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Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway Qiao Zhang a,b, Can Cui a, Cong-Qin Chen a, Xiao-Long Hu a, Ya-Hui Liu a, Yan-Hua Fan a, Wei-Hong Meng b, Qing-Chun Zhao b,n a b
Department of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 November 2014 Received in revised form 9 March 2015 Accepted 13 March 2015
Ethnopharmacological relevance: Fructus Alpiniae oxyphyllae (A. oxyphylla) is a traditional herb which is widely used in East Asian for the treatment of dyspepsia, diarrhea, abdominal pain, poor memory, inflammatory conditions and cancer. Materials and methods: The cytotoxic activities of ethanol extract (EE) and five extract layers including petroleum ether (PE), dichloromethane (DCLM), acetoacetate (EtOAc), n-Butanol (n-Bu) and water fractions (WF) of A. oxyphylla were tested on HepG2, SW480, MCF-7, K562 and HUVEC cell lines using MTT assay and LDH release assay. The component analysis was performed on HPLC with gradient elution. Hoechst 33342 staining, DCFH-DA fluorescence microscopy, flow cytometry analysis, western blot and migration assays were carried out to determine the anti-cancer mechanisms of PE. Results: MTT analysis showed that EE, PE and DCLM could inhibit cell proliferation on HepG2, SW480, MCF-7, K562 and HUVEC cell lines, especially PE fraction. HPLC analysis pointed out five main components which may contribute to the anti-proliferative activity of PE. Further study showed that PE increased LDH release, induced apoptosis, disrupted mitochondrial membrane potential and elevated intracellular reactive oxygen species (ROS) in HepG2 cells, whereas the antioxidant N-acetylcysteine (NAC) prevented PE-induced ROS generation. The results of western blot revealed that PE induced apoptosis in HepG2 cells by enhancing Bax/Bcl-2 ratio, increasing cytochrome c in cytosol and ase-3/9. Meanwhile, high levels of ROS could induce DNA damage-mediated protein expression, AKT, ERK inactivation and SAPKs activation. Furthermore, PE conspicuously blocked the migration of HUVEC cells. Conclusion: The present results demonstrated that PE induced apoptosis in HepG2 cells may be via a ROS-mediated signaling pathway. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alpinia oxyplylla Cytotoxicity Apoptosis ROS HepG2 Chemical compounds studied in this article: Yakuchinone A (PubChem CID:133145) Tectochrysin (PubChem CID:5281954) Chrysin (PubChem CID:5281607)
1. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases and the second leading cause of cancer related death around the world (Ferlay et al., 2010; Jemal et al., 2011). More than 600,000 people die from HCC each year. Many molecules and signaling pathways have been found to be involved in the development of HCC, including the Bcl-2, AKT/mTOR, RAF/ MEK/ERK and EGFR pathways (Blechacz and Gores, 2008; Ho et al., 2012). Chemotherapy, referred to the treatment of cancer using
pharmacological or natural agents to impede, arrest or reverse the carcinogenic process, remains the most practical and promising approach for the management of cancer patients (Link et al., 2010; Sporn and Suh, 2002). It is increasingly accepted that natural products are used as an alternative therapy globally because it generally has high therapeutic effectiveness as well as low side effects (Newman et al., 2003; Park et al., 2012). Fructus Alpiniae oxyphyllae (A. oxyphylla) is an important herb commonly used in East Asian traditional medicine. Its fruits were extensively used as a traditional Chinese medicine for curing
Abbreviations: Aq, aqueous fraction.; BuOH, n-Butanol; BSA, bovine serum albumin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; DCLM, dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FBS, fetal bovine serum; HepG2cells, human hepatic carcinoma cells; Hex, n-hexane; HUVEC, Human Umbilical Vein Endothelial Cells; K562, human leukemia cells; MCF-7, Michigan Cancer Foundation-7 breast cancercell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acety-l-cystein; PE, petroleum ether extract.; PI, PIpropidium iodide; P/S, penicillin-streptomycin; PBS, phosphate buffer saline; ROS, reactive oxygen species n Corresponding author. Tel./fax: þ 86 24 28851126. E-mail address: [email protected] (Q.-C. Zhao). http://dx.doi.org/10.1016/j.jep.2015.03.073 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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diarrhea, intestinal disorders, diuresis and poor memory (But et al., 1997; Pharmacopoeia Committee, 2005). Modern pharmacological studies have revealed that A. oxyphylla has many effects such as anti-neoplastic, vasodilating, anti-ulcer and neuroprotective (Itokawa et al., 1979; Lee et al., 1998; An et al., 2006; But et al., 1997). Several studies have reported the anti-proliferative property of A. oxyphylla against different cancer cells. The ethanol crude extract (EE) and its fractions, petroleum ether fraction (PE) and acetoacetate fraction (EtOAc) of A. oxyphylla, exhibited potent anti-proliferative effects on six cancer cell lines (A549, HepG2, SW480, HeLa, MCF-7 and MNK-45 cells) with IC50 values ranging from 40.1 to 166.3 mg/mL (Wang et al., 2013). The ethanol extract of A. oxyphylla suppressed skin carcinogenesis in mice and induced apoptotic death in human promyelocytic leukemia (HL)-60 cells (Lee et al., 1998). The Hex (n-hexane) and EtOAc fractions of A. oxyphylla could inhibit the proliferation of HepG2 cells probably through anti-angiogenesis (He et al., 2010). Although A. oxyphylla has been reported to exert anti-tumor activity, the reports on solid and hematological malignant tumor cells of humans such as HepG2, SW480, MCF-7 and K562 cell lines are relatively limited. Moreover, the pro-apoptotic effect and the underlying molecular mechanisms of A. oxyphylla on human cancer cells remain unclear and need deeply explored. This study was aimed at investigating the cytotoxic activity of A. oxyphylla extracts on several cells and the pro-apoptotic effect of PE on HepG2 cells, also we tried to explore the active components and action mechanisms of PE.
2. Materials and methods 2.1. Reagents Antibodies for cytochrome c (A-8), Bcl-2 (C21), Bax (P-19), Caspase3/9, Cleaved-Caspase-3/9 and β-actin (C4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p53, Bad, PARP, Cleaved-PARP, anti-phospho-AKT (Thr308), anti-AKT, anti-phosphop44/42(ERK1/2) (Thr202/Tyr204), anti-SAPK/JNK, anti-phospho-SAPK/ JNK (Thr183/Tyr185), anti-p38, anti-phospho-p38 and anti-p44/ 42MAPK (ERK1/2) (Thr202/Tyr204), were purchased from Cell Signaling Technology Inc. (USA). The secondary antibodies were purchased from ZSGB-BIO (Beijing, China). The enhanced chemiluminescent (ECL) plus reagent kit, Reactive oxygen species (ROS) detection kits and Rhodamine123 (Rh123) were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). HPLC-grade methanol and acetonitrile were purchased from Merck Company Inc. (Merck, Darmstadt, Germany). 2.2. Preparation of plant extracts and fractionation The fruit of Fructus A. oxyphylla was purchased from Shenyang Medicinal Material Co. (Shenyang, China), identified by Dr. QingChun Zhao (Department of Chinese Medicine, Shenyang Pharmaceutical University) and deposited in the herbarium of Department of Pharmacy, General Hospital of Shenyang Military Area Command (Voucher specimen number: ZQAO2013). The fruit of A. oxyphylla 5 kg was extracted with 50 L of 95% ethanol three times under reflux for 1 h each. These three filtrates were combined and concentrated in a rotary evaporator at 65 1C until dried to afford the crude extract 310 g. The crude extract was suspended in 2 L water, and then partitioned exhaustively with equal volume of petroleum ether, dichloromethane, acetoacetate and n-Butanol, respectively. The obtained ethanol extract (EE) and fractions such as PE (petroleum ether), DCLM (dichloromethane), EtOAc (acetoacetate), n-Bu (n-Butanol) and WF (water fractions) were stored in dark bottles at 4 1C after solvent evaporation and
