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Antitumor activity of tatariside F isolated from roots of Fagopyrum tataricum (L.) Gaertn against H22 hepatocellular carcinoma via up-regulation of p53.

ABSTRACT

Background: Fagopyrum tataricum (L.) Gaertn is a famous drinking food and herbal medicine in China, and have been commonly used for treating various diseases.

Purpose: This study was designed to investigate the antitumor effect of tatariside F (TF) isolated from the roots of F. tataricum against H22 hepatocellular carcinoma (HCC) in vitro and in vivo and explore the possible mechanisms.

Methods: In our present study, the anti-proliferative effect of TF against H22 cells was evaluated by MTT method. Furthermore, a mice xenograft model was established to investigate the antitumor effect of TF on HCC in vivo, and the possible mechanisms were determined by western blot and fluorescence polarization binding assay. In addition, the protective effect of TF on liver was also investigated by examining the histopathological changes and determining the liver biochemical parameters.

Results: Our results demonstrated that TF possessed notable antitumor effect against HCC both in vivo and in vitro, and the possible mechanism

might be related to up-regulation of the protein expressions of Bax and p53, and down-regulation of Bcl-2. What's more, TF also exhibited protective effects against CTX (cyclophosphamide)-induced liver damages when co-administrated with CTX.

Conclusion: Our study suggested that TF possess notable antitumor effects against HCC and might play a favorable role in drug combination therapy against tumors with protective effect on liver.

Keywords:

Tatariside F

Fagopyrum tataricum (L.) Gaertn

Hepatocellular carcinoma

H22 cell line

Apoptosis

p53

Introduction

Currently, cancer has become one of the most common and serious problems of people's health all over the world (Siegel et al., 2014). Hepatocellular carcinoma (HCC) is reported to be one of the most common and deadly malignant cancers with an increasing incidence rate, especially in Europe and East Asia (Katsuta et al., 2014; Lafaro et al., 2015; Qi et al., 2010); in addition, previous investigations also indicated that HCC is the most severe complication of chronic liver disease in the world (Hsu et al., 2010). Besides surgery, cytotoxicity remained as the conventional and commonly used strategy for curing HCC. Although these treatments have improved greatly, the 5-year survival rate of HCC patients is still poor (Mansour et al., 2014). Additionally, these current chemotherapy by synthetic drug alone commonly results in serious side effects (Kasaiet et al., 2008; Thomas, 2009). Therefore, finding new anti-HCC agents with notable therapeutic effect that can be used in combination with current existing drugs might be a feasible approach to treatment of HCC.

Traditional Chinese medicines (TCM) are well known to be effective in treatment of several diseases, especially the diseases which cannot be cured by modern synthetic drugs (Mohan et al., 2012; Peng et al., 2013). In addition, TCMs have been considered as a new source of anticancer agents or chemotherapy adjuvant to intensify the efficacy of chemotherapy and alleviate its toxic effects (Xu et al., 2013). Fagopyrum tataricum (L.) Gaertn (also called tartary buckwheat), a famous drinking food and herbal medicine in China, have been commonly used to treat lots of stubborn and chronic diseases, including cancers, rheumatic disorders and general debility (Guo, 2003; Guo et al., 2006). In our previous investigation, we have reported that a series of new compounds with significant antitumor activities were isolated from the roots of F. tataricum (Zheng et al., 2012; Li et al., 2014). As part of our continuing study, we further investigated the anticancer effect against HCC of tatariside F (TF, Fig. 1 A), which is one of the new compounds with large quantity. Furthermore, the protective effect of this compound on liver was also investigated in order to provide a scientific basis for the clinical use of TF.

[FIGURE 1 MITTED]

Materials and methods

Plant material

The roots of F. tataricum were collected from Jiande, Zhejiang Province, China in August 2010 and were identified by Prof. Lu-Ping Qjn (Department of Pharmacology, Second Military Medical University). A voucher specimen (#20100816) was deposited in the Department of Pharmacology, Second Military Medical University, Shanghai, P.R. China.

Animals

Experimental groups consisted of ICR mice (Institute of Cancer Research) (20 [+ or -] 2 g), which were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). They were housed at 21 [+ or -] 1[degrees]C under a 12 h light/dark cycle and had free access to standard pellet diet (Purina chow) and tap water. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Animal Experimentation Ethics Committee of Second Military Medical University.

