药学学报  2016, Vol. 51 Issue (7): 1091-1097   PDF    
三七皂苷Ft1抑制乳腺癌细胞增殖、迁移及促进凋亡的机制研究
邱水平, 李鸿丽, 石海莲 , 吴辉, 黄菲, 张蓓蓓, 吴晓俊, 王峥涛     
上海中医药大学中药研究所暨上海市复方中药重点实验室, 上海 201203
摘要: 研究三七皂苷Ft1抑制乳腺癌细胞增殖、迁移,促凋亡的作用及分子机制。CCK-8法、EdU染色、Hoechst 33258染色和单细胞迁移实验分别检测细胞活力、增殖、凋亡和迁移能力;Western blot检测凋亡相关蛋白、HIF-1α、PI3K/Akt/mTOR/p70S6K和MAPK通路蛋白表达。结果表明,Ft1能降低乳腺癌细胞MDA-MB-231和MCF-7的活力,抑制MDA-MB-231和BT-549细胞的增殖。Ft1也可诱导MDA-MB-231细胞核DNA高度聚集,并且在作用12 h后,下调HIF-1α和Bcl-2,上调cleaved caspase 3蛋白表达。进一步研究发现,Ft1在降低HIF-1α蛋白表达的同时,显著减少p-Akt、p-mTOR、p-p70S6K及p-ERK1/2的蛋白表达,但增加p-JNK蛋白水平。Ft1能够显著抑制EGFR的Tyr1068和Ser1046/1047两个位点的磷酸化。在不影响HS578T细胞活力的浓度下,Ft1能够显著抑制其迁移路径长度和速度。因此,Ft1具有抑制乳腺癌细胞增殖、迁移和促进凋亡作用,其作用可能与其调控Akt/mTOR/p70S6K和MAPK通路,下调HIF-1α,抑制Bcl-2和上调cleaved caspase 3蛋白有关。
关键词: 乳腺癌     三七皂苷Ft1     增殖     凋亡     缺氧诱导因子-1α    
Notoginsenoside Ft1 down-regulates HIF-1α, inhibits cell proliferation, decreases migration and promotes apoptosis in breast cancer cells
QIU Shui-ping, LI Hong-li, SHI Hai-lian , WU Hui, HUANG Fei, ZHANG Bei-bei, WU Xiao-jun, WANG Zheng-tao     
Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Abstract: To investigate the effect of notoginsenoside Ft1(Ft1) on proliferation, migration and apoptosis of breast cancer cells, we conducted several assays including CCK-8 assay, EdU staining, single cell migration assay and Hoechst 33258 staining. The effect of Ft1 on expression of apoptosis related proteins, HIF-1α, PI3K/Akt/mTOR/p70S6K and MAPK pathways was examined with Western blot. Ft1 could significantly reduce cell survival and inhibit cell proliferation in breast cancer cells in a dose-dependent manner. Ft1 also increased chromatin condensation of MDA-MB-231 cells. Furthermore, Ft1 decreased protein expression of Bcl-2 and HIF-1α and increased expression of cleaved caspase 3 in MDA-MB-231 cells after 12 h treatment. Ft1 significantly down-regulated the levels of p-Akt, p-mTOR and p-p70S6K as well as p-ERK1/2, but up-regulated that of p-JNK. Ft1 significantly inhibited the level of p-EGFR (Tyr1068) and p-EGFR (Ser1046/1047) in MDA-MB-231 cells. Finally, Ft1 significantly inhibited the migration path length and velocity of HS578T cells when used at the concentration without affecting cell viability. Thus, Ft1 exhibited multiple antitumor effects including inhibition of cell survival and migration, promotion of cell apoptosis in breast cancer cells. Suppression of HIF-1α via Akt/mTOR/p70S6K and MAPK pathways may be involved in the pharmacological effect of Ft1 on cell proliferation and apoptosis of breast cancer cells.
Key words: breast cancer     notoginsenoside Ft1     proliferation     apoptosis     HIF-1α    

