Telmisartan induces growth inhibition, DNA double-strand breaks and apoptosis in human endometrial cancer cells

Abstract

Telmisartan, an angiotensin II receptor type 1 blocker, is often used as an antihypertension drug, and it has also been characterized as a peroxisome proliferator-activated receptor-gamma (PPARγ) ligand. The purpose of this study was to elucidate the antitumor effects of telmisartan on endometrial cancer cells. We treated three endometrial cancer cell lines with various concentrations of telmisartan, and we investigated the effects of the telmisartan on the cell proliferation, apoptosis, and their related measurements in vitro. We also administered telmisartan to nude mice with experimental tumors to determine its in vivo effects and toxicity. All three endometrial cancer cell lines were sensitive to the growth-inhibitory effect of telmisartan. The induction of apoptosis was confirmed in concert with the altered expression of genes and proteins related to the apoptosis. We also observed that DNA double-strand breaks (DSBs) were induced in HHUA (human endometrial cancer) cells by telmisartan treatment. In addition, experiments in nude mice showed that telmisartan significantly inhibited human endometrial tumor growth, without toxic side effects. Our results suggest that telmisartan might be a new therapeutic option for the treatment of endometrial cancers.

Figure 1. The effect of the ARBs telmisartan, valsartan, losartan and candesartan on the proliferation of endometrial cancer cell lines in vitro.

Anticancer effects of telmisartan via the PPARγ-dependent pathway. (A–D) Ishikawa, HEC-59, and HHUA endometrial cancer cell lines were treated with telmisartan, candesartan, losartan or valsartan at various concentrations (1–100 μM) or the vehicle (control) for 48 h, and proliferation (% of control) was measured in a WST-1 assay. Results are means ± SD of three independent experiments with triplicate dishes. *P<0.05 vs. control, **P<0.01 vs. control. (E) Effect of GW9662 on telmisartan inhibition of HHUA cell proliferation. HHUA cells were incubated with telmisartan (10 or 50 μM) for 48 h followed by preincubation with GW9662 (10 μM) for 30 min. We found that the addition of GW9662 inhibited the anticancer effects of telmisartan at 10 or 50 μM. Results represent the means ± SD of three independent experiments. Columns, means; bars, SDs. *P<0.05 vs. control, **P<0.01 vs. control.

Figure 2. Expression of the apoptosis-related genes and proteins treated with telmisartan in endometrial cancer cells.

HHUA cells were treated with telmisartan at 10 or 100 μM, and cell lysates were harvested after 24 and 48 h. A Western blot analysis was performed with a series of antibodies (Bcl-2, Bcl-xL, Bax, cleaved caspase-3 and GAPDH). Control cells were treated with vehicle alone.

Figure 3. Expression of the cleaved PARP treated with telmisartan in endometrial cancer cells.

The protein expression of cleaved PARP was measured by ELISAs. HHUA cells were treated with 100 μM telmisartan for 48 h. Control cells were treated with vehicle alone. Results represent the means ± SD of three independent experiments. Columns, means; bars, SDs. **P<0.01 vs. control.

Figure 4. The activities of caspase-3 and caspase-7 treated with telmisartan in endometrial cancer cells.

The activities of caspase-3 and caspase-7 were assessed by Caspase-Glo 3/7 Assays. HHUA cells were treated with 100 μM telmisartan for 48 h. Control cells were treated with vehicle alone. Results represent the means ± SD of three independent experiments. Columns, means; bars, SDs. **P<0.01 vs. control.

Figure 5. Analysis of telmisartan-induced DSB formation in HHUA cells.

DSB formation was analyzed by PFGE. HHUA cells collected into agarose plugs, and their DNA was separated by size on an agarose gel. Under the electrophoresis conditions used, high-molecular-weight genomic DNA remained in the well, whereas lower-molecular-weight DNA fragments (several Mbp to 500 kbp) migrated into the gel and were compacted into a signal band.

Figure 6. Immunofluorescent staining of γ-H2AX.

(A) Time and dose dependency of histone H2AX phosphorylation by telmisartan. HHUA cells were treated with 10 or 100 μM telmisartan for indicated times followed by immunostaining using an anti-γ-H2AX antibody. Their nuclei were revealed by DAPI staining. (B) Percentage of γ-H2AX-positive cells. HHUA cells were treated with 10 or 100 μM telmisartan for 24 h or 48 h. Control cells were treated with vehicle alone. The telmisartan-treated cells had significantly higher levels of fluorescence compared to the untreated cells. Results = means ± SD of three independent experiments. Columns, means; bars, SDs. *P<0.05 vs. control, **P<0.01 vs. control.

Figure 7. HHUA tumors in nude mice treated with telmisartan.

(A) HHUA cells (5×10⁶) were bilaterally subcutaneously injected into the trunks of nude mice, forming two tumors per mouse. The mice were divided randomly into control and experimental groups. Telmisartan (100 μg/mouse) or diluent (control) was administered intraperitoneally for 5 days a week for 7 weeks. After 7 weeks of therapy, the tumors were removed from each mouse and weighed. The tumor weights in the two groups were significantly different. Columns, means; bars, SDs. **P<0.01 vs. control. (B) The effect of telmisartan on HHUA tumors in nude mice. The endometrial carcinoma cells from mice treated with telmisartan showed significantly weaker staining for Ki-67 compared to the control endometrial carcinoma cells. (C) Endometrial carcinoma cells from the mice treated with telmisartan underwent apoptosis (TUNEL-positive). All assays were performed three times. Columns, means; bars, SDs. **P<0.01 vs. control.