Inhibitiont of Obazema on Proliferation of Gastric Cancer BGC-823 Cells and Its

Ying LI1,2, Risha WEIZE1, Hairong ZHONG1, Jixiu SHEN1, Yisong LI1, Yuan LIU

1. College of Pharmacy, Southwest Minzu University, Chengdu 610041, China; 2. Ethnic Medicine Institute, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] This study aimed to investigate the inhibiting effect of Obazema on proliferation of gastric cancer BGC-823 cells and its mechanism. [Methods] BGC-823 cells were treated with high, medium and low concentrations of drug-containing serum (0.316%, 0.158% and 0.079%) for 0, 48, 72 and 96 h, respectively. Then, the proliferation of the cells was detected with CCK-8 method, and the expression of related proteins, B lymphocytoma-2 (Bcl-2), phosphorylated protein kinase B (p-Akt), protein kinase B (Akt) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was detected using Western blotting. [Results] The proliferation of the BGC-823 cells was significantly inhibited with different doses of Boenninghausenia albiflora (Hook.) Reichb. ex Meisn. var. albiflora (CH) and B. sessilicarpa Lévl. (S) (P<0.01), in dose-dependent and time-dependent manners. The inhibition of high-dose S on cell proliferation was similar to that of CTX 48 h after administration; the inhibition of high-dose CH on cell proliferation was significantly stronger than that of CTX (P<0.01); different doses of drug administration groups significantly inhibited the expression of p-Akt and Bcl-2 in the BGC-823 cells; the inhibition of high-dose CH on the expression of P-Akt and Bcl-2 and the inhibition of medium-dose CH on the expression of Bcl-2 were significantly stronger than that of CTX (P<0.05, P<0.01), in a certain dose-dependent manner; at the same dose, the inhibition of CH on the expression of the proteins was stronger than that of S (P<0.05, P<0.01); administration of S and CH significantly inhibited the expression of GAPDH compared with CTX (P<0.05, P<0.01). [Conclusions] Obazema has the capacity to inhibit the proliferation of BGC-823 cells. The mechanism may be achieved by inhibiting the expression of p-Akt and Bcl-2, and GAPDH may be the target gene of its anti-tumor mechanism. The inhibiting effect of CH on BGC-823 cells was more significantly than that of S.

Key words Yi Medicine, Obazema, BGC-823 cell, Boenninghausenia albiflora (Hook.) Reichb. ex Meisn. var. albiflora, Boenninghausenia sessilicarpa Lévl.

1 Introduction

The latest national cancer statistics released by the National Cancer Center of China in January, 2019 showed that gastric cancer was the third biggest cause of death among cancers, and it was also the third largest malignant tumor that harms human health[1-2]. Across the whole world, nearly 50% of new gastric cancer patients and deaths occur in China, and four-fifths of the patients with gastric cancer are diagnosed with advanced stages[3]. Patients with advanced gastric cancer usually have a poor prognosis, leading to clinical treatment failure, relapse and death[2-3]. Chemotherapy drugs are not ideal in clinical efficacy and adverse reactions. Mortality remains high, with a five-year survival rate of less than 30%[4]. Therefore, finding effective and safe anti-cancer drugs from traditional Chinese medicine has become a research hotspot for people to overcome cancers[5].

Obazema is a Yi Medicine, which is the root or whole grass ofBoenninghauseniaalbiflora(Hook.) Reichb. ex Meisn. var.albifloraandBoenninghauseniasessilicarpaLévl[6-7], also known as Chouchongcao, Yuxiangcao, Yanjiaocao or Songfengcao[6, 8-10]. It is a commonly used medicine in Yi Medicine, fresh or dry. At the same time, it is also a famous aromatic plant of Himalayan. It is first recorded in Southern Yunnan Materia Medica, mainly for treating chest pain, heart coldness, stomach pain, abdominal distension and sore-toxin. According toYizuYiyaoHuicui, Obzema can be used to treat stomach pain, acute nephritis, cystitis and acute gastroenteritis. It was ever included in the 1977 edition of theChinesePharmacopoeia, and currently,B.sessilicarpaLévl is recorded inStandardsforTraditionalChineseMedicineinYunnan(Vol.2):YiMedicine(2005 edition). Due to the wide distribution of this medicinal material, it is also included in books on local Chinese medicine and flora in Sichuan, Guizhou, Yunnan, Jiangxi, Zhejiang and Taiwan, China.

