龔 舒,段承剛,陶忠樺,劉曉燕,傅俊江,甘 淋
(西南醫(yī)科大學(xué):1病理生理學(xué)教研室;2公共衛(wèi)生學(xué)院;3醫(yī)學(xué)基礎(chǔ)研究中心;4生物化學(xué)教研室,四川瀘州 646000)
紅景天苷對(duì)人乳腺癌MDA-MB-435
細(xì)胞功能的作用*
龔 舒1,段承剛1,陶忠樺2,劉曉燕3,傅俊江3,甘 淋4
(西南醫(yī)科大學(xué):1病理生理學(xué)教研室;2公共衛(wèi)生學(xué)院;3醫(yī)學(xué)基礎(chǔ)研究中心;4生物化學(xué)教研室,四川瀘州 646000)
目的:通過檢測(cè)紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的增殖、凋亡、周期及運(yùn)動(dòng)侵襲的影響,探討紅景天苷抗人乳腺癌的作用。方法:體外培養(yǎng)MDA-MB-435細(xì)胞,CCK-8法篩查紅景天苷對(duì)細(xì)胞的半數(shù)有效抑制濃度(IC50)。以不加紅景天苷組為對(duì)照組,以IC50濃度的紅景天苷組為實(shí)驗(yàn)組,在光鏡下觀察其形態(tài)改變,CCK-8法檢測(cè)細(xì)胞增殖抑制率,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡和細(xì)胞周期,Transwell小室模型觀察細(xì)胞運(yùn)動(dòng)和侵襲能力的改變。結(jié)果:紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的IC50為4 mg/mL;光鏡下見紅景天苷組細(xì)胞呈現(xiàn)壞死和凋亡形態(tài);CCK-8法顯示紅景天苷組在作用24 h、48 h和72 h三個(gè)時(shí)間點(diǎn)均能抑制細(xì)胞的生長(zhǎng)增殖,抑制率均高于對(duì)照組,且具有顯著的時(shí)間-效應(yīng)依賴性(P<0.05);流式細(xì)胞術(shù)顯示紅景天苷組早期凋亡率為(3.31±0.21)%、晚期凋亡和壞死率為(28.80±2.15)%,高于對(duì)照組(1.26±0.13)%和(5.56±0.78)%,差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05);細(xì)胞周期檢測(cè)發(fā)現(xiàn)紅景天苷組細(xì)胞較對(duì)照組在G0/G1期的比例增加,停滯在S期的比例減少,G2/M期的比例減少;運(yùn)動(dòng)實(shí)驗(yàn)結(jié)果顯示,紅景天苷作用72 h后,紅景天苷組每個(gè)視野跨膜細(xì)胞數(shù)為(39.60±3.37)個(gè),遠(yuǎn)低于對(duì)照組(142.20±12.99)個(gè),差異具有統(tǒng)計(jì)學(xué)意義(P<0.01);侵襲實(shí)驗(yàn)結(jié)果示紅景天苷組每個(gè)視野跨膜細(xì)胞數(shù)為(4.20±1.55)個(gè),也低于對(duì)照組(13.00±3.09)個(gè),差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論:4 mg/mL紅景天苷作用人乳腺癌MDA-MB-435細(xì)胞,可抑制生長(zhǎng)增殖、誘導(dǎo)細(xì)胞凋亡和壞死發(fā)生、阻滯細(xì)胞周期、有效抑制腫瘤細(xì)胞的運(yùn)動(dòng)和侵襲。
紅景天苷;乳腺癌;增殖;凋亡;侵襲
乳腺癌是女性最為多發(fā)的惡性腫瘤之一,近年的發(fā)病率與死亡率有顯著上升[1]。紅景天含有40多種化合物,其中紅景天苷(Salidrosied,Sal)是迄今研究最多的有效成分之一[2],已證明具有抗缺氧、抗疲勞、抗輻射、抗紫外線、抗病毒及對(duì)神經(jīng)、內(nèi)分泌系統(tǒng)的雙向調(diào)節(jié)作用[3]。最近研究發(fā)現(xiàn),多個(gè)品種紅景天具有抗腫瘤作用[4],其機(jī)理可能為抑制腫瘤細(xì)胞增殖、誘導(dǎo)細(xì)胞分化和凋亡、阻滯細(xì)胞周期、抑制腫瘤新生血管生成等。故本研究從細(xì)胞水平出發(fā),采用分子生物學(xué)實(shí)驗(yàn)方法,研究紅景天苷對(duì)高侵襲的人乳腺癌MDA-MB-435細(xì)胞的增殖、凋亡、周期和運(yùn)動(dòng)侵襲能力的作用。