最新肿瘤流行病学统计数据显示,乳腺癌发病率已跃居全球女性肿瘤第一,占女性肿瘤的29%,致死率已跃居第二,占15%[1]。虽然目前在乳腺癌的治疗方法上有了新的进展,但在美国女性中乳腺癌的死亡率在众多癌症中依然高居第二位[2]。因此寻找有效的抗乳腺癌药物依然为当前研究的热点。

天然产物是新药研发的重要来源。三七 [Panax notoginseng (Burk) F.H.Chen] 为五加科植物,干燥根入药,又称田七、岑三七等,是我国传统中药材,主产于云南和广西等地。三七具有促凝血和抗血栓的 双向调节作用[3, 4],自古用于创伤止血,被明代著名药学家李时珍誉为“金不换”,在心脑血管疾病中疗效显著。皂苷是三七的主要活性成分,目前已分离到的皂苷成分有70多种。大部分皂苷成分均从三七根中分离得到,而三七皂苷Ft1首次从三七叶中分离得到[5],后续的研究结果显示,Ft1可促进创伤愈合[6],并经由二磷酸腺苷受体 (adenosine phosphate receptor) P2Y12介导促进血小板的聚集[7],但对于其抗肿瘤活性报道甚少。本课题组以往研究发现Ft1能够显著抑制神经母细胞瘤细胞的生长,并促进细胞凋亡[8],但其是否对其他肿瘤细胞也有抑制作用目前尚不清楚。本文旨在研究Ft1对乳腺癌细胞增殖、迁移和凋亡的作用,并探讨其分子机制,以期为其潜在的临床应用于乳腺癌治疗提供理论依据。

材料与方法 细胞株

人乳腺癌细胞MDA-MB-231细胞由上海中医药大学中药学院沈凯凯馈赠,人BT549细胞、HS578T细胞和MCF-7细胞由美国肯塔基大学徐韧教授馈赠。

主要试剂

三七皂苷Ft1由上海中医药大学中药研究所分离获得,经HPLC和质谱鉴定确定纯度 ( > 98%) 和结构。DMEM/High Glucose培养基、RPMI 1640培养基、0.25% 胰酶和胎牛血清 (FBS) 购买于Gibco公司。DMEM/F12 Mixture 培养液、二甲基亚砜 (DMSO) 购于Sigma公司,CCK-8 (Cell Counting Kit-8) 购于同仁化学公司。本研究所用抗体均购于CST公司。

仪器

电子天平购自Sartorius (BP211D),振荡仪购自Qilinbeier (VORTEX-5),台式高速离心机 (Centrifuge 5810R) 和高速离心机 (5415R) 均购自Eppendorf公司,荧光显微镜 (CKX41) 购自Olympus公司,酶标仪 (Varioskan Flash)、CO2培养箱 (HERA cell 150i) 和超净工作台 (1300 SERIES A2) 均购自Thermo公司,细胞计数仪 (Moxiz) 购自Orflo公司,电热恒温水浴锅购自北京市长风仪器仪表公司。

细胞培养和给药

MDA-MB-231细胞、MCF-7细胞和HS-578T细胞于含10% 胎牛血清、100 u∙mL−1青霉素和0.1 mg∙mL−1链霉素的DMEM培养液中,BT-549细胞于含10% 胎牛血清、100 u∙mL−1青霉素和0.1 mg∙mL−1链霉素的RPMI 1640培养液中,置于37 ℃、5% CO2细胞培养箱内培养。实验时,接种细胞数为每毫升1.0×105个,96、24和6孔板分别接种100 μL、400 μL和2 mL,使其贴壁过夜,给予不同 浓度Ft1作用于细胞,每个浓度3个复孔,药物孵育48 h后进行各项检查。

细胞增殖活力检测

弃原培养液并加入100 μL新鲜培养基后,加入CCK-8 10 μL,继续孵育1 h,用Thermo Fisher Varioskan Flash酶标仪检测450 nm处吸光度 (A),细胞存活率 = (A给药组/A对照组) × 100% 计算,实验重复3次。

