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五味子乙素對人乳腺癌細胞MCF-7增殖和Notch-1信號通路的影響

2019-11-15 12:11:50 中國中醫藥信息雜志 2019年10期

趙煒 趙志正

摘要:目的? 觀察五味子乙素對人乳腺癌細胞增殖及Notch-1信號通路的影響,探討其抑制乳腺癌發展的可能分子機制。方法? 不同濃度五味子乙素(0、20、40、80 μmol/L)處理乳腺癌細胞MCF-7,作用24、48、72 h后,CCK-8法檢測MCF-7細胞增殖。裸鼠皮下接種MCF-7細胞建立裸鼠乳腺癌移植瘤模型,隨機分為對照組和五味子乙素組,每4 d測量腫瘤體積并繪制腫瘤生長曲線,32 d后取腫瘤稱重。Western blot檢測MCF-7細胞和腫瘤組織Notch-1及其下游分子Hes-1、Hey-1蛋白水平;免疫組化檢測小鼠腫瘤組織Notch-1的表達。結果? 與空白組比較,五味子乙素顯著抑制MCF-7細胞生長,且抑制作用具有時間和濃度依賴性,同時顯著降低MCF-7細胞Notch-1及其下游分子Hes-1、Hey-1蛋白的表達。與對照組比較,五味子乙素組裸鼠腫瘤體積及質量明顯受抑制,且腫瘤組織Notch-1及其下游分子Hes-1、Hey-1蛋白表達顯著降低。結論? 五味子乙素可抑制乳腺癌發展,其機制可能與抑制乳腺癌細胞MCF-7及其裸鼠模型移植瘤的生長、降低Notch-1信號通路相關因子表達有關。

關鍵詞:五味子乙素;細胞增殖;Notch-1;移植瘤;乳腺癌細胞MCF-7;小鼠

中圖分類號:R285.5??? 文獻標識碼:A??? 文章編號:1005-5304(2019)10-0055-05

DOI:10.3969/j.issn.1005-5304.2019.10.013????? 開放科學(資源服務)標識碼(OSID):

Abstract: Objective To explore the effects of schisandrin B on the growth of MCF-7 and Notch-1 signaling pathway in breast cancer; To explore possible molecular mechanism that inhibit the development of breast cancer. Methods Human breast cancer cell MCF-7 was treated with different concentrations of schisandrin B (0, 20, 40, 80 μmol/L) for 24, 48, 72 h. CCK-8 assay was used to detect cell growth. Nude mice breast cancer xenograft models were established by subcutaneous inoculation of MCF-7 cells in nude mice and were randomly divided into the control group and the schisandrin B treatment group. Tumor volume was measured every 4 days and the tumor growth curve was drawn. After 32 days, the tumor was weighed. The expressions of Notch-1, Hes-1, Hey-1 in MCF-7 cells and tumor tissues were detected by Western blot. Immunohistochemistry was used to detect the expression of Notch-1 in tumor tissues. Results Compared with the blank group, schisandrin B significantly inhibited the growth of MCF-7 cells in a time- and concentration-dependent manner, and significantly decreased the expression of Notch-1 and its downstream molecules Hes-1 and Hey-1 in MCF-7 cells. Compared with the control group, the tumor volume and quality of the schisandrin B treatment group were significantly inhibited, and the expression of Notch-1 and its downstream molecules Hes-1 and Hey-1 significantly decreased. Conclusion Schisandrin B can inhibit the growth of breast cancer, and its mechanism can be related to inhibiting the growth of breast cancer cells MCF-7 and its nude mouse model, and decreasing the expression of Notch-1 signaling pathway.

