Effect of Extracts from Bark of Juglansmandshurica on Proliferation and Cell Cycle of SMMC-7721 Cells

LIANG Qichao, LIU Shuang, ZHANG Zhaoli*

(1. Mudanjiang Medical College, Mudanjiang 157011, China; 2. Mudanjiang Cardiovascular Hospital, Mudanjiang 157011, China)



Effect of Extracts from Bark ofJuglansmandshuricaon Proliferation and Cell Cycle of SMMC-7721 Cells

LIANG Qichao1, LIU Shuang2, ZHANG Zhaoli1*

(1. Mudanjiang Medical College, Mudanjiang 157011, China; 2. Mudanjiang Cardiovascular Hospital, Mudanjiang 157011, China)

LIANG Qichao

To study effect of extracts from bark ofJuglansmandshurica(EMBJM) on proliferation and cell cycle of SMMC-7721 cells, the SMMC-7721 cells in culture mediuminvitrowere given different concentrations of EMBJM. The inhibition rate of EMBJM on the SMMC-7721 cell was detected by MTT assay. The cell cycle analyzed was detected by flow cytometry (FCM). The EMBJM exhibited a significant inhibition on SMMC-7721 cells time-and dose-dependently. The cell cycle detection results suggested that EMBJM could arrest SMMC-7721 cells specifically in growth 2/mitosis(G2/M) and synthesis(S) phase cell cycle in a dose-dependent manner. Compared with control group (8.34%±1.42%; 26.80%±1.76%), the G2/M and S phase of SMMC-7721 cells exposed to EMBJM ( 13.89%±2.46%, 39.87%±2.83% (8 mg/L);(14.35%±2.68%, 44.81%±2.93% (16 mg/L)) were significantly increased (P<0.05,n=3). EMBJM can effectively inhibited proliferation in SMMC-7721 cells and effected cell cycle distribution.

bark ofJuglansmandshurica; cytotoxicity; cell cycle; SMMC-7721 cells

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide and is currently the second common type of cancer death in China[1]. Despite of the different clinical progress achieved in recent years, the overall outcome of HCC remains very poor, which is largely the result of a high ratio of recurrence or metastasis after operation[2-3]. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and noncytotoxic chemical agents for chemoprevention and treatment. Since only few synthetic antineoplastic compounds are effective for the treatment of this disease, the focus of more and more studies has shifted recently to study natural active agents for cancer chemoprevention and treatment.JuglansmandshuricaMaxim. is a member of the Juglandaceae plant family, native to the Eastern Asiatic Region (China, Russian Far East, North Korea and South Korea)[4]. Its roots, barks, leaves, and seeds have been used in folk medicine over a long period of time, particularly in Asia and Europe[5]. In recent years it has been widely used in the clinical treatment of esophageal, gastric, cardiac, and lung cancer[6-8]. Most reports onJ.mandshuricawere focused largely on some small molecule compounds, such as quinones, tetralones, flavonoids, diarylheptanoid, and polyphenols[9-16]. They have shown antioxidant, antitumor, anti-inflammatory and antimicrobial activity[17-21]. In this study, we researched the cytotoxic effect of EMBJM on the SMMC-7721 cell by MTT assay, and evaluated the effects of cell cycle of EMBJM on human hepatoma SMMC-7721 cells. EMBJM could cause SMMC-7721 cell cycle to be stagnatedinvitroin a dose dependent manner. EMBJM can effectively inhibited proliferation in SMMC-7721 cells and effected cell cycle distribution.

1 Materials and Methods

1.1 Materials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. RPMI1640 medium were obtained from Gibco. Fetal bovine serum (FBS) purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., and propidium iodide (PI) was purchased from KeyGen Biotech Corporation.

Spectrophotometer (Cary100, Varian, USA), HPLC (1100, Agilent, USA), Microplate reader (680, Bio-Rad, USA), Flow Cytometer (BD FACS Calibur, BD Biosciences, USA).

1.2 Preparation of EMBJM

Thefresh bark ofJuglansmandshuricaMaxim. was obtained from a mountain district (Mudanjiang, Heilongjiang Province), dried at room temperature for 3 weeks. The material was ground down powder and screened by a 0.425mm sieve. 500 g of sample powder was ground with ethyl ether for 24 h at room temperature, and discarded the ether layer. Then, sample powder was extracted with methanol there times by using Soxhlet apparatus for 6 h. The methanol solution was evaporated to dryness (49 g) and loaded on a silica gel column. The column was eluted with CHCl3-MeOH-H2O (60∶29∶6) to give three major fractions. Fraction 3 consisted of EMBJM (9.72 g), which is an amorphous brown powder. The purity of Juglone in EMBJM was 32.16% as determined by HPLC. It was dissolved in dimethyl sulfoxide (DMSO) and added to the experimental media to give the final concentrations till the final concentration of DMSO was less than 0.3%.

