GADD45β induction by S-adenosylmethionine inhibits hepatocellular carcinoma cell proliferation during acute ischemia-hypoxia

Abstract

Growth arrest DNA damage-inducible gene 45β (GADD45β), which influences cell growth, apoptosis and cellular response to DNA damage, is downregulated in hepatocellular carcinoma (HCC). S-adenosylmethionine (SAMe) serves as an essential methyl donor in multiple metabolic pathways and is a polyamine and glutathione (GSH) precursor. In this study, we assessed the roles of GADD45β and SAMe in cell survival during acute ischemia-hypoxia (I/H). SAMe treatment induced growth of HL-7702 normal hepatic cells, but decreased the viability of HepG2 (p53 wild-type) and Hep3B (p53 null) HCC cells. Cells were exposed to I/H with or without SAMe pre-treatment. I/H exposure alone triggered HCC cell proliferation promoted by autophagy. SAMe pre-treatment restored GADD45β expression and activated HCC cell apoptosis and eliminated I/H-induced HCC cell proliferation. p53 loss blunted the response to SAMe and I/H exposure in Hep3B cells; thus, the inhibitory effect of SAMe on cell proliferation may be reduced in p53-null cells as compared to wild-type cells. These results indicate that GADD45β induction by SAMe inhibits HCC cell proliferation during I/H as a result of increased apoptosis, and that SAMe also protects normal hepatocytes from apoptotic cell death and promotes normal cell regeneration. SAMe should be considered a potential therapeutic agent for the management of HCC.

Keywords: GADD45β; S-adenosylmethionine; apoptosis; hepatocellular carcinoma; ischemia-hypoxia.

Figure 1. Effects of SAMe on cell viability

Treatment with low SAMe concentrations (0-10 mmol/L) for 24 h induced HL-7702 cell growth, with an optimal increase in viability at 5 mmol/L. Higher concentrations (10-20 mmol/L) inhibited HL-7702 cell growth. SAMe treatment resulted in a dose-dependent inhibition of HepG2 and Hep3B cell proliferation.

Figure 2. Effects of acute I/H on cell proliferation with/without SAMe pre-treatment

Comparison of cell proliferation among Control (C), SAMe treatment (S), Ischemia (I), Ischemia-Hypoxia (I/H) and SAMe pre-treatment (S-I/H) groups. NS p>0.05; *p<0.05; **p<0.01

Figure 3. AVOs in cells during I/H, with/without SAMe pre-treatment

Qualitative AVO analyses A. Staining of live HepG2 and Hep3B cells for AVOs revealed the formation of LC3 puncta. Quantitative AVO analyses B. AVOs were quantitatively assessed according to the red-to-green fluorescence ratio obtained using Photoshop software. NS p>0.05; *p<0.05.

Figure 4. Beclin 1 expression during I/H, with/without SAMe pre-treatment

Beclin 1 levels in HepG2 (p53 wild type) A. Hep3B (p53 null) B. and HL-7702 (normal liver) C. cells in all treatment groups. NS p>0.05; *p<0.05; **p<0.01.

Figure 5. Effects of autophagy on HCC cell proliferation during acute I/H with/without SAMe pre-treatment

Beclin 1 levels A. and cell proliferation rates B. in all groups after autophagy inhibition via ATG7 siRNA, as compared to un-transfected cells. HepG2-si: ATG7 siRNA interference in HpeG2 cells. Hep3B-si: ATG7 siRNA interference in Hep3B cells. NS p>0.05; *p<0.05; **p<0.01.

Figure 6. HCC cell apoptosis during acute I/H, with/without SAMe pre-treatment

HepG2 and Hep3B cell apoptosis was investigated using Annexin V-FITC and PI. (AnV⁺) PI⁻ cells were considered early apoptotic and (AnV⁺) PI⁺ cells were considered late apoptotic and necrotic. NS p>0.05; *p<0.05; **p<0.01.

Figure 7. Influence of I/H on GADD45β expression with/without SAMe pre-treatment

(A) GADD45β levels in HepG2 A. Hep3B B. and HL-7702 C. cells in all groups.NS p>0.05; *p<0.05; **p<0.01.