Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells
Abstract
Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy.
Figures 
Fig. 1
(A) High-performance chromatogram of the…
Fig. 1
(A) High-performance chromatogram of the purified compound antcin K (retention time 6.5 minutes).…
Fig. 1 (A) High-performance chromatogram of the purified compound antcin K (retention time 6.5 minutes). Conditions: column, COSMOSIL 5C18-AR-II RP-C18; flow rate, 1 mL/min; detector, 254 nm; mobile phase, methanol (70%)/water (30%). (B) Effect of antcin K on cell viability in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 24 hours and 48 hours, cell viability was determined by MTT assay. Data are expressed as percentage of negative control (0.2% DMSO) and mean ± SD from one of three independent experiments. (C) Effect of antcin K on the degree of LC3-II fluorescence intensity in Hep3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 48 hours, the degree of LC3-II fluorescence intensity was analyzed by flow cytometry. (D) Effect of antcin K on degree of cell disruption in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K and lysis solution for 48 hours, the degree of cell disruption was determined by LDH leakage assay. Data are expressed as percentage between positive control (lysis solution) and negative control (0.2% DMSO) and mean ± SD from one of three independent experiments, and analyzed statistically using one-way ANOVA and Duncan's test. Different letters (a–d) represent statistically significant differences among treatments (p < 0.05). ANOVA = analysis of variance; DMSO = dimethyl sulfoxide; LDH = lactate dehydrogenase; SD = standard deviation.
Fig. 2
(A) Detection of chromatin condensation…
Fig. 2
(A) Detection of chromatin condensation in Hep 3B cells by DAPI staining. Typical…
Fig. 2 (A) Detection of chromatin condensation in Hep 3B cells by DAPI staining. Typical apoptotic changes comprise condensation of chromatin, its presence along the periphery of the nucleus, and segmentation of the nucleus. Cells were treated with 0μM, 80μM, 100μM, and 125μM antcin K for 24 hours. DAPI fluorescence of nuclei was visualized by excitation at 330–385 nm with a 420 nm barrier filter. The experiments were repeated two times with similar results. (B) Detection of DNA damage in Hep 3B cells by comet assay. Cells were treated with 0μM, 80μM, 100μM, and 125μM antcin K for 24 hours. EtBr fluorescence of DNA was visualized by excitation at 510–550 nm with a 590 nm barrier filter. The experiments were repeated two times with similar results. (C) Effect of antcin K on calcium ion mobilization in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 30 minutes, Fluo 3-Ca complex was analyzed by flow cytometry. (D) Effect of antcin K on mitochondrial calcium distribution in Hep 3B cells. After incubation of the cells with 0μM and 125μM antcin K for 30 minutes, mitochondrial calcium distribution was observed by immunofluorescence. The cells were fixed and stained with Rhod-2-AM (red fluorescence). Nuclear DNA in the cells was then counterstained with DAPI (blue fluorescence). The fluorescent images were recorded and superimposed (merged). Data are expressed as mean ± SD from one of three independent experiments and analyzed statistically using one-way ANOVA and Duncan's test. Different letters (a–c) represent statistically significant differences among treatments (p < 0.05). ANOVA = analysis of variance; DAPI = 4,6-diamidini-2-phenylindole; MFI = mean fluorescence intensity; Rhod-2-AM = Rhod-2-acetoxymethyl ester; SD = standard deviation.
Fig. 3
(A) Percentage of apoptotic cell…
Fig. 3
(A) Percentage of apoptotic cell induction by antcin K-treated Hep 3B cells. Cells…
Fig. 3 (A) Percentage of apoptotic cell induction by antcin K-treated Hep 3B cells. Cells were exposed to 0μM, 80μM, 100μM, and 125μM antcin K for 24 hours, and the distribution of apoptosis (lower right panel) was assessed by annexin V-FITC/PI assay. Effect of antcin K on ROS generation and ADP/ATP ratio in Hep 3B cells. (B) After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 24 hours, the fluorescence intensity of DCFH-DA was analyzed by flow cytometry. (C) Effect of antcin K on the degree of fluorescence intensity of DiOC6 in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 48 hours, the mitochondrial membrane potential was analyzed by flow cytometry. (D) After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 48 hours, the ADP/ATP ratio was determined by bioluminescence. Data are expressed as mean ± SD from one of three independent experiments and analyzed statistically using one-way ANOVA and Duncan's test. Different letters (a–d) represent statistically significant differences among treatments (p < 0.05). ADP/ATP = adenosine diphosphate/adenosine triphosphate; ANOVA = analysis of variance; DCFH-DA = dihydrodichlorofluorescein diacetate; DiOC6 = 3,3′-dihexyloxacarbocyanine iodide; MFI = mean fluorescence intensity; PI = propidium iodide; ROS = reactive oxygen species; SD = standard deviation.
Fig. 4
(A) Effects of antcin K…
Fig. 4
(A) Effects of antcin K on the expressions of mitochondria and ER stress-mediated…
Fig. 4 (A) Effects of antcin K on the expressions of mitochondria and ER stress-mediated apoptosis-related protein in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 48 hours, expressions of mitochondria and ER stress-mediated apoptosis-related protein were assessed by western blotting. The experiments were repeated at least two times with similar results, and β-actin was used as a loading control. Protein levels are expressed as multiples of negative control (0.2% DMSO) by β-actin-normalized densitometry and shown at the bottom of each band. Effects of antcin K on expressions of (B) caspase-dependent and (C) caspase-independent apoptosis-related proteins in Hep 3B cells. After incubation of the cells with 0μM, 80μM, 100μM, and 125μM antcin K for 48 hours, expressions of proteins were assessed by western blotting. The experiments were repeated at least two times with similar results, and β-actin was used as a loading control. Protein levels are expressed as multiples of negative control (0.2% DMSO) by β-actin-normalized densitometry and shown at the bottom of each band. CHOP = C/EBP homologous protein; Bcl-xL = B-cell lymphoma 2; Bax = B-cell lymphoma—extra-large; DMSO = dimethyl sulfoxide; ER = endoplasmic reticulum.
Fig. 5
Possible cell death mechanism pathway…
Fig. 5
Possible cell death mechanism pathway of antcin K in human liver cancer Hep…
Fig. 5 Possible cell death mechanism pathway of antcin K in human liver cancer Hep 3B cells. ADP/ATP = adenosine diphosphate/adenosine triphosphate; AIF = apoptotic-induced factor; CHOP = C/EBP homologous protein; Bcl-xL = B-cell lymphoma 2; Bax = B-cell lymphoma—extra-large; Endo-G = endonuclease G; ROS = reactive oxygen species.