Chemicals

Dimethyl sulfoxide (DMSO) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (MO, USA); the DMEM media and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, California, CA, USA); p53, Bcl-2, Bax, and [beta]-actin antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA); BCA protein assay reagent was purchased from Beyotime (Haimen, China); commercial kits for determination of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were purchased form the Nanjing Jiancheng Bioengineering Institute (Nanjing, China); Silica-gel (100-200, 200-300 mesh) was purchased from Qingdao Haiyang Chemical Co. (Qingdao, China); Sephadex LH-20 was purchased from GE Healthcare Co.(Beijing, China); all other chemicals used in this study were of analytical reagent grade.

Extraction, isolation and preparation of TF

The extraction of TF was carried out according to the method described in our previous paper (Zheng et al., 2012). Briefly, the roots of F. tataricum were roughly powdered and extracted three times with 80% ethanol (v/v) by reflux for 2 h each time. The solvent was evaporated under vacuum to afford crude extract. Then the extract was suspended in water and partitioned with petroleum ether (PE), dichloromethane ([CH.sub.2][Cl.sub.2]), ethyl acetate (EtOAc), and n-butanol (n-BuOH) successively. The EtOAc fraction was subjected to repeated column chromatography over silica gel (100-200 mesh) column chromatography and eluted with [CH.sub.2][Cl.sub.2]-MeOH (30:1~3:1). Combination of similar fractions on the basis of TLC analysis afforded 3 fractions (I-III). By using a series of chromatographic techniques, such as silica gel column chromatography (200-300 mesh) and Sephadex LH-20 chromatography, TF was isolated from fractions III. In addition, NMR and HPLC are used to identify authenticity and purity of TF, and the results demonstrated that the purity of TF was over 98.0% (Fig. 1B).

Cell culture

Human hepatocellular carcinoma cell line H22 was purchased from the American Type Culture Collection (MD, USA). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 [micro]g/ml streptomycin). The cell lines were cultured at 37[degrees]C in 5% C[O.sub.2]/95% air.

Determination of cell viability

Cell viability was performed using MTT assay according to reported method (Peng et al., 2015). Briefly, cells (1 x [10.sup.5] cells/ml) were plated and cultured in 96-well plates for 24 h, and subsequently treated with TF at series concentrations (0, 0.001, 0.01, 0.1,1,10 and 100 [micro]g/ml, and 0.1% DMSO was used to enhance the solubility of TF) for 24 h. Then, MTT assay was carried out to assay cell proliferation inhibition (%) (n = 4) by detecting the optical density (OD) at 570 nm. Then, the half maximal inhibitory concentration ([IC.sub.50)] value was calculated. The inhibition rate was calculated by using the following formula: ([OD.sub.control] - [OD.sub.treatment])/[OD.sub.control] x 100%.

Xenograft model in mice

In order to evaluate the antitumor effects of TF against HCC, four groups were considered: control (model group), cyclophosphamide (CTX) (25 mg/kg), TF (10 mg/kg) and CTX + TF (25 mg + 10 mg/kg) (n = 8). TF and CTX were prepared as emulsion by using egg lecithin, vegetable oil, and oleic acid. Mice were subcutaneously injected in the right back with H22 cells (1 x [10.sup.7]/per mouse/0.2 ml). When the tumors grew to approximate 2-3 mm in diameter, the mice were obtained respective treatments, and equal volume of solvent control. Mice were observed for 15 days, and tumor sizes were measured at 6, 8, 10, 12, and 14 days after the tumor inoculation. Tumor diameters were determined by using a vernier caliper, and subsequently tumor volumes were calculated as the formula: Volume = ([width.sup.2] x length)/2 (Zhou et al., 2013). Mice were sacrificed immediately after 15 days, and tumors tissues were collected and homogenized for further analysis. In addition, the liver tissues were also collected for histopathological examinations.

Histopathological examinations

Tissue sections was dissected and fixed in 10% formalin, which were subsequently embedded in paraffin, sectioned to 5 [micro]m thickness, de-paraffinized, re-hydrated using standard techniques, stained with hematoxylin and eosin (H8*E) (Wang et al., 2011). The histopathological changes in liver tissues were examined under a microscope (Olympus, Japan).