1.1 細(xì)胞株
人乳腺癌細(xì)胞株(MDA-MB-435)由美國貝勒醫(yī)學(xué)院徐建明教授饋贈(zèng),西南醫(yī)科大學(xué)醫(yī)學(xué)基礎(chǔ)研究中心保存。
1.2 藥物與試劑
紅景天苷(Sal)購自鄭州豐耀農(nóng)業(yè)科技有限公司;Cell Counting Kit-8(CCK-8)試劑盒購自日本同仁化學(xué)研究所;細(xì)胞DNA含量檢測(cè)試劑盒和Annexinv-FITC/PI細(xì)胞凋亡試劑盒購自南京凱基生物公司;Matrigel基質(zhì)膠購自美國BD公司。
1.3 主要儀器
二氧化碳培養(yǎng)箱、酶標(biāo)儀(美國Thermo公司);正置熒光顯微鏡(德國Leica公司);流式細(xì)胞儀(美國BD公司);Transwell小室(美國Corning公司)。
2.1 紅景天苷溶液的配制
蒸餾水5 mL高溫滅菌,將1 g/瓶的紅景天苷粉劑配制成濃度為200 mg/mL的溶液,正壓濾過除菌,-20℃儲(chǔ)存?zhèn)溆?,用時(shí)現(xiàn)配。
2.2 半數(shù)有效抑制濃度(half maximal inhibitory concentration,IC50)的篩查和計(jì)算
調(diào)整細(xì)胞密度至5×104個(gè)/mL,接種于96孔板中,每孔100 μL。24 h后,以不加紅景天苷的細(xì)胞為對(duì)照組,以不同濃度(0.01 mg/mL、0.1 mg/mL、1 mg/ mL、10 mg/mL、100 mg/mL)紅景天苷作用的細(xì)胞為實(shí)驗(yàn)組(Sal組),每組設(shè)4個(gè)平行孔。24 h后,每孔加入10 μL的CCK-8溶液,培養(yǎng)1 h后,置于酶標(biāo)儀波長(zhǎng)450 nm處測(cè)出各孔的吸光度(OD)值。改良寇式法公式計(jì)算半數(shù)有效抑制濃度lg(IC 50)=Xm-I [P-(3-Pm-Pn)/4]。此IC 50用于后續(xù)的紅景天苷實(shí)驗(yàn)組濃度。
2.3 CCK-8法檢測(cè)紅景天苷對(duì)細(xì)胞增殖的影響
調(diào)整細(xì)胞密度至5×104個(gè)/mL,接種于96孔板中,每孔100 μL。24 h后加入紅景天苷,實(shí)驗(yàn)組和對(duì)照組均設(shè)4個(gè)平行孔,分別培養(yǎng)24 h、48 h和72 h。各個(gè)時(shí)間點(diǎn)時(shí)在每孔加入10 μL的CCK-8溶液,培養(yǎng)1 h后,置于酶標(biāo)儀波長(zhǎng)450 nm處測(cè)出各孔的吸光度(OD)值。計(jì)算藥物對(duì)細(xì)胞生長(zhǎng)抑制率(IR)=(1-加藥組OD值/空白對(duì)照組OD值)×100%。
2.4 流式細(xì)胞術(shù)(FCM)檢測(cè)細(xì)胞凋亡和周期
2.4.1 細(xì)胞凋亡檢測(cè)
調(diào)整細(xì)胞密度至1×105個(gè)/mL,24 h后加入紅景天苷。作用24 h后,2 000 r/min離心收集細(xì)胞并調(diào)整密度至1×106個(gè)/mL。加Binding Buffer 500 μL懸浮細(xì)胞,再依次加入Annexin V-FITC、Propidium Iodide各5 μL混勻。置室溫避光反應(yīng)10 min后上機(jī)檢測(cè)細(xì)胞凋亡。
2.4.2 細(xì)胞周期檢測(cè)
同2.4.1離心收集細(xì)胞,冰乙醇固定,調(diào)整密度至1×106個(gè)/mL。每管加含0.5%EDTA、100 μg/mL RNaseA的PBS液1 mL,37℃水浴30 min。通過200目篩網(wǎng)過濾,加入PI染色劑至終濃度50 μg/ mL,避光孵育10 min后上機(jī)檢測(cè)細(xì)胞周期。