freeze-drying. On the basis of NMR analysis, the structures of isolated reference standards (9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin) were confirmed and their purities were 98.0% or greater as determined by HPLC-PDA. 2.3. Cell culture and treatments Human liver carcinoma cell line (HepG2), human colon cancer cell line (SW480), human breast cancer cell line (MCF-7), human leukemia cell line (K562) and human umbilical vein endothelial cell line (HUVEC) were obtained from Shenyang Pharmaceutical University (Shenyang, China). Cells were maintained in DMEM medium supplemented with 10% FBS in a humidified atmosphere with 5% CO2 in the air at 37 1C. Cells were seeded in 96-well plates (1 104 cells/well) and 6-well plates (1 105 cells/well) and allowed to adhere for 12 h under the same condition. Then, different concentrations of samples (50, 75, 100 mg/mL) or vehicle control (untreated medium containing vehicle 0.1% DMSO) were added and cells were treated for 24 h for the following experiments, except the ROS generation and the migration assay. 2.4. Cell viability assay The anti-proliferative effects of EE and five fraction layers on HepG2, SW480, MCF-7, K562 and HUVEC cells were examined by using MTT assay (Ma et al., 2014). Cells were seeded and dispersed evenly in medium (containing 10% FBS) in 96-well plates at a density of 1 104 cells per well and allowed to adhere for 12 h under the same condition. Then, cells were treated with different concentrations of samples for 24 h, the MTT stock solution was added into each well (final concentration: 0.5 mg/mL) and the cells were incubated at the same conditions for another 4 h. Then, after discarding the supernatant, 150 mL DMSO was added and the OD value of each well was determined at the wavelength of 490 nm using a Microplate Reader (Elx800 Bio-Tek, USA). 2.5. Lactate dehydrogenase (LDH) release assay The cell membrane integrity of HepG2 cells was evaluated by determining the activity of LDH leaking out of the cells according to the manufacturer's instructions (LDH detection kit, A020-2, Njjcbio, Nanjing). Briefly, cells were seeded in 96-well plates and afterwards exposed to different concentrations of PE for 24 h, the free supernatant was transferred into centrifuge tubes for testing. Following the sequences of the instruction, the LDH-assay reaction mixtures were transferred into wells of 96-well plates. The optical density of the color generated was determined at a wavelength of 450 nm using a Microplate Reader (Elx800 Bio-Tek, USA). 2.6. High performance liquid chromatography (HPLC) analysis The HPLC analytical method was applied for analysis of PE, EE and DCLM. The analysis was performed by high performance liquid chromatography coupled with a photodiode array detector (Waters 2996), a quaternary pump controller (Waters 600), an Auto Sampler injector (Waters 717) and LC solution software. The mobile phase was A (water) and B (acetonitrile). The elution conditions applied were: 0–30 min, linear gradient 30–40% B; 30–45 min, linear gradient 40–45% B; 45–50 min, linear gradient 45–50% B; 50–60 min, linear gradient 50–35% B; 60–70 min, linear gradient 35–30% B. The C18 column (5 mm, 250 mm 2.1 mm I.D, Agilent Technologies) was used and the column temperature was maintained at 30 1C. The flow-rate was 1.0 ml/min and 10 mL of the sample was delivered to the HPLC. The absorbance was monitored
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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with a PAD detector at wavelength of 260 nm. Five standards isolated from A. oxyphylla were dissolved in methanol. 2.7. Hoechst 33342 staining assay To examine cell apoptosis, the Hoechst 33342 staining assay was performed as previously reported (Gao et al., 2011) with minor revision. HepG2 cells were seeded into 6-well plates for 12 h and then treated with different concentrations of PE for 24 h. Subsequently, the culture medium was removed and the HepG2 cells were stained with Hoechst 33342 (final concentration 1.0 mg/mL) for 20 min, the apoptotic cell changes in morphology such as chromatin condensation and nuclear shrinking could be characterized. The fluorescence was observed under fluorescence microscope and images were captured with an electronic camera (Olympus, Tokyo; 100 magnification). 2.8. Annexin V-FITC/PI double staining assay In short, cells were seeded into 6-well plates for 12 h and exposed to different concentrations of PE for 24 h. Subsequently, according to the manufacturer's instructions of a commercially available kit (BD Pharmingen, CA, USA), the resuspended cells (1 105 cells/100 mL) were treated with PI (5 mL) and Annexin VFITC (5 mL) at room temperature for 15 min in the dark. Cells were analyzed using Becton Dickinson FACS Calibur System (NJ, USA) with emission filters of wavelength 515–545 nm for FITC (green) and 600 nm for PI (red). 2.9. ROS generation assay Intracellular ROS generation was detected by a fluorescence microscope with 20 ,70 -dihydrofluorescein-diacetate (DCFH-DA) according to the manufacturer's instructions of ROS detection kit (S0033, Beyotime, Jiangsu, China). Briefly, HepG2 cells were seeded in 6-well plates for 12 h and then incubated with fresh incubated medium containing 10 μM DCFH-DA at 37 1C in the dark for 20 min. Subsequently, cells were treated with or without 10 mM N-acetyl-L-cysteine (NAC) for 1 h and co-incubated with different concentrations of PE for 30 min. Then cells were washed with PBS buffer and the DCF fluorescence intensity was measured by the fluorescence microscope. Images were captured with an electronic camera (Olympus, Tokyo; 100 magnification).
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2.11. Western blot analysis To investigate the expression changes of the apoptosis-related protein in HepG2 cells after exposed to PE, cells were cultured in 75 mm2 dish (5 106 cells) for 12 h and then exposed to PE for 24 h. Then cells were collected, washed twice with PBS and centrifuged. Subsequently, each samples were added into 50 mL RIPA Lysis buffer (containing 1% Phenylmethanesulfonyl fluoride) and incubated on ice for 30 min. Then, samples were clarified by centrifugation at 12,000 rpm for 10 min at 4 1C, and protein concentrations were determined using Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were denatured by boiling for 5 min, equal amounts of protein (20 μg/lane) were loaded on 10% sodium dodecyl sulfate/polyacrylamide gels (Beyotime, Jiangsu, China) and blotted onto Polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris Buffered Saline Tween (TBST) for 2 h at room temperature and then incubated primary antibodies with 5% nonfat dry milk/TBST overnight at 4 1C. The membranes were washed three times with TBST for 5 min and subsequently incubated in secondary antibody with 5% nonfat dry milk/TBST for 1 h at room temperature. The membranes were washed as described above and detected using the ECL detection system. Blotted antibodies were visualized by chemiluminescence method (Beyotime, Jiangsu, China).
2.12. Migration assay Since Hex (n-hexane) fractions of A. oxyphylla, containing similar chemical components to PE, showed anti-angiogenic potentials both in vivo and in vitro models (He et al., 2010), we speculated PE may demonstrated potential anti-angiogenic effect. Then we attempted to examine the effect of PE on cell migration of HUVECs using the wound healing assay as previously reported (Wang et al., 2014). Cells (5 105 cells/well) were seeded in a 6-well plate for 12 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, Japan). After the treatment of PE (25 mg/mL) 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 (day 0). The images were analyzed using computing software (Image J 1.48q).
2.10. Mitochondrial membrane potential (MMP) disruption assay The MMP (Δψm) was monitored by Rh123 staining as previous reported (Ma et al., 2014). In short, HepG2 cells were seeded in 6-well plates for 12 h and incubated with different concentrations of PE for 24 h. Then cells were harvested, washed twice with PBS, re-suspended and stained with fresh incubated medium containing 1.0 mM Rh123 at 37 1C for 30 min. The fluorescence intensity of the cells was analyzed by flow cytometer (Becton Dickinson, NJ, USA).
2.13. Statistical analysis Experimental values were expressed as mean 7standard deviation of at least three experiments in triplicate. Data were analyzed using Sigmaplot 12.0 software and the statistical significance was assessed by one-way ANOVA using SPSS 21.2 software. The level of Po 0.05 was accepted to determine the statistical significance.
