- Email: [email protected]
Isoorientin from Gypsophila elegans induces apoptosis in liver cancer cells via mitochondrial-mediated pathway
Journal of Ethnopharmacology 187 (2016) 187–194
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Isoorientin from Gypsophila elegans induces apoptosis in liver cancer cells via mitochondrial-mediated pathway Xing Lin a, Jinbin Wei a, Yongxin Chen a,b, Ping He a, Jun Lin a, Shimei Tan a, Jinlan Nie a, Shengjuan Lu a, Min He a, Zhongpeng Lu b,c, Quanfang Huang b,n a
Guangxi Medical University, Nanning 530021, China The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China c Department of Biochemistry, University of Arkansas Medical School, 4301 W. Markham, Little Rock, AR 72205-7199, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 July 2015 Received in revised form 12 April 2016 Accepted 26 April 2016 Available online 27 April 2016
Ethnopharmacological relevance: Gypsophila elegans has been used as a traditional herbal medicine for treating immune disorders and chronic liver diseases in China. The aim of this study is to isolate an active ingredient from this herb and investigate its anti-tumor activity. Materials and methods: An active ingredient was isolated from the ethanol extract using bioassay-guided screening. And its anti-tumor activity was analyzed by testing the cytotoxicity, lactate dehydrogenase (LDH) release, clonogenecity and migration in HepG2 cells. To investigate its potential mechanism, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS), cytochrome c, mitochondria membrane potential (MMP) and caspase level were determined in liver cancer cell line HepG2. Results: A flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2″-O-α-L-arabinopyranosyl. Our results showed that GEI significantly inhibited the viability and proliferation of HepG2 cells in a dose- and time-dependent manner, and its cytotoxic effect was also confirmed by the elevated level of LDH. GEI treatment could markedly inhibit the clonogenicity and migration of HepG2 cells. Moreover, GEI induced remarkable apoptotic death of HepG2 cells through cell cycle arrest at G1 phase via the regulation of cell cycle-related genes, such as cyclin D, cyclin E and CDK2. Further study showed that GEI treatment significantly elevated ROS formation, followed by attenuation of MMP via up-regulation of Bax and down-regulation of Bcl-2, accompanied by cytochrome c release to the cytosol. In addition, GEI treatment resulted in a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. Conclusion: The present study demonstrates that the ability of GEI to induce apoptosis against HepG2 cells mediated by mitochondrial-mediated pathway. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytotoxicity Gypsophila elegans Isoorientin Mitochondrial-mediated pathway Reactive oxygen species
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with very high morbidity and mortality rates, and a poor prognosis. The high incidence of this kind of liver cancer has been caused by factors such as persistent infection with hepatitis B virus and contact with hepatocarcinogens such as nitrosamines, aflatoxins and alcohol (Henry et al., 2002). Despite extensive efforts by many investigators, systemic chemotherapy
Abbreviations: GEI, G. elegans isoorientin; ROS, reactive oxygen species; LDH, lactate dehydrogenase; FBS, fetal bovine serum; DCFH-DA, 2′,7′-dihydrofluoresceindiacetate; MMP, Mitochondria membrane potential; TBS, Tris-buffer saline n Correspondence to: Department of the Pharmacy, the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University, 89-9 Dongge Road, Nanning, Guangxi 530023, China. E-mail address: [email protected] (Q. Huang). http://dx.doi.org/10.1016/j.jep.2016.04.050 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
for HCC has been quite ineffective, as demonstrated by low response rates and no survival benefits (França et al., 2004; Rougier et al., 2007). Apoptosis, or programmed cell death, is thought to play a key role in the development and regulation of growth of both normal and cancerous cells. The hallmark of cancer cells is the dysregulation of cell proliferation and apoptosis. The tumor growth depends on the cell proliferation rate and apoptosis. Therefore, induction of apoptosis of tumor cells has become a strategy in cancer treatment (Youn et al., 2008; Tan et al., 2009). Generally, apoptosis may occur via the mitochondrial (intrinsic) pathway or the death receptor (extrinsic) pathway (Li et al., 1997; Nagata, 1997). In the mitochondrial (intrinsic) pathway, the regulation of apoptosis involves a large set of proteins including Bcl-2, an anti-apoptotic protein (Luna-More et al., 2004), which disrupts the mitochondria membrane potential, resulting in release of apoptogenic factors from the mitochondria to the cytoplasm.
188
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Natural products, a rich source of compounds with enormous structural diversity, have been extensively explored in the field of drug discovery. A large number of different sources of natural compounds present anti-cancer effects both in vitro and in vivo (Huang et al., 2012a; Lu et al., 2012, 2013). Therefore, it is vital to screen efficient natural compounds for hepatocellular carcinoma treatment. Caryophyllaceae Gypsophila elegans has been widely used in traditional Chinese medicine with a record of safety and efficacy in the treatment of immune disorders and liver diseases (Cao, 2007). In our study, a flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2″-O-α-L-arabinopyranosyl. Our results revealed that GEI significantly attenuated liver injury and fibrosis induced by chronic alcohol. The preliminary exploration of the underlying mechanisms indicated its protection against hepatic injury by radical scavenging action, antioxidant activity, as well as its ability to attenuate HSCs activation (Huang et al., 2012b). To further understand the anti-tumor effects of GEI, the present study was designed to investigate the effects of GEI on proliferation and apoptosis of human hepatocarcinoma cells (HepG2 cell). We found that GEI induces apoptosis in HepG2 cells, and the mitochondrial-mediated pathway may contribute to GEI-induced apoptosis.
2. Materials and methods 2.1. Materials G. elegans herb was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd (Nanning, China). Fetal bovine serum was purchased from Life Sciences (Grand Island, NY). MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, U.S.A). The antibodies including cytochrome c, Bcl-2, Bax and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences Pharmigen (San Diego, CA). 2.2. Preparation of GEI GEI was isolated from G. elegans according to our previous study (Huang et al., 2012b) with some modification. In the present study, the result indicated that the yield of GEI was higher than that of our previous study. Briefly, the dried powder of G. elegans herb (10 kg) was extracted with 80 L 70% ethanol under reflux for 2 h two times. The extract was negative-pressure filtered, and the filtrate was evaporated to a fluid extract by removing the ethanol under reduced pressure in a rotary evaporator at 60 °C. The crude extract was successively extracted with ethyl acetate and watersaturated n-butanol, and the later extract was then subjected to chromatography on a silicagel column (200–300 mesh, Yantai, PR China; Ø10 cm 300 cm) eluting with a gradient mixture of CHCl3 and MeOH (0–100% MeOH, 1500 mL each fraction). The CH3Cl3–MeOH (35: 65) fraction yielded a yellow powder after concentration, which was purified by Sephadex LH-20 and preparative HPLC to produce compound (27.82 g). Its structure was elucidated on the basis of physicochemical properties and spectral data: ESI-MS(m/z): 604 [MþNa] þ ; 1H NMR (500 MHz, CD3OD) δ: 7.32, 7.29, 6.88, 6.49, 6.40, 4.98, 4.37; 13C NMR(125 MHz, CD3OD) δ: 165.9, 104.7, 185.0, 159.8, 110.3, 166.1, 95.9, 163.7, 105.9, 123.0, 114.8, 148.0, 152.1, 117.9, 121.2, 73.9, 82.2, 74.8, 73.1, 83.1, 63.9, 110.2, 71.9, 81.1, 68.3, 66.8. These results indicated that the compound is isoorientin-2″- O-α-L- arabinopyranosyl, i.e., G. elegans isoorientin (GEI), with a chemical formula of C26O15H28 and a molecular weight of 580.24. Its chemical structure is shown in
OH OH HO HO HO
HO HO O
HO
O
O O
OH O
OH Fig. 1. The chemical structure of GEI.
Fig. 1. 2.3. Cell line and cytotoxicity assay Human liver carcinoma cell line (HepG2 cells) was obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in a humidified incubator with 5% CO2 and 95% oxygen at 37 °C. Cytotoxicity was measured by MTT method. Briefly, exponentially growing cells were seeded in 96-well plates and cultured until 50% confluence. Then, various concentrations of GEI were added and the final concentration in each well was 5, 10, 20, 40, 80 and 160 μmol/L, respectively. Each treatment was tested in tetrad wells and the untreated control group was administered culture medium containing no drug. All of the above plates were placed in a CO2 humidified-atmosphere incubator at 37 °C for 24, 48 and 72 h. At the end of exposure, 20 μL MTT (5 mg/mL) was added to each well and the plates were incubated at 37 °C for 4 h. Then, all culture medium supernatant was removed from the wells and replaced with 200 μL DMSO. The absorbance of each well was measured by standard enzyme-linked immunosorbant assay at 570 nm. The cell viability was calculated based on the following formula: cell viability (%) ¼average A570 nm of treated group/ average A570 nm of untreated control group 100% (Xie et al., 2012). 2.4. Lactate dehydrogenase (LDH) release assay LDH released into the culture supernatants was measured using Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific™, Pittsburgh, PA, USA) following the manufacturer's instructions. In brief, cells were seeded in 96-well plates and then treated with GEI at different concentrations (5, 10 or 20 μmol/L) for 48 h. The supernatant was transferred into 96-well plate to assess the LDH activity. Triton X-100 (2%) served as a positive control was used to completely lyse the cells and release the maximum LDH. Next, the LDH reaction solution (100 μL) was added to the cells for 30 min The optical density of the color generated was determined at a wave length of 490 nm using a Microplate Reader (Tecan, Männedorf, Switzerland). The results were expressed as percentage of Triton X-100-induced LDH release. 2.5. Clonogenicity assay The clonogenicity test was performed according to the previous study (Franken et al., 2006). HepG2 cells were seeded at a density of 1 106 cells in 6-well plates. After 24 h of incubation, the cells were treated with 5, 10 or 20 μmol/L of GEI for 144 h. The cells were fixed and stained with 0.1% crystal violet to visualize colonies.