2.5 細(xì)胞運(yùn)動(dòng)、侵襲實(shí)驗(yàn)
2.5.1 細(xì)胞運(yùn)動(dòng)實(shí)驗(yàn)
調(diào)整細(xì)胞密度至1×106個(gè)/mL。加200 μL細(xì)胞懸液至Transwell侵襲小室的上室中,在下室中加入600 μL含20%FBS的培養(yǎng)基。培養(yǎng)72 h后取出上室,經(jīng)甲醇固定、結(jié)晶紫染色,顯微鏡下成像和計(jì)數(shù)細(xì)胞。
2.5.2 細(xì)胞侵襲實(shí)驗(yàn)
4℃溶解Matrigel基質(zhì)膠,用預(yù)冷的不含F(xiàn)BS的培養(yǎng)基稀釋Matrigel基質(zhì)膠至200 μg/mL。在侵襲小室的上室中每孔加入100 μL的稀釋基質(zhì)膠,置培養(yǎng)箱4 h后,余步驟同2.5.1。
2.6 數(shù)據(jù)分析
表1 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的IC50篩查(,n=4)
表1 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的IC50篩查(,n=4)
組別對(duì)照組Sal組濃度(mg/mL)0 0.01 0.1 1 10 100 OD值0.92±0.23 0.97±0.16 0.91±0.28 0.85±0.19 0.27±0.01 0.19±0.02抑制率(%)0 -5.21±2.83 1.83±4.85 8.14±3.19 70.45±0.12 79.78±0.24
圖1 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的IC50篩查曲線
圖2 兩組人乳腺癌MDA-MB-435細(xì)胞形態(tài)對(duì)比(×100)
3.1 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的半數(shù)有效抑制濃度(IC50)篩查
用改良寇式法算出紅景天苷對(duì)MDA-MB-435細(xì)胞的IC50為4 mg/mL。以細(xì)胞抑制率為縱坐標(biāo)、紅景天苷濃度為橫坐標(biāo)作標(biāo)準(zhǔn)曲線,可見紅景天苷對(duì)MDA-MB-435細(xì)胞的IC50約為4 mg/mL,同改良寇式法公式計(jì)算結(jié)果一致。當(dāng)紅景天苷終濃度≤0.1 mg/mL時(shí),抑制率呈負(fù)數(shù),提示其可輕度促進(jìn)MDA-MB-435細(xì)胞增殖。當(dāng)紅景天苷終濃度>0.1 mg/mL以上,抑制率隨藥物濃度增大而升高 (見表1、圖1)。
3.2 紅景天苷作用人乳腺癌MDA-MB-435細(xì)胞的細(xì)胞形態(tài)改變
光鏡下見對(duì)照組細(xì)胞貼壁生長(zhǎng)、分布均勻、形態(tài)飽滿、無色透明、折光性好。紅景天苷組(4 mg/mL)見大部分細(xì)胞的體積變小、皺縮變圓、形態(tài)不典型、與鄰近細(xì)胞分離、失去貼壁特性,呈現(xiàn)增殖抑制作用與細(xì)胞壞死和凋亡特征(見圖2)。3.3 CCK-8比色法觀察紅景天苷對(duì)人乳腺癌MDAMB-435細(xì)胞增殖的影響
CCK-8檢測(cè)表明,紅景天苷對(duì)MDA-MB-435細(xì)胞的增殖有抑制作用。與對(duì)照組比較,紅景天苷組抑制率隨時(shí)間增加而上升。以細(xì)胞抑制率為縱坐標(biāo)、紅景天苷作用時(shí)間為橫坐標(biāo)作標(biāo)準(zhǔn)曲線顯示,4 mg/ mL的紅景天苷作用時(shí)間越長(zhǎng),細(xì)胞抑制率上升越明顯,各時(shí)間點(diǎn)組別差異有統(tǒng)計(jì)學(xué)意義(F=36.73,P<0.05)。紅景天苷對(duì)MDA-MB-435細(xì)胞的抑制作用具有顯著的時(shí)間-效應(yīng)依賴關(guān)系(見表2、圖3)。