Table 1 Cytotoxic activity of Fructus Alpiniae oxyphyllae on human cancer cells. Extracts
EE PE DCLM
HepG2
100.81 7 0.72 52.59 7 1.31 154.127 2.64
Cell lines, IC50 value (mg/ml)
HUVEC
MCF-7
K562
SW480
4200 97.82 7 1.15 4200
121.377 0.97 60.25 7 2.12 106.667 1.48
4200 53.18 71.35 4200
52.87 7 1.64 49.217 0.62 57.147 1.67
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 1. PE exhibited cytotoxic activity on HepG2 cells. (A) MTT results showed PE inhibited the growth of HepG2 cells with a dose-dependent manner. (B) PE increased the LDH release of HepG2 cells with a dose-dependent manner. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
3. Result 3.1. The extraction of A. oxyphylla suppressed the proliferation of human cancer cells The MTT assay was used to evaluate the cytotoxicity of EE, PE, DCLM, EtOAc, n-Bu and WF on human cell lines. The results showed that PE caused significant decreases in HepG2, SW480, MCF-7, K562 and HUVEC cells proliferation. EE and DCLM showed weak cytotoxicity on these cells, while no significant growth inhibitory activity was detected on other extract layers under 200 mg/mL (Data were not listed). PE showed the lowest IC50 value of 52.59 71.31 mg/mL than EE (100.81 70.72%) and DCLM (154.12 72.64%) against HepG2 cells (Table 1). All concentrations of PE (50, 75, 100 mg/mL) resulted in significant decrease in cell viability of HepG2 cells by 60.2474.30%, 37.30 7 1.71% and 4.48 71.20%, respectively (Fig. 1A). 3.2. PE induced LDH release from HepG2 cells LDH release level was measured by detecting changes in optical densities. PE increased the LDH activity significantly in HepG2 cells in a dose-dependent manner. Compared with the control group (101.67 75.51%), all concentrations of PE (50, 75, 100 mg/mL) resulted in significant increase in LDH release by 167.98 717.97%, 198.48 710.43% and 226.98 71.83%, respectively (Fig. 1B). 3.3. HPLC analysis of PE, EE and DCLM Based on the absorption profile, the retention time and spiking tests of PE, there were at least 18 distinct peaks eluted from PE using the optimized mobile phase and wavelength. 9-hydroxynootkatone (1), Yakuchinone B (2), Yakuchinone A (3), Oxyphyllacinol (4) and Tectochrysin (5) were identified as major components and their structures are shown in Fig. 2. Amongst them, Tectochrysin may be the maximum flavonoids components existed in PE according to the HPLC results (Fig. 3A). Other peaks that were not identified may include chrysin, oxyphyllenone, izalpinin and neonootkato. EE and DCLM were analyzed to compare the contents of main components under the same concentrations. The results showed that the contents of 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin in PE were significantly higher than EE (Fig. 3A and B), while these components were almost undetected in DCLM (Fig. 3C). 3.4. PE induced apoptosis of HepG2 cells As shown in Fig. 4A, cells of the control group had normal nuclear morphology under fluorescent microscope after Hoechst
Fig. 2. Structures of five main components of PE. 9-hydroxynootkatone (1), Yakuchinone B (2), Yakuchinone A (3), Oxyphyllacinol (4) and Tectochrysin (5).
33342 staining, indicating that the chromatin was equivalently distributed in the nucleus. The test group marked with nuclear fragmentation, condensation of chromatin and the morphological characteristics of apoptosis, which include emitting brighter fluorescence, were detected after treatment with different concentrations of PE for 24 h. These results indicated that PE is capable of inducing apoptosis in HepG2 cells in a dose-dependent manner. In order to further evaluate PE-induced apoptosis in HepG2 cells, cells were treated with the stain of AnnexinV-FITC/PI and detected by flow cytometry. The results revealed that PE dramatically triggered apoptotic response in HepG2 cells in a dosedependent manner after 24 h treatment compared with the control group. The percentage of early apoptotic cells (right low quadrant) increased from 2.53 71.30% to 11.85 73.64%, and the percentage of late apoptotic and necrotic cells (right upper quadrant) was also increased from 3.73 70.73% to 18.39 73.87% (Fig. 4B and C). Taken together, these results confirmed the proapoptotic effect of PE. 3.5. PE increased reactive oxygen species (ROS) formation Since ROS generation plays an important role in the proapoptotic activities, we investigated whether ROS generation was involved in PE-induced apoptosis. As shown in Fig. 5A, HepG2 cells were pre-incubated with or without NAC for 1 h and treated with different concentrations of PE for 30 min. Here, we must note that the treatment time of PE was chosen as 30 min, as we detected the largest amounts of ROS generation at that time (Data were not listed). Compared with the control group (100.01 7 0.25%), all concentrations of PE (50, 75, 100 mg/mL) significantly increased the intracellular ROS level by 216.61 70.20%, 363.23 70.32% and 507.97 70.08%, respectively (Fig. 5B).
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 3. LC chromatograms of A. oxyphylla extraction and fractions: (A) PE; (B) EE; (C) DCLM.
Compared with the cells treated only with 100 mg/mL PE (507.9770.08%), ROS generation decreased to 195.7670.13% when treated with 10 mM NAC followed by 100 mg/mL PE (Fig. 5C). While cells only treated with 10 mM NAC (92.0270.03%) showed no significant difference with cells of the control group. These results suggested that PE likely induced oxidative injury in HepG2 cell lines, whereas NAC could prevent PE-induced ROS generation. 3.6. PE decreased the mitochondrial membrane potential (MMP) of HepG2 cells We analyzed the integrity of mitochondrial function after treatment with or without PE for 24 h with the stain of Rh123 by flow cytometry. Compared with the control group (95.7671.24%), all concentrations of PE (50, 75, 100 mg/mL) resulted in a significant decrease of fluorescence intensity by 81.5373.08%, 73.1774.35% and 52.0771.56% (Fig. 6A and B). These results revealed that PE dramatically disrupted the MMP in HepG2 cells in a dose-dependent manner after 24 h treatment. 3.7. PE induced apoptosis through mitochondrial pathway Western blot analysis revealed that all concentrations of PE (50, 75, 100 mg/mL) resulted in a significant increase of p53 and cytochrome c (cytosolic) expression. The expression levels of caspase-3/9, PARP and Bad were decreased after 24 h treatment,
and remarkable cleavage of caspases-3/9 and PARP were detected compared with the control group (Fig. 7A). Mitochondrial dysfunction is regulated by Bcl-2 family proteins, thus the Bcl-2 family proteins were examined in the study. PE caused a significant reduction in Bcl-2 expression whereas the expression of Bax was significantly increased (Fig. 7B). Treatment with PE led to changes in Bax/Bcl-2 expression in a concentrationdependent manner (Fig. 7C). Thus, PE could induce apoptosis in HepG2 cells with mitochondrial pathway involved. 3.8. PE induced apoptosis via AKT, ERK inactivation and SAPKs activation Large amounts of AKT phosphorylation were observed in the control cells while PE (50, 75 and 100 mg/mL) significantly inhibited their phosphorylation in a dose-dependent manner (Fig. 8A). Moreover, PE significantly induced the phosphorylation of p38 and JNK in a dose-dependent manner, whereas the phosphorylation of ERK was significantly decreased (Fig. 8B). These results suggested that PE up-regulated p-p38, p-JNK and down-regulated p-ERK protein activities, which may acted as pro-apoptotic inducers in HepG2 cells. 3.9. PE inhibited the migration of HUVEC cells Wound-scratch assays were performed to compare the migratory potential of HUVEC cells induced by PE. After the treatment
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 4. PE induced apoptotic response in HepG2 cells. (A) Morphological changes of HepG2 cells after PE treatment and Hoechst staining. Irregular nuclei, crescent-shaped nuclei, chromatin margination and condensation, and apoptotic bodies were shown by arrows. (B) PE increased early and late apoptosis of HepG2 cells in a dose-dependent manner. (C) Column chart shows mean values of three experiments. All analyses were performed in triplicate. Data were expressed as mean 7SD. nP o0.05 vs. control. Numbers indicate the percentage of cells in each quadrant.
with PE (25 mg/mL) or vehicle control (DMSO) for different intervals (0, 24, 48 h), cells of the control group moved fast into the scraped area over time with migration rate of 100.43 72.89%, 71.14 75.02% and 49.63 75.24%, respectively. While PE treatment resulted in significant decrease in migration ability from 100.67 72.08% to 96.12 73.46% and 87.677 4.40%, respectively. These results revealed that PE could inhibit the migration of HUVEC cells in a time-dependent manner (Fig. 9A). Quantitative results were shown as migration ratio, which indicated differences in the migration ratio over the control groups (Fig. 9B).
