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).

References 37

1	Xia CF, Dong XS, Li H, et al. Cancer statistics in China and United States, 2022: profiles, trends, and determinants[J]. Chin Med J, 2022, 135(5): 584-90.
2	Wang SN, Wu XW, Wu XM, et al. Systematic analysis of the role of LDHs subtype in pan-cancer demonstrates the importance of LDHD in the prognosis of hepatocellular carcinoma patients[J]. BMC Cancer, 2024, 24(1): 156.
3	谢思雨, 李淼生, 江峰乐, 等. EHHADH是肝细胞癌脂肪酸代谢通路的关键基因: 基于转录组分分析[J]. 南方医科大学学报, 2023, 43(5): 680-93. DOI: 10.12122/j.issn.1673-4254.2023.05.02
4	Metur SP, Lei YC, Zhang ZH, et al. Regulation of autophagy gene expression and its implications in cancer[J]. J Cell Sci, 2023, 136(10): jcs260631.
5	Jain V, Singh MP, Amaravadi RK. Recent advances in targeting autophagy in cancer[J]. Trends Pharmacol Sci, 2023, 44(5): 290-302.
6	Chow AK, Yau SW, Ng L. Novel molecular targets in hepatocellular carcinoma[J]. World J Clin Oncol, 2020, 11(8): 589-605.
7	Wang Y, Deng BC. Hepatocellular carcinoma: molecular mechanism, targeted therapy, and biomarkers[J]. Cancer Metastasis Rev, 2023, 42(3): 629-52.
8	Glaviano A, Foo ASC, Lam HY, et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer[J]. Mol Cancer, 2023, 22(1): 138.
9	He PZ, He Y, Ma JJ, et al. Thymoquinone induces apoptosis and protective autophagy in gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway[J]. Phytother Res, 2023, 37(8): 3467-80.
10	Lee J, Roh JL. SLC7A11 as a gateway of metabolic perturbation and ferroptosis vulnerability in cancer[J]. Antioxidants, 2022, 11(12): 2444.
11	Liu JY, Xia XJ, Huang P. xCT: a critical molecule that links cancer metabolism to redox signaling[J]. Mol Ther, 2020, 28(11): 2358-66.
12	Huang Y, Dai ZY, Barbacioru C, et al. Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance[J]. Cancer Res, 2005, 65(16): 7446-54.
13	Li SJ, Lu ZY, Sun RB, et al. The role of SLC7A11 in cancer: friend or foe[J]? Cancers, 2022, 14(13): 3059.
14	Zhang W, Liu Y, Liao Y, et al. GPX4, ferroptosis, and diseases[J]. Biomed Pharmacother, 2024, 174: 116512.
15	Jia CX, Zhang X, Qu TT, et al. Depletion of PSMD14 suppresses bladder cancer proliferation by regulating GPX4[J]. PeerJ, 2023, 11: e14654.
16	Tang X, Ding H, Liang ML, et al. Curcumin induces ferroptosis in non-small-cell lung cancer via activating autophagy[J]. Thorac Cancer, 2021, 12(8): 1219-30.
17	Le YF, Guo JN, Liu ZJ, et al. Calenduloside E ameliorates non-alcoholic fatty liver disease via modulating a pyroptosis-dependent pathway[J]. J Ethnopharmacol, 2024, 319(Pt 2): 117239.
18	Li JX, Bu YJ, Li B, et al. Calenduloside E alleviates cerebral ischemia/reperfusion injury by preserving mitochondrial function[J]. J Mol Histol, 2022, 53(4): 713-27.
19	汤托, 王胜男, 蔡田雨, 等. 金盏花苷E通过ROS介导的JAK1-stat3信号途径抑制LPS诱发的炎症反应[J]. 南方医科大学学报, 2019, 39(8): 904-10. DOI: 10.12122/j.issn.1673-4254.2019.08.05
20	Wang SN, Chen XL, Cheng J, et al. Calunduloside E inhibits HepG2 cell proliferation and migration via p38/JNK-HMGB1 signalling axis[J]. J Pharmacol Sci, 2021, 147(1): 18-26.
21	Zhang W, Jiang BP, Liu YX, et al. Bufotalin induces ferroptosis in non-small cell lung cancer cells by facilitating the ubiquitination and degradation of GPX4[J]. Free Radic Biol Med, 2022, 180: 75-84.
22	Li DB, Wang YH, Dong C, et al. CST1 inhibits ferroptosis and promotes gastric cancer metastasis by regulating GPX4 protein stability via OTUB1[J]. Oncogene, 2023, 42(2): 83-98.
23	Wang XB, Chen YQ, Wang XD, et al. Stem cell factor SOX2 confers ferroptosis resistance in lung cancer via upregulation of SLC7A11[J]. Cancer Res, 2021, 81(20): 5217-29.
24	Hanzl A, Winter GE. Targeted protein degradation: current and future challenges[J]. Curr Opin Chem Biol, 2020, 56: 35-41.
25	Wang Y, Le WD. Autophagy and ubiquitin-proteasome system[J]. Adv Exp Med Biol, 2019, 1206: 527-50.
26	Xu ZR, Han X, Ou DM, et al. Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy[J]. Appl Microbiol Biotechnol, 2020, 104(2): 575-87.
27	Zhang M, Liu SH, Chua MS, et al. SOCS5 inhibition induces autophagy to impair metastasis in hepatocellular carcinoma cells via the PI3K/Akt/mTOR pathway[J]. Cell Death Dis, 2019, 10(8): 612.
28	Li H, Zhao SF, Shen LW, et al. E2F2 inhibition induces autophagy via the PI3K/Akt/mTOR pathway in gastric cancer[J]. Aging, 2021, 13(10): 13626-43.
29	Zhang JH, Fan JJ, Zeng X, et al. Targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive gastric cancer[J]. Cell Death Dis, 2021, 12(4): 288.
30	Yi H, Wang K, Du BY, et al. Aleuritolic acid impaired autophagic flux and induced apoptosis in hepatocellular carcinoma HepG2 cells[J]. Molecules, 2018, 23(6): 1338.
31	Li JK, Zhu PL, Wang Y, et al. Gracillin exerts anti-melanoma effects in vitro and in vivo: role of DNA damage, apoptosis and autophagy[J]. Phytomedicine, 2023, 108: 154526.
32	Yu WX, Lu C, Wang B, et al. Effects of rapamycin on osteosarcoma cell proliferation and apoptosis by inducing autophagy[J]. Eur Rev Med Pharmacol Sci, 2020, 24(2): 915-21.
33	Chen WS, Xian GY, Gu MH, et al. Autophagy inhibitors 3-MA and LY294002 repress osteoclastogenesis and titanium particle-stimulated osteolysis[J]. Biomater Sci, 2021, 9(14): 4922-35.
34	Ouyang SM, Li HX, Lou LL, et al. Inhibition of STAT3-ferroptosis negative regulatory axis suppresses tumor growth and alleviates chemoresistance in gastric cancer[J]. Redox Biol, 2022, 52: 102317.
35	Zeng C, Lin J, Zhang KT, et al. SHARPIN promotes cell proliferation of cholangiocarcinoma and inhibits ferroptosis via p53/SLC7A11/GPX4 signaling[J]. Cancer Sci, 2022, 113(11): 3766-75.
36	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5): 1060-72.
37	Mou YH, Wang J, Wu JC, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer[J]. J Hematol Oncol, 2019, 12(1): 34.

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