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
2.6. Migration assay Cell migration was assessed using a wound healing assay as previously reported (Wang et al., 2014). HepG2 cells were seeded in 6-well plates (5 105 cells per well) for 24 h to form a monolayer. Then, the medium was replaced with fresh incubated medium without fetal bovine serum and subjected to scratch assay and photographed with microscope (Olympus Optical, Tokyo, Japan). After the treatment of GEI (20 μmol/L) for 24 h and 48 h, the microscopic imaging was taken respectively. Percentage of the closed area was measured and compared with the value obtained before treatment. 2.7. Annexin V-FITC/PI assay Apoptotic cells were quantified by flow cytometry using the Phosphatidyl Serine Detection kit (Becton Dickinson). Briefly, HepG2 cells were incubated with GEI (5, 10 or 20 μmol/L) for 24 h. Then the cells were washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 106 cells/mL. Annexin V-FITC (10 μL) and PI (10 μL) were added to 100 μL of cells and then incubated for 20 min at room temperature in the dark. At the end of incubation, 400 μL of binding buffer was added, and the cells were analyzed immediately by FACScan flow cytometer (Becton Dickinson) using CellQuest software.
189
harvested, washed twice with PBS, re-suspended and stained with fresh incubated medium containing 1.0 μM Rhodamine 123 at 37 °C for 30 min The fluorescence intensity of the cells was analyzed by flow cytometer (Becton Dickinson, USA). 2.11. Caspase-3, -8 and -9 activities assay Caspases activities were measured using Caspase Activity Assay Kit (BioVision Research Products, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, 5 106 HepG2 cells were treated with 5, 10 or 20 μmol/L GEI for 24 h. Then cells were washed with cold PBS twice, resuspended in lysis buffer and left on ice for 20 min The lysate was centrifuged at 16,000 g at 4 °C for 20 min Supernatants were collected and protein concentrations were measured with a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). Caspase-3, -8 and -9 activities were measured by reaction buffer and caspase substrate peptides Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA, respectively. The release of p-nitroanilide (pNA) was qualified by determining the absorbance with a microplate reader (BioTek, Winooski, VT, USA) at 405 nm. The fold increase in absorbance was calculated by the formula [ODi/OD0], where OD0 is the absorbance without GEI treatment (untreated control) and ODi is the absorbance with GEI treatment at the indicated concentration. 2.12. Western blot analysis
2.8. Cell cycle analysis Cell cycle was analyzed by flow cytometry (FAC sort, Becton Dickinson). Briefly, HepG2 cells (1 106 cells) were seeded into a 100 mm2 dish. After 24 h of cultivation, the cells were treated with GEI (5, 10 or 20 μmol/L) for 48 h. Cells were harvested and fixed with 70% ice-cold ethanol. Cells were washed and the pellet was suspended in trypsin buffer and left for 10 min at room temperature. RNAase buffer (1%) was added after addition of trypsin inhibitor and incubated for 10 min, followed by the addition of 100 μg/mL propidium iodide (PI). Samples were incubated in the dark for 30 min at 4 °C. The fluorescence was measured using a FACScan flow cytometer. Data collection and analysis of the cell cycle distribution were performed using CellQuest and the Modfit software (Becton Dickinson). 2.9. Reactive oxygen species (ROS) assay The production of intracellular ROS was detected using the specific fluorogenic probe 2′,7′-dihydrofluorescein-diacetate (DCFH-DA). This probe diffuses across the cell membrane and is hydrolysed to DCFH, which is rapidly oxidised to the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF) in the presence of ROS (Hempel et al., 1999). The DCF fluorescence intensity is proportional to the ROS level. In this study, intracellular ROS generation was detected according to the manufacturer's instructions of ROS detection kit (Beyotime, Jiangsu, China). Briefly, HepG2 cells were seeded in 6-well plates for 12 h and then incubated with fresh medium containing10 μM DCFH-DA at 37 °C in the dark for 20 min Subsequently, cells were incubated with 5, 10 or 20 μmol/L GEI for 30 min Then cells were washed with PBS buffer and the DCF fluorescence intensity was measured by the fluorescence microscope.
HepG2 cells were cultured in 75 mm2 dish (5 105 cells) for 12 h and then exposed to GEI (5, 10 or 20 μmol/L) for 24 h. Then cells were collected, washed twice with PBS and centrifuged. Subsequently, each sample was added into 50 μL RIPA Lysis buffer (containing 1% Phenylmethanesulfonyl fluoride) and incubated on ice for 30 min Then, samples were clarified by centrifugation at 12,000 g for 10 min at 4 °C, and protein concentrations were determined using BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were separated on 15% SDS-polyacrylamide gels and electrotransferred to Immobilon-P membranes (Millipore). After blocking with Tris-buffer saline (TBS) containing 0.05% Tween 20% and 5% nonfat powdered milk, the membranes were incubated with specific primary antibodies against cytochrome c, Bcl-2, Bax and β-actin (Santa Cruz, USA) at 4 °C for 12 h. After three washes with TBS for 10 min each, the membranes were incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The membranes were washed and detection was carried out with an ECL Western blotting kit according to the manufacturer's instructions. 2.13. Real-time quantitative PCR assay HepG2 cells were treated with 5, 10 or 20 μmol/L GEI for 72 h. Total RNAwas extracted from cells with TRIzol reagent according the manufacturer's instructions. The concentration of total RNA in the final eluates was determined by spectrophotometry. Total RNA was heated at 65 °C for 10 min and then chilled on ice. Each sample was reverse-transcribed to cDNA at 37 °C for 90 min using a cDNA synthesis kit (Amersham Pharmacia Biotech). PCR was performed with specific primers which are listed in Table 1. PCR products were electrophoresed on 2% agarose gels and visualized by staining with ethidium bromide. In this study, β-actin was used as an internal control.
2.10. Mitochondria membrane potential (MMP) disruption assay The MMP (Δψm) was monitored by Rhodamine123 staining as previous reported (Ma et al., 2014). Briefly, HepG2 cells were seeded in 6-well plate (1 105 cells per well) for 12 h and incubated with GEI (5, 10 or 20 μmol/L) for 24 h. Then cells were
2.14. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post hoc
190
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Table 1 Primer sequence used in this study. Genes
Forward primer (5′-3′)
Reverse primer (5′-3′)
Cyclin D Cyclin E CDK2 Bcl-2 Bax β-actin
CTG GCC ATG AAC TAC CTG GA TTC TCG GCT CGC TCC AGG AA AGA TTC TTC TGG GCT GCA AG TTC TTT GAG TTC GGT GGG GTC GAC ATG TTT TCT GAC GGC AA CAA GAG ATG GCC ACG GCT GCT
CCA GGA AAT CAT GTG CAA TC TGG AGG ATA GAT TTC CTC AGA TCC GGA AGA GTT GGT CA TGC ATA TTT GTT TGG GGC AGG CCC AAA GTA GGA GAG GAG GC TCC TTC TGC ATC CTG TCG GCA
multiple comparisons. The data are presented as the means 7SD. A p-value o0.05 was considered to be statistically significant.
