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Fatsioside A‑induced apoptotic death of HepG2 cells requires activation of AMP‑activated protein kinase.

Zheng YS, Zhang JY, Zhang DH - Mol Med Rep (2015)

Bottom Line: The results of the present study demonstrated that fatsioside A induced apoptotic death of the human HepG2 HCC cells, which was associated with a marked activation of AMPK and increased expression of the downstream acetyl‑CoA carboxylase carboxylase.Inhibition of AMPK by RNA interference or by its inhibitor, compound C, suppressed fatsioside A‑induced caspase‑3 cleavage and apoptosis in the HepG2 cells, while AICAR, the AMPK activator, elicited marked cytotoxic effects.Together, these results suggested that fatsioside A‑induced apoptotic death requires AMPK activation in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Liver Diseases, Hospital for Infectious Diseases of Jining, Jining, Shandong 272031, P.R. China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most malignant types of human primary tumor and has a poor prognosis, therefore, the development of novel therapeutic modalities is necessary. Fatsioside A is a novel baccharane‑type triterpenoid glycoside, which is extracted from the fruits of Fatsia japonica. Previous data has revealed that fatsioside A can exert growth inhibition, cell cycle arrest and induce apoptosis in human glioma cells. However, no detailed investigations have been performed to determine its action on human hepatocellular cells, and the exact mechanisms underlying the induction of apoptosis remain to be elucidated. The aim of the present study was to investigate the anticancer effect of fatsioside A in the HepG2 human HCC cell line, and to investigate the underlying mechanisms by focusing on the AMP‑activated protein kinase (AMPK) signaling cascade. The results of the present study demonstrated that fatsioside A induced apoptotic death of the human HepG2 HCC cells, which was associated with a marked activation of AMPK and increased expression of the downstream acetyl‑CoA carboxylase carboxylase. Inhibition of AMPK by RNA interference or by its inhibitor, compound C, suppressed fatsioside A‑induced caspase‑3 cleavage and apoptosis in the HepG2 cells, while AICAR, the AMPK activator, elicited marked cytotoxic effects. Together, these results suggested that fatsioside A‑induced apoptotic death requires AMPK activation in HepG2 cells.

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AMPK inhibition suppresses fatsioside A-induced cell viability loss in HepG2 cells. (A) HepG2 cells were either left untreated or were treated with different concentrations of fatsioside A (40 or 80 µM) for 24 h. The phosphorylation of AMPKα and ACC were then assessed using western blot analysis. (B) HepG2 cells were pre-treated with the AMPK inhibitor, compound C (10 µM), for 1 h, followed by fatsioside A (80 µM) stimulation, and were further cultured for 24 h prior to assessment of cell viability using an MTT assay. (C) Scramble control (sc)RNAi- or AMPKα RNAi-transfected HepG2 cells were either left untreated or were treated with fatsioside A (80 µM), and were further cultured for 24 h prior to cell viability assessment using an MTT assay. (D) Expression levels of AMPKα in these cells were assessed using western blot analysis (upper) and were also assessed for cell apoptosis using Annexin V fluorescence-activated cell sorting 24 h, following treatment with fatsioside A (80 µM).*P<0.05, vs. fatsioside A + scRNAi group. (E) Expression levels of cleaved caspase-3 in the cells were examined using western blot analysis. Experiments were repeated three times and similar results were obtained. Ctrl, untreated control; FA, fatsopside A; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase carboxylase; p-, phosphorylated; RNAi, RNA interference; DMSO, dimethyl sulfoxide.
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f3-mmr-12-04-5679: AMPK inhibition suppresses fatsioside A-induced cell viability loss in HepG2 cells. (A) HepG2 cells were either left untreated or were treated with different concentrations of fatsioside A (40 or 80 µM) for 24 h. The phosphorylation of AMPKα and ACC were then assessed using western blot analysis. (B) HepG2 cells were pre-treated with the AMPK inhibitor, compound C (10 µM), for 1 h, followed by fatsioside A (80 µM) stimulation, and were further cultured for 24 h prior to assessment of cell viability using an MTT assay. (C) Scramble control (sc)RNAi- or AMPKα RNAi-transfected HepG2 cells were either left untreated or were treated with fatsioside A (80 µM), and were further cultured for 24 h prior to cell viability assessment using an MTT assay. (D) Expression levels of AMPKα in these cells were assessed using western blot analysis (upper) and were also assessed for cell apoptosis using Annexin V fluorescence-activated cell sorting 24 h, following treatment with fatsioside A (80 µM).*P<0.05, vs. fatsioside A + scRNAi group. (E) Expression levels of cleaved caspase-3 in the cells were examined using western blot analysis. Experiments were repeated three times and similar results were obtained. Ctrl, untreated control; FA, fatsopside A; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase carboxylase; p-, phosphorylated; RNAi, RNA interference; DMSO, dimethyl sulfoxide.

Mentions: As shown in Fig. 3A, fatsioside A induced significant AMPK activation in the HepG2 cells, as the expression levels of p-AMPKα and the downstream ACC in HepG2 cells were significantly increased following fatsioside A treatment. Notably, AMPK inhibition by its inhibitor, compound C or by AMPKα-RNAi suppressed fatsioside A-induced loss of cell viability (Fig. 3B and C). Fatsioside A-induced apoptosis and cleavage of caspase-3 was also inhibited by AMPK inhibition (Fig. 3D and E). The AICAR AMPK activator also inhibited HepG2 cell survival (Fig. 4A and B). The above results indicated that activation of AMPK was involved in fatsioside A-induced cytotoxicity in HepG2 cells.


