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Dioscin suppresses hepatocellular carcinoma tumor growth by inducing apoptosis and regulation of TP53, BAX, BCL2 and cleaved CASP3.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver, occurs frequently in the setting of chronic liver injury. Although multiple therapeutic approaches are available, the prognosis of patients with HCC remains poor. Dioscin is a natural steroid saponin that presents in various plants. The anti-cancer and anti-fibrotic effects have been extensively reported. However, the effect of dioscin on HCC remains unclear. We aimed to investigate the anti-HCC properties of dioscin in vitro and in vivo.

Methods: MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) assay was used to analyze the growth inhibition activity of Dioscin in human cell lines, Bel-7402, HepG2, Lovo, and EAhy926. Antitumor activity through induction of apoptosis was evaluated by flow cytometry using Annexin-V and propidium iodide (PI) staining, laser scanning confocal microscopy (LSCM) analysis with Hochest33342 and PI labeling, and DNA fragmentation analysis. The expression of apoptosis-related proteins tumor protein p53 (TP53), BCL2-associated X protein (BAX), B-Cell CLL/Lymphoma 2 (BCL2) and Caspase 3 (CASP3) was measured by Western blot. Nude mice bearing Bel-7402 were administered intraperitoneally at different doses of dioscin and 5-FU (5-Fluorouracil) treatment was used as a control. Tumor volume and tumor weight of each mouse were then measured.

Results: We demonstrated that Dioscin inhibited proliferation of HCC cell lines in a dose-dependent manner. Dioscin also significantly induced morphological changes during death by apoptosis and increased DNA damage of Bel-7402 cells. Moreover, we demonstrated that Dioscin displayed anticancer activity via up-regulating expression of TP53, BAX and CASP3 protein, as well as down-regulating BCL2 in Bel-7402 cells. Notably, the in vivo anticancer activity of Dioscin was further assessed and achieved greater inhibition efficiency at the concentration increased to 24 mg/kg/day than 5-FU at dose of 10mg/kg/day in nude mice bearing Bel-7402 cells.

Conclusions: Dioscin inhibited tumor growth via inducing apoptosis, which was accompanied by altered expression of apoptotic pathway proteins, such as TP53, BAX, BCL2 and CASP3. Our findings indicate that further evaluation of dioscin as a novel therapeutic approach for HCC is warranted.

Keywords:

Dioscin

Hepatocellular carcinoma

Apoptosis

Introduction

Hepatocellular carcinoma (HCC) is the most frequent primary neoplasm of the liver. HCC contributed to approximately 790,000 new liver cancer cases and 818,000 cancer deaths worldwide in 2013 (Fitzmaurice et al., 2015). Therapeutic options for HCC include liver resection, percutaneous ablation, palliative intra-arterial therapies and transplantation (Mauer et al., 2015). Approximately 25% of HCC patients are thought suitable for curative surgical treatment (Ma, 2013). The overall survival time for inoperable patients is only a few months. The long-term prognosis of HCC remains poor, which is largely attributable to the high tumor recurrence rate after curative surgery and the chemotherapy-resistant nature of this cancer (Bruix and Sherman, 2005).

Apoptosis is one of the most clearly characterized cell death processes (Estaquier et al., 2012). Dysregulation of the balance between cell proliferation and death is considered to be a protumorigenic process during hepatic carcinogenesis (Fabregat, 2009). The development of novel strategies to specifically trigger apoptosis would be valuable for the treatment of various types of cancer. Therefore, there is an increasing interest in identifying novel drugs with apoptosis-inducing properties in cancers of various origins. In particular, natural products from plants and Chinese herbal medicine, as promising antitumor agents, are currently and predominantly being studied due to low toxicity and great medicinal value. For example, recent studies have reported that paclitaxel (D'Anneoet al., 2010) Tanshinone IIA (Jiao and Wen, 2011), resveratrol (Atten et al., 2005), indirubin (Nam et al., 2005), arsenic (Rocha et al., 2011) can induce apoptosis of tumor cells in vitro as well as be potential antitumor agents in the clinic.

