2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway

doi:10.19852/j.cnki.jtcm.20230904.005

Abstract

Abstract:

OBJECTIVE: To investigate the anti-liver cancer effect of 2-hydroxy-3-methyl anthraquinone (HMA) and the specific mechanism based on nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1 (SIRT1)/cellular tumor antigen p53 (p53) pathway.

METHODS: Cell counting kit-8 method was used to observe the effect of HMA on the activity of human hepatocellular carcinoma cells (HepG2) cells. At 72 h and 80 μL HMA, the apoptosis rate of HepG2 cells in each group was measured by flow cytometry. Transwell was used to assay for cell invasion. The protein expression levels of SIRT1, p53, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-9 (CASP9) and caspase-3 (CASP3) were detected by Western Blot.

RESULTS: HMA significantly inhibited the proliferation of HepG2 cells, The half inhibiting concentration (IC50) of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively. Compared with the control group, the apoptosis rate of HMA, Selisistat (EX527), and HMA+ EX527 groups enhanced, while the apoptosis rate of SRT1720 diminished, demonstrating that inhibition of SIRT1 can lead to apoptosis of HepG2 cells. HMA+ EX527 group had the highest apoptosis rate, the lowest expression of SIRT1 and Bcl-2, and the highest expression of p53, Bax, CASP9 and CASP3. The number of invasions of HepG2 was significantly reduced after HMA and EX527 intervened. Western blot shows HMA could inhibit SIRT1, promote the expression of p53, and decrease the ratio of Bcl-2/Bax.

CONCLUSIONS: HMA induced apoptosis in HepG2 cells, while inhibiting proliferation and invasion. The mechanism of HMA against HCC may be related to the SIRT1/p53 pathway.

Key words: 2-hydroxy-3-methylanthraquinone, carcinoma, hepatocellular, apoptosis, invasion, sirtuin 1, genes p53

WU Shuang, LI Qiao, ZHU Xieying, ZHANG Taoyuan. 2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway[J]. Journal of Traditional Chinese Medicine, 2024, 44(6): 1104-1110.

Figures/Tables 4

Figure 1 Effects of HMA on the viability of HepG2 cells A: molecular formula of HMA; B: cell viability at different HMA doses and administration times. HMA with 0, 20, 40, 60, 80, 100 and 120 μMol/L for 24, 48 and 72 h to treatment HepG2 cells. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. Compare with 0 μM, aP < 0.001; compare with same time, bP < 0.001. Data were presented as mean ± standard deviation (n = 3).

Figure 2 Apoptosis rate of HepG2 after treatment by flow cytometry A-F: flow cytometry; A: Control group: Solvent treatment 72 h; B: HMA group: 80 μmol/L HMA treatment 72 h; C: HMA+EX527 group: 80 μmol/L HMA and 1 μmol/L EX527 treatment 72 h; D: HMA+SRT1720 group: 80 μmol/L HMA and 1 μmol/L SRT1720 treatment 72 h; E: EX527 group: 1 μmol/L EX527 treatment 72 h; F: SRT1720 group: 1 μmol/L SRT1720 treatment 72 h; G: comparison of apoptosis rates. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. aP < 0.001, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

Figure 3 Number of invasion of HepG2 after intervention by Transwell A-F: the number of invasion; A: Control group: Solvent treatment 72 h; B: HMA group: 80 μmol/L HMA treatment 72 h; C: HMA + EX527 group: 80 μmol/L HMA and 1 μmol/L EX527 treatment 72 h; D: HMA+SRT1720 group: 80 μmol/L HMA and 1 μmol/L SRT1720 treatment 72 h; E: EX527 group: 1 μmol/L EX527 treatment 72 h; F: SRT1720 group: 1 μmol/L SRT1720 treatment 72 h. Bar = 100 μm. G: comparison of number of invasion. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. aP < 0.001, bP < 0.01, cP < 0.05, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

Figure 4 HMA via SIRT1 / p53 activates apoptotic pathway A: protein levels by Western blot; ‘+’ represent treatment, ‘+’ represent no-treatment; B: SIRT1 protein expression; C: p53 protein expression; D: Bcl-2/Bax protein expression; E: Bcl-2 protein expression; F: Bax protein expression; G: CASP3 protein expression; H: CASP9 protein expression. Control: Solvent treatment 72 h; HMA: 80 μmol/L HMA treatment 72 h; HMA+EX527: 80 μmol/L HMA and 1 μmol/L EX527 treatment 72 h; HMA+SRT1720: 80 μmol/L HMA and 1 μmol/L SRT1720 treatment 72 h; EX527: 1 μmol/L EX527 treatment 72 h; SRT1720: 1 μmol/L SRT1720 treatment 72 h. HepG2: human hepatocellular carcinoma cells; HMA: 2-hydroxy-3-methyl anthraquinone; Selisistat: EX527. SIRT1: nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1; p53: cellular tumor antigen p53; Bcl-2: B-cell lymphoma-2; Bax: anti-Bcl-2 associated X protein; CASP9: caspase-9; CASP3: caspase-3. One-way analysis of variance followed by Tukey's multiple comparisons test, Graphpad 9.0.0. aP < 0.001, bP < 0.05, compare with Control group. Data were presented as mean ± standard deviation (n = 3).

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