EdU染色法检测细胞增殖

从4 ℃取出液态的matrigel胶,取24孔培养板每孔加入matrigel原液120 μL,在孔底部涂抹均匀,避免气泡产生,于37 ℃培养箱中孵育30 min。用0.25% 胰酶消化细胞至单 细胞悬液,调整细胞数至每毫升1.6×104个,悬液为含2% 血清的DMEM/F12 Mixture培养液,在每孔内接种细胞悬液250 μL,于37 ℃培养箱中孵育1 h,将药物稀释在上层胶 (DMEM/F12 Mixture + 2% FBS + 10% matrigel) 中配成不同浓度加入到每孔中。48 h后收集细胞 (收集细胞前18 h加入EdU染料),吸去培养基,用200 μL枪头将matrigel捣碎并均匀地涂 抹在载玻片上,待晾干后,加入4% 多聚甲醛 (para­formaldehyde,PFA) 室温固定30 min,3% BSA洗2遍。加入0.5% Triton X-100,室温孵育25 min,3% BSA洗2遍。加入Cu+ 和叠氮化合物混合的反应溶液,室温、避光30 min,用3% BSA和PBS各洗1遍,加入DAPI,室温避光5 min。PBS清洗,加一滴防淬灭液,最后封片,荧光显微镜下观察。

Hoechst 33258染色法检测细胞凋亡

弃去培养基,4% PFA固定10 min,PBS洗2遍,加入10 μg∙mL−1 Hoechst 33258染色液,染色15 min,PBS洗2遍,荧光显微镜下观察。

Western blot

收集细胞,加细胞裂解液150 μL,超声粉碎离心,取上清,每孔分别取20 μg样品蛋白上样,10% SDS聚丙烯酰胺凝胶电泳分离蛋白,然后4 ℃、100 V湿转120 min,5% BSA室温封闭2 h。分别用一抗 (1∶1 000) 4 ℃孵育过夜,HRP标记二抗 (1∶5 000) 室温孵育2 h,采用ECL显色液显色。

单细胞迁移实验

收集细胞前一天先用0.1 g∙L−1多聚赖氨酸包被玻璃培养皿 (培养皿中间分为4个区域),室温放置2 h,用无菌水洗2遍,置于超净台中干燥备用。用0.25% 胰酶消化各组细胞至单细 胞悬液,计数调整细胞数为每毫升2.0×104个细胞,每个1/4区域内加悬液500 μL,置于37 ℃培养箱内 继续培养3 h以上,待细胞贴壁后,将玻璃培养皿置于Nikon Biostation IM仪器中,实时监测细胞8 h。

统计学分析

所有数据均以± s表示,采用Graphpad Prism 5软件进行统计分析,多组间比较用One-way ANOVA Dunnett检验,P < 0.05表示差异有统计学意义。

结果 1 Ft1MDA-MB-231MCF-7细胞活力的影响

图 1所示,Ft1能显著抑制MDA-MB-231细胞 (P < 0.05,P < 0.001) 和MCF-7细胞的细胞活力 (P < 0.001)。MDA-MB-231细胞是三阴性乳腺癌,而MCF-7细胞 [雌激素受体 (ER)+,雄激素受体 (PR)+,人类表皮生长因子受体 (HER)] 是雌激素受体阳性细胞,临床恶性程度和转移能力较MDA-MB-231细胞弱,因此在后续机制研究中,主要以MDA-MB- 231细胞为主。

Figure 1 The effect of notoginsenoside Ft1 (Ft1) on cell viability of breast cancer cells (48 h) After treatment with Ft1 (10−200 μmol∙L−1) for 48 h,CCK-8 assay was used to determine the effect of Ft1 on cell viability of MDA-MB-231 and MCF-7 cells. n = 3−6,± s. P < 0.05,***P < 0.001 vs control group
2 Ft1对乳腺癌细胞增殖的影响

