Diabetes Metab J.  2015 Dec;39(6):518-527. 10.4093/dmj.2015.39.6.518.

Metformin Promotes Apoptosis but Suppresses Autophagy in Glucose-Deprived H4IIE Hepatocellular Carcinoma Cells

Affiliations
  • 1Department of Histology and Institute of Medical Science, Jeju National University School of Medicine, Jeju, Korea. parkdb@jejunu.ac.kr

Abstract

BACKGROUND
Metformin, a well-known anti-diabetic drug, has gained interest due to its association with the reduction of the prevalence of cancer in patients with type 2 diabetes and the anti-proliferative effect of metformin in several cancer cells. Here, we investigated the anti-proliferative effect of metformin with respect to apoptosis and autophagy in H4IIE hepatocellular carcinoma cells.
METHODS
H4IIE rat cells were treated with metformin in glucose-free medium for 24 hours and were then subjected to experiments examining the onset of apoptosis and/or autophagy as well as the related signaling pathways.
RESULTS
When H4IIE cells were incubated in glucose-free media for 24 hours, metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) reduced the viability of cells. Inhibition of AMP-activated protein kinase (AMPK) by compound C significantly blocked cell death induced by metformin or AICAR. Pro-apoptotic events (nuclear condensation, hydrolysis of intact poly ADP ribose polymerase and caspase-3) were stimulated by metformin and then suppressed by compound C. Interestingly, the formation of acidic intracellular vesicles, a marker of autophagy, was stimulated by compound C. Although the deprivation of amino acids in culture media also induced apoptosis, neither metformin nor compound C affected cell viability. The expression levels of all of the autophagy-related proteins examined decreased with metformin, and two proteins (light chain 3 and beclin-1) were sensitive to compound C. Among the tested inhibitors against MAP kinases and phosphatidylinositol-3-kinase/mammalian target of rapamycin, SB202190 (against p38MAP kinase) significantly interrupted the effects of metformin.
CONCLUSION
Our data suggest that metformin induces apoptosis, but suppresses autophagy, in hepatocellular carcinoma cells via signaling pathways, including AMPK and p38 mitogen-activated protein kinase.

Keyword

Apoptosis; Autophagy; H4IIE hepatocellular carcinoma cells; Metformin

MeSH Terms

Amino Acids
AMP-Activated Protein Kinases
Animals
Apoptosis*
Autophagy*
Carcinoma, Hepatocellular*
Cell Death
Cell Survival
Culture Media
Humans
Hydrolysis
Metformin*
Phosphotransferases
Poly(ADP-ribose) Polymerases
Prevalence
Protein Kinases
Rats
Sirolimus
AMP-Activated Protein Kinases
Amino Acids
Culture Media
Metformin
Phosphotransferases
Poly(ADP-ribose) Polymerases
Protein Kinases
Sirolimus

Figure

  • Fig. 1 Metformin (Met) inhibits cell viability and induces apoptosis in H4IIE cells. (A) H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in glucose-free DMEM (GFM) for 30 minutes. Cells were further treated with Met and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) for 4 hours (A) or 24 hours (B-E). The levels of phospho-AMP-activated protein kinase (p-AMPK) and actin were detected by Western blotting analyses, and the density of p-AMPK was normalized against actin. Each bar represents the average value of the duplicated experiments (n=2) (A). Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and each bar represents the mean±standard error (n=3) (B, C). The onset of apoptosis was detected using H33342 staining to observe nuclear chromatin condensation (D) and Western blotting analyses for poly ADP ribose polymerase (PARP) and cleaved caspase-3 (E). CTL, control. aP<0.001 vs. the non-treated control, bP<0.05 vs. cells treated with 1 mM AICAR, cP<0.001 vs. cells treated with 2 mM Met.

  • Fig. 2 Inhibition of AMP-activated protein kinase stimulates the formation of acidic intracellular vesicles, a marker of the onset of autophagy, and protected metformin-induced apoptosis in glucose-free DMEM (GFM), but not in amino acid (AA)-free medium. H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in fresh GFM (A, B), AA-free Hank's-balanced salt solution (1 g/L glucose) (C, D), or AA-containing DMEM (1 g/L glucose) (D) for 30 minutes. Cells were further treated with 2 mM metformin (Met) and 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, AIC) for 24 hours. After treatment, cells were stained with AO and H33342 (A-C) and subjected to the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (D). Arrows (B) indicates condensed chromatin (apoptotic bodies). Each bar represents the mean±standard error (n=3) (D). CTL, control. aP<0.05 vs. the non-treated control (dark bar), bP<0.05 vs. cells treated with 2 mM Met (dark bar), cP<0.01 vs. the non-treated control (white bar).

  • Fig. 3 Metformin decreases the expression levels of autophagy-related proteins. H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with compound C (CC, 10 µM) in glucose-free DMEM (GFM) for 30 minutes. Cells were further treated with metformin (Met, 0 to 2 mM) for 24 hours and subjected to Western blotting analyses. The density of light chain 3B (LC3B) or beclin-1 was normalized against actin. Each bar represents the average value of the duplicated experiments (n=2).

  • Fig. 4 Inhibition of p38 mitogen-activated protein kinase (p38MAPK) as well as AMP-activated protein kinase protected cells from apoptosis induced by metformin (Met). H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with various inhibitors against signaling proteins (20 µM compound C [CC], 50 µM SB202190, 50 µM SP600125, 10 µM U0126, 100 nM rapamycin, and 100 nM wortmannin) for 30 minutes. Cells were further treated 1 mM Met for 24 hours. After the treatments, the protein levels of cleaved caspase-3, beclin-1, light chain 3B (LC3B), and actin were detected using Western blotting analyses (A, C, D), and cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (B, E). Each bar represents the mean±standard error (n=3) (B, E). Rapa, rapamycin; SB, SB202190; SP, SP600125; U, U0126; Wt, wortmannin. aP<0.01 vs. cells treated with 2 mM Met (n=3) (B).


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