3. Results 3.1. Effects of GEI on the viability of HepG2 cells HepG2 cells were cultured with the various concentrations of GEI for 24, 48, and 72 h, and cytotoxicity were then determined using MTT assay. As shown in Fig. 2A, GEI inhibited the growth of HepG2 cells in a distinct dose- and time-dependent manner. The 50% inhibitory concentrations (IC50) were 70.06 79.13, 47.107 11.36 and 15.95 7 7.46 μmol/L after incubation for 24, 48 and 72 h, respectively. 3.2. Effect of GEI on LDH release A stable cytosolic enzyme of lactate dehydrogenase (LDH) catalyzes the oxidation of L-lactate to pyruvate. Upon membrane damage in cells, LDH enzyme is released into the culture medium, suggesting the loss of membrane integrity. Therefore, to further confirm the cytotoxic effect of GEI on HepG2 cells, LDH assay was also performed as another indicator of cytotoxicity. In the present study, LDH release level was measured by detecting changes in optical densities. As shown in Fig. 2B, treatment with GEI could significantly increase LDH activity in HepG2 cells in a dose-dependent manner. 3.3. Effects of GEI on the clonogenicity and migration of HepG2 cells Clonogenicity was determined by plating a fixed number of HepG2 cells onto multiple well tissue culture dishes. Cells were treated with GEI and maintained in culture for 144 h to allow formation of colonies. The size and number of colonies were
visually inspected by fixing and staining with 0.1% crystal violet. As shown in Fig. 3A, GEI treatment inhibited cell clonogenicity in human liver cancer HepG2 cells in a concentration-dependent manner. Wound-scratch assays were performed to investigate the effect of GEI on the migration of HepG2 cells. As shown in Fig. 3B and C, the cells of the untreated control group moved fast into the scraped area over time, while GEI treatment significantly inhibited the cells migration in a time-dependent manner. 3.4. Effect of GEI on cells apoptosis and cycle in HepG2 cells To explore the underlying basis for the cytotoxic effect of GEI in liver cancer cells, the level of cell apoptosis induced by GEI were analyzed in HepG2 cells. Cells were treated with GEI for 24 h, stained with annexin V-FITC and PI, and subsequently analyzed by flow cytometry. The results revealed that GEI significantly induced apoptosis of HepG2 cells in a dose-dependent manner after 24 h treatment when compared with the untreated control (Fig. 4A). In the cell cycle analysis, we observed increased accumulation of cells in G1 phase and decreased fractions of cells in G2 phase (Fig. 4B), suggesting that GEI induced the apoptotic death of HepG2 cells by cell cycle arrest at G1 phase. 3.5. Effect of GEI on cell cycle-related genes expression Previous studies have reported that cell cycle is controlled by cyclin-dependent kinases (CDKs), cyclin kinase inhibitors, and cyclins. In HepG2 cells, treatment with GEI resulted in a remarkable decrease in the transcription of genes, such as cyclin D gene, cyclin E gene and CDK2 gene, as assessed by RT-PCR analysis (Fig. 5A). 3.6. Effects of GEI on apoptosis-related genes and proteins In human, the Bcl-2 family has been identified and classified as anti-apoptotic protein (such as Bcl-2) and pro-apoptotic protein (such as Bax). The balance of the expressed levels of pro-apoptotic and anti-apoptotic proteins is critical for cell survival or death. In this study, the anti-apoptotic gene Bcl-2 was decreased by GEI in a dose-dependent manner, while the Bax, a pro-apoptotic gene, was significantly increased in GEI-treated HepG2 cells (Fig. 5B). Similarly, Western blot analysis showed a significant decrease in Bcl-2 protein expression, whereas an increase in Bax protein level in the GEI-treated cells (Fig. 5C).
Fig. 2. Effects of GEI on cell viability and LDH release in HepG2 cells. (A) MTT assay showed that GEI inhibited the viability of HepG2 cells in a dose- and time-dependent manner. (B) GEI significantly increased LDH activity in a dose-dependent manner. All analyses were performed in triplicate. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
191
Fig. 3. GEI inhibited the clonogenicity and migratory potential of HepG2 cells. (A) Clonogenecity assay revealed that GEI suppressed colony formation of HepG2 cells. (B) GEI treatment resulted in a significant decrease in migration ability in a time-dependent manner. (C) The representative bar charts of GEI mediated migration of HepG2 cells. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
3.7. Effect of GEI on mitochondria membrane potential (MMP) In recent years, mitochondrial pathway has been identified as a major mechanism for induction of apoptosis. Thus, the effect of GEI on integrity of mitochondrial function was analyzed using a stain of Rhodamine123 by flow cytometry. As shown in Fig. 6A and B, compared with the untreated control, GEI dramatically decreased the mitochondria membrane potential (MMP) of HepG2 cells in a dose-dependent manner. 3.8. Effect of GEI on reactive oxygen species (ROS) formation ROS generation can induce oxidative damage to vital cellular molecules and structures including lipids, proteins and DNA, activate the intrinsic apoptotic pathway due to mitochondrial dysfunction and finally lead to apoptosis. In this study, we investigated whether ROS generation was involved in GEI-induced apoptosis. As shown in Fig. 6C, compared with the untreated control, treatment with GEI resulted in a significant increase in ROS generation in HepG2 cells. 3.9. Effect of GEI on cytochrome c level In normal situation, cytochrome c resides in the mitochondrial intermembrane and serves as a transducer of electrons in the respiratory chain. However, the increment of ROS and subsequent mitochondrial dysfunction cause release of cytochrome c from mitochondria to cytosol. In this study, the cytosolic level of cytochrome c was measured to confirm apoptosis via the mitochondrial pathway in GEI-treated HepG2 cells. The result indicated that GEI treatment could result in a significant increase in the cytochrome c protein level when compared to the untreated control (Fig. 6D). 3.10. Effects of GEI on caspase-3, -8 and -9 activities The activation of cysteine proteases is an important feature of apoptotic cell death. We determined GEI-induced activation of the protease activities of caspase-3, caspase-8 and caspase-9. As shown in Fig. 7, GEI treatment caused a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. However, HepG2 cells treated with GEI displayed a slight increase in caspase-8 activity. 4. Discussion Apoptosis plays a crucial role in the cellular progress of
proliferation, differentiation, senescence and death. Nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptotic cells (Gao et al., 2011). Previous studies have confirmed that the occurrence and development of tumors is closely related to cell apoptosis (Krysko et al., 2008). The potential to induce apoptosis has therefore become an important topic in the study of anti-tumor drugs. In the present study, GEI strongly suppressed the viability and proliferation of HepG2 cells in a time- and dose-dependent manner. The cytotoxic effect of GEI was further confirmed by the significant increase in LDH activity of cell culture supernatants induced by GEI treatment. Flow cytometric analysis showed that GEI significantly induced apoptosis of HepG2 cells. Further study showed that the anti-proliferative effect of GEI could be attributed to cell cycle arrest that was induced by the ability of GEI to halt the cell cycle at G1 phase via the down-regulation of the cell cyclerelated genes transcription, such as cyclin D, cyclin E and CDK2. In addition, GEI treatment significantly inhibited the clonogenicity and migration of HepG2 cells. Based on the results obtained, we speculated that GEI may play a potential role in the treatment of hepatocellular carcinoma. Oxidative stress is considered to be important for the promotion of apoptosis in response to a variety of apoptotic stimuli. ROS play essential role in the oxidative stress response. Increasing evidences indicate that over-production of ROS can induce oxidative damage to vital cellular molecules and structures including lipids, proteins and DNA, activate the intrinsic apoptotic pathway due to mitochondrial dysfunction and finally lead to apoptosis (Circu and Aw, 2010). In the present study, our finding showed that GEI significantly increased the production of intracellular ROS in HepG2 cells, suggesting that excessive ROS generation is likely involved in GEI-induced apoptotic cell death in HepG2 cells. It is well known that apoptosis occurs via two primary pathways: the extrinsic pathway, which is associated with cell death receptors and their ligands on the cellular surface, and the intrinsic pathway, which is dependent on mitochondria. The latter has been considered to be the predominant apoptosis-inducing pathway (Fulda and Debatin, 2006; Wang et al., 2013a). Previous studies have demonstrated that mitochondrial dysfunction causes the collapse of mitochondria membrane potential (MMP), which results in mitochondrial permeability transition pore (MPTP) opening, enabling to the release of cytochrome c from mitochondria into cytosol (Chan et al., 2013; Wu et al., 2014). The mitochondria-dependent apoptotic pathway is regulated by Bcl-2 family of proteins, including Bax and Bcl-2 (Kong et al., 2013). Bax, which is a pro-apoptotic member of the Bcl-2 family, promotes the opening of the MPTP, causing the release of cytochrome c, whereas
192
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Fig. 4. Effects of GEI on the cells apoptosis and cycle in HepG2 cells. (A) Flow cytometric evaluation of annexin-V-FITC/PI-stained HepG2 cells showing the effect of different concentrations of GEI (5, 10 or 20 μmol/L) on the percentage of apoptotic cells 24 h after treatment. The data indicated that GEI significantly induced apoptosis of HepG2 cells in a dose-dependent manner. (B) The effect of GEI on cell cycle was assessed by flow cytometry. The result revealed that GEI treated HepG2 cells showed arrest in the G1 phase of the cell cycle. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
Bcl-2, which is an anti-apoptotic member of the Bcl-2 family, inhibits the formation of the MPTP, blocking cytochrome c release (Wang et al., 2013b). An increase in the Bax/Bcl-2 ratio has been demonstrated to promote apoptosis by directly activating the mitochondrial apoptotic pathway (Brunelle and Letai, 2009). In the present study, GEI treatment caused a loss of MMP in HepG2 cells and an obvious translocation of cytochrome c from mitochondria to cytosol. Furthermore, GEI treatment resulted in a significant decrease in Bcl-2 expression and a remarkable increase in Bax
level. These data indicate that GEI-induced apoptosis of HepG2 cells is mediated by the mitochondria-dependent pathway. A molecular hallmark of apoptosis is the activation of caspases, which has been demonstrated to play a central role during cellular apoptosis (Sun et al., 1999). Caspase-3, particularly, has a key role in executing apoptosis as it catalyzes specific cleavage of several important cellular proteins (Brentnall et al., 2013). Increase in caspase-3 activity is crucial for drug-induced apoptosis as the activation of caspase-3 is a hallmark of apoptosis and can be used in
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
193
Fig. 5. Effects of GEI on the cell cycle-related genes, and the apoptosis-related genes and proteins. (A) RT-PCR analysis showed that GEI repressed the transcription of cell cycle regulatory genes including cyclin D, cyclin E and CDK2. (B) GEI decreased the mRNA levels of anti-apoptosis gene Bcl-2, while enhanced the expression of pro-apoptotic gene Bax. (C) Western blot assay revealed that GEI treatment resulted in a decrease in the Bcl-2 protein expression and an increase in the Bax protein level. Lane-1, untreated control group; lane-2, 5 μmol/L GEI-treated group; lane-3, 10 μmol/L GEI-treated group; lane-4, 20 μmol/L GEI-treated group. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
quantifying the caspase cascade (Logue and Martin, 2008). Caspase-3 is triggered by either caspase-8 or caspase-9, which acts as a convergence point for different apoptosis signaling pathways. In the present study, the levels of caspase-3 and -9 were significantly
increased in GEI-treated HepG2 cells, confirming the involvement of mitochondria-dependent intrinsic pathway in the GEI-induced apoptotic events in HepG2 cells, whereas procaspase-8 did not change in HepG2 cells in response to GEI treatment, indicating no
Fig. 6. Effects of GEI on mitochondrial membrane potential (Δψm), ROS generation and cytochrome c. (A-B) GEI induced an obvious decrease of mitochondrial membrane potential (Δψm) in a dose-dependent manner. (C) GEI increased ROS generation in HepG2 cells. (D) GEI treatment resulted in an increase in the cytochrome c protein expression in cytosol. Lane-1, untreated control group; lane-2, 5 μmol/L GEI-treated group; lane-3, 10 μmol/L GEI-treated group; lane-4, 20 μmol/L GEI-treated group. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
194
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Fig. 7. Caspases activities in GEI-treated HepG2 cells. Cells were treated with 5, 10 or 20 μmol/L GEI for 24 h. Caspase activities in HepG2 were determined using the caspase-activity detection kits. Data were expressed as fold increase, and as means 7 SD. *Po 0.05 vs. untreated control.