4. Discussion Accumulating literatures have reported the anti-tumor effect of Fructus A. oxyphylla, while few studies have reported the proapoptotic effect and the action mechanism. In this study, we described for the first time the detailed pro-apoptotic activity and the action mechanism of A. oxyphylla on human solid and hematological malignant tumor cells. The obtained results showed that the cell viability of HepG2, SW480, MCF-7, K562 and HUVEC cell lines was reduced by ethanol extract (EE), petroleum ether fraction (PE), dichloromethane fraction (DCLM) of A. oxyphylla in a dose-dependent manner. PE, containing flavonoids, diarylheptanoids and sesquiterpenes, exerted the most significant antiproliferative and pro-apoptotic effects on HepG2 cells with IC50
value of 52.597 1.31 mg/mL at 24 h post-treatment. The LDH activity was increased significantly in a dose-dependent manner which supported the accuracy of cell viability results. These results were consistent with the previous researches of the anti-tumor effects of A. oxyphylla (Itokawa et al., 1979; Lee et al., 1998; Surh et al., 1998; He et al., 2010; Wang et al, 2013). The main chemical components of A. oxyphylla include flavonoids (e.g., Tectochrysin, Izalpinin, Chrysin, Apigenin-4', 7dimethylether and Kaempferide), diarylheptanoids (e.g., Yakuchinone A and B and Oxyphyllacinol) and sesquiterpenes (e.g., Nootkatone), which were regarded as representative constituents with putative pharmacological activities (Xu et al., 2009; Chen et al., 2014). Based on the literatures (Chun et al., 1999, 2002a, 2002b; Oh et al., 2014) and the results of our HPLC analysis, 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin mainly exist as major contents in PE due to their low polarity (Fig. 3A). Moreover, EE and DCLM fraction barely inhibited HepG2 cell growth at the same condition with IC50 value of 100.817 0.72 and 154.12 72.64, respectively (Table 1). Thus, we speculated that 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin might be involved and work in coordination with each other in the apoptosis-based cytotoxicity of PE against HepG2 cells. A similar study has reported the antioxidant and cytotoxic properties of ethanol extract of A. oxyphylla using Folin-Ciocalteu reagent, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 5. PE induced ROS generation in HepG2 cells. (A) Microscope results of ROS generation treated with PE or combined with NAC. (B) PE increased the ROS generation in HepG2 cells in a dose-dependent manner. (C) NAC inhibited PE-induced ROS generation in HepG2 cells. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
Fig. 6. PE induced the disruption of mitochondrial membrane potential (Δψm). (A) PE caused an obvious decrease of MMP in HepG2 cells in a dose-dependent manner. (B) Column chart showed mean values of three experiments ( 7 S.D.). All analyses were performed in triplicate. Data were expressed as mean 7SD. nP o 0.05 vs. control.
antioxidant capacity, reducing power and MTT assay. They concluded that PE showed significant anti-cancer capacity but very weak antioxidant activity, indicating that the main active compounds serving as anti-cancer agents in PE may not belong to polyphenols and deserve further study (Wang et al., 2013). Actually, we detected a significant promoting oxidation effects and overproduction of ROS after treatment of PE on HepG2 cells during the beginning few hours. Overproduction 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). Based on these findings, we proposed an intrinsic mechanism for the induction of apoptosis by PE in HepG2 cells. This activity was further confirmed through PE-induced ROS generation (Fig. 5), MMP disruption (Fig. 6), apoptosis related proteins expression changing and cytochrome c elevation in cytosol (Fig. 7), which eventually lead to induction of apoptosis. Apoptosis plays a crucial role in the cellular progress of proliferation, differentiation, senescence and death. Nuclear
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 7. Western blot analysis of the expression of apoptosis related protein. (A) The expression of caspase-3/9, cleaved-caspase-3/9, PARP, cleaved-PARP, Bad, p53 and cytochrome c; (B) PE reduced the expression of Bcl-2 while increased the expression of Bax in a dose-dependent manner. (C) The representative bar charts of PE mediated Bax/Bcl-2 expression. β-Actin was used as an internal control. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
Fig. 8. PE induced the expression changes of AKT, ERK and SAPKs. (A) PE inhibited the phosphorylation of AKT in a dose-dependent manner; (B) PE induced the phosphorylation of p38 and JNK and decrease the phosphorylation of ERK as compared with the control group. β-Actin was used as an internal control. All analyses were performed in triplicate.
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 9. PE induced the changes of migratory potential of HUVEC cells. (A) PE treatment resulted in a significant decrease in migration ability in a time-dependent manner. (B) The representative bar charts of PE mediated migration of HUVEC. All analyses were performed in triplicate. Data were expressed as mean 7 SD. *P o0.05 vs. control.
fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptotic cells (Gao et al., 2011). Our results of Hoechst staining revealed that irregular nuclei, crescent-shaped nuclei, chromatin migration and condensation were observed in HepG2 cells after treatment of PE (Fig. 4A). Annexin V-FITC/PI staining was used to explore whether PE promoted the early and late apoptosis of HepG2 cells. Compared with cells of the control group, fluorescence intensities of the treated cells were increased at concentrations from 50 to 100 m g/mL (Fig. 4B and C). These results showed that the pro-apoptotic activity of PE against HepG2 cells was remarkable. In this case, what the action mechanisms could be? Activation of mitochondria-mediated intrinsic apoptotic pathway is one of the key mechanisms involved in the function of anti-tumor drugs, which is governed by Bcl-2 family proteins. Bax/Bcl-2 regulates the release of cytochrome c from mitochondria into the cytosol and cytochrome c in the cytosol initiates caspases cascades (such as caspase-3/9), which leads to cell apoptosis (Chipuk et al., 2006). Moreover, PARP is generally cleaved as a substrate by caspase-3 in apoptosis (Zhang et al., 2005). Our results revealed that PE significantly inhibited Bcl-2 expression and promoted Bax expression in a concentration-dependent manner accompanied by an increase of cytochrome c in cytosol. These events caused the cleavage of 35 KD caspase-3 to generate 17 KD fragment and the cleavage of 46 KD caspase-9 to generate 35 KD fragment. PE also activated the cleavage of PARP in HepG2 cells in a dose-dependent manner (Fig. 7A). These results indicated that HepG2 cells treated with PE were subjected to apoptosis involving mitochondria pathway. Once intracellular ROS levels are too high, cell proliferation, even apoptosis will occur by ROS-mediated events such as DNA damage, the trigger of AKT and MAPKs signaling pathways (Chen and Wong, 2008; Huang et al., 2001). DNA damage-mediated AKT activation and p53 overexpression are important targets for treating various tumors because it regulates cells proliferation and apoptosis (Chao et al., 2010). AKT is a downstream target of PI3K and its phosphorylation has been considered as an important factor in the aggressiveness of cancer. Activated AKT can enhance cell survival by regulating downstream targets such as promoting the release of NF-κB and inhibiting the expression and activation of pro-apoptotic proteins including Bad, p53, Caspase-9 (del Peso et al., 1997; Johnson et al., 2003; Song et al., 2005; VelazquezGarcia et al., 2011; Wang et al., 2012). Our results demonstrated that PE down-regulated the phosphorylation of AKT and promoted
the expression of p53 and Bad in a dose-dependent manner, implying that PE-induced apoptosis is associated with the AKT pathway. MAPKs signal transduction pathways have recently gained great attention as tumor suppressors which are activated upon cellular stress and often engage pathways that can block proliferation or promote apoptosis (Dayem et al., 2010). Once activated, ERK plays an important role in anti-apoptotic activity, while JNK and p38 are significant in pro-apoptotic activity (Raingeaud et al., 1996; Sheth et al., 2001). Our results showed that PE led to a decrease in phosphorylation of anti-apoptotic kinases ERK and an increase in phosphorylation of pro-apoptotic kinases JNK and p38, indicating that MAPKs signaling pathways were triggered to induce apoptosis of HepG2 cells (Fig. 8B). We speculated that p38 MAPK and JNK may be involved in PE-induced growth inhibition and pro-apoptosis activities in HepG2 cells. It has been reported that the Hex (n-hexane) and EtOAc (acetoacetate) fractions of A. oxyphylla, containing a series of sesquiterpenes and diarylheptanoids, showed anti-angiogenic potentials both in vivo and in vitro models (He et al., 2010). Since PE has the similar chemical components to Hex and our results showed PE exerted significant anti-proliferative effect on HUVECs with IC50 value 49.21 70.62 mg/mL (Table 1), we tried to find out whether PE has the potential anti-angiogenic effects using the migration assay of HUVECs. Our results suggested that PE inhibited the migration of HUVEC cells in a time-dependent manner. Thus, we speculated that PE play a potential role of anti-angiogenesis which lends support to some of the anti-cancer properties of A. oxyphylla.
5. Conclusion In conclusion, our results showed that the extractions of Fructus A. oxyphyllae displayed cytotoxicity to HepG2, SW480, MCF-7, K562 and HUVEC cells. PE significantly inhibited HepG2 cell proliferation possibly through the induction of apoptosis mediated by inducing ROS formation, decreasing MMP, regulating Bcl-2 family protein and activating caspase-3/9. ROS induced DNA damage-mediated protein expression of p53, AKT, ERK inactivation and SAPKs activation, which contributed to apoptosis induced by PE. Furthermore, migration of HUVEC cells was blocked by PE. The present results demonstrated that PE induced apoptosis in HepG2
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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cells via a ROS-mediated signaling pathway. The obtained findings may provide new insights for the high potential use of low polarity components from A. oxyphylla as an anti-neoplastic agents.