Western blotting

Total proteins of the liver tissues were extracted, and the protein concentration was determined by using BCA protein assay reagent. Then equal amounts of proteins (35 [micro]g) were separated by 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), then blotted on PVDF membrane and probed with various primary antibodies, followed by incubation with horseradish-peroxidase-conjugated secondary antibody and chemiluminescence detection. To normalize for protein loading, antibodies directed against [beta]-actin was used, and the proteins expression levels were expressed as a relative value to that of [beta]-actin.

Fluorescence polarization binding assay

All fluorescence experiments were performed according to the method described in previous literature with minor modification (Moerke, 2009). Briefly, the fluorescence polarization experiments were read on Biotek Synergy H4 with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in black 96-well NBS assay plates at room temperature. The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P = (Intparallel - GIntperpedicular)/(Intparallel + GIntperpedicular), and the correction factor G (G = 0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP). The dose-dependent binding experiments were carried out with serial dilution in DMSO of compounds. A 5 ml sample of the tested sample and pre-incubated (for 30 min) MDM2 binding domain (1-118) (10 nM) and PMDM-F peptide (Anaspec) (10 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 mg/ml bovine gamma globulin; 0.02% sodium azide were added into microplates to produce a final volume of 115 [micro]L. For each assay, the controls included the MDM2 binding domain and PMDM-F. Plates were read at 1 h after mixing all assay components. Binding constant (Ki) and inhibition curves were fitted using the GraphPad Prism software. Nutlin-3a (Sigma-Aldrich), the first potent and specific non-peptide small-molecule MDM2 inhibitor was used as reference compound for validating the assay in each plate.

Determination of the liver biochemical parameters

Liver tissues were collected and homogenized, then centrifuged at 3000 rpm for 10 mins. After that, the supernatant was used for determining the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) following the instructions of the commercial kits.

Statistical analysis

Data are presented as mean [+ or -] S.D, and were evaluated with one-way ANOVA following by Dunnett multiple comparisons test between different groups. The statistical significance of differences was analyzed by using SPSS software (SPSS for Windows 18.0, SPSS Inc., USA), and the differences were considered significant at p < 0.05.

Results

TF possessed notable inhibitory effect against HCC both in vitro and in vivo

The anti-tumor activity of TF against H22 both in vivo and in vitro was evaluated in our present study. The results showed that TF possessed notable anti-proliferative effects against H22 cells with [IC.sub.50] value of 1.31 [micro]g/ml, demonstrating a significant concentration-dependent manner in the concentration range from 0.001 to 100 [micro]g/ml (Fig. 2A).

Furthermore, the antitumor activity of TF against HCC in vivo was further determined in a mice xenograft model. As shown in Fig. 2B and 2C, CTX (25 mg/kg) notably inhibited the tumor growth (p < 0.05). Similar to that of CTX, TF (10 mg/kg) also possessed significant inhibitory effect against both the weight and growth of the H22 tumors (p < 0.05). Interestingly, higher suppression against the tumor growth was observed in mice treated with drug combination CTX + TF (25 mg/kg + 10 mg/kg) (p < 0.01). Our results indicated that combination treatment of CTX + TF can achieve better therapeutic effect against HCC than the administration of CTX or TF alone (p < 0.05).

TF up-regulated p53 and Bax, and down-regulated Bcl-2

To investigate the possible mechanism, we determined the expressions of p53, Bcl-2, and Bax in tumor tissues. As shown in Fig. 3, the p53 proteins was up-regulated dramatically by treatment of CTX (25 mg/kg), TF (10 mg/kg), and CTX + TF (25 + 10 mg/kg) (p < 0.05, p < 0.05, and p < 0.01, respectively), compared with control. Similar to the expression of p53, the Bax protein was also significantly increased by administration of CTX (25 mg/kg), TF (10 mg/kg), and CTX + TF (25 + 10 mg/kg) (p < 0.05, p < 0.05, and p < 0.01, respectively); whereas the expression of Bcl-2 was obviously down-regulated by different treatments (p < 0.05, p < 0.05, and p < 0.01, respectively). What's more, the expressions of p53 and Bax in tumor tissues of CTX + TF (25 + 10 mg/kg) treatment of mice significantly increased compared with that of CTX (25 mg/kg) group (p < 0.05), whereas the Bcl-2 expression in CTX + TF (25 + 10 mg/kg) group markedly decreased compared with CTX alone treated mice (p < 0.05).