death receptor-mediated extrinsic caspase-8 pathway in GEI-induced HepG2 cells apoptosis. In summary, the anti-cancer potential of GEI was supported by the evidence provided in the present study, including lactate dehydrogenase leakage, reactive oxygen species generation, loss in mitochondrial membrane potential, increase in the level of cytochrome c, up-regulation of Bax, down-regulation of Bcl-2 and activation of initiator and executioner caspases. Our findings may provide a new insight for the potential use of GEI as chemotherapeutic and chemopreventive agent for the treatment of hepatocellular carcinoma.
Disclosure statement The authors declare that there are no conflicts of interest.
Acknowledgment The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (No. 81260674) and the Guangxi Natural Science Foundation (2014GXNSFAA118155; 2014GXNSFAA118154; 2012GXNSFCA053005).
References Brentnall, M., Rodriguez-Menocal, L., De Guevara, R.L., Cepero, E., Boise, L.H., 2013. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 14, 32. Brunelle, J.K., Letai, A., 2009. Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell Sci. 122, 437–441. Cao, G., 2007. The clinical application of Dong-Yao (Chinese Minorities medicine herb) Gypsophila elegans. J. Med. Pharm. Chin. Minor. 15, 24–25. Chan, S.H., Leu, W.J., Hsu, L.C., Chang, H.S., Hwang, T.L., Chen, I.S., Chen, C.S., Guh, J. H., 2013. Reevesioside F induces potent and efficient anti-proliferative and apoptotic activities through Na þ /K þ -ATPase alpha3 subunit-involved mitochondrial stress and amplification of caspase cascades. Biochem. Pharmacol. 86, 1564–1575. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and
apoptosis. Free Radic. Biol. Med. 48, 749–762. França, A.V.C., Elias Júnior, J., Lima, B.L.G., Martinelli, A.L.C., Carrilho, F.J., 2004. Diagnosis, staging and treatment of hepatocellular carcinoma. Braz. J. Med. Biol. Res. 37, 1689–1705. Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J., Van Bree, C., 2006. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319. Fulda, S., Debatin, K.M., 2006. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811. Gao, Y., Su, Y., Qu, L., Xu, S., Meng, L., Cai, S.Q., Shou, C., 2011. Mitochondrial apoptosis contributes to the anti-cancer effect of Smilax glabra Roxb. Toxicol. Lett. 207, 112–120. Hempel, S.L., Buettner, G.R., O’Malley, Y.Q., Wessels, D.A., Flaherty, D.M., 1999. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146–159. Henry, S.H., Bosch, F.X., Bowers, J., 2002. Aflatoxin, Hepatitis and Worldwide Liver Cancer Risks, Mycotoxins and Food Safety. Springer, pp. 229–233. Huang, M., Lu, J., Huang, M., Bao, J., Chen, X., Wang, Y., 2012a. Terpenoids: natural products for cancer therapy. Expert Opin. Investig. Drugs 21, 1801–1818. Huang, Q., Zhang, S., Zheng, L., Liao, M., He, M., Huang, R., Zhuo, L., Lin, X., 2012b. Protective effect of isoorientin-2″-O-α-L-arabinopyranosyl isolated from Gypsophila elegans on alcohol induced hepatic fibrosis in rats. Food Chem. Toxicol. 50, 1992–2001. Kong, D., Zheng, T., Zhang, M., Wang, D., Du, S., Li, X., Fang, J., Cao, X., 2013. Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways. PLoS One 8, e69403. Krysko, D.V., Vanden Berghe, T., D’Herde, K., Vandenabeele, P., 2008. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44, 205–221. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., Wang, X., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489. Logue, S.E., Martin, S.J., 2008. Caspase activation cascades in apoptosis. Biochem. Soc. Trans. 36, 1–9. Lu, J., Bao, J., Chen, X., Huang, M., Wang, Y., 2012. Alkaloids isolated from natural herbs as the anticancer agents. Evid. Based Complement. Altern. Med. 2012, 1–12. Lu, J., Bao, J., Wu, G., Xu, W., Huang, M., Chen, X., Wang, Y., 2013. Quinones derived from plant secondary metabolites as anti-cancer agents. Anticancer Agents Med. Chem. 13, 456–463. Luna-More, S., Weil, B., Bautista, D., Garrido, E., Florez, P., Martinez, C., 2004. Bcl-2 protein in normal, hyperplastic and neoplastic breast tissues. A metabolite of the putative stem-cell subpopulation of the mammary gland. Histol. Histopathol. 19, 457–463. Ma, W., Zou, Y., Wang, P., Yao, X., Sun, Y., Duan, M., Fu, Y., Yu, B., 2014. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. 70, 1–8. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365. Rougier, P., Mitry, E., Barbare, J.-C., Taieb, J., 2007. Hepatocellular carcinoma (HCC): an update. Semin. Oncol., S12–S20. Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R., Cohen, G.M., 1999. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053–5060. Tan, X., Xia, H., Xu, J., Cao, J., 2009. Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin. World J. Gastroenterol. 15, 2234–2239. Wang, J., Yang, Z., Wen, J., Ma, F., Wang, F., Yu, K., Tang, M., Wu, W., Dong, Y., Cheng, X., 2014. SKLB-M8 induces apoptosis through the AKT/mTOR signaling pathway in melanoma models and inhibits angiogenesis with decrease of ERK1/2 phosphorylation. J. Pharmacol. Sci. 126, 198–207. Wang, J., Yuan, L., Xiao, H., Xiao, C., Wang, Y., Liu, X., 2013a. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways. Apoptosis 18, 751–765. Wang, Y., Li, X., Wang, X., Lau, W., Wang, Y., Xing, Y., Zhang, X., Ma, X., Gao, F., 2013b. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/ GSK-3beta signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS One 8, e70956. Wu, W., Zhou, X., Liu, P., Fei, W., Li, L., Yun, H., 2014. Isoflurane reduces hypoxia/ reoxygenation-induced apoptosis and mitochondrial permeability transition in rat primary cultured cardiocytes. BMC Anesthesiol., 14–17. Xie, J., Que, W., Liu, H., Liu, M., Yang, A., Chen, M., 2012. Anti-proliferative effects of cucurmosin on human hepatoma HepG2 cells. Mol. Med. Rep. 5, 196–201. Youn, M., Kim, J., Park, S., Kim, Y., Kim, S., Lee, J., Chai, K., Kim, H., Cui, M., So, H., 2008. Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells. World J. Gastroenterol. 14, 511–517.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Isoorientin from Gypsophila elegans induces apoptosis in liver cancer cells via mitochondrial-mediated pathway Xing Lin a, Jinbin Wei a, Yongxin Chen a,b, Ping He a, Jun Lin a, Shimei Tan a, Jinlan Nie a, Shengjuan Lu a, Min He a, Zhongpeng Lu b,c, Quanfang Huang b,n a
Guangxi Medical University, Nanning 530021, China The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China c Department of Biochemistry, University of Arkansas Medical School, 4301 W. Markham, Little Rock, AR 72205-7199, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 July 2015 Received in revised form 12 April 2016 Accepted 26 April 2016 Available online 27 April 2016
Ethnopharmacological relevance: Gypsophila elegans has been used as a traditional herbal medicine for treating immune disorders and chronic liver diseases in China. The aim of this study is to isolate an active ingredient from this herb and investigate its anti-tumor activity. Materials and methods: An active ingredient was isolated from the ethanol extract using bioassay-guided screening. And its anti-tumor activity was analyzed by testing the cytotoxicity, lactate dehydrogenase (LDH) release, clonogenecity and migration in HepG2 cells. To investigate its potential mechanism, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS), cytochrome c, mitochondria membrane potential (MMP) and caspase level were determined in liver cancer cell line HepG2. Results: A flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2″-O-α-L-arabinopyranosyl. Our results showed that GEI significantly inhibited the viability and proliferation of HepG2 cells in a dose- and time-dependent manner, and its cytotoxic effect was also confirmed by the elevated level of LDH. GEI treatment could markedly inhibit the clonogenicity and migration of HepG2 cells. Moreover, GEI induced remarkable apoptotic death of HepG2 cells through cell cycle arrest at G1 phase via the regulation of cell cycle-related genes, such as cyclin D, cyclin E and CDK2. Further study showed that GEI treatment significantly elevated ROS formation, followed by attenuation of MMP via up-regulation of Bax and down-regulation of Bcl-2, accompanied by cytochrome c release to the cytosol. In addition, GEI treatment resulted in a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. Conclusion: The present study demonstrates that the ability of GEI to induce apoptosis against HepG2 cells mediated by mitochondrial-mediated pathway. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytotoxicity Gypsophila elegans Isoorientin Mitochondrial-mediated pathway Reactive oxygen species
