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Chinese medicine formula "Weikang Keli" induces autophagic cell death on human gastric cancer cell line SGC-7901.

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Keywords: Weikang Keli Gastric cancer Autophagy Herbal

ABSTRACT

Weikang Keli (constitutes of Root of Codonopsis pilosula, Rhizoma Atractylodis Macrocephalae, Rhizoma Curcumae Aeruginosae, Rhizoma Pine'hoe, Actinidia chinensis Planch, and Rhodiola rosea) is a well known Chinese herbal formula for gastric cancer therapy in clinical treatment. However, the detailed molecular mechanisms involved are still not fully understood. In this study, we found that Weikang Keli could induce patterns of autophagy in SGC-7901 cells, including intracellular vacuole formation, microtubule-associated protein 1 light chain 3 (LC3) conversion. Hoechst 33258 staining and Western blot analysis of apoptosis-related proteins showed that WK induced SGC-7901 cell death was not through apoptosis. In vivo study also revealed that i.g. administration of Weikang Keli once a day for 25 days could significantly reduce tumor volumes by about 50%. Collectively, the current data indicated that Weikang Kell induced gastric cancer cell death by autophagy effects.

[C] 2012 Elsevier GmbH. All rights reserved.

Introduction

Despite its declining incidence, gastric cancer is still the second most common cause of death from cancer in Asia and worldwide (Kamangar et al. 2006; Leung et al. 2008). Gastric cancer is a serious threat to people's lives and health. Surgery remains the mainstay of any curative treatment; however, approximately two-thirds of patients diagnosed with gastric cancer have high possibilities of recurrence and metastasis after surgeries (Hundahl et al. 2000). Meanwhile, gastric cancer is not sensitive to radiotherapy, turning out to reply on the major treatment approach of chemotherapy. Most patients with advanced gastric cancer need to accept cytotoxic chemotherapy as one of the main treatments. Recently, platinum compounds, 5-fluorouracil and taxanes have been widely used in treatment of gastric cancer. Various attempts have been made to improve the objective response rate to chemotherapy, including chemotherapeutic agents in combination, however, the optimal combination regimen has remained elusive, and standard treatment remains a matter of debate (Ajani 2005). It is necessary to find new compounds and optimized combinational treatment for gastric cancer.

As Traditional Chinese Medicine can improve human immunity, attenuation function and synergies, it has been widely used in adjuvant therapies for cancer. A common view holds that the therapy against cancer shall integrate surgery, radiotherapy, chemotherapy and Traditional Chinese Medicine (Zhu and Si 2005). According to the understanding of the pathogenesis of gastric cancer in Traditional Chinese Medicine, based on the aim of tonifying spleen, invigorating the circulation of blood and detoxifying (Fu et al. 2011: Xu et al. 2003), a Chinese herbal compound, Weikang Keli (WK), constitutes six ingredients including Root of Codonopsis pilosula, Rhizoma Atractylodis Macrocephalae, Rhizoma Curcumae Aeruginosae, Rhizoma Pinelliae, Actinidia chinensis Planch, and Rhodiola rosea, can be used in the therapy against cancer. Studies have verified that sesquiterpene compounds extracted from Rhizoma Atractylodis Macrocephalae can induce cell differentiation of melanoma B16 and suppress cell migration via Ras/Erk and PI3K/Akt signaling pathways (Yan et al. 2011). The essential ingredient in Rhizoma Curcumae Aeruginosae, curcumol, can induce the apoptosis of lung cancer cell ASTC-a-1 through caspase nondependent mitochondrial approach (Zhang et al. 2011). Normal butanol extracts of Actinidia chinensis Planch plays a key role in suppressing lung cancer cell A549, which is hold in the stage of GO-G1 with a significantly reduced ratio in the S stage (Wang et al. 2010). Ester plant hormone extracted from Rhodiola rosea plays a dual role of protection and treatment for cells. Experiments in vivo and in vitro have confirmed that it can suppress the proliferation, migration and invasion of breast cancer cells, and can induce the generation of autophagy vesicles of human breast cancer cell MDA-MB-231 and mouse breast cancer cell V14 while shunning away the impact on the normal breast epithelial cells (Tu et al. 2008). WK has performed excellent efficacy in the clinical prevention and treatment of gastric cancer. However, the specific mechanism of WK remains not clarified. In this experiment, the human gastric cancer cell line SGC-7901 was taken as the object of study. Through in vitro and in vivo study, this paper intends to explore the mechanism of WK on suppressing the growth of gastric cancer cells.