图 2所示,采用3D培养技术培养乳腺癌细胞BT-549和MCF-7细胞,EdU染色结果显示,Ft1可明显抑制3D培养下乳腺癌细胞的增殖。

Figure 2 The effect of Ft1 on cell proliferation of breast cancer cells (48 h). MCF-7 and BT-549 cells were cultured by 3D cultivation technology,and the effect of Ft1 on cell proliferation of MCF-7 and BT-549 cells was determined by using EdU staining method after treatment with Ft1 (20,40 μmol∙L−1) for 48 h. n = 5,± s. P < 0.05,**P < 0.01,***P < 0.001 vs control group
3 Ft1MDA-MB-231细胞凋亡的影响

在荧光显微镜下,正常活细胞呈弥散均匀荧光,出现细胞凋亡时,细胞核或细胞质内可见浓染致密的颗粒块状荧光。如图 3A~C所示,control组细胞 呈弥散均匀荧光,而Ft1 (40和80 μmol∙L−1) 作用MDA-MB-231细胞48 h后,均能诱导细胞出现浓染致密的颗粒块状高亮蓝色荧光。进一步Western blot检测结果 (图 3D) 显示,与control组细胞相比,Ft1能显著上调cleaved caspase 3的蛋白表达,下调缺氧诱导因子-1α (hypoxia inducible factor-1α,HIF-1α) 和Bcl-2蛋白的表达,提示Ft1能够诱导MDA-MB-231细胞的凋亡。

Figure 3 The effect of Ft1 on apoptosis of breast cancer cells (48 h) in MDA-MB-231 cells. After treatment with Ft1 (40 and 80 μmol∙L−1) for 48 h,typical morphology of apoptosis was observed by Hoechst 33258 staining (A−C). Meanwhile,as early as 6 h-treatment,Western blot results (D) showed that Ft1 could down-regulate protein expression of HIF-1α and Bcl-2,and up-regulate protein expression of cleaved caspase 3 in MDA- MB-231 cells. Scale bar: 200 μm
4 Ft1对乳腺癌细胞Akt/mTOR和丝裂原活化蛋白激酶 (MAPK) 信号通路蛋白表达的影响

图 4所示,时效实验结果表明,Ft1作用6 h以上能显著降低p-mTOR、p-Akt和p-p70S6K的蛋白表达。与此同时,如图 5所示,Ft1作用6 h后,即能显著抑制p-ERK1/2和p-JNK的蛋白表达,而作用24 h后,却能够显著上调p-JNK的蛋白表达。

Figure 4 The effect of Ft1 on protein expression of PI3K/Akt/ mTOR/p70S6K pathways in MDA-MB-231 cells. After treatment with Ft1 (80 μmol∙L−1) for 0,1,6,12 and 24 h,respectively,cells were collected and lysed with RIPA buffer,and expression of the key proteins from PI3K/Akt/mTOR/p70S6K pathways was determined by Western blot method. GAPDH was used as internal control

Figure 5 The effect of Ft1 on protein expression of MAPK pathways in MDA-MB-231 cells. Cells were treated with Ft1 (80 μmol∙L−1) for 12 h,and expression of the key proteins from MAPK pathways was determined by Western blot method. GAPDH was used as internal control

Ft1 (80 μmol∙L−1) 干预12 h后进行Western blot检测,结果如图 6显示,Ft1能够显著抑制Akt/mTOR/ p70S6K的蛋白磷酸化 (P < 0.05,P < 0.01,P < 0.001),同时能够抑制ERK1/2蛋白的磷酸化 (P < 0.05),而上调JNK的蛋白磷酸化 (P < 0.001),下调HIF-1α蛋白水平 (P < 0.01)。Ft1也有抑制p-p85 PI3K蛋白表达的趋势,但无统计学意义。