Dioscin (diosgenyl 2,4-di-0-[alpha]-l-rhamnopyranosyl-[beta]-d-glucopyranoside) is extracted from medicinal plants, such as Polygonatum zanlanscianense Pamp, Dioscorea nipponica Makino and Dioscorea zingiberensis Wright, which is used in China as a herb medicine for thousands of years, as a monomer form (Sun et al., 2003). Several studies have reported that dioscin has anti-inflammatory (Wu et al., 2015), lipid-lowering (Kaskiw et al., 2009), anticancer (Gao et al., 2011a), hepatoprotective effects (Lu et al., 2011) and anti-multidrug resistance (Sun et al., 2011). Dioscin also exerts anti-fibrotic effects in liver (Zhang et al., 2015). Interestingly, dioscin can be utilized as a raw material for the synthesis of steroid hormone drugs (Brautbar and Williams, 2002). In vitro studies have revealed that dioscin can induce apoptosis and inhibit proliferation of cancer cells, which may contribute to the antitumor activity of this natural drug (Gao et al., 2011a).

However, the effects of dioscin on the proliferation and apoptosis of HCC had not been fully evaluated. In this study, the effects of dioscin on tumor growth, cell proliferation and apoptosis were investigated as well as the potential involvement of p53 (TP53), BCL2-associated X protein (BAX), B-Cell CLL/Lymphoma 2 (BCL2) and Caspase 3 (CASP3) signaling pathway.

Materials and methods

Chemicals and reagents

Dioscin (CAS No.19057-60-4, 99% pure by HPLC, Fig. 1) was purchased from Ronghe Medical Science and Technology Development Company Limited (Cat No. 110602, Shanghai, China), and prepared with DMSO as 10 mmol/l stock solution. MTT, propidium iodide (PI), Hoechst 33,342, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reagents were purchased from Sigma (St. Louis, MO, USA). RPMI-1640, DMEM medium, Fetal Bovine Serum (FBS) and trypsin were purchased from Gibco BRL Life Technologies (Grand Island, New York, USA). Penicillin and streptomycin were purchased from Amresco Chemical Co. Ltd. (Solon, OH, USA). The Annexin V-FITC apoptosis detection kit was purchased from BioVision (Mountain View, CA, USA). The commercially available antibodies used for Western blotting were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Cell culture

The human cell line Bel-7402, LoVo, HepG2 and EAhy926 was obtained from the Chinese Type Culture Collection (Shanghai Institute of Cell Biology, Chinese Academy of Science, Shanghai, China), cells were incubated in culture medium (RPMI-1640 medium for Bel-7402 and LoVo. DMEM for HepG2 and EAhy926) supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 [micro]g/ml) at 37[degrees]C in a humidified atmosphere of 95% air and 5% C02. When the cultures were 80%-90% confluent, the cells were respectively washed with phosphate-buffered saline (PBS), detached with 0.25% trypsin, centrifuged and replated into 96-or 24-well plates at an appropriate density according to the experimental design.

Animals

60 male nude mice, weighing 18-22 g, were purchased from Experimental Animal Center of Guangzhou University of Chinese Medicine. All animal experiments were conducted in accordance with Chinese Council on Animal Care guidelines and approved by Guangzhou University of Chinese Medicine.

Cell growth inhibitory rate analysis (MTT Assay)

The microculture tetrazolium method was used to analyze the inhibition of the cell growth rate. Cells in logarithmic growth phase were collected, and 5 x [10.sup.3] cells/well were dispensed into 96-well culture plates and stimulated with vehicle (0.1% DMSO only) or various concentrations of dioscin (0.25, 1, 4, 16, 64 [micro]mol/l). Each treatment and vehicle group (0.1% DMSO only) contained six parallel wells. The culture plates were incubated for 24, 48, and 72 h prior to the addition of tetrazolium reagent. MTT working solution was prepared as follows: 5 mg MTT/ml phosphate-buffered saline (PBS) was sterilized by filtering with 0.45 [micro]m filters. After the stimulation period, MTT working solution (20 [micro]) was added and the plates were incubated for 4h, following centrifuged at 1000 rpm for 15 min. The culture medium supernatant was removed from the wells and replaced with DMSO (100 [micro]l). Following thorough solubilization, the absorbance (A value) of each well was measured using a microplate reader at 570 nm, with 630 nm as reference wavelength. The cell growth inhibition rate was calculated according to the following formula: Cell growth inhibition rate = [(A value of the control group--A value of the observed group)/(A value of the controlled group)] x 100%. In addition, the Bel-7402, LoVo, HepG2 and EAhy926 cells were treated with different concentrations of dioscin for 48 h and then observed under microscope to reveal cell growth states.