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with very high morbidity and mortality rates, and a poor prognosis. The high incidence of this kind of liver cancer has been caused by factors such as persistent infection with hepatitis B virus and contact with hepatocarcinogens such as nitrosamines, aflatoxins and alcohol (Henry et al., 2002). Despite extensive efforts by many investigators, systemic chemotherapy
Abbreviations: GEI, G. elegans isoorientin; ROS, reactive oxygen species; LDH, lactate dehydrogenase; FBS, fetal bovine serum; DCFH-DA, 2′,7′-dihydrofluoresceindiacetate; MMP, Mitochondria membrane potential; TBS, Tris-buffer saline n Correspondence to: Department of the Pharmacy, the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University, 89-9 Dongge Road, Nanning, Guangxi 530023, China. E-mail address: [email protected] (Q. Huang). http://dx.doi.org/10.1016/j.jep.2016.04.050 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
for HCC has been quite ineffective, as demonstrated by low response rates and no survival benefits (França et al., 2004; Rougier et al., 2007). Apoptosis, or programmed cell death, is thought to play a key role in the development and regulation of growth of both normal and cancerous cells. The hallmark of cancer cells is the dysregulation of cell proliferation and apoptosis. The tumor growth depends on the cell proliferation rate and apoptosis. Therefore, induction of apoptosis of tumor cells has become a strategy in cancer treatment (Youn et al., 2008; Tan et al., 2009). Generally, apoptosis may occur via the mitochondrial (intrinsic) pathway or the death receptor (extrinsic) pathway (Li et al., 1997; Nagata, 1997). In the mitochondrial (intrinsic) pathway, the regulation of apoptosis involves a large set of proteins including Bcl-2, an anti-apoptotic protein (Luna-More et al., 2004), which disrupts the mitochondria membrane potential, resulting in release of apoptogenic factors from the mitochondria to the cytoplasm.
188
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Natural products, a rich source of compounds with enormous structural diversity, have been extensively explored in the field of drug discovery. A large number of different sources of natural compounds present anti-cancer effects both in vitro and in vivo (Huang et al., 2012a; Lu et al., 2012, 2013). Therefore, it is vital to screen efficient natural compounds for hepatocellular carcinoma treatment. Caryophyllaceae Gypsophila elegans has been widely used in traditional Chinese medicine with a record of safety and efficacy in the treatment of immune disorders and liver diseases (Cao, 2007). In our study, a flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2″-O-α-L-arabinopyranosyl. Our results revealed that GEI significantly attenuated liver injury and fibrosis induced by chronic alcohol. The preliminary exploration of the underlying mechanisms indicated its protection against hepatic injury by radical scavenging action, antioxidant activity, as well as its ability to attenuate HSCs activation (Huang et al., 2012b). To further understand the anti-tumor effects of GEI, the present study was designed to investigate the effects of GEI on proliferation and apoptosis of human hepatocarcinoma cells (HepG2 cell). We found that GEI induces apoptosis in HepG2 cells, and the mitochondrial-mediated pathway may contribute to GEI-induced apoptosis.
2. Materials and methods 2.1. Materials G. elegans herb was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd (Nanning, China). Fetal bovine serum was purchased from Life Sciences (Grand Island, NY). MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, U.S.A). The antibodies including cytochrome c, Bcl-2, Bax and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences Pharmigen (San Diego, CA). 2.2. Preparation of GEI GEI was isolated from G. elegans according to our previous study (Huang et al., 2012b) with some modification. In the present study, the result indicated that the yield of GEI was higher than that of our previous study. Briefly, the dried powder of G. elegans herb (10 kg) was extracted with 80 L 70% ethanol under reflux for 2 h two times. The extract was negative-pressure filtered, and the filtrate was evaporated to a fluid extract by removing the ethanol under reduced pressure in a rotary evaporator at 60 °C. The crude extract was successively extracted with ethyl acetate and watersaturated n-butanol, and the later extract was then subjected to chromatography on a silicagel column (200–300 mesh, Yantai, PR China; Ø10 cm 300 cm) eluting with a gradient mixture of CHCl3 and MeOH (0–100% MeOH, 1500 mL each fraction). The CH3Cl3–MeOH (35: 65) fraction yielded a yellow powder after concentration, which was purified by Sephadex LH-20 and preparative HPLC to produce compound (27.82 g). Its structure was elucidated on the basis of physicochemical properties and spectral data: ESI-MS(m/z): 604 [MþNa] þ ; 1H NMR (500 MHz, CD3OD) δ: 7.32, 7.29, 6.88, 6.49, 6.40, 4.98, 4.37; 13C NMR(125 MHz, CD3OD) δ: 165.9, 104.7, 185.0, 159.8, 110.3, 166.1, 95.9, 163.7, 105.9, 123.0, 114.8, 148.0, 152.1, 117.9, 121.2, 73.9, 82.2, 74.8, 73.1, 83.1, 63.9, 110.2, 71.9, 81.1, 68.3, 66.8. These results indicated that the compound is isoorientin-2″- O-α-L- arabinopyranosyl, i.e., G. elegans isoorientin (GEI), with a chemical formula of C26O15H28 and a molecular weight of 580.24. Its chemical structure is shown in
OH OH HO HO HO
HO HO O
HO
O
O O
OH O
OH Fig. 1. The chemical structure of GEI.
Fig. 1. 2.3. Cell line and cytotoxicity assay Human liver carcinoma cell line (HepG2 cells) was obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China) and maintained in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in a humidified incubator with 5% CO2 and 95% oxygen at 37 °C. Cytotoxicity was measured by MTT method. Briefly, exponentially growing cells were seeded in 96-well plates and cultured until 50% confluence. Then, various concentrations of GEI were added and the final concentration in each well was 5, 10, 20, 40, 80 and 160 μmol/L, respectively. Each treatment was tested in tetrad wells and the untreated control group was administered culture medium containing no drug. All of the above plates were placed in a CO2 humidified-atmosphere incubator at 37 °C for 24, 48 and 72 h. At the end of exposure, 20 μL MTT (5 mg/mL) was added to each well and the plates were incubated at 37 °C for 4 h. Then, all culture medium supernatant was removed from the wells and replaced with 200 μL DMSO. The absorbance of each well was measured by standard enzyme-linked immunosorbant assay at 570 nm. The cell viability was calculated based on the following formula: cell viability (%) ¼average A570 nm of treated group/ average A570 nm of untreated control group 100% (Xie et al., 2012). 2.4. Lactate dehydrogenase (LDH) release assay LDH released into the culture supernatants was measured using Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific™, Pittsburgh, PA, USA) following the manufacturer's instructions. In brief, cells were seeded in 96-well plates and then treated with GEI at different concentrations (5, 10 or 20 μmol/L) for 48 h. The supernatant was transferred into 96-well plate to assess the LDH activity. Triton X-100 (2%) served as a positive control was used to completely lyse the cells and release the maximum LDH. Next, the LDH reaction solution (100 μL) was added to the cells for 30 min The optical density of the color generated was determined at a wave length of 490 nm using a Microplate Reader (Tecan, Männedorf, Switzerland). The results were expressed as percentage of Triton X-100-induced LDH release. 2.5. Clonogenicity assay The clonogenicity test was performed according to the previous study (Franken et al., 2006). HepG2 cells were seeded at a density of 1 106 cells in 6-well plates. After 24 h of incubation, the cells were treated with 5, 10 or 20 μmol/L of GEI for 144 h. The cells were fixed and stained with 0.1% crystal violet to visualize colonies.