According to the theory of Yi Medicine[11], Obazema belongs to the soil in the five elements. "Oba" means frog, describing the symptom of flatulence being treated. "Zema" means pepper, with spicy flavor, belonging to Qing (yang), and can be used to relieve diseases caused by cold coagulation. In Yi Medicine, Obazema has a wide range of clinical applications, for treatment of gastric cancer, gastroenteritis, cold, bronchitis, malaria and other symptoms. For the treatment of gastric cancer, Obazema removes blood stasis and promotes blood circulation by removing the cold coagulation in stomach to reinforce Qing (yang), and at the same time, it diminishes swelling and relieves pain, with good efficacy. However, the mechanism of action has not been reported.

In this experiment, the inhibiting effect of Obazema on gastric cancer BGC-823 cells was investigatedinvitro, and the expression of B lymphocytoma-2 (Bcl-2), phosphorylated protein kinase B (p-Akt), protein kinase B (Akt) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in BGC-823 cells was detected to study the anti-gastric cancer activity and mechanism of Obazema, with a view to providing a basis for clinical application.

2 Materials and methods

2.1 Materials

2.1.1Cells and experimental animals. BGC-823 cells were purchased from the Cell Bank (Shanghai), Chinese Academy of Sciences.

SPF-grade male SD rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (certificate No.SCXK[Sichuan]2015-024). After one week of adaptive feeding, the rats were provided with free access to standard pellet feed and purified UP water. The temperature and humidity of the animal room were controlled at 23-28 ℃and 40%-55%, respectively.

2.1.2Drugs and reagents. Obazema was identified as the dried whole grass ofB.sessilicarpaLévl andB.albiflora(Hook.) Reichb. ex Meisn. var. albiflora by Professor Liu Yuan and Dr. Li Ying of Southwest Minzu University. It was collected from Liukou Village (103°01′48.68″ E, 27°39′59.28″ N; 1 988.1 m asl), Diluo Township, Butuo County, Liangshan Yi Autonomous Prefecture on July 7, 2018.

DMEM high-sugar medium was purchased from the HyClone Laboratories Inc (USA). Fetal bovine serum (FBS) was purchased from GIBCO Inc (USA). Trypsin-EDTA digestion solution (0.25%), penicillin-streptomycin mixed solution (100 ×), RIPA tissue/cell rapid lysate, Tris-HCl (pH 8.8) running buffer, Tris-HCl (pH 6.8) running buffer, 10% SDS, 10% ammonium persulfate, TEMED, protein loading buffer (4 ×), skimmed milk powder, PBS phosphate buffer and Tween-20 were purchased from Beijing Solarbio Technology Co., Ltd (China). CCK-8 cell proliferation and toxicity analysis kit was purchased from American SAB Tech Inc (USA). BCA protein assay kit was purchased from Thermo Electron Corporation (USA). Protein pre-stained marker was purchased from Fermentas Inc (USA). NC film and illuminant were purchased from Millipore Corporation (USA). Bcl-2 antibody was purchased from Abcam Inc (USA). P-Akt antibody, Akt antibody and GAPDH antibody were purchased from the American CST Inc (USA). Goat anti-rabbit IgG secondary antibody (HRP) was purchased from Shanghai Beyotime Biotechnology Co., Ltd (China). The positive drug cyclophosphamide (CTX) for injection was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd.

