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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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Fatsioside A induces apoptotic and necrotic death of HepG2 cells. (A and B) HepG2 cells were treated with or without fatsioside A and were cultured in Dulbecco's modified Eagle's medium for 24 h, following which apoptotic and necrotic cell death was assessed using an Annexin V fluorescence-activated cell sorting. (C) Expression levels of cleaved caspase-3, Bcl-2 and β-actin were assessed using western blot analysis. (D) HepG2 cells were pre-treated with z-VAD-fmk (50 µM), a general caspase inhibitor, for 1 h, followed by fatsioside A (40 and 80 µM) stimulation and further culture for 24 h, Cell viability was then assessed using a 3-(4, 5-dimetryl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Experiments were repeated three times and similar results were obtained. Data are expressed as the mean ± standard deviation. *P<0.05, vs. Ctrl; #P<0.05, vs. fatsioside A group. Ctrl, untreated control; PI, propidium iodide; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; Bcl-2, B-cell lymphoma-2.
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f2-mmr-12-04-5679: Fatsioside A induces apoptotic and necrotic death of HepG2 cells. (A and B) HepG2 cells were treated with or without fatsioside A and were cultured in Dulbecco's modified Eagle's medium for 24 h, following which apoptotic and necrotic cell death was assessed using an Annexin V fluorescence-activated cell sorting. (C) Expression levels of cleaved caspase-3, Bcl-2 and β-actin were assessed using western blot analysis. (D) HepG2 cells were pre-treated with z-VAD-fmk (50 µM), a general caspase inhibitor, for 1 h, followed by fatsioside A (40 and 80 µM) stimulation and further culture for 24 h, Cell viability was then assessed using a 3-(4, 5-dimetryl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Experiments were repeated three times and similar results were obtained. Data are expressed as the mean ± standard deviation. *P<0.05, vs. Ctrl; #P<0.05, vs. fatsioside A group. Ctrl, untreated control; PI, propidium iodide; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; Bcl-2, B-cell lymphoma-2.

Mentions: The results described above indicated that fatsioside A inhibited HepG2 cell survival and proliferation; therefore, the present study subsequently assessed whether cell apoptosis was involved in this effect. As shown in Fig. 2A and B, fatsioside A (40 and 80 µM) induced early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptosis in the HepG2 cells. In addition, fatsioside A also caused caspase-3 cleavage and Bcl-2 degradation (Fig. 2C). Notably, fatsioside A also induced necrotic (Annexin V−/PI+) HepG2 cell death (Fig. 2A and B). Furthermore, the results of the cell viability assay results, as shown in Fig. 2D, demonstrated that z-VAD-fmk, a general caspase inhibitor, suppressed, but did not reverse, fatsioside A-induced HepG2 loss of viability, indicating that apoptotic and necrotic death accounted for the fatsioside A-induced cytotoxicity in the 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)

Fatsioside A induces apoptotic and necrotic death of HepG2 cells. (A and B) HepG2 cells were treated with or without fatsioside A and were cultured in Dulbecco's modified Eagle's medium for 24 h, following which apoptotic and necrotic cell death was assessed using an Annexin V fluorescence-activated cell sorting. (C) Expression levels of cleaved caspase-3, Bcl-2 and β-actin were assessed using western blot analysis. (D) HepG2 cells were pre-treated with z-VAD-fmk (50 µM), a general caspase inhibitor, for 1 h, followed by fatsioside A (40 and 80 µM) stimulation and further culture for 24 h, Cell viability was then assessed using a 3-(4, 5-dimetryl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Experiments were repeated three times and similar results were obtained. Data are expressed as the mean ± standard deviation. *P<0.05, vs. Ctrl; #P<0.05, vs. fatsioside A group. Ctrl, untreated control; PI, propidium iodide; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; Bcl-2, B-cell lymphoma-2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-12-04-5679: Fatsioside A induces apoptotic and necrotic death of HepG2 cells. (A and B) HepG2 cells were treated with or without fatsioside A and were cultured in Dulbecco's modified Eagle's medium for 24 h, following which apoptotic and necrotic cell death was assessed using an Annexin V fluorescence-activated cell sorting. (C) Expression levels of cleaved caspase-3, Bcl-2 and β-actin were assessed using western blot analysis. (D) HepG2 cells were pre-treated with z-VAD-fmk (50 µM), a general caspase inhibitor, for 1 h, followed by fatsioside A (40 and 80 µM) stimulation and further culture for 24 h, Cell viability was then assessed using a 3-(4, 5-dimetryl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Experiments were repeated three times and similar results were obtained. Data are expressed as the mean ± standard deviation. *P<0.05, vs. Ctrl; #P<0.05, vs. fatsioside A group. Ctrl, untreated control; PI, propidium iodide; FITC, fluorescein isothiocyanate; DMSO, dimethyl sulfoxide; Bcl-2, B-cell lymphoma-2.
Mentions: The results described above indicated that fatsioside A inhibited HepG2 cell survival and proliferation; therefore, the present study subsequently assessed whether cell apoptosis was involved in this effect. As shown in Fig. 2A and B, fatsioside A (40 and 80 µM) induced early (Annexin V+/PI−) and late (Annexin V+/PI+) apoptosis in the HepG2 cells. In addition, fatsioside A also caused caspase-3 cleavage and Bcl-2 degradation (Fig. 2C). Notably, fatsioside A also induced necrotic (Annexin V−/PI+) HepG2 cell death (Fig. 2A and B). Furthermore, the results of the cell viability assay results, as shown in Fig. 2D, demonstrated that z-VAD-fmk, a general caspase inhibitor, suppressed, but did not reverse, fatsioside A-induced HepG2 loss of viability, indicating that apoptotic and necrotic death accounted for the fatsioside A-induced cytotoxicity in the 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