Keywords: schisandrin B; cell proliferation; Notch-1; xenografted tumor ; breast cancer cell MCF-7; mice

乳腺癌是發生在女性中最常見腫瘤之一,是女性癌癥相關死亡的主要原因[1]。近年來,隨著診斷學尤其是癌癥分子生物學和基因組學領域的進步,乳腺癌檢測發生了革命性的變化[2]。目前,治療乳腺癌主要方法包括外科手術、化療、放療,大大改善了乳腺癌的預后,但仍有許多患者復發及在治療過程中出現不良反應和耐藥。因此,尋找有效治療乳腺癌的藥物,成為當前研究的重點。五味子乙素(Schisandrin B)是從五味子中分離得到的主要生物活性成分[3]。近年研究發現,五味子乙素在多種疾病中介導多種生物活性,包括抗炎、抗氧化和心血管保護作用[4-6]。有報道顯示,五味子乙素可抑制膽管癌、肺腺癌、膠質瘤、胃癌細胞增殖[7-10],誘導細胞凋亡,減弱腫瘤的侵襲和轉移[11]。本研究分別通過細胞與動物實驗,觀察五味子乙素對人乳腺癌細胞增殖及Notch-1信號通路表達的影響,探討其可能的分子機制。

1? 實驗材料

1.1? 動物

SPF級雌性Balb/C小鼠10只,6~8周,體質量18~20 g,購自廣東省醫學實驗動物中心。飼養于溫度(20±2)℃、相對濕度(50±5)%環境,日照時間12 h,自由攝食飲水。實驗前小鼠適應性喂養7 d。

1.2? 藥物

五味子乙素,南京道斯夫生物科技有限公司,溶于二甲基亞砜并配制成100 mmol/L貯備液保存。

1.3? 細胞

人乳腺癌細胞株MCF-7,中國科學院上海細胞庫提供,在本實驗室進行培養。

1.4? 主要試劑與儀器

杜爾伯科改良伊格爾(DMEM)培養基、胰蛋白酶和胎牛血清,美國Gibco公司;青/鏈霉素,美國Sigma公司;BCA法蛋白濃度檢測試劑盒,上海碧云天生物技術公司;抗Notch-1抗體、抗Hes-1抗體、抗Hey-1抗體、抗β-actin抗體和辣根過氧化物標記的二抗,英國Abcam公司;CCK-8試劑盒,上海博谷生物科技有限公司。超低溫冰箱(日本Sanyo公司),CO2細胞培養箱(美國Thermo公司),低速離心機(美國Thermo公司),電泳儀(北京六一生物科技有限公司),凝膠成像儀(美國Azure Biosystems公司)。

2? 實驗方法

2.1? 細胞培養

MCF-7細胞培養于含10%胎牛血清的DMEM培養基,置于37 ℃、含5%CO2且濕度完全飽和的恒溫培養箱中培養,定期取對數生長期細胞進行傳代。

2.2? CCK-8實驗

用于檢測細胞增殖并計算增殖抑制率。100 μL對數生長期MCF-7細胞(1×105/孔)接種于96孔板,每組設置4個復孔,共4組,加入終濃度為0、20、40、80 μmol/L五味子乙素,培養24、48、72 h,每孔加入CCK-8試劑10 μL,繼續培養4 h,使用分光光度計檢測波長450 nm處吸光度,計算細胞增殖抑制率。細胞增殖抑制率(%)=(1-給藥組A450÷對照組A450)×100%。

2.3? 乳腺癌細胞移植瘤裸鼠模型建立

用PBS將對數期乳腺癌細胞MCF-7濃度調整為2×107/mL,取0.1 mL懸液注射于小鼠右后側肢體皮下,接種后觀察1周成瘤,將10只成瘤小鼠隨機分為對照組和五味子乙素組,每組5只。五味子乙素組小鼠腹腔注射五味子乙素,每2 d注射1次,直至細胞接種后32 d,每次100 mg/kg,體積0.2 mL;對照組注射等體積PBS。每4 d用游標卡尺測量腫瘤長徑和短徑,腫瘤體積=長徑×短徑2÷2。細胞接種32 d,處死裸鼠,稱取瘤重,計算抑瘤率。抑瘤率(%)=(對照組瘤體平均質量-給藥組瘤體平均質量)÷對照組瘤體平均質量×100%。腫瘤組織置于液氮中保存備用。

