20(S)-Ginsenoside Rg3 Partially Induces Maturation of HepG2 Cells via the AMPK/HNF4A Pathway
Abstract
Background: We aimed to investigate whether 20(S)-ginsenoside Rg3 (Rg3) reduced heterogeneity by inducing the maturation of HepG2 cells via the AMP-activated protein kinase (AMPK)/hepatocyte nuclear factor 4A (HNF4A) pathway.
Methods: This in vitro cell research study was conducted under the guidance of Hebei Medical University, Hebei, China, from Sep 2022 to Dec 2023. HepG2 cells were treated with varying concentrations of Rg3 in a low glucose microenvironment. The mRNA expression of ALBUMIN (ALB, a marker for hepatocyte function) and HNF4A (a marker for differentiation of HCC cell), and AMPK protein levels were measured after significant changes in cell morphology were observed. Additionally, 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR) (an AMPK agonist) and Compound C (an AMPK inhibitor) were used to explore further the underlying mechanism.
Results: Under treatment of 5 μM, 10 μM, and 20 μM Rg3, some cells became flattened and larger, and there was an increase in the mRNA expression of ALB and HNF4A (P<0.05). However, there was a decreasing trend in AMPK protein content with 8 μM Rg3 (P<0.05). Compared to the control group, some cells exposed to 8 μM Rg3 exhibited pronounced morphological changes, along with upregulated expression of ALB and HNF4A mRNA. However, no such changes were observed when 8 μM Rg3 was combined with 1.6 mM AICAR. Compared to the control group, 10 μM Compound C or 8 μM Rg3 treatments led to similar changes in cell morphology and showed an increasing trend in HNF4A mRNA expression. Additionally, after treatment with Compound C, pHNF4A was mainly in the nucleus, while after Rg3 treatment, it was mostly in the cytoplasm.
Conclusion: Rg3 partially induced the maturation of HepG2 cells through the AMPK/HNF4A pathway.
Keywords: 20(S)-ginsenoside Rg3; HepG2; Maturation, Genetics
2. Moradi S, Moradi G, Piroozi B et al (2021). The Burden of Cancer in a Sample of Iranian Population. Iran J Public Health, 50(8):1687-96.
3. Ladd AD, Duarte S, Sahin I, et al (2024). Mechanisms of drug resistance in HCC. Hepatology, 79(4):926-40.
4. Chan LK, Tsui YM, Ho DW, et al (2022). Cellular heterogeneity and plasticity in liver cancer. Semin Cancer Biol, 82:134-49.
5. Zhang X, Zhu XJ, Zhong Z, et al (2022). Small Molecule-Induced Differentiation As a Potential Therapy for Liver Cancer. Adv Sci (Weinh), 9(15):e2103619.
6. Li Y, Zong Y, Xiao Z, et al (2016). Devel-opmental Stage-Specific Embryonic In-duction of HepG2 Cell Differentiation. Dig Dis Sci, 61(4):1098-106.
7. Zhou B, Yan Z, Liu R, et al (2016). Prospec-tive Study of Transcatheter Arterial Chemoembolization (TACE) with Gin-senoside Rg3 versus TACE Alone for the Treatment of Patients with Ad-vanced Hepatocellular Carcinoma. Radi-ology, 280(2):630-9.
8. Hwang SK, Jeong YJ, Cho HJ, et al (2022). Rg3-enriched red ginseng extract pro-motes lung cancer cell apoptosis and mitophagy by ROS production. J Ginseng Res, 46(1):138-46.
9. Xia J, Ma S, Zhu X, et al (2022). Versatile ginsenoside Rg3 liposomes inhibit tu-mor metastasis by capturing circulating tumor cells and destroying metastatic niches. Sci Adv, 8(6): eabj1262.
10. Sun D, Zou Y, Song L, et al (2022). A cy-clodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer. Acta Pharm Sin B, 12(1):378-93.
11. Cai X, Wang Z, Lin S, et al (2024). Ginseno-side Rg3 suppresses vasculogenic mim-icry by impairing DVL3-maintained stemness via PAAD cell-derived exoso-mal miR-204 in pancreatic adenocarci-noma. Phytomedicine, 126:155402.