表2 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞增殖的抑制作用(,n=4)
表2 紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞增殖的抑制作用(,n=4)
注:a與對(duì)照組相比,P<0.05
組別對(duì)照組Sal組抑制率(%)24 h 0 30.08±4.94a48 h 0 38.95±2.58a72 h 0 45.01±12.13a
3.4 紅景天苷誘導(dǎo)人乳腺癌MDA-MB-435細(xì)胞凋亡
流式檢測(cè)顯示:紅景天苷組(4 mg/mL)早期凋亡率為(3.31±0.21)%,晚期凋亡和壞死率為(28.80± 2.15)%,高于對(duì)照組的早期凋亡率(1.26±0.13)%、晚期凋亡和壞死率(5.56±0.78)%,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)??梢娫诩t景天苷干預(yù)下,誘導(dǎo)細(xì)胞早期凋亡、晚期凋亡和壞死的作用增強(qiáng)(見圖4)。
圖3 不同時(shí)間點(diǎn)紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞增殖的抑制曲線
圖4 兩組人乳腺癌MDA-MB-435細(xì)胞的細(xì)胞凋亡情況
3.5 紅景天苷影響人乳腺癌MDA-MB-435細(xì)胞周期
流式檢測(cè)顯示,與對(duì)照組比較,紅景天苷對(duì)MDA-MB-435細(xì)胞周期分布有影響,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。紅景天苷可致MDA-MB-435細(xì)胞的G0/G1期延長(zhǎng),停滯在S期的比例減少,在G2/M期的比例減少(見圖5,表3)。
圖5 兩組人乳腺癌MDA-MB-435細(xì)胞的細(xì)胞周期檢測(cè)
表3 紅景天苷影響人乳腺癌MDA-MB-435細(xì)胞的細(xì)胞周期分布(,n=4)
表3 紅景天苷影響人乳腺癌MDA-MB-435細(xì)胞的細(xì)胞周期分布(,n=4)
組別對(duì)照組Sal組t P G0/G1(%)36.11±4.32 53.09±3.64 4.98<0.05 S(%)54.44±5.12 42.59±2.59 5.18<0.05 G2/M(%)9.46±2.47 4.32±0.73 4.81<0.05
Transwell法結(jié)果顯示,在24 h和48 h時(shí)間點(diǎn),細(xì)胞運(yùn)動(dòng)和侵襲差異均不明顯。作用72 h后,紅景天苷組MDA-MB-435細(xì)胞的運(yùn)動(dòng)和侵襲能力較對(duì)照組顯著降低。紅景天苷組運(yùn)動(dòng)和侵襲實(shí)驗(yàn)中每個(gè)視野跨膜細(xì)胞數(shù)分別為(39.60±3.37)個(gè)和(4.20± 1.55)個(gè),顯著低于對(duì)照組運(yùn)動(dòng)與侵襲實(shí)驗(yàn)的(142.20 ±12.99)個(gè)和(13.00±3.09)個(gè),差異有統(tǒng)計(jì)學(xué)意義(P<0.05,見圖6,表4)。
圖6 紅景天苷抑制人乳腺癌MDA-MB-435細(xì)胞運(yùn)動(dòng)與侵襲能力情況
表4 Transwell法檢測(cè)紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的運(yùn)動(dòng)與侵襲能力(,n=4)
表4 Transwell法檢測(cè)紅景天苷對(duì)人乳腺癌MDA-MB-435細(xì)胞的運(yùn)動(dòng)與侵襲能力(,n=4)
組別對(duì)照組Sal組t P細(xì)胞運(yùn)動(dòng)142.20±12.99 39.60±3.37 14.37<0.01細(xì)胞侵襲13.00±3.09 4.20±1.55 5.32<0.05