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
2.6. Migration assay Cell migration was assessed using a wound healing assay as previously reported (Wang et al., 2014). HepG2 cells were seeded in 6-well plates (5 105 cells per well) for 24 h to form a monolayer. Then, the medium was replaced with fresh incubated medium without fetal bovine serum and subjected to scratch assay and photographed with microscope (Olympus Optical, Tokyo, Japan). After the treatment of GEI (20 μmol/L) for 24 h and 48 h, the microscopic imaging was taken respectively. Percentage of the closed area was measured and compared with the value obtained before treatment. 2.7. Annexin V-FITC/PI assay Apoptotic cells were quantified by flow cytometry using the Phosphatidyl Serine Detection kit (Becton Dickinson). Briefly, HepG2 cells were incubated with GEI (5, 10 or 20 μmol/L) for 24 h. Then the cells were washed twice with cold PBS and resuspended in binding buffer at a concentration of 1 106 cells/mL. Annexin V-FITC (10 μL) and PI (10 μL) were added to 100 μL of cells and then incubated for 20 min at room temperature in the dark. At the end of incubation, 400 μL of binding buffer was added, and the cells were analyzed immediately by FACScan flow cytometer (Becton Dickinson) using CellQuest software.
189
harvested, washed twice with PBS, re-suspended and stained with fresh incubated medium containing 1.0 μM Rhodamine 123 at 37 °C for 30 min The fluorescence intensity of the cells was analyzed by flow cytometer (Becton Dickinson, USA). 2.11. Caspase-3, -8 and -9 activities assay Caspases activities were measured using Caspase Activity Assay Kit (BioVision Research Products, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, 5 106 HepG2 cells were treated with 5, 10 or 20 μmol/L GEI for 24 h. Then cells were washed with cold PBS twice, resuspended in lysis buffer and left on ice for 20 min The lysate was centrifuged at 16,000 g at 4 °C for 20 min Supernatants were collected and protein concentrations were measured with a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). Caspase-3, -8 and -9 activities were measured by reaction buffer and caspase substrate peptides Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA, respectively. The release of p-nitroanilide (pNA) was qualified by determining the absorbance with a microplate reader (BioTek, Winooski, VT, USA) at 405 nm. The fold increase in absorbance was calculated by the formula [ODi/OD0], where OD0 is the absorbance without GEI treatment (untreated control) and ODi is the absorbance with GEI treatment at the indicated concentration. 2.12. Western blot analysis
2.8. Cell cycle analysis Cell cycle was analyzed by flow cytometry (FAC sort, Becton Dickinson). Briefly, HepG2 cells (1 106 cells) were seeded into a 100 mm2 dish. After 24 h of cultivation, the cells were treated with GEI (5, 10 or 20 μmol/L) for 48 h. Cells were harvested and fixed with 70% ice-cold ethanol. Cells were washed and the pellet was suspended in trypsin buffer and left for 10 min at room temperature. RNAase buffer (1%) was added after addition of trypsin inhibitor and incubated for 10 min, followed by the addition of 100 μg/mL propidium iodide (PI). Samples were incubated in the dark for 30 min at 4 °C. The fluorescence was measured using a FACScan flow cytometer. Data collection and analysis of the cell cycle distribution were performed using CellQuest and the Modfit software (Becton Dickinson). 2.9. Reactive oxygen species (ROS) assay The production of intracellular ROS was detected using the specific fluorogenic probe 2′,7′-dihydrofluorescein-diacetate (DCFH-DA). This probe diffuses across the cell membrane and is hydrolysed to DCFH, which is rapidly oxidised to the highly fluorescent compound 2′,7′-dichlorofluorescein (DCF) in the presence of ROS (Hempel et al., 1999). The DCF fluorescence intensity is proportional to the ROS level. In this study, intracellular ROS generation was detected according to the manufacturer's instructions of ROS detection kit (Beyotime, Jiangsu, China). Briefly, HepG2 cells were seeded in 6-well plates for 12 h and then incubated with fresh medium containing10 μM DCFH-DA at 37 °C in the dark for 20 min Subsequently, cells were incubated with 5, 10 or 20 μmol/L GEI for 30 min Then cells were washed with PBS buffer and the DCF fluorescence intensity was measured by the fluorescence microscope.
HepG2 cells were cultured in 75 mm2 dish (5 105 cells) for 12 h and then exposed to GEI (5, 10 or 20 μmol/L) for 24 h. Then cells were collected, washed twice with PBS and centrifuged. Subsequently, each sample was added into 50 μL RIPA Lysis buffer (containing 1% Phenylmethanesulfonyl fluoride) and incubated on ice for 30 min Then, samples were clarified by centrifugation at 12,000 g for 10 min at 4 °C, and protein concentrations were determined using BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were separated on 15% SDS-polyacrylamide gels and electrotransferred to Immobilon-P membranes (Millipore). After blocking with Tris-buffer saline (TBS) containing 0.05% Tween 20% and 5% nonfat powdered milk, the membranes were incubated with specific primary antibodies against cytochrome c, Bcl-2, Bax and β-actin (Santa Cruz, USA) at 4 °C for 12 h. After three washes with TBS for 10 min each, the membranes were incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The membranes were washed and detection was carried out with an ECL Western blotting kit according to the manufacturer's instructions. 2.13. Real-time quantitative PCR assay HepG2 cells were treated with 5, 10 or 20 μmol/L GEI for 72 h. Total RNAwas extracted from cells with TRIzol reagent according the manufacturer's instructions. The concentration of total RNA in the final eluates was determined by spectrophotometry. Total RNA was heated at 65 °C for 10 min and then chilled on ice. Each sample was reverse-transcribed to cDNA at 37 °C for 90 min using a cDNA synthesis kit (Amersham Pharmacia Biotech). PCR was performed with specific primers which are listed in Table 1. PCR products were electrophoresed on 2% agarose gels and visualized by staining with ethidium bromide. In this study, β-actin was used as an internal control.
2.10. Mitochondria membrane potential (MMP) disruption assay The MMP (Δψm) was monitored by Rhodamine123 staining as previous reported (Ma et al., 2014). Briefly, HepG2 cells were seeded in 6-well plate (1 105 cells per well) for 12 h and incubated with GEI (5, 10 or 20 μmol/L) for 24 h. Then cells were
2.14. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post hoc
190
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Table 1 Primer sequence used in this study. Genes
Forward primer (5′-3′)
Reverse primer (5′-3′)
Cyclin D Cyclin E CDK2 Bcl-2 Bax β-actin
CTG GCC ATG AAC TAC CTG GA TTC TCG GCT CGC TCC AGG AA AGA TTC TTC TGG GCT GCA AG TTC TTT GAG TTC GGT GGG GTC GAC ATG TTT TCT GAC GGC AA CAA GAG ATG GCC ACG GCT GCT
CCA GGA AAT CAT GTG CAA TC TGG AGG ATA GAT TTC CTC AGA TCC GGA AGA GTT GGT CA TGC ATA TTT GTT TGG GGC AGG CCC AAA GTA GGA GAG GAG GC TCC TTC TGC ATC CTG TCG GCA
multiple comparisons. The data are presented as the means 7SD. A p-value o0.05 was considered to be statistically significant.