Figure 6 The effect of Ft1 on protein expression of PI3K/Akt/mTOR and MAPK pathways in MDA-MB-231 cells.Cells were treated with Ft1 (80 μmol∙L−1) for 12 h,and expression of the key proteins from PI3K/Akt/mTOR/p70S6K and MAPK pathways was determined by Western blot method. GAPDH was used as internal control. n = 3,± s. P < 0.05,**P < 0.01,***P < 0.001 vs control group
5 Ft1对乳腺癌细胞EGFR磷酸化蛋白表达的影响

进一步就Ft1对Akt/mTOR和MAPK通路的上游通路EGFR蛋白磷酸化是否有抑制作用进行了考察。结果如图 7显示,80 μmol∙L−1 Ft1干预12 h均能显著抑制p-EGFR (Tyr1068) 和p-EGFR (Ser1046/1047)的蛋白表达 (P < 0.05)。

Figure 7 The effect of Ft1 on protein expression of p-EGFR (Tyr1068) and p-EGFR (Ser1046/1047) in MDA-MB-231 cells. Cells were treated with Ft1 (80 μmol∙L−1) for 12 h,and expression of p-EGFR (Tyr1068) and p-EGFR (Ser1046/1047) was determined by Western blot method. GAPDH was used as internal control. n = 3,± s. P < 0.05 vs control group
6 Ft1对乳腺癌细胞迁移能力的影响

图 8所示,单细胞迁移实验结果显示,Ft1干预48 h,能够显著抑制HS578T单细胞迁移路径的长度 (P < 0.01) 和速度 (P < 0.01),也有抑制HS578T细胞单细胞终点与起点的直线距离的趋势,但无统计学差异。

Figure 8 The effect of Ft1 on single cell migration of breast cancer cell HS578T. Single cell migration assay was used to detect the effect of Ft1 (20 μmol∙L−1) on the migration ability of HS578T cells for 48 h treatment. A: Effect of Ft1 on path length of single cell migration; B: Effect of Ft1 on distance from origin of single cell migration; C: Effect of Ft1 on velocity of single cell migration; D: Photograph of single cell migration path of control cells; E: Photograph of single cell migration path of Ft1-treated cells. In D and E,migration path with every colors showed every different single cell migration and there were about twenty cells,respectively. n = 50,± s. P < 0.05,**P < 0.01,***P < 0.001 vs control group
讨论

三七主要功用化瘀止血,消肿止痛。有关三七在心脑血管疾病和抗炎方面的研究比较多 [9, 10, 11]。近年来发现三七具有多种抗肿瘤活性。三七总皂苷能够抑制肝癌细胞SMMC-7721和CBRH791的生长并且能诱导SMMC-7721细胞的凋亡,作用呈时间和剂量依赖性[12, 13]。三七皂苷Rg1对S-180和H-22瘤细胞移植性肿瘤均有明显的抑瘤作用[14]。三七皂苷R1可以浓度依赖性地抑制HL-60细胞增殖及促进HL-60细胞凋亡[15]。Ft1最先是从三七叶中分离得到,本课题组前期研究发现Ft1可显著抑制神经母细胞瘤SH-SY5Y细胞的增殖并促进其细胞凋亡[8],但是Ft1对乳腺癌是否也有抑制作用,尚未见文献报道。本研究发现,Ft1可明显降低乳腺癌细胞的生存率并抑制其增殖,同时也可诱导凋亡。乳腺癌往往具有早期转移的特性,本研究同时也考察了Ft1对乳腺癌细胞迁移能力的影响。单细胞迁移实验结果显示,Ft1能够显著抑制乳腺癌细胞的迁移能力,提示其对乳腺癌的转移也有潜在的抑制作用。

据报道,PI3K/Akt/mTOR/p70S6K信号通路过 度激活贯穿肿瘤整个病理发展进程,在肿瘤细胞生存、增殖、凋亡和迁移中均发挥着广泛的病理生理作用[16]。因此,本研究在确定了Ft1抑制乳腺癌细胞活力和增殖并促进凋亡作用后,首先考察了其对PI3K/ Akt/mTOR/p70S6K信号通路的影响。研究表明,Ft1确实能够显著抑制Akt/mTOR/p70S6K信号通路的过度激活。