1.3 Cell culture conditions

Human hepatocarcinoma cell line (SMMC-7721) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), and 100 g/L penicillin-streptomycin at 37 ℃ in a 5% CO2/95% air humidified incubator. The cell culture medium was changed every other day. Exponentially growing cells in culture were used for these experiments.

1.4 MTT assay

The effect of EMBJM treatment on the proliferation of HCC cell line SMMC-7721 was measured by MTT assay. Briefly, cells attached on culture dishes were collected and planted at a density of 1×104cells per mL, 100 μL volumes in each well of 96-well plates. Next, the cells were treated with EMBJM for various concentrations (0.5, 1, 2, 4, 8 and 16 mg/L), and the cells were incubated after 24, 48 and 72 h of culture. Then, 20 μL MTT(5 g/L in PBS) was added to each well and continuously incubated at 37 ℃ for 4 h. At the end of this period, the MTT solutions were replaced with 150 μL dimethylsulfoxide and shaken for 10 min. Absorbance was measured by automatic microplate reader (680, Bio-Rad, USA) at 490 nm. Assays were performed in triplicate on three independent experiments.

1.5 Cell cycle by flow cytometry (FCM)

The cell cycle distribution was analyzed byflow cytometry. SMMC-7721 cells in exponential growth (1×106cells per well) were seeded into 6-well plate and treated with various concentrations EMBJM for 48 h. The treated cells were collected at designated time after treatments, washed twice by ice-cold PBS and fixed with 70% ethanol overnight at -20 ℃. The fixed cells were washed with cold PBS and stained with propidium iodide (PI) working solutions (0.1%TritonX-100, 100 g/L PI, 0.01 g/L RNase) for 30 min at 4 ℃ in darkness. The stained cells were subjected to a BD FACS Calibur Flow Cytometer (BD Biosciences, USA). The proportion of cells in growth 0/growth 1 (G0/G1), synthesis (S) and growth 2/mitosis (G2/M) phase was calculated using WinMDI 2.9 and Cylchred software, and was represented as DNA histograms. Each assay was replicated 3 times.

1.6 Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) for multiple comparisions. Data are expressed as means±SD. A probability of 0.05 or less was considered significant.

2 Results and Discussion

2.1Invitrocytotoxicity

Table 1 In vitro cytotoxicity of EMBJM against SMMC-7721 cells

MTT assay was used to assess theinvitrocytotoxicity of EMBJM compared to the free EMBJM solution,which was reflected by the percentage of relative reduction of cell viability, i.e. the percentage of inhibition rate. As shown in Table 1 and Fig.1, the inhibition rates from different concentration EMBJM of SMMC-7721 cells were in a time and dose dependent manner. At the same concen-tration, the EMBJM showed significantly higher inhibition rates against SMMC-7721 cells compared to free EMBJM solution.

Fig.1 Inhibition rate of EMBJM on the SMMC-7721 cells

2.2 Effect on the cell cycle

Table 2 and Fig.2 show the cell cycle histograms of the cell lines treated with EMBJM solution at different concentrations (2, 4, 8 and 16 mg/L) for 48 h. The results suggested that EMBJM could arrest SMMC-7721 cells specifically in G2/M and S phase cell cycle in a dose-dependent manner. Compared with control group (8.34%±1.42%), the G2/M phase (indicated as percentage) of SMMC-7721 cells exposed to EMBJM (11.01%±1.64%(2 mg/L), 11.78%±1.52%(4 mg/L), 13.89%±2.46%(8 mg/L),14.35%±2.68%(16 mg/L)) were significantly increased. The proportions of SMMC-7721 cells in S phase were 26.80%±1.76% in the control group, 29.37%±1.84% and 31.16%±1.79% and 39.87%±2.83% and 44.81%±2.93% in SMMC-7721 cells treated with 2 to 16 mg/L EMBJM, respectively. The proportions of cells in G2/M and S phases in the 8 and 16 mg/L EMBJM groups were significantly increased compared with the control group (P<0.05,n=3). EMBJM could cause SMMC-7721 cells cycle to be stagnatedinvitroin G2/M and S phases in a dose-dependent manner. More importantly, a sub-G1 peak was observed in SMMC-7721 cells treated with EMBJM (Fig.2). These results indicated that EMBJM might change the cell cycle distribution. These data suggested that EMBJM induced cell cycle arrest at G2/M and S phase to a greater extent than the free EMBJM solution at the same concentration.