Hoechst 33,342 and PI double staining for apoptosis detection

Bel-7402 cells were grown on glass bottom culture dishes and treated with vehicle (0.1% DMSO only), 5-FU or dioscin at different concentrations (2.5, 5, 7.5 and 10 [micro]mol/l). The culture medium was discarded after 24 h of incubation and the remained cells were washed with new culture medium (serum free). The cells were stained with Hoechst33342 (10 [micro]g/ml) in culture medium (serum free) at room temperature in the dark for 20 min. Subsequently, PI was added to a final concentration of 10[micro]g/ml and incubated for an additional 10 min in the dark. After staining, the cells were washed twice with 1 X PBS and immediately evaluated by a confocal laser scanning microscope (Olympus FluoView[R] FV1000).

Flow cytometric analysis of apoptosis

A fluorescein isothiocyanate (FITC) Annexin-V Apoptosis Detection Kit (Cat No.KlOl-25) was used according to the manufacturer's instructions to evaluate apoptosis. Bel-7402 cells were cultured in 6-well plates for 24 h, followed by incubation with vehicle, 5-FU or dioscin (1, 2.5, 5, 7.5 and 10 [micro]mol/l). After 24 h, the Bel-7402 cells were harvested and washed with cold PBS. The cells were resuspended in 1 ml of 1 x binding buffer. The resuspended cells (100 [micro]l) were transferred to a 5 ml culture tube, and Annexin V-FITC (5 [micro]l) and propidium iodide (PI, 5 [micro]l) were added. The cells were vortexed and incubated for 15 min in the dark. Binding buffer (400 [micro]l) was added to each tube. Flow cytometric analysis was performed immediately after staining. Data acquisition and analysis were performed by a fluorescence-activated cell scanner (FACS) flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells in the early stages of apoptosis were Annexin V-positive and Pi-negative, whereas cells in the late stages of apoptosis were positive for both annexin V and PI. PI stained the necrotic cells.

DNA ladder analysis

Bel-7402 cells (1.5 x [10.sup.6] cells/ml) were exposed to vehicle (0.1% DMSO only), various concentrations of dioscin or 5-FU for 24 h and harvested by centrifugation. Cell pellets were dissolved in 20 pi of lysis buffer (20 mmol//l EDTA, 100mmol/l Tris, pH8.0, 0.8%(w/v) SDS). Cell lysates were treated with 10mg/ml RNase A and incubated at 37[degrees]C for 1 h. The supernatant was incubated overnight at 50[degrees]C with 20 mg/ml proteinase K. The resultant DNA solution was transferred to 2% agarose gel and electrophoresis was performed at 35 V for 4 h with TAE (40mmol/l Tris, 20mmol/l sodium acetate, and 1 mmol/1 EDTA) as the running buffer. DNA was visualized with ethidium bromide (0.5 [micro]g/ml) under UV light.

Western blot analysis of TP53, BAX, BCL2 protein expression and CASP3 cleaved levels

Following treatment with different concentrations of dioscin for 24 h, the protein expression levels of TP53, BAX, BCL2 and CASP3 (P17) were detected by Western blotting. Bel-7402 cells (1 x [10.sup.6]) were washed twice with ice-cold 1 XPBS and lysed with cell lysis buffer at 4[degrees]C for 30 min. Cell debris was removed by centrifugation at 12,000 x g for 10 min at 4[degrees]C. Equal amounts of proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane, which was incubated in the blocking solution for 2 h at room temperature. The membranes were first incubated with primary antibodies at a dilution of 1:1000 for 12 h at 4[degrees]C, followed by extensive washing 2 times with PBS and TBST. The membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) and washed with TBST. GAPDH was used as an internal loading control. The immunoreactive proteins were detected with an ECL Western blot detection system.

In vivo antitumor reactivity

About 0.5 ml abdominal dropsy was collected from nude mice bearing Bel-7402 cells and centrifuged to discard the supernatant. The cells in the sediment were suspended and diluted with serum-free RPMI 1640 medium to 5 x [10.sup.6]/ml. Mice were subcutaneously injected with 0.1 ml cell suspension at axillary region. On the fifth day of inoculation, 50 mice with above-mentioned injection were randomly divided into 5 groups containing 10 animals per group. The mice were intraperitoneally administered with 0.2 ml normal saline daily as vehicle group, and the mice were injected with 5-FU injection 10mg/kg/day (0.2 ml, 1 mg/ml) as 5-FU group. The low-dose group mice, middle-dose group mice, and high-dose group mice were intraperitoneally administrated with dioscin (diluted in sterile saline to achieve desired final concentrations) 6mg/kg/day (0.2 ml 0.6 mg/ml), 12mg/kg/day (0.2 ml 1.2 mg/ml), and 24mg/kg/day (0.2 ml 2.4 mg/ml) respectively. Mice were euthanized at the 11th day after intraperitoneally injection and tumors were harvested, weighed, and photographed. The tumors were measured by vernier calipers and the tumor volume was calculated according to the following formula: Tumor volume = 1/2 x A x [B.sup.2], Where A was the largest and B was the smallest superficial diameter of the tumor. The inhibition rate of tumor growth was calculated using the following equations: Inhibition rate = (W-L)/Wx 100%, where W was tumor weight of vehicle group and L stood for tumor weight of experiment group. Formalin-fixed paraffin-embedded tumor sections were stained with hematoxylin-eosin (HE) for histology.