Materials and methods

Cell line and animals

Human gastric cancer cell line SGC-7901 from Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, were cultured in RPMI-1640 medium (Invitrogen Corp, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 100 Units/m1 penicillin, 10014/ml streptomycin at 37[degress]C in a humidified atmosphere of 95% air and 5% [CO.sub.2]. BALB/c-nu mice were purchased from Shanghai SLRC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed under specific pathogen-free conditions and provided with a standard rodent laboratory diet from Shanghai SLRC Laboratory Animal Co., Ltd.

Single herb and WK preparation

WK is composed of Root of Codonopsis pilosula (10 g), Rhizoma Atractylodis macrocephalae (15g), Rhizoma Curcumae aeruginosae (10 g), Rhizoma Pinelliae (10 g), Actinidia chinensis Planch (15 g), and Rhodiola rosea (15 g). All medicinal plants used to prepare formulae were provided by Jiangsu Province Hospital on Integration of Chinese and Western Medicine (Nanjing, Jiangsu, China), plant parts, and origin used in the formula as Table 1. Aqueous extract of single herb and WK were prepared according to the following procedure: single herb or six medicinal materials were homogenized with a Waring blender, then soaked in the tenfold double distilled water for 24 h. The mixture was heated to 100[degrees]C for 2 h, and the decoction was filtrated. The filtrates obtained from 2 cycles of the procedures were mixed, concentrated by heating and granulated by lyophilization. Total yield of the WK extract is 200 g lyophilized powder from water extract of 1 kg raw mixed herb. WK and its preparations were standardized, regulated, and quality-controlled according to the guidelines defined by Chinese State Food and Drug Administration (SFDA). Standardization of herbal extracts was done using I-IPLC fingerprinting with chemical standards (adenosine and lobetyolin) purchased from National Institute for the Control of Pharmaceutical and Biological Products in China.

Table 1
The composition of WK.

Species         Chinese    Plant  Origin     Crams,      %
                name       part                   g

Root of         Dang shen  Root   Cansu,         10   13.3
Codonopsis                        China
pilosula

Rhizoma         Sheng bai  Root,  Zhejiang,      15   20.0
Atractytodis    zhu        stem   China
Macrocephatae

Rhizoma         E zhu      Root,  Guangxi,       10   13.3
Curcumae                   stem   China
Aeruginosae

Rhizoma         Fa ban     Tuber  Sichuan,       10   13.3
Pinelliae       xia               China

Actinidia       Teng li    Root   Yunnan,        15   20.0
chinensis       gen               China
Planch

Rhodiola rosea  Hongjing   Root,  Xizang,        15   20.0
                tian       stem   China

Total amount                                     75  100.0

Table 2
Characterization of compounds in WK by HPLC.

Peak  RT(tR,  Compound name
no.     min)

1      13.38  Adenosine

2      27.35  Di-(2-ethylhexyl) phthalate

3      34.30  5-Hydroxy4',7-dimethoxy-flavone

4      35.95  (--)-Epicatechin

5      36.24  Salidioside

6      48.31  Stearic acid

7      49.43  n-Heneicosane

8      54.74  Lobetyolin

9      73.23  Zeylanine

10     89.42  Codonolactone

m     105.49  Zedoarolide A

Peak  Molecular formula                       Classification
no.                                                      (a)

1     C.sub.10][H.sub.13][N.sub.5][O.sub.4]                4

2     [C.sub.24][H.sub.34][D.sub.4][O.sub.4]               1

3     [C.sub.17][H.sub.14][O.sub.5]                        1

4     [C.sub.15][H.sub.14][O.sub.6]

5     [C.sub.14][H.sub.20][O.sub.7]                        6

6     [C.sub.18][H.sub.36][O.sub.2]                        1

7     [C.sub.21][H.sub.44]                                 1

8     [C.sub.20][H.sub.28][O.sub.8]                        1

9     [C.sub.15][H.sub.20][O.sub.4]                        2

10    [C.sub.15][H.sub.20][O.sub.3]                        1

m     [C.sub.15][H.sub.20][O.sub.6]                        3

a (1 )Roor of Codonopsis pilosula: (2) Rhizoma Atractylodis
macrocephcdae: (3) Rhizoma Curcumae aeruginosae: (4) Rhizoma
Pinelliae; (5)Acrinidia chinensis Planch; (6) Rhodiola rosea.


