mTOR pathway was determined. C. The antiproliferative effects of telmisartan or the control in combination with various concentrations of compound C were assessed in OE19 cells for 48 h. D. Western blot analysis of cyclin D1 and cyclin E in OE19 cells treated with control, telmisartan alone, compound C alone, or telmisartan combined with compound C for 48 h. n.s., not significant; *, P<0.05.

Figure 4

A. The template indicates the locations of tyrosine kinase antibodies spotted onto a human phospho-RTK array. B. Representative expression of various phosphorylated tyrosine kinase receptors in OE19 cells treated with or without 100 μM telmisartan at 24 h. C. Densitometry indicated that the ratios of p-EGFR and ERBB2 spots of telmisartan-treated to untreated cells were 11.6% and 17.5%, respectively. D. Western blot analysis of Akt and p-Akt (Ser473), which are downstream of EGFR signaling, in EAC cells treated with 100 μM telmisartan. E. The antiproliferative effects of telmisartan or the control in combination with various concentrations of MK-2206 were assessed in OE19 cells for 48 h. (D) Western blot analysis of cyclin D1 and cyclin E in OE19 cells treated with the control, telmisartan alone, MK-2206 alone, or telmisartan combined with MK-2206 for 48 h. *,P<0.05.

Figure 5. The growth of OE19-derived tumors in mice treated with telmisartan is inhibited

A. Representative images of gross OE19 tumors from nude mice treated with vehicle (i) or 50 μg of telmisartan. (ii). B. Histological examination using H and E staining. C. Fibrotic tissue as determined by Azan staining of subcutaneous xenograft tumors 22 days after telmisartan injection. D. Tumor growth curves of the control and telmisartan groups. Tumor volume (mm3) was calculated as (tumor length (mm) × tumor width (mm)2)/2. The tumors were significantly smaller in the telmisartan-treated mice than those in the vehicle-treated mice. Each point represents the mean ± standard deviation of 7 animals. P = 0.0007 by two-way ANOVA.

Figure 6. Schematic model for telmisartan inhibition of cell proliferation and G1 cell cycle progression in EAC cells

The activation of AMPKα in human EAC cells inhibits mTOR and p70S6K, inducing G1 cell cycle arrest. Telmisartan may affect the cell cycle via the phosphorylation of EGFR and Akt/mTOR.