TF showed excellent p53-MDM2 inhibitory activity

The binding [K.sub.i] constant of TF was measured by fluorescence polarization (FP) binding assay. According to the results of our present investigation (Fig. 4), TF displayed excellent p53-MDM2 inhibitory activity with [K.sub.i] values of 5.9 [micro]M.

TF possessed significant protective effect on liver tissues

The histopathological changes of the liver tissues are showed in the Fig. 5A. In control group, hepatocytes were well-preserved and uniform cytoplasm, prominent nucleus, nucleolus and central veins were visible. Similar to the control group, no obvious changed was observed in the TF (10 mg/kg) treated mice, and only moderate inflammatory cell infiltration existed. In contrary, liver tissues of mice treated CTX (25 mg/kg) showed extensive liver injuries, characterized by severe hepatocellular degeneration and necrosis, fatty changes, serious inflammatory cell infiltration. Interestingly, the damage of liver tissues in the group of CTX + TF (25 mg/kg + 10 mg/kg) improved greatly compared with the mice treated CTX alone. No obvious necrosis and damage were observed in the mice treated CTX + TF, and just moderate inflammatory cell infiltration presented.

TF can decrease the levels of GSH, SOD, and MDA in liver

As shown in Fig. 5B, effects of TF on the levels of GSH, SOD and MDA in liver tissues were represented. The levels of SOD in TF (10 mg/kg) and CTX + TF (25 mg/kg + 10 mg/kg) groups were increased significantly compared with the control mice (p < 0.05), whereas the levels of MDA were obviously decreased (p < 0.01). In addition, the level of MDA in CTX + TF group significantly decreased compared with mice treated CTX alone (p < 0.05).

Discussion

To the best of our knowledge, this is the first report on the antitumor effect of Tatariside F (TF) from F. tataricum against HCC probably via the up-regulation of p53 and Bax, and down-regulation of Bcl-2. In addition, TF also possessed notable hepatoprotective effect, which can prevent the liver damage induced by CTX.

In our present study, we found thatTF possessed notable cytotoxic effect against HCC with the [IC.sub.50] value of 1.31 [micro]g/ml. In addition, we also determined the effect of TF on HUVECs (a normal human cell line) according to previous report (Peng et al., 2015). The results revealed that no obvious cytotoxic effect on HUVECs was observed for TF below the concentration of 100 [micro]g/ml, with cell proliferation inhibition rate < 15% (Fig. S1). Since 1979, the p53 has been considered to be a pivotal anti-cancer gene and a mainstay of our own natural anticancer defense, and the p53 gene can eliminate and suppress abnormal cells' proliferation, thus preventing the development of cancer (Gasco et al., 2002; Rong et al., 2010; Levine & Oren, 2009). p53 can be activated by a series of cellular stresses, such as DNA damage and abnormal proliferation of cells (Moll & Petrenko, 2004; Vousden & Lan, 2007). The activated p53 can act as a transcriptional factor to regulate gene expressions, resulting in apoptosis and cell cycle arrest (Riley et al., 2008). In addition, previous investigations also reported that p53 can directly activate the pro-apoptotic proteins including Bax, which can also inactivate the anti-apoptotic proteins such as Bcl-2 (Gasco et al., 2002; Tweddle et al., 2003). Therefore, p53 is regarded as a promising target for cancer treatment, and plenty of studies have demonstrated that up-regulating the expression of p53 is beneficial for treating cancers (Kim et al., 2013; Selivanova 8) Wiman, 2007; Zhao et al., 2012). In our present work, we demonstrated that TF can significantly up-regulate the protein expression of p53 in tumor tissues. It is known that p53 is maintained at a low level under normal condition via mediated by the negative regulators MDM2 and MDM4 (Rong et al., 2010; Stommel & Wahl, 2004). Therefore, if the p53-MDM2 interaction could be disrupted or prevented, the p53 will then be released and suppress tumor growth. Our results also demonstrated that TF showed excellent p53-MDM2 inhibitory activity with [K.sub.i] values of 5.9 [micro]M. Both of the in vivo and in vitro results indicated that TF is a promising potential promoter for the up-regulation of p53.