HPLC analysis

HPLC fingerprint of single herb and WK was performed as following procedure: Extracts were separated by a Waters 2695 HPLC instrument (Waters, USA) equipped with a Alltima C18 (250 mm x 4.6 mm, 5 [mu]m) column. The mobile phase consisted of water (I) and acetonitrile (II) at a flow rate of 1.0 ml [mm.sup.-1]. A gradient program was used as follows: 0 min, 5% II; 17 min, 10% II; 36 min, 17% II; 100 min, 60% II; 110 min, 100% II; 120 min, 100% II. The PDA detector scanned from 200 nm to 600 nm, the monitor wave length was set at 254 nm. Fig. 1 showed the HPLC fingerprint of WK extracts (Table 2).

Cell morphological assessment

SGC-7901 cells were cultured in RPMI-1640 till mid-log phase. Negative control ([H.sub.20]), positive control (hydroxychloroquine, HCQ), WK (0.005,0.01 and 0.02 giml) was then added to the culture medium. The morphology of cells was monitored under an inverted microscope (Zeiss Axio Observer Al) at 2h, 6h, 12h and 24h.

Cytotoxicity assay

SGC-7901 cells were seeded in 96 well plates, 1 x [10.sup.4] cells per well. The medium was replaced by serum-free medium containing different concentration of WK. After 24h, 48h and 72h, the viability of SGC-7901 cells were analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu m bromide] assay as described previously (Cai et al. 2011). The percent inhibition of the treated cells was calculated by the formula: % inhibition =1--([0D.sub.570nm]--[OD.sub.630nm])treated/([0D.sub.570nm]--[OD.sub.630 nm])control x 100%.

Immunofluorescent analysis

WK induced autophagy on SGC-7901 cells was examined using an immunocytochemical method. SGC-7901 cells were plated in 24 well plates at a density of 5 x [10.sup.4] cells per well. The next clay, medium was changed to serum-free medium for 12h before stimulating with 0.02 g/m1 WK for 2h or 50[mu],M HCQ for 24h. Treated cells were terminated by washing with ice cold PBS followed by fixation with 4% formaldehyde for 30 min at 4[degrees]C. Cells were permeabilized with 0.05% NP-40 (Amresco, USA) for 30 min at room temperature, washed with PBS and blocked with 1% bovine serum albumin (BSA) for 1h. Rabbit anti-LC3 antibody was added and incubated overnight at 4[degrees]C. After washing with PBS for three times, goat anti-rabbit IgG-FITC was added and incubated for 1h at room temperature. After that, cells were incubated with Hoechst 333258 for 15 min at room temperature. Fluorescence cells were observed and photographed under a fluorescence microscope.

Western blotting analysis

For preparation of cell extracts, cells were treated as described in the figure legends and lysed with lysis buffer (Beyotime, China) on ice. Lysate was centrifuged at 13,000 x g for 5 min at 4[degrees]C. The concentration of protein in the supernatants was detected by Nanodrop 1000 Spectrophotometer (Thermo, NH, USA). Equal amount of protein was separated on 13% SDS-polyacrylamide gels (SDS-PAGE) and transferred onto the PVDF membranes (Millipore, MA, USA). Proteins were detected using specific primary antibodies (Mcl-1, Bc1-2, Bcl-[X.sub.L], Bax, PARP, LC3, GAPDH) (Mcl-1, Bcl-2, Bc1-[X.sub.L], PARP, LC3 antibodies were obtained from Cell Signal Technology, Bax antibody was purchased from BD Biosciences, GAPDH antibodies were obtained from Santa Cruz Biotechnology) for 2h at room temperature, followed by 1-IRP conjugated secondary antibodies for 1h at room temperature. Detection was performed by the enhanced chemiluminescence (Luminata Crescendo Western FIRP substrate, Millipore, USA). Membranes were then exposed to film.

In vivo tumor xenograft experiment in nude mice

BALB/c-nu mice at 4 weeks of age, weighting 14-15g were used after 1-week's compatible raise. A 200 lii cell suspension of 2 x [10.sup.6] SGC-7901 cells, 1:1 mixed with matrigel (BD Biosciences, USA), were subcutaneously injected into the right flank of nude mice. Model animals with a tumor volume of 100 [mm.sup.3] were selected and randomly divided into five groups of ten mice. Three groups were received different dosages of WI< treatment (i.g.) daily: 2.4 g/kg, 4.8 g/kg, and 9.6 g/kg body weight, respectively. The two control group received 0.9% normal saline or 15 mg/kg 5-Fu (i.p.). Tumor volume and weight were recorded every 3 days, the tumor volume was calculated using the formula: Tumor volume ([mm.sup.3]) = 1/2 (length x [width.sup.2]). After 4 weeks treatment, all mice were killed and then tumor was segregated and weighed.