Fatsioside A‑induced apoptotic death of HepG2 cells requires activation of AMP‑activated protein kinase.

Zheng YS, Zhang JY, Zhang DH - Mol Med Rep (2015)

AMPK inhibition suppresses fatsioside A-induced cell viability loss in HepG2 cells. (A) HepG2 cells were either left untreated or were treated with different concentrations of fatsioside A (40 or 80 µM) for 24 h. The phosphorylation of AMPKα and ACC were then assessed using western blot analysis. (B) HepG2 cells were pre-treated with the AMPK inhibitor, compound C (10 µM), for 1 h, followed by fatsioside A (80 µM) stimulation, and were further cultured for 24 h prior to assessment of cell viability using an MTT assay. (C) Scramble control (sc)RNAi- or AMPKα RNAi-transfected HepG2 cells were either left untreated or were treated with fatsioside A (80 µM), and were further cultured for 24 h prior to cell viability assessment using an MTT assay. (D) Expression levels of AMPKα in these cells were assessed using western blot analysis (upper) and were also assessed for cell apoptosis using Annexin V fluorescence-activated cell sorting 24 h, following treatment with fatsioside A (80 µM).*P<0.05, vs. fatsioside A + scRNAi group. (E) Expression levels of cleaved caspase-3 in the cells were examined using western blot analysis. Experiments were repeated three times and similar results were obtained. Ctrl, untreated control; FA, fatsopside A; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase carboxylase; p-, phosphorylated; RNAi, RNA interference; DMSO, dimethyl sulfoxide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581823&req=5

f3-mmr-12-04-5679: AMPK inhibition suppresses fatsioside A-induced cell viability loss in HepG2 cells. (A) HepG2 cells were either left untreated or were treated with different concentrations of fatsioside A (40 or 80 µM) for 24 h. The phosphorylation of AMPKα and ACC were then assessed using western blot analysis. (B) HepG2 cells were pre-treated with the AMPK inhibitor, compound C (10 µM), for 1 h, followed by fatsioside A (80 µM) stimulation, and were further cultured for 24 h prior to assessment of cell viability using an MTT assay. (C) Scramble control (sc)RNAi- or AMPKα RNAi-transfected HepG2 cells were either left untreated or were treated with fatsioside A (80 µM), and were further cultured for 24 h prior to cell viability assessment using an MTT assay. (D) Expression levels of AMPKα in these cells were assessed using western blot analysis (upper) and were also assessed for cell apoptosis using Annexin V fluorescence-activated cell sorting 24 h, following treatment with fatsioside A (80 µM).*P<0.05, vs. fatsioside A + scRNAi group. (E) Expression levels of cleaved caspase-3 in the cells were examined using western blot analysis. Experiments were repeated three times and similar results were obtained. Ctrl, untreated control; FA, fatsopside A; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase carboxylase; p-, phosphorylated; RNAi, RNA interference; DMSO, dimethyl sulfoxide.
Mentions: As shown in Fig. 3A, fatsioside A induced significant AMPK activation in the HepG2 cells, as the expression levels of p-AMPKα and the downstream ACC in HepG2 cells were significantly increased following fatsioside A treatment. Notably, AMPK inhibition by its inhibitor, compound C or by AMPKα-RNAi suppressed fatsioside A-induced loss of cell viability (Fig. 3B and C). Fatsioside A-induced apoptosis and cleavage of caspase-3 was also inhibited by AMPK inhibition (Fig. 3D and E). The AICAR AMPK activator also inhibited HepG2 cell survival (Fig. 4A and B). The above results indicated that activation of AMPK was involved in fatsioside A-induced cytotoxicity in HepG2 cells.

Bottom Line: The results of the present study demonstrated that fatsioside A induced apoptotic death of the human HepG2 HCC cells, which was associated with a marked activation of AMPK and increased expression of the downstream acetyl‑CoA carboxylase carboxylase.Inhibition of AMPK by RNA interference or by its inhibitor, compound C, suppressed fatsioside A‑induced caspase‑3 cleavage and apoptosis in the HepG2 cells, while AICAR, the AMPK activator, elicited marked cytotoxic effects.Together, these results suggested that fatsioside A‑induced apoptotic death requires AMPK activation in HepG2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Liver Diseases, Hospital for Infectious Diseases of Jining, Jining, Shandong 272031, P.R. China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most malignant types of human primary tumor and has a poor prognosis, therefore, the development of novel therapeutic modalities is necessary. Fatsioside A is a novel baccharane‑type triterpenoid glycoside, which is extracted from the fruits of Fatsia japonica. Previous data has revealed that fatsioside A can exert growth inhibition, cell cycle arrest and induce apoptosis in human glioma cells. However, no detailed investigations have been performed to determine its action on human hepatocellular cells, and the exact mechanisms underlying the induction of apoptosis remain to be elucidated. The aim of the present study was to investigate the anticancer effect of fatsioside A in the HepG2 human HCC cell line, and to investigate the underlying mechanisms by focusing on the AMP‑activated protein kinase (AMPK) signaling cascade. The results of the present study demonstrated that fatsioside A induced apoptotic death of the human HepG2 HCC cells, which was associated with a marked activation of AMPK and increased expression of the downstream acetyl‑CoA carboxylase carboxylase. Inhibition of AMPK by RNA interference or by its inhibitor, compound C, suppressed fatsioside A‑induced caspase‑3 cleavage and apoptosis in the HepG2 cells, while AICAR, the AMPK activator, elicited marked cytotoxic effects. Together, these results suggested that fatsioside A‑induced apoptotic death requires AMPK activation in HepG2 cells.

Show MeSH
Related in: MedlinePlus