Calenduloside E inhibits hepatocellular carcinoma cell proliferation and migration by down-regulating GPX4 and SLC7A11 expression through the autophagy pathway

doi:10.12122/j.issn.1673-4254.2024.07.12

Abstract

Abstract:

Objective To investigate the molecular mechanism through which calenduloside E inhibits hepatocellular carcinoma (HCC) cell proliferation and migration. Methods HCC cell lines HepG2 and Huh7 treated with calenduloside E were examined for changes in cell viability using CCK-8 assay and expressions of GPX4, SLC7A11, LC3, P62 and phosphorylation of Akt/mTOR using Western blotting. The effects LY294002 and Rapamycin (the inhibitor and activator of autophagy, respectively) on proliferation and migration of calenduloside E-treated HCC cells were evaluated using EdU and Transwell assays. The TCGA database was used to explore the expression levels of GPX4 and SLC7A11 in HCC and normal liver tissues and their correlation with the patients' survival outcomes. GPX4 and SLC7A11 expressions were also detected in HCC cells and normal hepatocytes using RT-qPCR and Western blotting. Results Calenduloside E obviously inhibited the viability of HCC cells. GPX4 and SLC7A11 were highly expressed in HCC tissues and cell lines, and their expression levels were negatively correlated with the patients' survival. In HCC cell lines, calenduloside E significantly inhibited the expressions of GPX4 and SLC7A11 proteins, activated the Akt-mTOR pathway, and enhanced the expression of LC3 II. The inhibitory effect of calenduloside E on GPX4 and SLC7A11 expressions was significantly enhanced by rapamycin but attenuated by LY294002. Inhibiting the autophagy pathway obviously diminished the inhibitory effect of calenduloside E on proliferation and migration of HCC cells, while activating this pathway produced the opposite effect. Conclusion Calenduside E inhibits the proliferation and migration of HCC cells by down-regulating GPX4 and SLC7A11 expression via the autophagy pathway.

Key words: calenduside E, glutathione peroxidase 4, solute carrier family 7 member 11, autophagy, hepatocellular carcinoma

Qianyi CHEN, Shuhan SHANG, Huan LU, Sisi LI, Zhimian SUN, Xirui FAN, Zhilin QI. Calenduloside E inhibits hepatocellular carcinoma cell proliferation and migration by down-regulating GPX4 and SLC7A11 expression through the autophagy pathway[J]. Journal of Southern Medical University, 2024, 44(7): 1327-1335.

Figures/Tables 9

Tab.1 Primer sequences for RT-qPCR of SLC7A11, GPX4 and GAPDH

Primers	Sequence of primer
Primers	Forward (form 5′ to 3′)	Reverse (from 5′ to 3′)
SLC7A11	TCTCCAAAGGAGGTTACCTGC	AGACTCCCCTCAGTAAAGTGAC
GPX4	GAGGCAAGACCGAAGTAAACTAC	CCGAACTGGTTACACGGGAA
GAPDH	AAAGCCTGCCGGTGACTAA	AGAGTTAAAAGCAGCCCTGG

Fig.1 CE inhibits the viability of hepatocellular carcinoma (HCC) Huh7 cells (Mean±SD). *P<0.05, **P<0.01 vs 0 µg/mL group.

Fig.2 Expression levels of GPX4 and SLC7A11 in HCC tissues and cells and their correlation with the patients' survival (Mean±SD). A: Expression of GPX4 and SLC7A11 in HCC tissues and normal liver tissues. B: Correlation of GPX4 and SLC7A11 expressions with the patients' survival. The expressions of GPX4 and SLC7A11 mRNA (C) and protein (D) in HCC cells and normal liver cells. **P<0.01 vs HL-7702.

Fig.3 Effects of CE on GPX4 and SLC7A11 expressions in HepG2 (A, B) and Huh7 cells (C, D). Data are presented as Mean±SD. **P<0.01 vs 0 µg/mL. CE: Calenduloside E.

Fig.4 Effects of CE on GPX4 and SLC7A11 protein degradation in HepG2 (A, B) and Huh7 cells (C, D). Data are presented as Mean±SD. *P<0.05, **P<0.01 vs CHX group.

Fig.5 Effects of CE on the autophagy pathway in HepG2 (A) and Huh7 cells (B). Data are presented as Mean±SD. *P<0.05, **P<0.01 vs 0 µg/mL.

Fig.6 Effects of CE on autophagy of HepG2 (A) and Huh7 cells (B)(Original magnification: ×200).

Fig.7 Effects of autophagy inhibitor or activator on CE-induced GPX4 and SLC7A11 down-regulation in HepG2 (A) and Huh7 (B) cells (Mean±SD). *P<0.05, **P<0.01 vs Control; &P<0.05, &&P<0.01 vs CE.

Fig.8 Effects of autophagy inhibitor or activator on proliferation and migration of CE-treated HepG2 (A, B) and Huh7 (C, D) cells (×100).

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