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Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway Qiao Zhang a,b, Can Cui a, Cong-Qin Chen a, Xiao-Long Hu a, Ya-Hui Liu a, Yan-Hua Fan a, Wei-Hong Meng b, Qing-Chun Zhao b,n a b
Department of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 November 2014 Received in revised form 9 March 2015 Accepted 13 March 2015
Ethnopharmacological relevance: Fructus Alpiniae oxyphyllae (A. oxyphylla) is a traditional herb which is widely used in East Asian for the treatment of dyspepsia, diarrhea, abdominal pain, poor memory, inflammatory conditions and cancer. Materials and methods: The cytotoxic activities of ethanol extract (EE) and five extract layers including petroleum ether (PE), dichloromethane (DCLM), acetoacetate (EtOAc), n-Butanol (n-Bu) and water fractions (WF) of A. oxyphylla were tested on HepG2, SW480, MCF-7, K562 and HUVEC cell lines using MTT assay and LDH release assay. The component analysis was performed on HPLC with gradient elution. Hoechst 33342 staining, DCFH-DA fluorescence microscopy, flow cytometry analysis, western blot and migration assays were carried out to determine the anti-cancer mechanisms of PE. Results: MTT analysis showed that EE, PE and DCLM could inhibit cell proliferation on HepG2, SW480, MCF-7, K562 and HUVEC cell lines, especially PE fraction. HPLC analysis pointed out five main components which may contribute to the anti-proliferative activity of PE. Further study showed that PE increased LDH release, induced apoptosis, disrupted mitochondrial membrane potential and elevated intracellular reactive oxygen species (ROS) in HepG2 cells, whereas the antioxidant N-acetylcysteine (NAC) prevented PE-induced ROS generation. The results of western blot revealed that PE induced apoptosis in HepG2 cells by enhancing Bax/Bcl-2 ratio, increasing cytochrome c in cytosol and ase-3/9. Meanwhile, high levels of ROS could induce DNA damage-mediated protein expression, AKT, ERK inactivation and SAPKs activation. Furthermore, PE conspicuously blocked the migration of HUVEC cells. Conclusion: The present results demonstrated that PE induced apoptosis in HepG2 cells may be via a ROS-mediated signaling pathway. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alpinia oxyplylla Cytotoxicity Apoptosis ROS HepG2 Chemical compounds studied in this article: Yakuchinone A (PubChem CID:133145) Tectochrysin (PubChem CID:5281954) Chrysin (PubChem CID:5281607)
1. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases and the second leading cause of cancer related death around the world (Ferlay et al., 2010; Jemal et al., 2011). More than 600,000 people die from HCC each year. Many molecules and signaling pathways have been found to be involved in the development of HCC, including the Bcl-2, AKT/mTOR, RAF/ MEK/ERK and EGFR pathways (Blechacz and Gores, 2008; Ho et al., 2012). Chemotherapy, referred to the treatment of cancer using
pharmacological or natural agents to impede, arrest or reverse the carcinogenic process, remains the most practical and promising approach for the management of cancer patients (Link et al., 2010; Sporn and Suh, 2002). It is increasingly accepted that natural products are used as an alternative therapy globally because it generally has high therapeutic effectiveness as well as low side effects (Newman et al., 2003; Park et al., 2012). Fructus Alpiniae oxyphyllae (A. oxyphylla) is an important herb commonly used in East Asian traditional medicine. Its fruits were extensively used as a traditional Chinese medicine for curing
Abbreviations: Aq, aqueous fraction.; BuOH, n-Butanol; BSA, bovine serum albumin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; DCLM, dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FBS, fetal bovine serum; HepG2cells, human hepatic carcinoma cells; Hex, n-hexane; HUVEC, Human Umbilical Vein Endothelial Cells; K562, human leukemia cells; MCF-7, Michigan Cancer Foundation-7 breast cancercell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acety-l-cystein; PE, petroleum ether extract.; PI, PIpropidium iodide; P/S, penicillin-streptomycin; PBS, phosphate buffer saline; ROS, reactive oxygen species n Corresponding author. Tel./fax: þ 86 24 28851126. E-mail address: [email protected] (Q.-C. Zhao). http://dx.doi.org/10.1016/j.jep.2015.03.073 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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diarrhea, intestinal disorders, diuresis and poor memory (But et al., 1997; Pharmacopoeia Committee, 2005). Modern pharmacological studies have revealed that A. oxyphylla has many effects such as anti-neoplastic, vasodilating, anti-ulcer and neuroprotective (Itokawa et al., 1979; Lee et al., 1998; An et al., 2006; But et al., 1997). Several studies have reported the anti-proliferative property of A. oxyphylla against different cancer cells. The ethanol crude extract (EE) and its fractions, petroleum ether fraction (PE) and acetoacetate fraction (EtOAc) of A. oxyphylla, exhibited potent anti-proliferative effects on six cancer cell lines (A549, HepG2, SW480, HeLa, MCF-7 and MNK-45 cells) with IC50 values ranging from 40.1 to 166.3 mg/mL (Wang et al., 2013). The ethanol extract of A. oxyphylla suppressed skin carcinogenesis in mice and induced apoptotic death in human promyelocytic leukemia (HL)-60 cells (Lee et al., 1998). The Hex (n-hexane) and EtOAc fractions of A. oxyphylla could inhibit the proliferation of HepG2 cells probably through anti-angiogenesis (He et al., 2010). Although A. oxyphylla has been reported to exert anti-tumor activity, the reports on solid and hematological malignant tumor cells of humans such as HepG2, SW480, MCF-7 and K562 cell lines are relatively limited. Moreover, the pro-apoptotic effect and the underlying molecular mechanisms of A. oxyphylla on human cancer cells remain unclear and need deeply explored. This study was aimed at investigating the cytotoxic activity of A. oxyphylla extracts on several cells and the pro-apoptotic effect of PE on HepG2 cells, also we tried to explore the active components and action mechanisms of PE.
2. Materials and methods 2.1. Reagents Antibodies for cytochrome c (A-8), Bcl-2 (C21), Bax (P-19), Caspase3/9, Cleaved-Caspase-3/9 and β-actin (C4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p53, Bad, PARP, Cleaved-PARP, anti-phospho-AKT (Thr308), anti-AKT, anti-phosphop44/42(ERK1/2) (Thr202/Tyr204), anti-SAPK/JNK, anti-phospho-SAPK/ JNK (Thr183/Tyr185), anti-p38, anti-phospho-p38 and anti-p44/ 42MAPK (ERK1/2) (Thr202/Tyr204), were purchased from Cell Signaling Technology Inc. (USA). The secondary antibodies were purchased from ZSGB-BIO (Beijing, China). The enhanced chemiluminescent (ECL) plus reagent kit, Reactive oxygen species (ROS) detection kits and Rhodamine123 (Rh123) were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). HPLC-grade methanol and acetonitrile were purchased from Merck Company Inc. (Merck, Darmstadt, Germany). 2.2. Preparation of plant extracts and fractionation The fruit of Fructus A. oxyphylla was purchased from Shenyang Medicinal Material Co. (Shenyang, China), identified by Dr. QingChun Zhao (Department of Chinese Medicine, Shenyang Pharmaceutical University) and deposited in the herbarium of Department of Pharmacy, General Hospital of Shenyang Military Area Command (Voucher specimen number: ZQAO2013). The fruit of A. oxyphylla 5 kg was extracted with 50 L of 95% ethanol three times under reflux for 1 h each. These three filtrates were combined and concentrated in a rotary evaporator at 65 1C until dried to afford the crude extract 310 g. The crude extract was suspended in 2 L water, and then partitioned exhaustively with equal volume of petroleum ether, dichloromethane, acetoacetate and n-Butanol, respectively. The obtained ethanol extract (EE) and fractions such as PE (petroleum ether), DCLM (dichloromethane), EtOAc (acetoacetate), n-Bu (n-Butanol) and WF (water fractions) were stored in dark bottles at 4 1C after solvent evaporation and