2.1.3Instruments and equipment. The used instruments and equipment included TDZ4-WS centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., China), P-type pipette (Gilson, France), DNM-9602 enzyme-labeled analyzer (Beijing Prolong New Technology Co., Ltd., China), TR4001, TR4002, TR5000 cell culture dishes (Trueline, USA), BHC-1300IIB2 biological safety cabinet (Suzhou Jinjing Purification Equipment Company, China), Thermo Forma 3111 CO2constant-temperature incubator (Thermo, USA), XDS-500C inverted microscope (Shanghai Caikang Optical Instrument Co., Ltd.), Tanon-5200 imaging system (Shanghai Tianneng Technology Co., Ltd.), mini protean 3 cell electrophoresis instrument (BIO-RAD, USA), TE77XP electrorotation instrument (Hoefer, USA), MK3 microplate reader (Rabb, Finland), P-type pipette (Gilson, France) and HI1210 water bath (Leica, Germany).

2.2 Methods

2.2.1Preparation of test solutions. Combining the daily dose of Yi Medicine in clinics and the ratio of surface area of rat to human body, the intragastric administration dose was converted. Appropriate amounts of the powder ofB.sessilicarpaLévl andB.albiflora(Hook.) Reichb. ex Meisn. var.albiflorawere weighed, decocted with water, and centrifuged, and the supernatant was mixed. Then, the mixed supernatant was concentrated to 2.0 g/mL of crud drug, sealed and stored at 4 ℃ for use.

2.2.2Preparation of drug containing serum. Forty-eight male SD rats, weighing (200 ± 20) g were evenly and randomly divided into 8 groups (control group-A1; CTX group-B1; low-dose S group-C1; medium-dose S group-D1; high-dose S group-E1; low-dose CH group-F1, medium-dose CH group-G1 and high-dose CH group-H1). Before the beginning of the experiment, the rats were fastened for 12 h, but provided with free access to drinking water. Different doses (0.16, 0.32 and 0.64 g/200 g/d) ofB.sessilicarpaLévl. (S) andB.albiflora(Hook.) Reichb. ex Meisn. var.albiflora(CH) were given to the rats orally. The rats in the CTX group were injected intraperitoneally at a dose of 20 mg/200 g/d. The rats in the control group were given with an equal volume of normal saline once a day. The administration lasted for one week. Blood was collected from the posterior orbital venous plexus of each rat 0.5 h after the last administration. After standing for 1 h, the blood samples were centrifuged at 4 500 r/min for 10 min, and the serum was collected and preserved at -80 ℃ for analysis.

Just before the beginning of cell culture, the serum samples were taken out of the refrigerator, thawed at room temperature, inactivated in a 56 ℃ water bath for 30 min, prepared into DMEM medium containing 10% of serum and passed through 0.22-μm filter, respectively.

2.2.3Cell culture. The BGC-823 cells were inoculated into DMEM high-sugar culture medium containing 10% FBS and 1% penicillin-streptomycin mixed solution and incubated in an incubator (37 ℃, 5% CO2). The cultures were passaged when they reached 80%-90% confluence, that is, one every 2 d. The cells in the logarithmic growth phase and in good condition were selected for further analysis.

2.2.4Detection of cell proliferation with CCK-8 method. The cells in the logarithmic growth phase were selected, trypsinized, and diluted to 5×104cells/mL. A certain volume (100 μL) of each of the six cell suspensions in each group was transferred to one well of a 96-well cell culture plate (5×103cells/well), and then cultured at 37 ℃ overnight, with 100 μL of the culture medium as a blank control.

At 0, 48, 72 and 96 h, 10 μL of the culture in each well was pipetted out, transferred to one well of a new plate (one new plate for each time point), added with 90 μL of each of Cell Counting Kit -8 (CCK-8) and serum-free essential medium, and cultured in an incubator (37 ℃, 5% CO2) for 1 h, respectively. Then, the absorbances (A) of the new cultures were determined immediately using a microplate reader (λ = 450 nm), and the cell proliferation activity was calculated.

