Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata are mediated via down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell
Lan-Hsiang Chien (簡嵐翔 ), Yu-Yi Chan (詹于宜 ), Chun-Sheng Chang (張春生 ), Ting-Feng Wu* (吳定峰 )
Department of Biotechnology and Biotechnology Research Center, Southern-Taiwan University, Tainan, TaiwanAbstract Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that A. camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities. Three HPLC fractions, fractions 5 to 7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6 induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated the mitochondrial pathway and ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor (RhoGDI-) expression and subsequently activated c-Jun NH2-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target.
Introduction
Antrodia camphorata is the traditional Taiwan crude drug for treating diarrhea, hypertension , inflammation and cancer. Previous studies have shown that the Antrodia camphorata crude extract has anti-proliferation and anti-migration on the transitional cell carcinomas (Chiung, et al., 2007) and ethanol extract from wild fruiting bodies could induce leukemia HL-60 cells apoptosis ( Mei et al., 2008 ). Recent studies has identified that lanostane and ergostane - type triterpenes isolated from the fruiting bodies of A. camphorata could inhibit proliferation of human colon, live, breast, lung cancer cells ( Chi et al., 2009).
However, the wild fruiting bodies of A. camphorata are rarity in nature, very expensive and the failure of artificial cultivation . Therefore , using a submerged cultured method to obtain useful cellular materials or to produce effective substances from cultured mycelia , might be a possible way to overcome the disadvantage of the fruiting bodies ( Sone et al., Agricultural and Biological Chemistry , 1985 ). The mycelia of Antrodia camphorata in submerged culture induced apoptosis in human hepatoma cells ( Tuzz et al., Journal of Ethnopharmacology , 2005 ). A. camphorata inhibited proliferation and induced apoptosis of human breast cancer cells in vitro and in vivo ( Hseu et al., Food and Chemical Toxicology , 2008 ) . Our previous study demonstrates that ethanol extracts of A. camphorata cultivated by solid -state fermentation could effectively impede the proliferation of human lung cancer A549 cells and inhibit the expression of galectin-1, eIF-5a, RhoGDI-α and calpain-1/2 ( Hung et al., Proteomics, 2006). However, no information about their mechanism of action and significant compounds. To explore their significant compounds , the ethanol extracts of A. camphorata (AC-EtOH) were separated by HPLC and collect to one fraction every fifteen minutes. This study evaluates the whether the eight fractions also have the inhibitory effect on A549 cell line and explore their mechanism of action.
Results and discussion
Figure 1. Preparation for Antrodia camphorata extracts ( A ) The extraction flow chart ( B ) The ethanol extracts of A. camphorata were separated using HPLC column and the elute was collected every fifteen minutes. Totally eight fractions were obtained.
Methods
Preparation for Antrodia camphorata extracts
A. camphorata mycelia were provided by Biotechnology Center, Southern Taiwan University. A. camphorata mycelia (about 555 g) were sequentially extracted using the solvent with increasing polarity, including n-hexane, ethylacetate, ethanol and wtaer (Figure 1A). 20.1 g, 16 g and 127.6 g of the products were obtained respectively in the EtOAc, EtOH and water extraction experiments.
Separation of the ethanol extract of A. camphorata by high performance liquid chromatography (HPLC) The ethanol extracts were separated using HPLC column (250 x 4.6 ㎜ , Hypersil, 5μm ) with a linear solvent gradient elution system composed of solvents A and B ( A： H2O； B： 100 % acetonitrite ) The elute was collected every 15 minutes and totally eight fractions were obtained. Each fraction was dissolved in dimethyl sulfoxide ( DMSO ) and control cultures received the carrier solvent ( 0.1 % DMSO ).Cell viability Cell viability was measured by MTT assay.
Apoptosis analysis
SubG1 analysis and Annexin V/propidium iodide (PI) assay were used to observe human A549 cell apoptosis
Western blotting
After treatment as indicated above , cells were collected and lysed in the lysis buffer. Total cell lysates were separated by 12.5 % SDS- PAGE and transferred onto PVDF membranes. The membrane was blotted with primary antibody overnight , followed by incubation with HRP-conjugated secondary antibody and visualized using chemiluminescence.
