Berberine-induced apoptotic and autophagic death of HepG2 cells requires AMPK activation

Abstract

Background: Hepatocellular carcinoma (HCC), the primary liver cancer, is one of the most malignant human tumors with extremely poor prognosis. The aim of this study was to investigate the anti-cancer effect of berberine in a human hepatocellular carcinoma cell line (HepG2), and to study the underlying mechanisms by focusing on the AMP-activated protein kinase (AMPK) signaling cascade.

Results: We found that berberine induced both apoptotic and autophagic death of HepG2 cells, which was associated with a significant activation of AMPK and an increased expression of the inactive form of acetyl-CoA carboxylase (ACC). Inhibition of AMPK by RNA interference (RNAi) or by its inhibitor compound C suppressed berberine-induced caspase-3 cleavage, apoptosis and autophagy in HepG2 cells, while AICAR, the AMPK activator, possessed strong cytotoxic effects. In HepG2 cells, mammalian target of rapamycin complex 1 (mTORC1) activation was important for cell survival, and berberine inhibited mTORC1 via AMPK activation.

Conclusions: Together, these results suggested that berberine-induced both apoptotic and autophagic death requires AMPK activation in HepG2 cells.

Keywords: AMPK; Apoptosis; Autophagy and mTOR; Berberine; Hepatocellular carcinoma.

Figure 1

Berberine induces apoptotic and necrotic death of HepG2 cells. HepG2 cells were either left untreated or treated with described concentration of berberine, cells were further cultured in DMEM for 48 hours, the cell viability was tested by “MTT” assay (A), the percentage of trypan blue dye positive cells was recorded (B); HepG2 cell proliferation was analyzed by BrdU incorporation assay (C). HepG2 cells treated with or without berberine were cultured in DMEM for 24 hours, apoptotic and necrotic cell death was tested by Annexin V FACS assay (D and E), expressions of cleaved-caspase 3, Bcl-2 and β-actin were tested by western blots (F). HepG2 cells were pre-treated with z-VAD-fmk (50 μM) for 1 hour, followed by berberine (50 and 100 μM) stimulation, cells were further cultured for 48 hours before cell viability was tested (G). Experiments in this figure were repeated three times, and similar results were obtained. Data were expressed as mean ± SD. *p < 0.05 vs. Ctrl group (A and B). ^#p < 0.05 vs. berberine-treated group (C).

Figure 2

Berberine induces apoptotic and necrotic death of HepG2 cells. HepG2 cells were either left untreated or treated with described concentration of berberine (10, 50, 100 and 200 μM), cells were further cultured in DMEM (no serum) for 24 hours, expressions of Beclin-1, LC3B-II, Erk1/2 and β-actin were tested by western blots (A). The number of LC3-GFP puncta positive cells (autophagic cells) was counted (B).Cell viability of HepG2 cells with indicated treatment for 48 hours was tested by MTT assay (C). HepG2 cells transfected with scramble control siRNA, Beclin-1 siRNA or LC3B siRNA (100 nM each, for 48 hours) were either left untreated, or stimulated with berberine (100 μM) plus z-VAD-fmk (50 μM) (Berberine + ZVAD), cells were further cultured for 48 hours, expression of Beclin-1, LC3B and β-actin was tested by western blots (D), cell viability was also tested (E). Experiments in this figure were repeated three times, and similar results were obtained. Data were expressed as mean ± SD. ^#p < 0.05 vs. Ctrl group (B). *p < 0.05 (C and E).

Figure 3

Activation of AMPK is involved in berberine-induced cytotoxicity in HepG2 cells. HepG2 cells were either left untreated or treated with described concentration of berberine (10, 25, 50, 100 and 200 μM) for 4 hours, or treated with 100 μM of berberine for described time (15′, 30′, 1 h, 2 h and 4 h), phospho- and total AMPKα/ACC were tested by western blots (A and B). HepG2 cells were pre-treated with the AMPK inhibitor compound C (10 μM) for 1 hour, followed by berberine (100 μM) stimulation, cells were further cultured for 48 hours before cell viability was tested (C). Scramble control RNAi or AMPKα RNAi transfected HepG2 cells were either left untreated or treated with berberine (100 μM), cells were further cultured for 48 hours before cell viability was tested (D), expressions of AMPKα and β-actin in those cells were also tested by western blot (D, upper). Above cells were also tested for cell apoptosis 24 hours after stimulation (E), expressions of cleaved-caspase-3 and β-actin were examined (F), the number of LC3-GFP puncta positive cells were also recorded (G). HepG2 cells were either left untreated or treated with AICAR (1 mM), phospho- and total AMPKα/ACC were tested by western blots 2 hours after stimulation (H), and cell viability was examined by MTT assay after 48 hours incubation (I). Experiments in this figure were repeated three times, and similar results were obtained. *p < 0.05 (C and D). **p < 0.05 vs. berberine-treated group (G and E). ^#p < 0.05 vs. Ctrl group (I).

Figure 4

mTORC1 activation is required for HepG2 cell survival, inhibited by berberine. HepG2 cells were either left untreated or treated with described concentration of berberine (10, 25, 50, 100 and 200 μM) for 4 hours, or treated with 100 μM of berberine for described time, expressions of phospho- and total Akt, S6 and 4EBP1, as well as Erk1/2 were tested by western blots (A and B). HepG2 cells were either left untreated or stimulated with RAD001 (200 nM) or rapamycin (200 nM), Akt and S6 activations were tested by western blots 4 hours after stimulation (C), cell viability was analyzed by MTT assay 48 hours after stimulation (D). HepG2 cells were pre-treated with the AMPK inhibitor compound C (AMPKi, 10 μM) for 1 hour, followed by berberine (100 μM) stimulation for 4 hours, expressions of S6 (p- and t-), p-4E-BP1 and Erk1/2 were tested by western blots (E). Experiments in this figure were repeated three times, and similar results were obtained. Data were expressed as mean ± SD. *p < 0.05 vs. Ctrl group (D).