freeze-drying. On the basis of NMR analysis, the structures of isolated reference standards (9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin) were confirmed and their purities were 98.0% or greater as determined by HPLC-PDA. 2.3. Cell culture and treatments Human liver carcinoma cell line (HepG2), human colon cancer cell line (SW480), human breast cancer cell line (MCF-7), human leukemia cell line (K562) and human umbilical vein endothelial cell line (HUVEC) were obtained from Shenyang Pharmaceutical University (Shenyang, China). Cells were maintained in DMEM medium supplemented with 10% FBS in a humidified atmosphere with 5% CO2 in the air at 37 1C. Cells were seeded in 96-well plates (1 104 cells/well) and 6-well plates (1 105 cells/well) and allowed to adhere for 12 h under the same condition. Then, different concentrations of samples (50, 75, 100 mg/mL) or vehicle control (untreated medium containing vehicle 0.1% DMSO) were added and cells were treated for 24 h for the following experiments, except the ROS generation and the migration assay. 2.4. Cell viability assay The anti-proliferative effects of EE and five fraction layers on HepG2, SW480, MCF-7, K562 and HUVEC cells were examined by using MTT assay (Ma et al., 2014). Cells were seeded and dispersed evenly in medium (containing 10% FBS) in 96-well plates at a density of 1 104 cells per well and allowed to adhere for 12 h under the same condition. Then, cells were treated with different concentrations of samples for 24 h, the MTT stock solution was added into each well (final concentration: 0.5 mg/mL) and the cells were incubated at the same conditions for another 4 h. Then, after discarding the supernatant, 150 mL DMSO was added and the OD value of each well was determined at the wavelength of 490 nm using a Microplate Reader (Elx800 Bio-Tek, USA). 2.5. Lactate dehydrogenase (LDH) release assay The cell membrane integrity of HepG2 cells was evaluated by determining the activity of LDH leaking out of the cells according to the manufacturer's instructions (LDH detection kit, A020-2, Njjcbio, Nanjing). Briefly, cells were seeded in 96-well plates and afterwards exposed to different concentrations of PE for 24 h, the free supernatant was transferred into centrifuge tubes for testing. Following the sequences of the instruction, the LDH-assay reaction mixtures were transferred into wells of 96-well plates. The optical density of the color generated was determined at a wavelength of 450 nm using a Microplate Reader (Elx800 Bio-Tek, USA). 2.6. High performance liquid chromatography (HPLC) analysis The HPLC analytical method was applied for analysis of PE, EE and DCLM. The analysis was performed by high performance liquid chromatography coupled with a photodiode array detector (Waters 2996), a quaternary pump controller (Waters 600), an Auto Sampler injector (Waters 717) and LC solution software. The mobile phase was A (water) and B (acetonitrile). The elution conditions applied were: 0–30 min, linear gradient 30–40% B; 30–45 min, linear gradient 40–45% B; 45–50 min, linear gradient 45–50% B; 50–60 min, linear gradient 50–35% B; 60–70 min, linear gradient 35–30% B. The C18 column (5 mm, 250 mm 2.1 mm I.D, Agilent Technologies) was used and the column temperature was maintained at 30 1C. The flow-rate was 1.0 ml/min and 10 mL of the sample was delivered to the HPLC. The absorbance was monitored
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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with a PAD detector at wavelength of 260 nm. Five standards isolated from A. oxyphylla were dissolved in methanol. 2.7. Hoechst 33342 staining assay To examine cell apoptosis, the Hoechst 33342 staining assay was performed as previously reported (Gao et al., 2011) with minor revision. HepG2 cells were seeded into 6-well plates for 12 h and then treated with different concentrations of PE for 24 h. Subsequently, the culture medium was removed and the HepG2 cells were stained with Hoechst 33342 (final concentration 1.0 mg/mL) for 20 min, the apoptotic cell changes in morphology such as chromatin condensation and nuclear shrinking could be characterized. The fluorescence was observed under fluorescence microscope and images were captured with an electronic camera (Olympus, Tokyo; 100 magnification). 2.8. Annexin V-FITC/PI double staining assay In short, cells were seeded into 6-well plates for 12 h and exposed to different concentrations of PE for 24 h. Subsequently, according to the manufacturer's instructions of a commercially available kit (BD Pharmingen, CA, USA), the resuspended cells (1 105 cells/100 mL) were treated with PI (5 mL) and Annexin VFITC (5 mL) at room temperature for 15 min in the dark. Cells were analyzed using Becton Dickinson FACS Calibur System (NJ, USA) with emission filters of wavelength 515–545 nm for FITC (green) and 600 nm for PI (red). 2.9. ROS generation assay Intracellular ROS generation was detected by a fluorescence microscope with 20 ,70 -dihydrofluorescein-diacetate (DCFH-DA) according to the manufacturer's instructions of ROS detection kit (S0033, Beyotime, Jiangsu, China). Briefly, HepG2 cells were seeded in 6-well plates for 12 h and then incubated with fresh incubated medium containing 10 μM DCFH-DA at 37 1C in the dark for 20 min. Subsequently, cells were treated with or without 10 mM N-acetyl-L-cysteine (NAC) for 1 h and co-incubated with different concentrations of PE for 30 min. Then cells were washed with PBS buffer and the DCF fluorescence intensity was measured by the fluorescence microscope. Images were captured with an electronic camera (Olympus, Tokyo; 100 magnification).
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2.11. Western blot analysis To investigate the expression changes of the apoptosis-related protein in HepG2 cells after exposed to PE, cells were cultured in 75 mm2 dish (5 106 cells) for 12 h and then exposed to PE for 24 h. Then cells were collected, washed twice with PBS and centrifuged. Subsequently, each samples were added into 50 mL RIPA Lysis buffer (containing 1% Phenylmethanesulfonyl fluoride) and incubated on ice for 30 min. Then, samples were clarified by centrifugation at 12,000 rpm for 10 min at 4 1C, and protein concentrations were determined using Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were denatured by boiling for 5 min, equal amounts of protein (20 μg/lane) were loaded on 10% sodium dodecyl sulfate/polyacrylamide gels (Beyotime, Jiangsu, China) and blotted onto Polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris Buffered Saline Tween (TBST) for 2 h at room temperature and then incubated primary antibodies with 5% nonfat dry milk/TBST overnight at 4 1C. The membranes were washed three times with TBST for 5 min and subsequently incubated in secondary antibody with 5% nonfat dry milk/TBST for 1 h at room temperature. The membranes were washed as described above and detected using the ECL detection system. Blotted antibodies were visualized by chemiluminescence method (Beyotime, Jiangsu, China).
2.12. Migration assay Since Hex (n-hexane) fractions of A. oxyphylla, containing similar chemical components to PE, showed anti-angiogenic potentials both in vivo and in vitro models (He et al., 2010), we speculated PE may demonstrated potential anti-angiogenic effect. Then we attempted to examine the effect of PE on cell migration of HUVECs using the wound healing assay as previously reported (Wang et al., 2014). Cells (5 105 cells/well) were seeded in a 6-well plate for 12 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, Japan). After the treatment of PE (25 mg/mL) 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 (day 0). The images were analyzed using computing software (Image J 1.48q).
2.10. Mitochondrial membrane potential (MMP) disruption assay The MMP (Δψm) was monitored by Rh123 staining as previous reported (Ma et al., 2014). In short, HepG2 cells were seeded in 6-well plates for 12 h and incubated with different concentrations of PE for 24 h. Then cells were harvested, washed twice with PBS, re-suspended and stained with fresh incubated medium containing 1.0 mM Rh123 at 37 1C for 30 min. The fluorescence intensity of the cells was analyzed by flow cytometer (Becton Dickinson, NJ, USA).
2.13. Statistical analysis Experimental values were expressed as mean 7standard deviation of at least three experiments in triplicate. Data were analyzed using Sigmaplot 12.0 software and the statistical significance was assessed by one-way ANOVA using SPSS 21.2 software. The level of Po 0.05 was accepted to determine the statistical significance.