Statistical analysis

The results of multiple experiments are given as the mean [+ or -] SD. Statistical analysis was performed using the statistical software package SPSS 13.0. Analysis of variance and least significant difference-t (LSD-t) were used for multiple comparisons between mean. A P value of 0.05 (two-sided) was considered statistically significant.

Results

Dioscin inhibits HCC cell growth

To investigate the growth inhibition effects of dioscin, HCC cell lines, such as Bel-7402, HepG2, Lovo, and EAhy926 cells, were treated with various concentrations of dioscin for 48 h. As shown in Fig. 1C, the number of alive cells decreased, accompanied by increasing concentration of dioscin, suggesting dioscin inhibited those cell lines in dose-dependent manner. The IC50 of Bel-7402, HepG2, Lovo, and EAhy926 cells were 3.5uM, 6.4uM, 5.3uM, and 8.5uM respectively, which suggested Bel-7402 cells were most sensitive to Dioscin. Therefore, Bel-7402 cells were used for further investigation. Bel-7402 was treated with various concentrations of dioscin for 24, 48 and 72 h. As shown in Fig. IB, dioscin significantly inhibited Bel-7402 cell growth in a dose- and time-dependent manner. 1C50 values at 24, 48, and 72 h were (9.50 [+ or -] 0.03), (3.54 [+ or -] 0.03) and (1.56 [+ or -] 0.02) [micro]mol/l, respectively. The maximal growth inhibition rate approached 100% in Bel-7402 cells stimulated for 72 h with diosin (10 [micro]mol/l).

Dioscin-induced morphological alterations in Bel-7402 cells

Laser scanning confocal microscopic evaluation of Hoechst33342/PI double staining was performed to observe Bel-7402 cell apoptosis or necrosis after dioscin treatment. Bel-7402 cells exhibited fusiform or flat multilateral structures under differential interference contrast microscope (Fig. 2A). Normal Bel-7402 nuclei displayed uniform blue staining with shallow, deep inside particles (Fig. 2B). The apoptotic cells exhibited nuclear chromatin condensation and dense staining gathered at the nuclear membrane with pyknosis or fragmentation of apoptotic bodies (Fig. 2C). The necrotic cells were stained only with PI (Red, Fig. 2D). Dioscin at higher concentrations (5 [micro]mol/l) resulted in increasing of necrotic cells. These findings suggest that dioscin inhibited the growth rate in a dose- and time-dependent fashion, which was associated with increased apoptosis. Lower concentrations of dioscin induced early apoptosis while the higher concentrations resulted in increased levels of late apoptosis and necrosis.

Dioscin-induced apoptosis of Bel-7402 cells detected by flow cytometry

Following treatment of Bel-7402 cells with vehicle or dioscin (1, 2.5, 5, 7.5 and 10 [micro]mol/l) for 24 h, the proportion of apoptotic cells (M2 + M4) were (0.2 [+ or -] 0.2%), (37.6 [+ or -] 2.5%), (44.5 [+ or -] 2.7%), (45.0 [+ or -] 2.5%), (47.4 [+ or -] 3.2%) and (61.6 [+ or -] 4.1%), respectively (Fig. 3). The degree of apoptosis in each experimental group was significantly higher than that in the control group with increasing dosage of dioscin (P<0.01). The results indicated that with increasing concentration of dioscin, the number of early apoptotic cells (M4) decreased while the number of middle or late apoptotic cells (M2) gradually increased, which were consistent with the Hoechst33342/PI double staining.

Dioscin stimulated DNA fragmentation in Bel-7402 cells

Cells were incubated with series concentrations of dioscin (1, 2.5, 5. 7.5 and 10 [micro]mol/l) in culture medium for 24 h. DNA laddering was observed at 7.5 and 10 [micro]mol/1 of Dioscin (Fig. 4).