12. Wang J, Tian L, Khan MN, et al (2018). Ginsenoside Rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of NF-κB mediated epithelial-mesenchymal transition and stemness. Cancer Lett, 415:73-85.
13. Peng M, Li X, Zhang T, et al (2016). Stere-oselective pharmacokinetic and metabo-lism studies of 20(S)- and 20(R)-ginsenoside Rg₃ epimers in rat plasma by liquid chromatography-electrospray ionization mass spectrometry. J Pharm Biomed Anal, 121:215-24.
14. Cheng Z, He Z, Cai Y, et al (2019). Conver-sion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors. Cell Res, 29(2):124-35.
15. Li Y, Liu D, Zong Y, et al (2015). Develop-mental Stage-Specific Hepatocytes In-duce Maturation of HepG2 Cells by Re-building the Regulatory Circuit. Mol Med, 21(1):285-95.
16. Li X, Feng L, Kuang Q, et al (2024). Micro-plastics cause hepatotoxicity in diabetic mice by disrupting glucolipid metabo-lism via PP2A/AMPK/HNF4A and promoting fibrosis via the Wnt/β-catenin pathway. Environ Toxicol, 39(2):1018-30.
17. Zhong Y, Qi H, Li X, et al (2020). Tumor supernatant derived from hepatocellular carcinoma cells treated with vincristine sulfate have therapeutic activity. Eur J Pharm Sci, 155:105557.
18. Li Y, Xiao Z, Li B, et al (2016). Ginsenoside exhibits concentration-dependent dual effects on HepG2 cell proliferation via regulation of c-Myc and HNF-4α. Eur J Pharmacol, 792:26-32.
19. Jo H, Lee J, Jeon J, et al (2020). The critical role of glucose deprivation in epithelial-mesenchymal transition in hepatocellu-lar carcinoma under hypoxia. Sci Rep, 10(1):1538.
20. Zhao W, Ma J, Zhang Q, et al (2024). Gin-senoside Rg3 overcomes tamoxifen re-sistance through inhibiting glycolysis in breast cancer cells. Cell Biol Int, 48(4):496-509.
21. Liu H, Xie T, Liu Y (2023). Ginsenoside Rg3 inhibits the malignant progression of cervical cancer cell by regulating AKT2 expression. Heliyon, 9(8):e19045.
22. Li J, Yang B (2023). Ginsenoside Rg3 en-hances the radiosensitivity of lung can-cer A549 and H1299 cells via the PI3K/AKT signaling pathway. In Vitro Cell Dev Biol Anim, 59(1):19-30.
23. Huang Y, Xian L, Liu Z, et al (2022). AMPKα2/HNF4A/BORIS/GLUT4 pathway promotes hepatocellular carci-noma cell invasion and metastasis in low glucose microenviroment. Biochem Pharmacol, 203:115198.
24. Hong YH, Varanasi US, Yang W, et al (2003). AMP-activated protein kinase regulates HNF4alpha transcriptional ac-tivity by inhibiting dimer formation and decreasing protein stability. J Biol Chem, 278(30):27495-501.
25. Prabhakar B, Lee S, Bochanis A, et al (2021). lnc-RHL, a novel long non-coding RNA required for the differenti-ation of hepatocytes from human bipo-tent progenitor cells. Cell Prolif, 54(2):e12978.
26. Rodríguez-Aguilera JR, Ecsedi S, Gold-smith C, et al (2020). Genome-wide 5-hydroxymethylcytosine (5hmC) emerges at early stage of in vitro differentiation of a putative hepatocyte progenitor. Sci Rep, 10(1):7822.
27. Schonfeld M, Averilla J, Gunewardena S, et al (2022). Male-Specific Activation of Ly-sine Demethylases 5B and 5C Mediates Alcohol-Induced Liver Injury and Hepatocyte Dedifferentiation. Hepatol Commun, 6(6):1373-91.
28. Vető B, Bojcsuk D, Bacquet C, et al (2017). The transcriptional activity of hepato-cyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2. PLoS One, 12(2):e0172020.
29. Xu Y, Zhou Z, Kang X, et al (2022). Mettl3-mediated mRNA m6A modification controls postnatal liver development by modulating the transcription factor Hnf4a. Nat Commun, 13(1):4555.
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