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. 2015 May 1;10(5):e0125727.
doi: 10.1371/journal.pone.0125727. eCollection 2015.

Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721

Zheng Lu  1 , Shengbo Cao  1 , Hongbo Zhou  1 , Ling Hua  2 , Shishuo Zhang  1 , Jiyue Cao  1
Affiliations

Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721

Zheng Lu et al. PLoS One. .

Abstract

Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cytotoxic effect of ARG on HCC cells and normal hepatic cells.
(A) Hep G2, SMMC7721 and LO2 cell lines were treated with ARG at 5, 10, 20, 50, 80, 100 μM for 24 h. (B) Hep G2 cells were exposed to a gradient dose of ARG for the indicated time period and IC50 value was calculated for each time point. Cell viability inhibition was assessed by MTT assay. Each value is the mean ± SD of five independent experiments. *p<0.05, **p<0.01, ***p<0.0001 significant difference between control and ARG-treated cells in each cell line, as analyzed by Dunnett’s Multiple Comparion Test.
Fig 2
Fig 2. ARG promotes HCC cells to undergo apoptosis.
(A) Hep G2, SMMC7721 and LO2 cells treated with ARG (0, 1.56, 12.5, 100 μM) for 24 h were double-stained with Annexin V-FITC/PI and analyzed by flow cytometry. Quantification of population rate of early apoptotic cells (Annexin V+/PI− cells, lower right quadrant) and late apoptotic cells (Annexin V+/PI+ cells, upper right quadrants) were shown. Data represent the mean ± SD of triplicate experiments. Significant differences from vehicle-treated control were indicated as *p<0.05, **P<0.01, ***P<0.0001 in each cell line. (B) HepG2 and SMMC7721 cells were treated with ARG at the concentration of 20 μM for 48 h. The nuclei morphology changes were analyzed by Hoechst 33342 staining and the immunofluorescence analysis of cleaved caspase-3 are visualized using the cleaved caspase-3 antibody. The cellular fluorescent changes were observed by fluorescence microscope. At least 200 cells were counted to score the percentage of apoptotic cells which contained both cleaved caspase-3 and condensed and highly fluorescent nuclei fragments in each treatment group. Significant differences from control group were indicated as ***P<0.0001 in each cell line.
Fig 3
Fig 3. ARG caused the disfunction of mitochondrial membrane in HCC cells.
Hep G2 and SMMC7721 cells were exposed to ARG (0, 1.56, 12.5, 100 μM) for 12 h and 24 h respectively. (A) Flow cytometric detection of ΔΨ m in HCC cells using JC-1. (B) The expression of Bcl-2 and Bax were analyzed by western blot with the loading control of β-actin. (C) Mitochondrial and cytoplasmic extracts were measured for Cyt c levels. Purity of the extracts was verified by β-actin (cytoplasmic) and COX IV (mitochondrial) expressions. Data were represented as mean ± SD of three separate experiments. Significant differences from control were indicated *p<0.05, **p<0.01, ***p<0.0001.
Fig 4
Fig 4. ARG induced the changes of apoptotic proteins related to extrinsic pathways.
Hep G2 and SMMC7721 cells were incubated with ARG (0, 1.56, 12.5, 100 μM) for 12 h and 24 h respectively, and then harvested for investigating the TNF-α, Fas and FasL expression level by western blot. The data represent mean ± SD of three separate experiments. Significant differences from control were indicated as *p<0.05, **p<0.01, ***p<0.0001.
Fig 5
Fig 5. Effect of ARG on the activation of caspases.
After Hep G2 and SMMC7721 cells were treated with ARG (0, 1.56, 12.5, 100 μM) for 12 h and 24 h respectively. The protein levels of caspase-3, cleaved PARP-1, pro-caspase -8, -9 were analyzed by western blot analysis (A) and the activities of caspase-8, -9 were detected by Caspase Colorimetric Assay Kits (B). The data represent mean ± SD (n = 3). *p< 0.05, **p< 0.01, ***p< 0.0001 vs. untreated control.
Fig 6
Fig 6. Effect of ARG on NF-κB, p53 and PI3K/AKT pathways in HCC cells.
Hep G2 and SMMC7721 cells were exposed to ARG (0, 1.56, 12.5, 100 μM) for 12 h and 24 h respectively. Western blot analysis was performed. (A) NF-κB (p65) and p53 in nuclear, total NF-κB (p65) and p53 expression levels were shown. (B) PI3K, Akt and p-Akt expression levels were shown. Histone 3 was used as a loading control for nuclear protein, and β-actin for whole cell protein. The data shown are the representative image from three independent experiments. *p<0.05, **p<0.01, ***p<0.0001 significant differences from control.
Fig 7
Fig 7. The apoptotic pathways induced by ARG in Hep G2 cell line.
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