3. Results 3.1. Effects of GEI on the viability of HepG2 cells HepG2 cells were cultured with the various concentrations of GEI for 24, 48, and 72 h, and cytotoxicity were then determined using MTT assay. As shown in Fig. 2A, GEI inhibited the growth of HepG2 cells in a distinct dose- and time-dependent manner. The 50% inhibitory concentrations (IC50) were 70.06 79.13, 47.107 11.36 and 15.95 7 7.46 μmol/L after incubation for 24, 48 and 72 h, respectively. 3.2. Effect of GEI on LDH release A stable cytosolic enzyme of lactate dehydrogenase (LDH) catalyzes the oxidation of L-lactate to pyruvate. Upon membrane damage in cells, LDH enzyme is released into the culture medium, suggesting the loss of membrane integrity. Therefore, to further confirm the cytotoxic effect of GEI on HepG2 cells, LDH assay was also performed as another indicator of cytotoxicity. In the present study, LDH release level was measured by detecting changes in optical densities. As shown in Fig. 2B, treatment with GEI could significantly increase LDH activity in HepG2 cells in a dose-dependent manner. 3.3. Effects of GEI on the clonogenicity and migration of HepG2 cells Clonogenicity was determined by plating a fixed number of HepG2 cells onto multiple well tissue culture dishes. Cells were treated with GEI and maintained in culture for 144 h to allow formation of colonies. The size and number of colonies were
visually inspected by fixing and staining with 0.1% crystal violet. As shown in Fig. 3A, GEI treatment inhibited cell clonogenicity in human liver cancer HepG2 cells in a concentration-dependent manner. Wound-scratch assays were performed to investigate the effect of GEI on the migration of HepG2 cells. As shown in Fig. 3B and C, the cells of the untreated control group moved fast into the scraped area over time, while GEI treatment significantly inhibited the cells migration in a time-dependent manner. 3.4. Effect of GEI on cells apoptosis and cycle in HepG2 cells To explore the underlying basis for the cytotoxic effect of GEI in liver cancer cells, the level of cell apoptosis induced by GEI were analyzed in HepG2 cells. Cells were treated with GEI for 24 h, stained with annexin V-FITC and PI, and subsequently analyzed by flow cytometry. The results revealed that GEI significantly induced apoptosis of HepG2 cells in a dose-dependent manner after 24 h treatment when compared with the untreated control (Fig. 4A). In the cell cycle analysis, we observed increased accumulation of cells in G1 phase and decreased fractions of cells in G2 phase (Fig. 4B), suggesting that GEI induced the apoptotic death of HepG2 cells by cell cycle arrest at G1 phase. 3.5. Effect of GEI on cell cycle-related genes expression Previous studies have reported that cell cycle is controlled by cyclin-dependent kinases (CDKs), cyclin kinase inhibitors, and cyclins. In HepG2 cells, treatment with GEI resulted in a remarkable decrease in the transcription of genes, such as cyclin D gene, cyclin E gene and CDK2 gene, as assessed by RT-PCR analysis (Fig. 5A). 3.6. Effects of GEI on apoptosis-related genes and proteins In human, the Bcl-2 family has been identified and classified as anti-apoptotic protein (such as Bcl-2) and pro-apoptotic protein (such as Bax). The balance of the expressed levels of pro-apoptotic and anti-apoptotic proteins is critical for cell survival or death. In this study, the anti-apoptotic gene Bcl-2 was decreased by GEI in a dose-dependent manner, while the Bax, a pro-apoptotic gene, was significantly increased in GEI-treated HepG2 cells (Fig. 5B). Similarly, Western blot analysis showed a significant decrease in Bcl-2 protein expression, whereas an increase in Bax protein level in the GEI-treated cells (Fig. 5C).
Fig. 2. Effects of GEI on cell viability and LDH release in HepG2 cells. (A) MTT assay showed that GEI inhibited the viability of HepG2 cells in a dose- and time-dependent manner. (B) GEI significantly increased LDH activity in a dose-dependent manner. All analyses were performed in triplicate. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
191
Fig. 3. GEI inhibited the clonogenicity and migratory potential of HepG2 cells. (A) Clonogenecity assay revealed that GEI suppressed colony formation of HepG2 cells. (B) GEI treatment resulted in a significant decrease in migration ability in a time-dependent manner. (C) The representative bar charts of GEI mediated migration of HepG2 cells. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
3.7. Effect of GEI on mitochondria membrane potential (MMP) In recent years, mitochondrial pathway has been identified as a major mechanism for induction of apoptosis. Thus, the effect of GEI on integrity of mitochondrial function was analyzed using a stain of Rhodamine123 by flow cytometry. As shown in Fig. 6A and B, compared with the untreated control, GEI dramatically decreased the mitochondria membrane potential (MMP) of HepG2 cells in a dose-dependent manner. 3.8. Effect of GEI on reactive oxygen species (ROS) formation ROS generation can induce oxidative damage to vital cellular molecules and structures including lipids, proteins and DNA, activate the intrinsic apoptotic pathway due to mitochondrial dysfunction and finally lead to apoptosis. In this study, we investigated whether ROS generation was involved in GEI-induced apoptosis. As shown in Fig. 6C, compared with the untreated control, treatment with GEI resulted in a significant increase in ROS generation in HepG2 cells. 3.9. Effect of GEI on cytochrome c level In normal situation, cytochrome c resides in the mitochondrial intermembrane and serves as a transducer of electrons in the respiratory chain. However, the increment of ROS and subsequent mitochondrial dysfunction cause release of cytochrome c from mitochondria to cytosol. In this study, the cytosolic level of cytochrome c was measured to confirm apoptosis via the mitochondrial pathway in GEI-treated HepG2 cells. The result indicated that GEI treatment could result in a significant increase in the cytochrome c protein level when compared to the untreated control (Fig. 6D). 3.10. Effects of GEI on caspase-3, -8 and -9 activities The activation of cysteine proteases is an important feature of apoptotic cell death. We determined GEI-induced activation of the protease activities of caspase-3, caspase-8 and caspase-9. As shown in Fig. 7, GEI treatment caused a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. However, HepG2 cells treated with GEI displayed a slight increase in caspase-8 activity. 4. Discussion Apoptosis plays a crucial role in the cellular progress of
proliferation, differentiation, senescence and death. Nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptotic cells (Gao et al., 2011). Previous studies have confirmed that the occurrence and development of tumors is closely related to cell apoptosis (Krysko et al., 2008). The potential to induce apoptosis has therefore become an important topic in the study of anti-tumor drugs. In the present study, GEI strongly suppressed the viability and proliferation of HepG2 cells in a time- and dose-dependent manner. The cytotoxic effect of GEI was further confirmed by the significant increase in LDH activity of cell culture supernatants induced by GEI treatment. Flow cytometric analysis showed that GEI significantly induced apoptosis of HepG2 cells. Further study showed that the anti-proliferative effect of GEI could be attributed to cell cycle arrest that was induced by the ability of GEI to halt the cell cycle at G1 phase via the down-regulation of the cell cyclerelated genes transcription, such as cyclin D, cyclin E and CDK2. In addition, GEI treatment significantly inhibited the clonogenicity and migration of HepG2 cells. Based on the results obtained, we speculated that GEI may play a potential role in the treatment of hepatocellular carcinoma. Oxidative stress is considered to be important for the promotion of apoptosis in response to a variety of apoptotic stimuli. ROS play essential role in the oxidative stress response. Increasing evidences indicate that over-production of ROS can induce oxidative damage to vital cellular molecules and structures including lipids, proteins and DNA, activate the intrinsic apoptotic pathway due to mitochondrial dysfunction and finally lead to apoptosis (Circu and Aw, 2010). In the present study, our finding showed that GEI significantly increased the production of intracellular ROS in HepG2 cells, suggesting that excessive ROS generation is likely involved in GEI-induced apoptotic cell death in HepG2 cells. It is well known that apoptosis occurs via two primary pathways: the extrinsic pathway, which is associated with cell death receptors and their ligands on the cellular surface, and the intrinsic pathway, which is dependent on mitochondria. The latter has been considered to be the predominant apoptosis-inducing pathway (Fulda and Debatin, 2006; Wang et al., 2013a). Previous studies have demonstrated that mitochondrial dysfunction causes the collapse of mitochondria membrane potential (MMP), which results in mitochondrial permeability transition pore (MPTP) opening, enabling to the release of cytochrome c from mitochondria into cytosol (Chan et al., 2013; Wu et al., 2014). The mitochondria-dependent apoptotic pathway is regulated by Bcl-2 family of proteins, including Bax and Bcl-2 (Kong et al., 2013). Bax, which is a pro-apoptotic member of the Bcl-2 family, promotes the opening of the MPTP, causing the release of cytochrome c, whereas
192
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Fig. 4. Effects of GEI on the cells apoptosis and cycle in HepG2 cells. (A) Flow cytometric evaluation of annexin-V-FITC/PI-stained HepG2 cells showing the effect of different concentrations of GEI (5, 10 or 20 μmol/L) on the percentage of apoptotic cells 24 h after treatment. The data indicated that GEI significantly induced apoptosis of HepG2 cells in a dose-dependent manner. (B) The effect of GEI on cell cycle was assessed by flow cytometry. The result revealed that GEI treated HepG2 cells showed arrest in the G1 phase of the cell cycle. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
Bcl-2, which is an anti-apoptotic member of the Bcl-2 family, inhibits the formation of the MPTP, blocking cytochrome c release (Wang et al., 2013b). An increase in the Bax/Bcl-2 ratio has been demonstrated to promote apoptosis by directly activating the mitochondrial apoptotic pathway (Brunelle and Letai, 2009). In the present study, GEI treatment caused a loss of MMP in HepG2 cells and an obvious translocation of cytochrome c from mitochondria to cytosol. Furthermore, GEI treatment resulted in a significant decrease in Bcl-2 expression and a remarkable increase in Bax
level. These data indicate that GEI-induced apoptosis of HepG2 cells is mediated by the mitochondria-dependent pathway. A molecular hallmark of apoptosis is the activation of caspases, which has been demonstrated to play a central role during cellular apoptosis (Sun et al., 1999). Caspase-3, particularly, has a key role in executing apoptosis as it catalyzes specific cleavage of several important cellular proteins (Brentnall et al., 2013). Increase in caspase-3 activity is crucial for drug-induced apoptosis as the activation of caspase-3 is a hallmark of apoptosis and can be used in
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
193
Fig. 5. Effects of GEI on the cell cycle-related genes, and the apoptosis-related genes and proteins. (A) RT-PCR analysis showed that GEI repressed the transcription of cell cycle regulatory genes including cyclin D, cyclin E and CDK2. (B) GEI decreased the mRNA levels of anti-apoptosis gene Bcl-2, while enhanced the expression of pro-apoptotic gene Bax. (C) Western blot assay revealed that GEI treatment resulted in a decrease in the Bcl-2 protein expression and an increase in the Bax protein level. Lane-1, untreated control group; lane-2, 5 μmol/L GEI-treated group; lane-3, 10 μmol/L GEI-treated group; lane-4, 20 μmol/L GEI-treated group. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
quantifying the caspase cascade (Logue and Martin, 2008). Caspase-3 is triggered by either caspase-8 or caspase-9, which acts as a convergence point for different apoptosis signaling pathways. In the present study, the levels of caspase-3 and -9 were significantly
increased in GEI-treated HepG2 cells, confirming the involvement of mitochondria-dependent intrinsic pathway in the GEI-induced apoptotic events in HepG2 cells, whereas procaspase-8 did not change in HepG2 cells in response to GEI treatment, indicating no
Fig. 6. Effects of GEI on mitochondrial membrane potential (Δψm), ROS generation and cytochrome c. (A-B) GEI induced an obvious decrease of mitochondrial membrane potential (Δψm) in a dose-dependent manner. (C) GEI increased ROS generation in HepG2 cells. (D) GEI treatment resulted in an increase in the cytochrome c protein expression in cytosol. Lane-1, untreated control group; lane-2, 5 μmol/L GEI-treated group; lane-3, 10 μmol/L GEI-treated group; lane-4, 20 μmol/L GEI-treated group. Data were expressed as means 7 SD. *Po 0.05 vs. untreated control.