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Figure 2. The inhibitory effects of HPLC fractions on human A549 cells. (A) Treatment with 200μg/ml for 72 h, the maximum inhibitory effect on proliferation was fractions 5-7. ( B ) Treatment with various concentration of fraction 5-7 for 72 h, 25 μg / ml fraction 6 inhibited cell proliferation at 72 % , indicating that human A549 cell was more sensitive to fraction 6 than to fraction 5 and 7. ( C ) Treatment with 50μg / ml of fraction 6 for 0~72 h, which inhibited cell proliferation at 80% and the result was similar with Fig. 2B. (D) Treatment with various concentration of fraction 6 on MRC-5 cell, the inhibitory effect was observed with 100μg/ml. These results suggested that normal ( MRC-5 ) and cancer (A549) cell lines exhibit differential sensitivity to fraction 6.
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Figure 3. Apoptosis assay ( A ) , ( B ) The remarkable accumulation of sub-G1 was considered apoptosis. Fraction- 6 (50μg/ml) elevated the percentage of human A549 cells in the sub-G1 phase from 24% to 34%, 64% (24h~72h) when compared with the control group. (C),(D) Compared with vehicle-treated cells, 50μg / ml fraction 6 induced 17%, 29%, 13% of early apoptosis cells and 6%, 11%, 75% of late apoptosis cells at 24h, 48h and 72 h. These results suggest edthat fraction 6 could induce apoptosis on A549 cell. However, cytometric analysis could not determine whether Fr-6 leaded to the apoptosis by death receptor signaling, mitochondrial damage or ER stress. To explore the mechanism by which Fr-6 invoked the apoptosis, western blotting with anti-cappase-3, -12 and bax/bcl-2 were carried out in Fig. 4.
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Figure 4. Effects of fraction 6 on caspase-3, caspase-12 activation and Bax, Bcl-2 expression ( A ) A549 cells treated with fraction 6 caused a time-dependent decrease in pro-caspase3. (B) Bax could induce apoptosis, but Bcl-2 could inhibit apoptosis on the mitochondria pathway. The up-regulation of Bax / Bcl-2 expression was detected at time-dependent after treatment. (C) ER-induced apoptosis is associated with the activation of caspase12. Fraction 6 also caused a time-dependent decrease in pro-caspase12. These results suggested that fraction 6 could induce apoptosis on A549 cells through mitochondria pathway and ER-induced pathway. Our previous examinations suggested that the ethanol extracts of A. camphorata (SACE) can evoke the apoptosis of human A549 cells possibly by the down-regulation of human galectin-1, human eIF5A, human Rho GDI-, human calpain small (regulatory) subunit cells (Wu et al., 2006).
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Figure 5. Effect of fraction 6 on galectin-1, calpain-1/2, RhoGDI-α and eIF-5a expression To investigate if Fr-6 had the same effect on the expression of these four tumor-related genes, western blotting with the antibody against each of these four proteins was carried out. (A), (B), (C), (D) Fraction 6 (50μg / ml) caused a time-dependent decrease in galectine-1, calpain-1 / 2, RhoGDI-α and eIF-5a expression after treatment. These results suggest ed that fraction 6 could reduce the expression of the proteins as indicated above on A549 cell.
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Figure 6. Effect of fraction 6 on ERK and JNK activation. Recent findings showed that the recruiting of H-Ras to the cell membrane is dependent on galectin-1 and galectin-1 binds H-Ras to stimulate the transformation (Belanis et al., 2008; Paz et al., 2001). Since Ras take part in Erk pathway, western blotting with the antibodies against Erk and phospho-Erk (p-Erk) to examine if Fr-6 could interfere with Erk pathway. (A), (B) The down-regulation of ERK and phospho-ERK expression were detected at time - dependent after treatment. Recent investigations showed that he inhibition of RhoGDI- production invokes the apoptosis of insulin-secreting cells by the activation of c-Jun NH2-terminal kinase (JNK) pathway. (C) The down-regulation of JNK expression was detected at time-dependent after treatment. (D) The up-regulation of phospho-JNK expression was detected at time-dependent after treatment. These results suggest that fraction 6 could prevent the activation of ERK, but promote the activation of JNK.
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Figure 7. Effect of fraction 6 on activated JNK expression in relation to RhoGDI-α Cells were treated with 50μg / ml fraction 6 alone or 15μM JNK inhibitor ( SP600125 ) for the time periods indicated. (A), (B) The SP600125 was administered to cells 24 h prior to fraction- 6 treatment. Pre-treatment with JNK inhibitor could prevent the activation of JNK and the reduction in RhoGDI-α expression caused by fraction 6. ( C ) Viability of cells treated with both fraction 6 and the JNK inhibitor was 42 % greater than that of cells treated with fraction 6 alone at 72 h. These results suggested that fraction 6-induced cell death might occur through an increase in the levels of activated JNK.