Statistical evaluation

All results shown represent the mean [+ or -]S.E.M. from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistical analyses were performed using an unpaired, two-tailed Student's t-test.

Results

WK suppressed the proliferation of gastric cancer cells SGC-7901

WIT assay was applied to analyze the inhibition effect of WK on cell growth. Fig. 2 showed that WK inhibited proliferation of SGC-7901 cells in a dose-dependent, time-independent manner with the [IC.sub.50] of 0.02 g/ml. Interestingly, marketable vacuoles in the cytoplasm of WK-treated cells were observed under an inverted light microscope (Fig. 3C-E). However, this phenomenon was not observed in negative control (Fig. 3A). In order to clarify whether this phenomenon was related with autophagy, HCQ was used as a positive control. HCQ a lysosomal alkalization agent, could prevent autophagosome fusion with lysosomes, which leading to increased accumulation of autophagosome. Fortunately, the same phenomenon occurred in HCQ-treated group (Fig. 3B). These observations suggested that WK can possibly induce autophagic cell death, which should be validated by further experiments.

WK induced cell death in a non-apoptotic approach

Apoptosis was controlled by regulators, which have either an inhibitory effect on programmed cell death (anti-apoptotic) or block the protective effect of inhibitors (pro-apoptotic) (Vaux 1993; Wang et al. 1996). After treatment with WK, whole cell lysis extraction was prepared to detect the anti-apoptotic proteins (Bcl-2, Bcl-[X.sub.L], Mcl-1) and pro-apoptotic protein (Bax). As shown in Fig. 4, those Bcl-2 family proteins did not present significant change. If cells triggered caspase-dependent apoptosis pathway, PARP will be cut to inactivation (Yu et at. 2006). Cleavage occurs at aspartic acid 214 and glycine 215, separating PARP into a 24 kDa and 89 kDa segment. Neither of these two bands was detected in Western blot analysis. In summary, we believe that WK was not caused cell death by apoptotic pathway.

WK induced autophagy on SGC-7901 cells

To confirm that WK-induced cell death was largely non-apoptotic, nuclear morphology was evaluated by Hoechst 33258 staining. SGC-7901 cells were treated with vehicle control, HCQ or WK. The nuclei of HCQ-treated and WK-treated cells appeared similar to control 1-120-treated cells (Fig. 5). LC3 protein, which is a biochemical marker for autophagic cells (Tanida 2011), was used to obtain independent evidence supporting the conclusion that WK triggers autophagocytosis. WK-treated cells demonstrated an intense, punctuate fluorescence pattern as positive control HCQ. In contrast, vehicle control cells did not have this fluorescence pattern.

Western blotting was used to detect the impact of WK on the cleavage of protein LC3. LC3 has two forms, LC3-I and LC3-II. The latter is located on the autophagosomes, and always remains on the membrane. LC3-11 is considered as the sign molecule of autophagy. As shown in Fig. 6, 24h exposure to different concentrations of WK, the expression of LC3-1 protein reduced while that of LC3-II increased. After 2 h, 12 h and 24 h treatment with 0.02 g/m1 WK, the expression of LC3-II was significantly higher than that of the negative control group. In the positive control group, 24 h treatment with 50 [micro]M HCQ the same phenomenon was observed, indicating that WK could induce autophagy on SGC-7901 cells.

Significantly reduced tumor volume in nude mice gastric cancer model by WK

In order to confirm the antineoplastic effects of WK for gastric cancer in vivo, SGC-7901 cells were injected in nude mice and treated with 2.4, 4.8, 9.6g/m1 WK for 25 days starting after tumor growth to 100 [mm.sup.3]. 5-Fu used as positive control. As shown in Fig. 7, after administration of water extraction of WI< to nude mice, successfully decreased the weight and volume of tumor (p < 0.05). Vehicle treated animals exhibited a median tumor weight of 3.307 [+ or -] 1.213g, whereas 2.4, 4.8, 9.6 g/ml WK-treated animals revealed tumor weight of 1.888 [+ or -] 0.711, 1.486 [+ or -] 0.524, 1.406 [+ or -] 0.680, reflecting 43%, 55%, and 57% reduction, respectively. 5-Fu reduced tumor weight by 51%.