Dioscin increased TP53, BAX and cleaved caspase3 protein expression

The protein expression of TP53, BAX and CASP3 protein were evaluated by western blot analysis. Following treatment with dioscin, the expression of TP53, BAX, and cleaved caspase3 were clearly increased, whereas BCL2 decreased, compared to the control (Fig. 5). These results suggest that TP53, BAX, BCL2 and cleaved caspase3 may play a critical role in modulating dioscin-induced apoptosis in HCC.

Dioscin inhibited tumor growth in vivo

As illustrated in Figs. 6-8, the tumor size and tumor weight of mice in 5-FU, low-dose (6mg/kg), middle-dose (12 mg/kg), and high-dose group (24 mg/kg) of dioscin declined significantly compared to control group. The tumor inhibition rate was 76.49%, 49.51%, 79.50% and 86.68% respectively. Therefore, the tumor inhibition rate of high-dose group (24 mg/kg) is higher than 5-FU. Compared to 5-FU group, the tumor size and weight of mice in low-dose (6 mg/kg) group increased significantly, whereas middle-dose (12 mg/kg) and high-dose group (24 mg/kg) mice had no significant difference with 5-FU group. Histology of tumor sections showed necrosis and collapse of tumor cells enlarged with the increase of dioscin dose (Fig. 6B).

Discussion

Many natural products from plants that exert anticancer activity have recently been identified (Hu et al., 2016). Dioscin is a traditional Chinese anti-snake venom medicine that has been shown to have anticancer properties in other cancer cell types, such as human gastric cancer SGC-7901 cells (Gao et al., 2011a), human ovarian cancer SKOV3 cells (Gao et al., 2011a; Man et al., 2010) and human cervical cancer HeLa (Kaskiw et al., 2009), MCF-7 and MDA-MB-231 cells (Aumsuwan et al., 2015). However, the effects of diosin in HCC remain unclear. In the current study, we found that dioscin inhibited the growth of HCC cells, stimulated the induction of apoptosis and inhibited the tumor growth in vivo. In addition, dioscin increased the protein expression of TP53, BAX and cleaved caspase3, which play an important role in apoptosis.

MTT analysis indicated that dioscin inhibited the growth of Bel-7402 cells in both time and concentration dependent manner. Similar effects were also observed in gastric cancer SGC-7901 cells, human ovarian cancer SKOV3 cells treated by dioscin (Gao et al., 2011a, b; Man et al., 2010). Therefore, the participation of apoptosis in dioscin-induced growth inhibition of Bel-7402 cells was evaluated. Morphological changes including nuclear chromatin condensation, dense staining gathered at the nuclear membrane, pyknosis or fragmentation of apoptotic bodies were observed by LSCM for qualitative analysis. Similar nuclear morphological changes was also observed in the SGC-7901 and SKOV3 cells treated by dioscin (Gao et al., 2011a,b; Man et al., 2010). Quantitative analysis of apoptosis by FACS indicated that dioscin induced apoptosis at significantly higher rate than that observed in the control group. In addition as the dosage of dioscin increased, the apoptotic rate gradually increased from early to late stage apoptosis. These findings are different from SKOV3 cells treated with dioscin, which showed an increase of early stage apoptosis without significant increases at late stage apoptosis (Gao et al., 2011b). Furthermore, G2/M phase cell cycle arrest and cyclins B1/CDK1 down-regulation, that occurs in dioscin-induced apoptosis of SGC-7901 (Gao et al., 2011a), was not observed in Bel-7402 cells treated with dioscin (data not shown). DNA ladder analysis indicated DNA extracted from the dioscin treatment groups displayed characteristic DNA fragmentation found in apoptotic cells. These data suggest that dioscin at low doses induces apoptosis and at relatively higher doses triggers late stage apoptosis and/or necrosis in HCC Bel-7402 cells.

