Suppression of AGTR1 Induces Cellular Senescence in Hepatocellular Carcinoma Through Inactivating ERK Signaling

Abstract

Objective: Cellular senescence is an effective barrier against tumorigenesis. Hence, it is of significance to characterize key features of cellular senescence and the induction of senescence in hepatocellular carcinoma (HCC) cells via pharmacological interventions. Our study determined the biological roles as well as mechanisms of angiotensin II type I receptor (AGTR1) on cellular senescence in HCC. Methods: Lentivirus vector-mediated overexpression or knockdown of AGTR1 was conducted in HCC cells, respectively. A volume of 8 μM sorafenib was used to induce cellular senescence, and ERK was activated by 30 ng/ml ERK agonist EGF. Proliferation was evaluated via clone formation assay. HCC cell senescence was examined by flow cytometry for cell cycle, senescence-associated β-galactosidase (SA-β-gal) staining, and senescence-associated heterochromatin foci (SAHF) analysis. AGTR1, p53, p21, extracellular signal-regulated kinase (ERK), and p-ERK expression were assessed through Western blot or immunofluorescence. Results: AGTR1-knockout HCC cells displayed the attenuated proliferative capacity, G2-M phase arrest, increased expression of p53 and p21, and elevated percentages of SA-β-gal- and SAHF-positive cells. In sorafenib-exposed HCC cells, overexpressed AGTR1 enhanced the proliferative capacity and alleviated G2-M phase arrest as well as decreased p53 and p21 expression and the proportions of SA-β-gal- and SAHF-positive cells. Moreover, AGTR1 knockdown attenuated the activity of p-ERK in HCC cells, and ERK agonist ameliorated AGTR1 knockdown-induced cellular senescence. Conclusion: This study demonstrates that suppression of AGTR1 induces cellular senescence in HCC through inactivating ERK signaling. The significant synergistic effect of AGTR1 suppression and sorafenib might represent a potential combination therapy for HCC.

Keywords: AGTR1; ERK signaling; cellular senescence; hepatocellular carcinoma; sorafenib.

FIGURE 1

AGTR1 knockdown alleviates proliferation and induces growth arrest for HCC cells. (A,B) AGTR1 protein expression in L-02, HepG2 along with Huh7 cell lines. (C–E) AGTR1 protein expression in HepG2 and Huh7 cell lines with sh-AGTR1 lentivirus transduction. (F–H) Number of colonies in HCC cell lines with sh-AGTR1 lentivirus transduction. (I–K) Cell cycle distribution of HCC cell lines with sh-AGTR1 lentivirus transduction.

FIGURE 2

Suppression of AGTR1 triggers cellular senescence of HCC cells via p53/p21 signaling. (A–E) Expression of p53, p-p53, p21, and p-p21 in HepG2 and Huh7 cell lines with sh-AGTR1 lentivirus transduction. (F,G) SA-β-Gal activity of HCC cell lines with sh-AGTR1 lentivirus transduction. Scale bar, 10 μm. (H,I) SAHF activity of HCC cell lines with sh-AGTR1 lentivirus transduction. Scale bar, 10 μm.

FIGURE 3

Upregulated AGTR1 increases proliferation and weakens growth arrest for sorafenib-treated HCC cells. (A,B) AGTR1 protein expression in sorafenib-treated HepG2 and Huh7 cell lines under AGTR1 lentivirus transduction. (C,D) Number of colonies of sorafenib-treated HCC cells with AGTR1 lentivirus transduction. (E–G) Cell cycle of sorafenib-treated HCC cells with AGTR1 lentivirus transduction.

FIGURE 4

Upregulated AGTR1 weakens cellular senescence of sorafenib-treated HCC cells. (A–C) p53 and p21 protein expression in sorafenib-treated HepG2 along with Huh7 cell lines under AGTR1 lentivirus transduction. (D,E) SA-β-Gal activity of HCC cell lines with AGTR1 lentivirus transduction. Scale bar, 10 μm. (F,G) SAHF activity of HCC cell lines with AGTR1 lentivirus transduction. Scale bar, 10 μm.

FIGURE 5

Suppression of AGTR1 weakens ERK activity in HCC cells. (A–C) ERK and p-ERK levels in HepG2 and Huh7 cell lines with sh-AGTR1 lentivirus transduction. (D,E) Immunofluorescent staining of p-ERK in HCC cell lines with sh-AGTR1 lentivirus transduction. Scale bar, 10 μm.

FIGURE 6

AGTR1 knockdown suppresses proliferation and triggers growth arrest for HCC cells through inactivating ERK signaling. (A–C) Number of colonies of HepG2 and Huh7 cell lines with sh-AGTR1 lentivirus transduction or ERK agonist EGF. (D–F) Cell cycle of HCC cell lines with sh-AGTR1 lentivirus transduction or ERK agonist EGF.

FIGURE 7

AGTR1 knockdown triggers cellular senescence of HCC cells in an ERK-dependent pathway. (A–E) p53 and p21 protein expressions in HepG2 along with Huh7 cell lines with sh-AGTR1 lentivirus transduction or ERK agonist EGF. (F–H) SA-β-Gal activity of HCC cell lines with sh-AGTR1 lentivirus transduction or ERK agonist EGF. Scale bar, 10 μm. (I–K) SAHF activity of HCC cell lines with sh-AGTR1 lentivirus transduction or ERK agonist EGF. Scale bar, 10 μm.