Telmisartan inhibits hepatocellular carcinoma cell proliferation in vitro by inducing cell cycle arrest

Abstract

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related death. Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB) that might inhibit cancer cell proliferation, but the mechanisms through which telmisartan affects various cancers remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human HCC and to assess the expression of microRNAs (miRNAs). We studied the effects of telmisartan on HCC cells using the HLF, HLE, HepG2, HuH-7 and PLC/PRF/5 cell lines. In our experiments, telmisartan inhibited the proliferation of HLF, HLE and HepG2 cells, which represent poorly differentiated types of HCC cells. However, HuH-7 and PLC/PRF/5 cells, which represent well-differentiated types of HCC cells, were not sensitive to telmisartan. Telmisartan induced G0/G1 cell cycle arrest of HLF cells by inhibiting the G0-to-G1 cell cycle transition. This blockade was accompanied by a marked decrease in the levels of cyclin D1, cyclin E and other cell cycle-related proteins. Notably, the activity of the AMP-activated protein kinase (AMPK) pathway was increased, and the mammalian target of rapamycin (mTOR) pathway was inhibited by telmisartan treatment. Additionally, telmisartan increased the level of caspase-cleaved cytokeratin 18 (cCK18), partially contributed to the induction of apoptosis in HLF cells and reduced the phosphorylation of ErbB3 in HLF cells. Furthermore, miRNA expression was markedly altered by telmisartan in vitro. In conclusion, telmisartan inhibits human HCC cell proliferation by inducing cell cycle arrest.

Figures

Figure 1.

Telmisartan inhibits the proliferation of HLF cells. (A) Effects of the ARBs (telmisartan, valsartan, losartan and irbesartan) on the proliferation of HLF cells. (B) Effects of telmisartan on the proliferation of HCC cells (HLF, HLE, HepG2, HuH-7 and PLC/PRF/5). (**P

Figure 2.

The antiproliferative effects of telmisartan on HCC cells are mediated by the induction of cell cycle arrest. (A) Cell cycle analysis of HLF cells after telmisartan treatment (*P

Figure 3.

Telmisartan induces the phosphorylation of AMPKα, and the levels of the p-mTOR and p-p70S6K proteins were decreased in HLF cells treated with telmisartan.

Figure 4.

Telmisartan partially contributes to the induction of apoptosis in HLF cells. (A) Flow cytometry assessment of apoptosis in telmisartan-treated HLF cells. (B) Average number of Annexin V positive cells from a triplicate assessment. (C) The levels of cCK18 were measured by ELISA (*P

Figure 5.

Telmisartan increases the level of phospho-p53 (ser392) and decreases the level of survivin. (A) Template showing the location of apoptosis-related proteins on the human apoptosis antibody array. (B) Representative expression levels of various apoptosis-related proteins in HLF cells treated with or without telmisartan. (C) The densitometric ratio of the telmisartan-treated group to the non-treated group for the phospho-p53 (ser392) and survivin spots (*P

Figure 6.

Telmisartan reduces the levels of p-ErbB3 in HLF cells. (A) Template showing the locations of the tyrosine kinase antibodies on the human p-RTK array. (B) Representative expression of various p-RTKs in HLF cells treated with or without telmisartan. (C) The densitometric ratio of the telmisartan-treated group to the non-treated group for the p-ErbB3 spots (**P

Figure 7.

Telmisartan increases the levels of bFGF in HLF cells. (A) Template showing the locations of angiogenesis-related proteins spotted on the human angiogenesis array. (B) Representative expression levels of various angiogenesis-related proteins in HLF cells treated with or without telmisartan. (C) The densitometric ratio of the telmisartan-treated group to the non-treated group for the bFGF spots (**P

Figure 8.

Telmisartan affects miRNA expression in HLF cells. (A) Hierarchical clustering of HLF cells cultured with or without telmisartan according to the expression profiles of numerous differentially expressed miRNAs. The miRNA clustering color scale presented at the top indicates the relative miRNA expression levels, with red and blue representing high and low expression levels, respectively (P102−ΔCt value for each miRNA was used to create the figure; the lines represent averages with interquartile ranges (**P<0.01).