Cell proliferation activity = (Adrug-Ablank)/(Acontrol-Ablank)

2.2.5Expression detection of Bcl-2, P-Akt and Akt proteins in cells using Western blotting. The cells in the logarithmic growth phase were selected and prepared into cell suspensions of 7×105cells/mL. The cells were inoculated into 6-well culture plates, and after a 24-h culture, the cells were re-inoculated into the drug-containing serum and cultured for 24 h. Three replicates were arranged for each group. After the culture ended, the cells were collected on ice to extract total protein. The cells of each replicate were added with lysate according to the dose of 150 μL of lysate per 20 mg of cells, homogenized, and centrifuged at 12 000 r/min for 15 min at 4 ℃, and the supernatant was collected. After protein concentration was determined with the BCA protein assay kit, the supernatant was stored at -80 ℃.

In a centrifuge tube, 20 μg of each of the protein samples was added, followed by 4×protein loading buffer. After denaturing in a boiling water batch for 10 min, the mixtures were centrifuged, and the supernatant was collected for loading. SDS-PAGE electrophoresis was performed at 80 V for 120 min (spacer gel, 8 V for 20 min; separating gel, 120 V for 60 min). The proteins were transferred to a NC membrane using the semi-dry method (25 V, 30 min). The NC membrane was then soaked in 5% skimmed milk powder solution (BSA for phosphorylated protein) at 4 ℃ overnight. The primary antibody was diluted to the required concentration with the blocking solution according to the instruction, and the membrane was incubated with the diluted primary antibody at room temperature for 2 h. The membrane was then washed three times with TBST, 5 min/time. The HRP-labeled secondary antibody was diluted according to the ratio of 1∶1 000. The membrane was then incubated with the diluted secondary antibody at 37 ℃ for 1 h. The membrane was washed three times with TBST, 5 min/time. ECL color-developing solution was prepared in the dark. The front of the membrane was added with a certain volume of the ECL color-developing solution and stood in dark for 5 min. The color-developing solution was discarded, and the remaining on the surface of the membrane was dried with paper carefully. After covering a flat layer of transparent paper, the membrane was transferred to the imaging system for scanning. The protein band images obtained were analyzed using a gel image processing system.

3 Results and analysis

3.1 Effects on cell proliferationAs shown in Table 1 and Fig.1, compared with the control group, all the administration groups and CTX group showed significant inhibiting effects on the proliferation on BGC-823 cells (P<0.01), and the inhibiting effects of the S and CH administration groups on the proliferation of the cancer cells showed obvious dose-dependent and time-dependent relationships. The inhibiting effect of High-dose S on the proliferation of the cancer cells was similar to that of CTX 48 h after the administration (P>0.05), and the inhibiting effects of the other doses of S were significantly weaker than those of CTX (P<0.05,P<0.01). The activity of low (excepting 96 h after the administration) and medium-dose (excepting 72 h after the administration) CH was similar to that of low and medium-dose S (P>0.05), but it was all weaker than that of CTX (P<0.01). The inhibiting effect of high-dose CH on the proliferation of the BGC-823 cells was significantly stronger than that of CTX (P<0.01).

GroupDetechtion time∥h0487296A10.253±0.030.511±0.040.631±0.020.826±0.08B10.254±0.030.474±0.03aa0.531±0.05aa0.615±0.012aaC10.255±0.020.494±0.01aa,bb0.564±0.02aa,bb0.698±0.007aa,bbD10.255±0.020.482±0.02aa,bb,cc0.541±0.01aa,bb,cc0.671±0.06aa,bb,ccE10.254±0.020.479±0.02aa,cc0.537±0.01aa,b,cc0.636±0.07aa,bb,cc,ddF10.257±0.010.492±0.01aa,bb,dd,ee0.565±0.05aa,bb,dd,ee0.682±0.03aa,bb,c,dd,eeG10.255±0.030.485±0.04aa,bb,cc,e0.547±0.02aa,bb,cc,d,ee0.650±0.06aa,bb,ccH10.252±0.010.467±0.04aa,bb,cc,dd,ee0.510±0.02aa,bb,cc,dd,ee0.536±0.010aa,bb,cc,dd,ee