Table 1 Cytotoxic activity of Fructus Alpiniae oxyphyllae on human cancer cells. Extracts
EE PE DCLM
HepG2
100.81 7 0.72 52.59 7 1.31 154.127 2.64
Cell lines, IC50 value (mg/ml)
HUVEC
MCF-7
K562
SW480
4200 97.82 7 1.15 4200
121.377 0.97 60.25 7 2.12 106.667 1.48
4200 53.18 71.35 4200
52.87 7 1.64 49.217 0.62 57.147 1.67
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 1. PE exhibited cytotoxic activity on HepG2 cells. (A) MTT results showed PE inhibited the growth of HepG2 cells with a dose-dependent manner. (B) PE increased the LDH release of HepG2 cells with a dose-dependent manner. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
3. Result 3.1. The extraction of A. oxyphylla suppressed the proliferation of human cancer cells The MTT assay was used to evaluate the cytotoxicity of EE, PE, DCLM, EtOAc, n-Bu and WF on human cell lines. The results showed that PE caused significant decreases in HepG2, SW480, MCF-7, K562 and HUVEC cells proliferation. EE and DCLM showed weak cytotoxicity on these cells, while no significant growth inhibitory activity was detected on other extract layers under 200 mg/mL (Data were not listed). PE showed the lowest IC50 value of 52.59 71.31 mg/mL than EE (100.81 70.72%) and DCLM (154.12 72.64%) against HepG2 cells (Table 1). All concentrations of PE (50, 75, 100 mg/mL) resulted in significant decrease in cell viability of HepG2 cells by 60.2474.30%, 37.30 7 1.71% and 4.48 71.20%, respectively (Fig. 1A). 3.2. PE induced LDH release from HepG2 cells LDH release level was measured by detecting changes in optical densities. PE increased the LDH activity significantly in HepG2 cells in a dose-dependent manner. Compared with the control group (101.67 75.51%), all concentrations of PE (50, 75, 100 mg/mL) resulted in significant increase in LDH release by 167.98 717.97%, 198.48 710.43% and 226.98 71.83%, respectively (Fig. 1B). 3.3. HPLC analysis of PE, EE and DCLM Based on the absorption profile, the retention time and spiking tests of PE, there were at least 18 distinct peaks eluted from PE using the optimized mobile phase and wavelength. 9-hydroxynootkatone (1), Yakuchinone B (2), Yakuchinone A (3), Oxyphyllacinol (4) and Tectochrysin (5) were identified as major components and their structures are shown in Fig. 2. Amongst them, Tectochrysin may be the maximum flavonoids components existed in PE according to the HPLC results (Fig. 3A). Other peaks that were not identified may include chrysin, oxyphyllenone, izalpinin and neonootkato. EE and DCLM were analyzed to compare the contents of main components under the same concentrations. The results showed that the contents of 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin in PE were significantly higher than EE (Fig. 3A and B), while these components were almost undetected in DCLM (Fig. 3C). 3.4. PE induced apoptosis of HepG2 cells As shown in Fig. 4A, cells of the control group had normal nuclear morphology under fluorescent microscope after Hoechst
Fig. 2. Structures of five main components of PE. 9-hydroxynootkatone (1), Yakuchinone B (2), Yakuchinone A (3), Oxyphyllacinol (4) and Tectochrysin (5).
33342 staining, indicating that the chromatin was equivalently distributed in the nucleus. The test group marked with nuclear fragmentation, condensation of chromatin and the morphological characteristics of apoptosis, which include emitting brighter fluorescence, were detected after treatment with different concentrations of PE for 24 h. These results indicated that PE is capable of inducing apoptosis in HepG2 cells in a dose-dependent manner. In order to further evaluate PE-induced apoptosis in HepG2 cells, cells were treated with the stain of AnnexinV-FITC/PI and detected by flow cytometry. The results revealed that PE dramatically triggered apoptotic response in HepG2 cells in a dosedependent manner after 24 h treatment compared with the control group. The percentage of early apoptotic cells (right low quadrant) increased from 2.53 71.30% to 11.85 73.64%, and the percentage of late apoptotic and necrotic cells (right upper quadrant) was also increased from 3.73 70.73% to 18.39 73.87% (Fig. 4B and C). Taken together, these results confirmed the proapoptotic effect of PE. 3.5. PE increased reactive oxygen species (ROS) formation Since ROS generation plays an important role in the proapoptotic activities, we investigated whether ROS generation was involved in PE-induced apoptosis. As shown in Fig. 5A, HepG2 cells were pre-incubated with or without NAC for 1 h and treated with different concentrations of PE for 30 min. Here, we must note that the treatment time of PE was chosen as 30 min, as we detected the largest amounts of ROS generation at that time (Data were not listed). Compared with the control group (100.01 7 0.25%), all concentrations of PE (50, 75, 100 mg/mL) significantly increased the intracellular ROS level by 216.61 70.20%, 363.23 70.32% and 507.97 70.08%, respectively (Fig. 5B).
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 3. LC chromatograms of A. oxyphylla extraction and fractions: (A) PE; (B) EE; (C) DCLM.
Compared with the cells treated only with 100 mg/mL PE (507.9770.08%), ROS generation decreased to 195.7670.13% when treated with 10 mM NAC followed by 100 mg/mL PE (Fig. 5C). While cells only treated with 10 mM NAC (92.0270.03%) showed no significant difference with cells of the control group. These results suggested that PE likely induced oxidative injury in HepG2 cell lines, whereas NAC could prevent PE-induced ROS generation. 3.6. PE decreased the mitochondrial membrane potential (MMP) of HepG2 cells We analyzed the integrity of mitochondrial function after treatment with or without PE for 24 h with the stain of Rh123 by flow cytometry. Compared with the control group (95.7671.24%), all concentrations of PE (50, 75, 100 mg/mL) resulted in a significant decrease of fluorescence intensity by 81.5373.08%, 73.1774.35% and 52.0771.56% (Fig. 6A and B). These results revealed that PE dramatically disrupted the MMP in HepG2 cells in a dose-dependent manner after 24 h treatment. 3.7. PE induced apoptosis through mitochondrial pathway Western blot analysis revealed that all concentrations of PE (50, 75, 100 mg/mL) resulted in a significant increase of p53 and cytochrome c (cytosolic) expression. The expression levels of caspase-3/9, PARP and Bad were decreased after 24 h treatment,
and remarkable cleavage of caspases-3/9 and PARP were detected compared with the control group (Fig. 7A). Mitochondrial dysfunction is regulated by Bcl-2 family proteins, thus the Bcl-2 family proteins were examined in the study. PE caused a significant reduction in Bcl-2 expression whereas the expression of Bax was significantly increased (Fig. 7B). Treatment with PE led to changes in Bax/Bcl-2 expression in a concentrationdependent manner (Fig. 7C). Thus, PE could induce apoptosis in HepG2 cells with mitochondrial pathway involved. 3.8. PE induced apoptosis via AKT, ERK inactivation and SAPKs activation Large amounts of AKT phosphorylation were observed in the control cells while PE (50, 75 and 100 mg/mL) significantly inhibited their phosphorylation in a dose-dependent manner (Fig. 8A). Moreover, PE significantly induced the phosphorylation of p38 and JNK in a dose-dependent manner, whereas the phosphorylation of ERK was significantly decreased (Fig. 8B). These results suggested that PE up-regulated p-p38, p-JNK and down-regulated p-ERK protein activities, which may acted as pro-apoptotic inducers in HepG2 cells. 3.9. PE inhibited the migration of HUVEC cells Wound-scratch assays were performed to compare the migratory potential of HUVEC cells induced by PE. After the treatment
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 4. PE induced apoptotic response in HepG2 cells. (A) Morphological changes of HepG2 cells after PE treatment and Hoechst staining. Irregular nuclei, crescent-shaped nuclei, chromatin margination and condensation, and apoptotic bodies were shown by arrows. (B) PE increased early and late apoptosis of HepG2 cells in a dose-dependent manner. (C) Column chart shows mean values of three experiments. All analyses were performed in triplicate. Data were expressed as mean 7SD. nP o0.05 vs. control. Numbers indicate the percentage of cells in each quadrant.
with PE (25 mg/mL) or vehicle control (DMSO) for different intervals (0, 24, 48 h), cells of the control group moved fast into the scraped area over time with migration rate of 100.43 72.89%, 71.14 75.02% and 49.63 75.24%, respectively. While PE treatment resulted in significant decrease in migration ability from 100.67 72.08% to 96.12 73.46% and 87.677 4.40%, respectively. These results revealed that PE could inhibit the migration of HUVEC cells in a time-dependent manner (Fig. 9A). Quantitative results were shown as migration ratio, which indicated differences in the migration ratio over the control groups (Fig. 9B).