Apoptosis, a programmed cell death, is a tightly regulated process by several proteins (Liu et al., 2010). Bcl-2 family proteins play crucial roles in the intrinsic apoptosis, and are recognized as the first regulatory step for inducing mitochondrial apoptosis (Okada & Mak, 2014). Previous researches on Bcl-2 family proteins revealed that they present a complex network to regulate apoptosis (Okada & Mak, 2014). Additionally, Bcl-2 family proteins are commonly divided into two types, including pro-apoptotic members: Bax, Bak and Bd-xs, and anti-apoptotic members: Bcl-2, Bcl-xl, and Mcl-1 (Yang et al., 2014). Among them, Bcl-2, an anti-apoptotic protein, is reported to prevent the release of cytochrome C into cytoplasm, leading to anti-apoptosis, whereas, Bax, apro-apoptotic protein, promotes the apoptosis. The ratio of Bax and Bcl-2 plays a key role in inducing mitochondrial-mediated apoptosis (Park et al., 2013). In our investigation, the expression of Bax was up-regulated obviously by treatment of TF, whereas Bcl-2 was significantly downregulated, which revealed that the antitumor effect of TF against HCC might be related to the mitochondrial-mediated apoptosis.

Currently, besides surgery, the first option for treating HCC is chemotherapy with chemical drugs, which often results in serious side-effects, such as organ damages (Xu et al., 2013). In particular, liver is an indispensable organ for primary metabolism and detoxification (Li et al., 2013). Therefore, protecting the liver functions is urgent and important for HCC patients. In our present study, cyclophosphamide (CTX), a commonly used antitumor drug, resulted in serious damages in liver tissues of tumor-bearing mice. Interestingly, TF can alleviate this damage dramatically, indicating that TF is an ideal candidate agent for treating HCC with low toxicity on liver. Previous works reported that oxidative stress plays an important role in liver damage caused by drugs, alcohol, and etc. (Conde et al. 2008; Wang et al., 2011). In hepatic oxidative stress, MDA is one of the main end products of lipid peroxidation (LPO), and is commonly used as an indicator of tissue damage. In addition, SOD is a crucial antioxidative enzyme in mammalian cells (Wang et al., 2011). Our results also demonstrated that TF can also increase the level of SOD, and decrease the level of MDA in liver tissues, suggesting that TF exerted hepatoprotective effect by decreasing liver oxidative stress level.

In conclusion, our present study demonstrated that TF possessed notable antitumor effect against HCC, and the possible mechanism might be related to up-regulation of the protein expressions of p53 and Bax, and down-regulation of Bcl-2. In addition, TF also showed protective effects against CTX-induced liver damages when co-administrated with CTX, indicating that TF may play a favorable role in drug combination therapy against tumors with protective effect on liver.

Conflict of interest

The authors declare no conflict of interest in this paper.

Abbreviations: C[H.sup.2][Cl.sup.2], dichloromethane; CTX, cyclophosphamide; DMSO, dimethyl sulfoxide; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; EtOAc, ethyl acetate; FBS, fetal bovine serum; FP, fluorescence polarization; GSH, glutathione; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; ICR mice, Institute of Cancer Research mice; MDA, malondialdehyde; n-BuOH, n-butanol; OD, optical density; PE, petroleum ether; SDS/PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; SOD, superoxide dismutase; TCM, traditional Chinese medicines; TF, tatariside F.

ARTICLE INFO

Article history:

Received 14 April 2015

Accepted 28 April 2015

Acknowledgments

This study was supported by Outstanding Youth Program of Shanghai Medical System (XYQ2013100).

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.05.003.

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Wei Peng (a,b), (1), Changling Hu (a,c), (1), Zhiheng Shu (a), Ting Han (a), Luping Qin (a) **, Chengjian Zheng (a), *

(a) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Cuohe Road, Shanghai 200433, P.R. China

(b) College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, P.R. China

(c) Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, P.R. China

* Corresponding author. Tel.: (+86) 21 81871309; fax: (+86) 21 81871309.

** Corresponding author. Tel.: (+86) 21 81871300; fax: (+86) 21 81871300.

E-mail addresses: qinsmmu@126.com (L. Qin), zhengchengjian@smmu.edu.cn (C. Zheng).

(1) These authors contributed equally to this manuscript.

http://dx.doi.Org/10.1016/j.phymed.2015.05.003
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Author:Peng, Wei; Hu, Changling; Shu, Zhiheng; Han, Ting; Qin, Luping; Zheng, Chengjian
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jul 15, 2015
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