Note: Compared with the control group:aaP<0.01; compared with CTX group:bP<0.05,bbP<0.01; compared with low-dose S group:cP<0.05,ccP<0.01; compared with medium-dose S group:dP<0.05,ddP<0.01; and compared with high-dose S group:eP<0.05. The same blow. A1. control group; B1. CTX group; C1. low-dose S group; D1. medium-dose S group; E1. high-dose S group; F1. low-dose CH group; G1. medium-dose CH group; H1. high-dose CH group. The same below.

Fig.1 BGC-823 cells in the groups(×100)

3.2 Effects on the expression of related proteins in Bcl-2, P-Akt and Akt signaling pathways of cellsAs shown in Table 2 and Fig.2, compared with the control group, after CTX intervention, the expression levels of p-Akt and Bcl-2 in the BGC-823 cells significantly reduced (P<0.01), and the expression levels of Akt and GAPDH changed insignificantly (P>0.05). Different doses of S and CH all significantly downregulated the expression levels of p-Akt and Bcl-2 (P<0.05,P<0.01), while the expression levels of Akt and GAPDH did not change significantly (P>0.05). The inhibiting effect of high-dose CH on the expression of p-Akt was more significant than that of CTX (P<0.01); the inhibiting effects of medium and high-dose CH on the expression of Bcl-2 were more significantly than that of CTX (P<0.05,P<0.01); the influence of low-dose S on the expression of GAPDH was similar to that of CTX (P>0.05); the other doses of S and CH significantly inhibited the expression of the proteins compared with CTX (P<0.05,P<0.01). The influence of S and CH on the expression of the proteins showed obvious dose-dependent relationships. At the same dose, the inhibition of CH on the expression of the proteins was stronger than that of S (P<0.05,P<0.01).

GroupRelative expression levelp-AktBcl-2AktGAPDHA13 183.30±35.745 256.56±326.023 346.76±300.394 932.83±170.44B11 944.36±176.86aa3 724.10±128.75aa3 711.26±219.96 5 770.36±186.60C12 096.99±131.02aa3 996.94±151.23aa3 455.59±345.375 329.36±383.70D12 019.36±301.13aa3 914.64±341.29aa3 775.97±272.19 5 134.34±362.28bE11 865.91±272.64aa3 626.67±567.91aa3 736.16±488.814 646.87±551.48bb,cF12 385.51±73.02a,b,d,ee3 974.68±349.75aa,ee3 469.71±385.08 4 768.15±307.94bb,cG11 833.90±191.40aa,ff3 066.12±148.34aa,b,cc,dd,e,ff3 349.99±483.13 4 632.11±376.40bb,cH11 166.31±148.76aa,bb,cc,dd,ee,ff,gg2 333.17±183.09aa,bb,cc,dd,ee,ff,g3 097.67±308.06d,e4 620.50±344.63bb,c

Fig.2 Effects of Obazema on expression of p-Akt, Bcl-2, Akt and GAPDH in BGC-823 cells

4 Discussion

Gastric cancer is an malignant tumor that originates from the gastric mucosal epithelium. It is one of the most common malignant tumors, and is also one of the major diseases that threaten the health of Chinese people. According to the pathogenesis theory of Yi Medicine, cancers are caused by cold. The clinical manifestation of gastric cancer in Yi Medicine is "cold coagulation and blood stasis".B.sessilicarpaLévl. belongs to Qing, and is distributed to spleen and stomach channels. In the body, it is spread to the entire digestive tract from the umbilical bottom through the stomach along with Qingqi (CosmicHumanism). It can relieve cold coagulation in spleen and stomach to promote blood circulation to promote the generation of qi, and the circulation of qi will promote the generation of turbidity (ChorographyofYiPeopleinSouthwestChina). Through the qi-clearing and metaplastic effects, it promotes the digestion and absorption of food, generation and circulation of blood, and generation and circulation of body fluid, replenishes turbid gas (material basis-essence of water and food), nourishes the whole body, and is applicable to symptoms such as epigastric pain caused by cold coagulation.