Although dioscin could be used as a raw material to synthesize steroid hormone drugs, dioscin did not affect the expression of receptors of steroid hormone, such as estrogen, androgen and progesterone, in Bel-7402 cells (data not shown). Several mechanisms regulating dioscin-inhibited growth and -induced apoptosis have been reported for other cancer cell lines (Liu et al., 2004). The mitochondrial pathway apoptosis pathway was induced by dioscin in Hela cells, which was associated with activation of caspase3, caspase9 and down-regulation BCL2 protein expression (Cai et al., 2002). Another study detected that reactive oxygen species (ROS) generation is the primary mechanism responsible for dioscin-induced proapoptotic activity. The key targets in dioscin-induced apoptosis were PRDX1 and PRDX6 (Wang et al., 2012). Dioscin can also induce dose-dependent [[Ca.sup.2+]] influx and might trigger apoptosis and/or necrosis via calcium ion overload (Gao et al., 2011 a,b). A recent study reported that the death receptor pathway (Fas, FasL, TNF-[alpha], TNFR1, TRAF-1 and FADD) was involved in dioscin-induced apoptosis in SGC7901 (Gao et al., 2011a). The tumor suppressor gene TP53 is a crucial regulator of apoptosis in a number of different cell types (Hong et al., 2014). This current study demonstrated that TP53 expression in the dioscin treatment groups increased significantly. TP53 can regulate expression of genes involving in various processes such as cell cycle arrest, apoptosis and others mechanisms (Hong et al., 2014). TP53 also plays a crucial role in mitochondrial membrane stability and promotes apoptosis (Rich et al., 2000; Vousden, 2000) independent of its transcriptional activity (Caelles et al., 1994). Wild-type TP53 can bind the BAX gene promoter region and regulate BAX gene transcription (Kitada et al., 1996; Moroni et al., 2001). BAX is a pro-apoptotic member of the BCL2 family and a TP53 target (Huang et al., 2007). BAX forms heterodimers with BCL2, which inhibits its activity (Walensky and Gavathiotis, 2011) and promotes the release of cytochrome c from mitochondria into the cytosol, where they facilitate caspase activation and execution of apoptosis (Priault et al., 2003). CASP3 is a major mediator of apoptotic and necrotic cell death (Behar et al., 2011). Both extrinsic and intrinsic pathways converge at CASP3 together with other effector caspases. The results of the current study demonstrate that dioscin upregulates TP53, which may in turn increases BAX protein expression, activates CASP3 protein expression and subsequently induces apoptosis. These data suggest that dioscin may induce apoptosis of HCC Bel-7402 cells via TP53-BAX-caspase3 signaling pathway.

In conclusion, our findings indicate that dioscin could inhibit the growth of HCC cell line and inhibit the tumor growth in vivo, possibly by inducing apoptosis via the TP53, BAX, and caspase3 related mitochondria-mediated pathway. Our findings also imply dioscin could be developed as an anti-cancer drug for HCC patients in the future.

ARTICLE INFO

Article history:

Received 12 March 2016

Revised 28 June 2016

Accepted 3 July 2016

Conflict of interest

We declare that these are no conflict of interest associated with this publication and we have no financial relationships that can inappropriately influence its outcome.

Acknowledgments

This study was partly supported by National Natural Science Foundation of China (No. 81072906, No. 30973811), Guangdong Natural Science Foundation (No.10451130001004472), Zengcheng Science and Technology Innovation Support Funds (No. ZC201004).

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Guangxian Zhang (a), (1), Xiancheng Zeng (b), (1), Ren Zhang (a), (1), Juan Liu (a), Weici Zhang (c), Yujun Zhao (b), Xiaoyuan Zhang (a), Zhixue Wu (a), Yuhui Tan (a), Yingya Wu (a), Biaoyan Du (a), *

(a) School of Fundamental Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

(b) Department of General Surgery, The Second People's Hospital of Guangdong Province, Guangzhou 510317, China

(c) Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, University of California Davis, School of Medicine, Davis, CA 95616, USA

Abbreviations: HCC, Hepatocellular carcinoma: MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide; PI, propidium iodide; LSCM. laser scanning confocal microscopy; BAX. BCL2-associated X protein; BCL2. B-Cell CLL/Lymphoma 2; CASP3, Caspase 3; 5-FU, 5-Fluorouracil; RPMI-1640. Roswell Park Memorial Institute tissue culture medium 1640, DM EM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.

* Corresponding author at: School of Fundamental Medical Sciences. Guangzhou Higher Education Mega Center, 232 Waihuan Rd E. Guangzhou 510006, China. Fax: +86-20-39358020.

E-mail address: dubiaoyan@gzucm.edu.cn (B. Du).

(1) These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.phymed.2016.07.003

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Title Annotation:Original article
Author:Zhang, Guangxian; Zeng, Xiancheng; Zhang, Ren; Liu, Juan; Zhang, Weici; Zhao, Yujun; Zhang, Xiaoyuan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Nov 15, 2016
Words:5542
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