Table 2 The cell cycle distribution of the cell treated with different concentration EMBJM

1)*：P<0.05, vs. control

Fig.2 Inhibition of cell-cycle progress in SMMC-7721cells by treatment with EMBJM for 48 h

3 Conclusions

3.1 We examined the cytotoxic effect of EMBJM on the SMMC-7721 cell by MTT assay. Under the experimental conditions, EMBJM was found to have a higher effective in inhibiting cell proliferation in a dose-and time-dependent manner. As shown in Fig.1, inhibition rates of EMBJM (16 mg/L) were determined as 44.09%±3.74%, 67.74%±3.54% and 72.72%±3.21% in the SMMC-7721 with 24, 48 and 72h treatment, respectively. EMBJM was found to potently inhibit the growth of SMMC-7721 cellsinvitro.

3.2 In order to decipher the suppressive mechanisms of EMBJM on the SMMC-7721 cell, we monitored changes in cell-cycle distribution by flow cytometry. Compared with control group (8.34%±1.42%; 26.80%±1.76%), the G2/M and S phase of SMMC-7721 cells exposed to EMBJM (13.89%±2.46%, 39.87%±2.83%(8 mg/L);14.35%±2.68%, 44.81%±2.93%(16 mg/L)) were significantly increased (P<0.05,n=3). The results confirmed the occurrence of G2/M and S phase arrest in SMMC-7721 cells treated with EMBJM. These results and the emerging evidence of its anticancer effects make EMBJM a very promising candidate for being an effective anticancer agent.

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2016- 09-25

黑龙江省自然科学基金项目(H201692)；黑龙江省教育厅科学技术研究项目(12531742)；牡丹江医学院科学技术研究项目(ZS201515)

梁启超 (1985— ), 男, 黑龙江佳木斯人，硕士，实验师，主要从事天然产物化学研究；E-mail: xiaochao9199@163.com

梁启超，刘爽，张朝立.核桃楸皮提取物对人肝癌细胞SMMC-7721的增殖抑制作用及对细胞周期的影响(英文)[J].林产化学与工业，2017，37(3)：136-140.

核桃楸皮提取物对人肝癌细胞SMMC-7721的增殖抑制作用及对细胞周期的影响

梁启超1，刘 爽2，张朝立1

(1．牡丹江医学院，黑龙江 牡丹江 157011； 2．牡丹江心血管病医院，黑龙江 牡丹江 157011)

研究核桃楸皮提取物(EMBJM)对人肝癌细胞SMMC-7721的增殖抑制作用及对细胞周期的影响。体外培养SMMC-7721细胞，并用不同浓度核桃楸皮甲醇提取物处理。通过MTT法研究EMBJM对SMMC-7721细胞增殖活性的影响；采用流式细胞仪分析EMBJM对SMMC-7721细胞周期的影响。EMBJM可以显著抑制SMMC-7721细胞生长，具有时间和计量依赖性。细胞周期实验表明EMBJM将SMMC-7721细胞阻滞在G2/M和S期，并具有剂量依赖性。EMBJM质量浓度为8和16mg/L时, G2/M期细胞所占百分比分别为13.89%±2.46%和14.35%±2.68%, 与空白对照组(8.34%±1.42%)比较，差异均有统计学意义(P<0.05)；同等浓度范围内S期细胞所占百分比分别为39.87%±2.83%和44.81%±2.93%，与空白对照组(26.80%±1.76%)比较, 差异均有统计学意义(P<0.05)。桃楸皮甲醇提取物可以影响SMMC-7721细胞周期分布和有效抑制SMMC-7721细胞生长。

核桃楸皮；细胞毒性；细胞周期；SMMC-7721细胞

10.3969/j.issn.0253-2417.2017.03.019

*通讯作者：张朝立(1979— )，男，讲师，博士，研究领域为天然产物化学研究；E-mail: chaolizhang79@163.com。

TQ35 Document code:A Article ID:0253-2417(2017)03- 0136-05