This study found that the two origins [B.sessilicarpaLévl. (S) andB.albiflora(Hook.) Reichb. ex Meisn. var.albiflora(CH)] of Obazema are effective in inhibiting the proliferation of BGC-823 cells in dose and time-dependent manners. Cyclophosphamide (CTX) is a chemotherapy drug with significant cytotoxicity, and is widely used in clinical treatment of cancers including gastric cancer. Inhibition of high-dose S on the proliferation of the GCB-823 cells was similar to that of CTX 48 h after the administration, and inhibition of high-dose CH on the cell proliferation was stronger than that of CTX.

The Akt signal transduction pathways, such as the PI3K/Akt signaling pathway, have multiple functions. They participate in regulating the survival of tumor cells, regulating cell proliferation, apoptosis, invasion and metastasis, etc., and are closely related to the occurrence, development and prognosis of tumors[12-13]. Akt is the most critical targeting regulatory molecule downstream of the signaling pathways, and is seen as an important target for treating tumors. After the upstream molecules are activated, Akt will transfer from plasm to membrane, promoting the activation of Akt. Activated Akt transmits biological signals in the cytoplasm through phosphorylation[14-15], further activating the downstream factor Bcl-2, thereby regulating cell proliferation, protein synthesis, metabolism, apoptosis and other processes. GAPDH is a key enzyme for glycolysis. Upregulation of its expression with the increase of glycolysis during tumor cell proliferation may be a compensatory response during tumorigenesis[16-18].

In this study, Western blotting technology was used to detect the effects of different origins of Obazema and different drug-containing sera on the expression of key proteins in the Akt signalling pathways in BGC-823 cells. The results of this study show that compared with the control group, the drug administration groups all significantly inhibited the expression of p-Akt and Bcl-2 in BGC-823 cells. The inhibition of high-dose CH on the expression of p-Akt and Bcl-2 and the inhibition of medium-dose CH on the expression of Bcl-2 were stronger than that of CTX, in a certain dose-dependent manner. Compared with CTX, the expression of GAPDH was inhibited except that in the low-dose S administration group. At the drug concentration in blood, the inhibiting effect of CH on the expression of the proteins was much stronger than that of S. The inhibiting effect of CH on the proliferation of gastric cancer BGC-823 cells was stronger than that of S.

This study found that Obazema down-regulated the expression of p-Akt and Bcl-2 proteins through inhibiting the activation of the Akt signal transduction pathways, thus achieving the effect of inhibiting the proliferation of BGC-823 cells. Compared with CTX, Obazema effectively inhibited the expression of GAPDH in BGC-823 cells. It is speculated that GAPDH may be a potential marker for Obazema’s proliferations on BGC-823 cells, and it is also a target gene for its antitumor mechanism.

The previous research results of this research group show that the antioxidative activity of the ethanol extract of skimmed Obazema is significantly higher than that of Vc, chlorogenic acid and citric acid, and the antioxidant effect can reduce the reactive oxygen free radicals of carcinogens, and so, Obazema may be one of the material foundations for treatment of cancers. However, the specific regulation mechanism needs further research and exploration to provide reference for clinical application and development.

The CCK-8 (Cell Counting Kit-8) method has many advantages such as high sensitivity, reliable data, good reproducibility, easy operation, water solubility and low toxicity to cells compared with the traditional MTT method, so it has been widely used in anti-tumor drug screening, cell proliferation test and cytotoxicity test. In this study, CCK-8 method was used to detect cell proliferation.