Discussion

WK is a prescription of Jiangsu Province Institute of Traditional Chinese Medicine, which has been validated for many years in clinical treatment (Xu et al. 2003). In the prescription, Root of Codonopsis pilosula mainly helps tonifying spleen, supplemented by Rhizoma Atractylodis Macrocephalae. Rhizoma Pinelliae reduces phlegm and tonifies the stomach. Rhodiola rosea promotes the circulation of blood while Actinidia chinensis Planch promotes the detoxification, reduces dampness and clears the heat. Rhizoma Curcumae Aerugi-nosae expels stasis. All these ingredients are integrated in WK with enhanced functions. WK has performed outstanding efficacy in improving the immunity, the anticancer functions of chemotherapy drugs and life quality of patients. In addition, ingredients such as Rhizoma Atractylodis Macrocephalae, Rhizoma Curcumae Aerug-inosae, Actinidia chinensis Planch and Rhodiola rosea have been proved to be effective in inhibiting tumor growth, reducing invasion, promoting apoptosis and regulating the immunity of patients (Liu et at. 2008; Tu et al. 2008; Wang et al. 2010; Yan et at. 2011; Zhang et al. 2011).

Programmed cell death was generally divided into apoptosis, autophagy and necrosis. Current studies focused on the promotion of tumor cells apoptosis by monomer of Traditional Chinese Medicine while a few reported the promotion of autophagy. No report or literature has addressed that Chinese herbal compound can induce autophagy of tumor cells.

We firstly explored the mechanism of inhibition effect of WK on SGC-7901 cells growth from the apoptosis aspect. Bc1-2, Mcl-1, Bcl-[X.sub.L] and Bax are members of Bc1-2 family, and can regulate mitochondrial apoptosis with P53 (Wang et al. 2011). PARP is the segment substrate of caspase, a core member of apoptosis and plays an important role in apoptosis. In this study, different concentration of WK was applied to SGC-7901 cells. The expression of apoptosis-related proteins was analyzed by Western blot. The results showed no significant changes between vehicle control group and WK-treated group, which indicated that WK induced cell death of SGC-7901 with a non-apoptotic approach.

In the observation of cell growth under inverted light microscope, we found that 2 h treatment with the water extract of WK, vacuolar changes were observed, similar to autophagy. Then, researches on autophagy were conducted. LC3, as the sign molecule of autophagy of mammalian cells, can be detected by immunoblotting, immunoprecipitation, or immunofluorescence detection methods. After the treatment of WK, green punctate fluorescent granules were observed scattering in the cytoplasm, similar to cells treated by HCQ. In negative control group, few green spots were observed. Western blotting analysis found that the expression of LC3-1protein reduced while that of LC3-ll increased in SGC-7901 cells after exposure to WK.

Further animal experiments successfully constructed in vivo transplantation gastric cancer model. The weight and volume of tumor in both WK treatment group and 5-Fu group decreased, yet showing no significant difference (p > 0.05). However, the movements and responsive sensitiveness and food-intake of nude mice in the WK treatment group were obviously better than those of the vehicle control group and 5-Fu group.

In summary, WK can possibly inhibited the growth of SGC-7901 cells in vitro and in vivo through autophagy, meanwhile, improve the life quality of nude mice. However, the specific signaling pathways of autophagy need to be further studied.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 81202967, 81274150 and 30873410) and Jiangsu Province's Outstanding Leader Program of Traditional Chinese Medicine.

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* Corresponding author at: Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, #100 Shizi Street, Hongshan Road, Nanjing, Jiangsu, China. Tel.: +86 25 85608666; fax: +86 25 85608666.

** Corresponding author.

E-mail addresses: wxn002005@126.com (X. Wang), pcao79@yahoo.com (P. Cao).

1 These authors contributed equally to this work.

Jiege Huo (a), (1) Fengxia Qin (b), (1), Xueting Cai (c), Jianming Ju (c), Chunping Hu (c), Zhigang Wang (c), Wuguang Lu (c), Xiaoning Wang (a), **, Peng Cao (c), *

(a.) Department of Oncology, Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing 210028, Jiangsu, China

(b.) Department of Emergency, Jiangsu Province Hospital on Integration of Chinese and Western Medicine, Nanjing 210028, Jiangsu, China

(c.) Laboratoiy of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, Jiangsu, China
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Author:Huo, Jiege; Qin, Fengxia; Caic, Xueting; Ju, Jianming; Hu, Chunping; Wang, Zhigang; Lu, Wuguang; Wan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jan 15, 2013
Words:4039
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