194
X. Lin et al. / Journal of Ethnopharmacology 187 (2016) 187–194
Fig. 7. Caspases activities in GEI-treated HepG2 cells. Cells were treated with 5, 10 or 20 μmol/L GEI for 24 h. Caspase activities in HepG2 were determined using the caspase-activity detection kits. Data were expressed as fold increase, and as means 7 SD. *Po 0.05 vs. untreated control.
death receptor-mediated extrinsic caspase-8 pathway in GEI-induced HepG2 cells apoptosis. In summary, the anti-cancer potential of GEI was supported by the evidence provided in the present study, including lactate dehydrogenase leakage, reactive oxygen species generation, loss in mitochondrial membrane potential, increase in the level of cytochrome c, up-regulation of Bax, down-regulation of Bcl-2 and activation of initiator and executioner caspases. Our findings may provide a new insight for the potential use of GEI as chemotherapeutic and chemopreventive agent for the treatment of hepatocellular carcinoma.
Disclosure statement The authors declare that there are no conflicts of interest.
Acknowledgment The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (No. 81260674) and the Guangxi Natural Science Foundation (2014GXNSFAA118155; 2014GXNSFAA118154; 2012GXNSFCA053005).
References Brentnall, M., Rodriguez-Menocal, L., De Guevara, R.L., Cepero, E., Boise, L.H., 2013. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 14, 32. Brunelle, J.K., Letai, A., 2009. Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell Sci. 122, 437–441. Cao, G., 2007. The clinical application of Dong-Yao (Chinese Minorities medicine herb) Gypsophila elegans. J. Med. Pharm. Chin. Minor. 15, 24–25. Chan, S.H., Leu, W.J., Hsu, L.C., Chang, H.S., Hwang, T.L., Chen, I.S., Chen, C.S., Guh, J. H., 2013. Reevesioside F induces potent and efficient anti-proliferative and apoptotic activities through Na þ /K þ -ATPase alpha3 subunit-involved mitochondrial stress and amplification of caspase cascades. Biochem. Pharmacol. 86, 1564–1575. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and
apoptosis. Free Radic. Biol. Med. 48, 749–762. França, A.V.C., Elias Júnior, J., Lima, B.L.G., Martinelli, A.L.C., Carrilho, F.J., 2004. Diagnosis, staging and treatment of hepatocellular carcinoma. Braz. J. Med. Biol. Res. 37, 1689–1705. Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J., Van Bree, C., 2006. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319. Fulda, S., Debatin, K.M., 2006. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811. Gao, Y., Su, Y., Qu, L., Xu, S., Meng, L., Cai, S.Q., Shou, C., 2011. Mitochondrial apoptosis contributes to the anti-cancer effect of Smilax glabra Roxb. Toxicol. Lett. 207, 112–120. Hempel, S.L., Buettner, G.R., O’Malley, Y.Q., Wessels, D.A., Flaherty, D.M., 1999. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146–159. Henry, S.H., Bosch, F.X., Bowers, J., 2002. Aflatoxin, Hepatitis and Worldwide Liver Cancer Risks, Mycotoxins and Food Safety. Springer, pp. 229–233. Huang, M., Lu, J., Huang, M., Bao, J., Chen, X., Wang, Y., 2012a. Terpenoids: natural products for cancer therapy. Expert Opin. Investig. Drugs 21, 1801–1818. Huang, Q., Zhang, S., Zheng, L., Liao, M., He, M., Huang, R., Zhuo, L., Lin, X., 2012b. Protective effect of isoorientin-2″-O-α-L-arabinopyranosyl isolated from Gypsophila elegans on alcohol induced hepatic fibrosis in rats. Food Chem. Toxicol. 50, 1992–2001. Kong, D., Zheng, T., Zhang, M., Wang, D., Du, S., Li, X., Fang, J., Cao, X., 2013. Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways. PLoS One 8, e69403. Krysko, D.V., Vanden Berghe, T., D’Herde, K., Vandenabeele, P., 2008. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44, 205–221. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., Wang, X., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489. Logue, S.E., Martin, S.J., 2008. Caspase activation cascades in apoptosis. Biochem. Soc. Trans. 36, 1–9. Lu, J., Bao, J., Chen, X., Huang, M., Wang, Y., 2012. Alkaloids isolated from natural herbs as the anticancer agents. Evid. Based Complement. Altern. Med. 2012, 1–12. Lu, J., Bao, J., Wu, G., Xu, W., Huang, M., Chen, X., Wang, Y., 2013. Quinones derived from plant secondary metabolites as anti-cancer agents. Anticancer Agents Med. Chem. 13, 456–463. Luna-More, S., Weil, B., Bautista, D., Garrido, E., Florez, P., Martinez, C., 2004. Bcl-2 protein in normal, hyperplastic and neoplastic breast tissues. A metabolite of the putative stem-cell subpopulation of the mammary gland. Histol. Histopathol. 19, 457–463. Ma, W., Zou, Y., Wang, P., Yao, X., Sun, Y., Duan, M., Fu, Y., Yu, B., 2014. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. 70, 1–8. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365. Rougier, P., Mitry, E., Barbare, J.-C., Taieb, J., 2007. Hepatocellular carcinoma (HCC): an update. Semin. Oncol., S12–S20. Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R., Cohen, G.M., 1999. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053–5060. Tan, X., Xia, H., Xu, J., Cao, J., 2009. Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin. World J. Gastroenterol. 15, 2234–2239. Wang, J., Yang, Z., Wen, J., Ma, F., Wang, F., Yu, K., Tang, M., Wu, W., Dong, Y., Cheng, X., 2014. SKLB-M8 induces apoptosis through the AKT/mTOR signaling pathway in melanoma models and inhibits angiogenesis with decrease of ERK1/2 phosphorylation. J. Pharmacol. Sci. 126, 198–207. Wang, J., Yuan, L., Xiao, H., Xiao, C., Wang, Y., Liu, X., 2013a. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways. Apoptosis 18, 751–765. Wang, Y., Li, X., Wang, X., Lau, W., Wang, Y., Xing, Y., Zhang, X., Ma, X., Gao, F., 2013b. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/ GSK-3beta signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS One 8, e70956. Wu, W., Zhou, X., Liu, P., Fei, W., Li, L., Yun, H., 2014. Isoflurane reduces hypoxia/ reoxygenation-induced apoptosis and mitochondrial permeability transition in rat primary cultured cardiocytes. BMC Anesthesiol., 14–17. Xie, J., Que, W., Liu, H., Liu, M., Yang, A., Chen, M., 2012. Anti-proliferative effects of cucurmosin on human hepatoma HepG2 cells. Mol. Med. Rep. 5, 196–201. Youn, M., Kim, J., Park, S., Kim, Y., Kim, S., Lee, J., Chai, K., Kim, H., Cui, M., So, H., 2008. Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells. World J. Gastroenterol. 14, 511–517.