2.4? Western blot檢測

MCF-7細胞用預冷的細胞裂解液裂解15 min,4 ℃、12 000 r/min離心5 min,取上清液,收取蛋白。腫瘤組織先用液氮進行研磨,然后加入細胞裂解液,離心后獲得腫瘤組織蛋白。BCA比色法計算蛋白濃度,通過SDS-PAGE將蛋白進行分離后,將蛋白轉移至PVDF膜,隨后用5%脫脂奶粉封閉液封閉2 h,根據說明書稀釋一抗,利用封閉液將抗體稀釋到所需濃度,4 ℃孵育過夜,用TBST洗滌3次×5 min,隨后根據用量,稀釋HRP標記的二抗,37 ℃孵育1 h。用TBST洗滌3次×5 min。用ECL化學發光檢測,用Image J對圖像進行灰度分析。

2.5? 免疫組化檢測

脫蠟、水化后切片,經1%Triton X-100處理15 min,再用3%H2O2處理10 min,隨后加入Notch-1抗體孵育,置于濕盒4 ℃過夜,第2日清洗后孵育對應的二抗,室溫30 min,最后進行染色封片。染色結果采用DAB顯色法進行觀察,使用蘇木精進行對比染色。

3? 統計學方法

采用SPSS17.0統計軟件進行分析。實驗數據以—x±s表示,用方差分析進行比較,兩組間比較用t檢驗。P<0.05表示差異有統計學意義。

4? 結果

4.1? 五味子乙素對乳腺癌MCF-7細胞增殖的影響

經不同濃度五味子乙素(0、20、40、80 μmol/L)處理的乳腺癌MCF-7細胞,其對乳腺癌MCF-7細胞增殖的抑制作用具有時間和濃度依賴性,細胞抑制率隨五味子乙素濃度增加和處理時間的延長而增加。結果見表1。

4.2? 五味子乙素對乳腺癌MCF-7細胞Notch-1信號通路相關因子表達的影響

[6] THANDAVARAYAN R A, GIRIDHARAN V V, ARUMUGAM S, et al. Schisandrin B prevents doxorubicin induced cardiac dysfunction by modulation of DNA damage, oxidative stress and inflammation through inhibition of MAPK/p53 signaling[J]. PLoS One,2015,10(3):e0119214.

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[11] DAI X, YIN C, GUO G, et al. Schisandrin B exhibits potent anticancer activity in triple negative breast cancer by inhibiting STAT3[J]. Toxicology and Applied Pharmacology,2018, 358:110-119.

[12] NIE J, ZHAO C, DENG L, et al. Efficacy of traditional Chinese medicine in treating cancer[J]. Biomedical Reports,2016,4(1):3-14.

[13] LI X, TSAUO J, GENG C, et al. Ginsenoside Rg3 decreases NHE1? expression via inhibiting EGF-EGFR-ERK1/2-HIF-1α pathway in hepatocellular carcinoma:A novel antitumor mechanism[J]. The American Journal of Chinese Medicine,2018,46(8):1915-1931.

[14] ZHOU R, CHEN H, CHEN J, et al. Extract from Astragalus membranaceus inhibit breast cancer cells proliferation via PI3K/AKT/mTOR signaling pathway[J]. BMC Complementary and Alternative Medicine,2018,18(1):83.

[15] LI Q, LU X H, CAI L, et al. Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells[J]. Cancer Cell International,2015,15(1):12.

[16] WU Y F, CAO M F, GAO Y P, et al. Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells[J]. World Journal of Gastroenterology,2004,10(20):2944.

[17] XIANG S S, WANG X A, LI H F, et al. Schisandrin B induces apoptosis and cell cycle arrest of gallbladder cancer cells[J]. Molecules,2014,19(9):13235-13250.

[18] LIU Z, ZHANG B, LIU K, et al. Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial-mesenchymal transition[J]. PLoS One,2012,7(7):e40480.

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