PI3K和P70S6K的靶蛋白在细胞生存和增殖方面扮演着重要的角色[17]。HIF-1α为其靶蛋白之一,它通过调控其下游基因如骨髓细胞瘤细胞原癌基因 (cellular-myelocytomatosis,c-MYC)、胰岛素样生长因子-2 (insulin-like growth factor 2,IGF-2)、血管内皮生长因子 (vascular endothelial growth factor,VEGF)、丙酮酸脱氢酶激酶同工酶1 (pyruvate dehydrogenase kinase isozyme 1,PDK1) 和乳酸脱氢酶A (lactate dehydrogenase A,LDHA) 等,参与调控肿瘤细胞增殖、凋亡和迁移等。HIF-1α的激活受与细胞骨架蛋白tensin同源的在肿瘤的10号染色体有缺失的磷脂酶 (phosphatase and tensin homolog deleted on chro­mosome ten,PTEN) 调控,后者负反馈调控PI3K/Akt,而PI3K/Akt又参与调控HIF-1α的合成并介导VEGF的生成[18, 19]。因此PI3K/Akt/mTOR信号通路可以直接或间接参与调控HIF-1α的合成和激活,从而调控肿瘤细胞的活力、增殖、凋亡和迁移。另有研究也证明,PI3K/Akt/mTOR/p70S6K和MAPK信号通路均参与调控HIF-1α的表达,从而调控肿瘤细胞的生长[20]。EGFR是PI3K/Akt/mTOR/p70S6K和MAPK信号通路的上游信号分子。EGFR通路可以通过促进HIF-1的表达和活性,促进肿瘤发展进程[21]。因此本研究同时也考察了Ft1对EGFR磷酸化的抑制作用,结果显示,Ft1能显著抑制乳腺癌细胞MDA-MB-231细胞p-EGFR (Tyr1068) 和p-EGFR (Ser1046/1047) 的蛋白表达。因此,本研究结果提示,Ft1可能通过抑制EGFR的激活,进而抑制Akt/mTOR/p70S6K和MAPK (ERK1/2) 通路的激活,下调HIF-1α的蛋白表达,从而抑制乳腺癌细胞的增殖并促进其凋亡。

Bcl-2是凋亡通路的关键调节分子,在多种肿瘤细胞中均高表达,并通过抑制Bax转位入线粒体,从而下调cleaved caspase 3的表达,抑制肿瘤细胞的凋亡。Akt通过调控凋亡相关蛋白如Bcl-2,Bax和cleaved caspase 3等,参与抑制肿瘤细胞的凋亡[16]。此外,MAPK信号通路 (包括ERK、SAPK/JNK和p38 MAPK),积极参与了药物诱导的肿瘤细胞凋亡作用[22]。同时,MAPK通路也参与调控caspase 3活性和Bcl-2的蛋白表达,从而调控肿瘤细胞的凋亡[8]。本研究结果显示,Ft1可上调cleaved caspase 3,下调Bcl-2,同时Ft1能够显著抑制MDA-MB-231细胞ERK1/2的磷酸 化,进一步提示Ft1可能通过调控Akt/mTOR/p70S6K和MAPK通路从而促进细胞的凋亡。尽管如此,Akt/mTOR/p70S6K和MAPK通路、HIF-1α、Bcl-2和cleaved caspase 3蛋白在介导Ft1抑制乳腺癌作用中的相互关系尚需要进一步深入研究。

综上所述,Ft1可能通过抑制EGFR的激活,从 而对AKT/mTOR/p70S6K和MAPK (ERK1/2) 两条信号通路的双重抑制,下调HIF-1α蛋白表达,从而抑制Bcl-2的蛋白表达,促进cleaved caspase 3的蛋白表达,最终抑制乳腺癌细胞增殖和促进细胞凋亡,并在抑制肿瘤生长的同时抑制肿瘤的转移,提示Ft1在抗乳腺癌化疗潜在候选药物中的有效性,值得进一步深入研究。

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