4. Discussion Accumulating literatures have reported the anti-tumor effect of Fructus A. oxyphylla, while few studies have reported the proapoptotic effect and the action mechanism. In this study, we described for the first time the detailed pro-apoptotic activity and the action mechanism of A. oxyphylla on human solid and hematological malignant tumor cells. The obtained results showed that the cell viability of HepG2, SW480, MCF-7, K562 and HUVEC cell lines was reduced by ethanol extract (EE), petroleum ether fraction (PE), dichloromethane fraction (DCLM) of A. oxyphylla in a dose-dependent manner. PE, containing flavonoids, diarylheptanoids and sesquiterpenes, exerted the most significant antiproliferative and pro-apoptotic effects on HepG2 cells with IC50
value of 52.597 1.31 mg/mL at 24 h post-treatment. The LDH activity was increased significantly in a dose-dependent manner which supported the accuracy of cell viability results. These results were consistent with the previous researches of the anti-tumor effects of A. oxyphylla (Itokawa et al., 1979; Lee et al., 1998; Surh et al., 1998; He et al., 2010; Wang et al, 2013). The main chemical components of A. oxyphylla include flavonoids (e.g., Tectochrysin, Izalpinin, Chrysin, Apigenin-4', 7dimethylether and Kaempferide), diarylheptanoids (e.g., Yakuchinone A and B and Oxyphyllacinol) and sesquiterpenes (e.g., Nootkatone), which were regarded as representative constituents with putative pharmacological activities (Xu et al., 2009; Chen et al., 2014). Based on the literatures (Chun et al., 1999, 2002a, 2002b; Oh et al., 2014) and the results of our HPLC analysis, 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin mainly exist as major contents in PE due to their low polarity (Fig. 3A). Moreover, EE and DCLM fraction barely inhibited HepG2 cell growth at the same condition with IC50 value of 100.817 0.72 and 154.12 72.64, respectively (Table 1). Thus, we speculated that 9-hydroxynootkatone, Yakuchinone B, Yakuchinone A, Oxyphyllacinol and Tectochrysin might be involved and work in coordination with each other in the apoptosis-based cytotoxicity of PE against HepG2 cells. A similar study has reported the antioxidant and cytotoxic properties of ethanol extract of A. oxyphylla using Folin-Ciocalteu reagent, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 5. PE induced ROS generation in HepG2 cells. (A) Microscope results of ROS generation treated with PE or combined with NAC. (B) PE increased the ROS generation in HepG2 cells in a dose-dependent manner. (C) NAC inhibited PE-induced ROS generation in HepG2 cells. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
Fig. 6. PE induced the disruption of mitochondrial membrane potential (Δψm). (A) PE caused an obvious decrease of MMP in HepG2 cells in a dose-dependent manner. (B) Column chart showed mean values of three experiments ( 7 S.D.). All analyses were performed in triplicate. Data were expressed as mean 7SD. nP o 0.05 vs. control.
antioxidant capacity, reducing power and MTT assay. They concluded that PE showed significant anti-cancer capacity but very weak antioxidant activity, indicating that the main active compounds serving as anti-cancer agents in PE may not belong to polyphenols and deserve further study (Wang et al., 2013). Actually, we detected a significant promoting oxidation effects and overproduction of ROS after treatment of PE on HepG2 cells during the beginning few hours. Overproduction 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). Based on these findings, we proposed an intrinsic mechanism for the induction of apoptosis by PE in HepG2 cells. This activity was further confirmed through PE-induced ROS generation (Fig. 5), MMP disruption (Fig. 6), apoptosis related proteins expression changing and cytochrome c elevation in cytosol (Fig. 7), which eventually lead to induction of apoptosis. Apoptosis plays a crucial role in the cellular progress of proliferation, differentiation, senescence and death. Nuclear
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 7. Western blot analysis of the expression of apoptosis related protein. (A) The expression of caspase-3/9, cleaved-caspase-3/9, PARP, cleaved-PARP, Bad, p53 and cytochrome c; (B) PE reduced the expression of Bcl-2 while increased the expression of Bax in a dose-dependent manner. (C) The representative bar charts of PE mediated Bax/Bcl-2 expression. β-Actin was used as an internal control. All analyses were performed in triplicate. Data were expressed as mean 7 SD. nPo 0.05 vs. control.
Fig. 8. PE induced the expression changes of AKT, ERK and SAPKs. (A) PE inhibited the phosphorylation of AKT in a dose-dependent manner; (B) PE induced the phosphorylation of p38 and JNK and decrease the phosphorylation of ERK as compared with the control group. β-Actin was used as an internal control. All analyses were performed in triplicate.
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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Fig. 9. PE induced the changes of migratory potential of HUVEC cells. (A) PE treatment resulted in a significant decrease in migration ability in a time-dependent manner. (B) The representative bar charts of PE mediated migration of HUVEC. All analyses were performed in triplicate. Data were expressed as mean 7 SD. *P o0.05 vs. control.
fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptotic cells (Gao et al., 2011). Our results of Hoechst staining revealed that irregular nuclei, crescent-shaped nuclei, chromatin migration and condensation were observed in HepG2 cells after treatment of PE (Fig. 4A). Annexin V-FITC/PI staining was used to explore whether PE promoted the early and late apoptosis of HepG2 cells. Compared with cells of the control group, fluorescence intensities of the treated cells were increased at concentrations from 50 to 100 m g/mL (Fig. 4B and C). These results showed that the pro-apoptotic activity of PE against HepG2 cells was remarkable. In this case, what the action mechanisms could be? Activation of mitochondria-mediated intrinsic apoptotic pathway is one of the key mechanisms involved in the function of anti-tumor drugs, which is governed by Bcl-2 family proteins. Bax/Bcl-2 regulates the release of cytochrome c from mitochondria into the cytosol and cytochrome c in the cytosol initiates caspases cascades (such as caspase-3/9), which leads to cell apoptosis (Chipuk et al., 2006). Moreover, PARP is generally cleaved as a substrate by caspase-3 in apoptosis (Zhang et al., 2005). Our results revealed that PE significantly inhibited Bcl-2 expression and promoted Bax expression in a concentration-dependent manner accompanied by an increase of cytochrome c in cytosol. These events caused the cleavage of 35 KD caspase-3 to generate 17 KD fragment and the cleavage of 46 KD caspase-9 to generate 35 KD fragment. PE also activated the cleavage of PARP in HepG2 cells in a dose-dependent manner (Fig. 7A). These results indicated that HepG2 cells treated with PE were subjected to apoptosis involving mitochondria pathway. Once intracellular ROS levels are too high, cell proliferation, even apoptosis will occur by ROS-mediated events such as DNA damage, the trigger of AKT and MAPKs signaling pathways (Chen and Wong, 2008; Huang et al., 2001). DNA damage-mediated AKT activation and p53 overexpression are important targets for treating various tumors because it regulates cells proliferation and apoptosis (Chao et al., 2010). AKT is a downstream target of PI3K and its phosphorylation has been considered as an important factor in the aggressiveness of cancer. Activated AKT can enhance cell survival by regulating downstream targets such as promoting the release of NF-κB and inhibiting the expression and activation of pro-apoptotic proteins including Bad, p53, Caspase-9 (del Peso et al., 1997; Johnson et al., 2003; Song et al., 2005; VelazquezGarcia et al., 2011; Wang et al., 2012). Our results demonstrated that PE down-regulated the phosphorylation of AKT and promoted
the expression of p53 and Bad in a dose-dependent manner, implying that PE-induced apoptosis is associated with the AKT pathway. MAPKs signal transduction pathways have recently gained great attention as tumor suppressors which are activated upon cellular stress and often engage pathways that can block proliferation or promote apoptosis (Dayem et al., 2010). Once activated, ERK plays an important role in anti-apoptotic activity, while JNK and p38 are significant in pro-apoptotic activity (Raingeaud et al., 1996; Sheth et al., 2001). Our results showed that PE led to a decrease in phosphorylation of anti-apoptotic kinases ERK and an increase in phosphorylation of pro-apoptotic kinases JNK and p38, indicating that MAPKs signaling pathways were triggered to induce apoptosis of HepG2 cells (Fig. 8B). We speculated that p38 MAPK and JNK may be involved in PE-induced growth inhibition and pro-apoptosis activities in HepG2 cells. It has been reported that the Hex (n-hexane) and EtOAc (acetoacetate) fractions of A. oxyphylla, containing a series of sesquiterpenes and diarylheptanoids, showed anti-angiogenic potentials both in vivo and in vitro models (He et al., 2010). Since PE has the similar chemical components to Hex and our results showed PE exerted significant anti-proliferative effect on HUVECs with IC50 value 49.21 70.62 mg/mL (Table 1), we tried to find out whether PE has the potential anti-angiogenic effects using the migration assay of HUVECs. Our results suggested that PE inhibited the migration of HUVEC cells in a time-dependent manner. Thus, we speculated that PE play a potential role of anti-angiogenesis which lends support to some of the anti-cancer properties of A. oxyphylla.
5. Conclusion In conclusion, our results showed that the extractions of Fructus A. oxyphyllae displayed cytotoxicity to HepG2, SW480, MCF-7, K562 and HUVEC cells. PE significantly inhibited HepG2 cell proliferation possibly through the induction of apoptosis mediated by inducing ROS formation, decreasing MMP, regulating Bcl-2 family protein and activating caspase-3/9. ROS induced DNA damage-mediated protein expression of p53, AKT, ERK inactivation and SAPKs activation, which contributed to apoptosis induced by PE. Furthermore, migration of HUVEC cells was blocked by PE. The present results demonstrated that PE induced apoptosis in HepG2
Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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cells via a ROS-mediated signaling pathway. The obtained findings may provide new insights for the high potential use of low polarity components from A. oxyphylla as an anti-neoplastic agents.
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Please cite this article as: Zhang, Q., et al., Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.073i
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