中醫(yī)認(rèn)為,正虛邪實(shí)是乳腺癌發(fā)病的總病機(jī),組方原則以健脾益氣為主,以軟堅(jiān)散結(jié)、疏肝理氣、清熱解毒為佐[5]。因化學(xué)合成藥物的毒副作用和耐藥性,篩選天然藥物作為治療腫瘤的候選和輔助藥物成為研究熱點(diǎn)[6]??拱┲兴帉?duì)乳腺癌細(xì)胞的殺傷作用,可通過阻滯細(xì)胞周期、誘導(dǎo)細(xì)胞凋亡、遏制細(xì)胞遷移、逆轉(zhuǎn)細(xì)胞耐藥性以及干擾基因表達(dá)等多種途徑實(shí)現(xiàn),具有廣大的應(yīng)用前景[7]。文獻(xiàn)證實(shí)[8],紅景天苷對(duì)乳腺癌的生長(zhǎng)及癌細(xì)胞的轉(zhuǎn)移均有有效抑制作用,具有潛在的臨床藥用開發(fā)價(jià)值。Hu等[9]認(rèn)為紅景天苷對(duì)多種腫瘤細(xì)胞生長(zhǎng)均有抑制,是由于紅景天苷通過上調(diào)細(xì)胞周期蛋白依賴激酶抑制因子-p27Kip1與p21Cip1的表達(dá),下調(diào)細(xì)胞周期蛋白依賴激酶CDK4和Cdc2的表達(dá),抑制細(xì)胞周期蛋白D1和B1的表達(dá),進(jìn)而抑制細(xì)胞周期蛋白依賴激酶CDK4/細(xì)胞周期蛋白D1的相關(guān)通路,阻滯癌細(xì)胞周期于G1期;抑制細(xì)胞周期蛋白依賴激酶Cdc2/細(xì)胞周期蛋白B1通路,阻滯癌細(xì)胞周期于G2期。雖然目前研究提示紅景天苷可能對(duì)乳腺癌治療有一定作用,但其調(diào)
3.6 紅景天苷抑制人乳腺癌MDA-MB-435細(xì)胞的運(yùn)動(dòng)侵襲控癌細(xì)胞增殖、凋亡、周期和運(yùn)動(dòng)侵襲的詳細(xì)機(jī)制尚不清楚。本課題組就紅景天苷的上述作用做出初步研究,結(jié)果發(fā)現(xiàn),4 mg/mL紅景天苷作用人乳腺癌MDA-MB-435細(xì)胞,可抑制其生長(zhǎng)增殖、誘導(dǎo)細(xì)胞凋亡和壞死發(fā)生、阻滯細(xì)胞周期、有效抑制腫瘤細(xì)胞的運(yùn)動(dòng)和侵襲,為下一步探討紅景天苷成為抗乳腺癌靶點(diǎn)藥物的分子機(jī)制提供了實(shí)驗(yàn)基礎(chǔ)。
1.Higgins MJ,Baselga J.Targeted therapies for breast cancer [J].J Clin Invest,2011,121(10):3797-3803.
2.中國科學(xué)院中國植物志編輯委員會(huì).中國植物志[M].第五十四卷.北京,中國科學(xué)出版社,2013:82-85.
3.莊?,?紅景天作用及機(jī)制的研究進(jìn)展[J].河北中醫(yī),2012,34(4):617-619.
4.Sun KX,Xia HW,Xia RL.Anticancer effect of Salidroside on colon cancer through inhibiting JAK2/STAT3 signaling pathway[J].Int J Clin Exp Pathol,2015,8(1):615-621.
5.奚燕,楊銘,許麗雯.我院抗腫瘤中藥處方分析[J].中國醫(yī)藥導(dǎo)刊,2015,17(1):61-62,64.
6.Sertel S,Plinkert PK,Efferth T.Natural products derived from traditional chinese medicine as novel inhibitors of the epidermal growth factor receptor[J].Comb Chem High T-hroughput Screen,2010,13(10):849-854.
7.孫放,孫行云,劉占春,等.中醫(yī)藥治療乳腺癌的現(xiàn)代生物學(xué)研究進(jìn)展[J].云南中醫(yī)學(xué)院學(xué)報(bào),2014,37(6):85-88,92.
8.韓雪嬌,郭娜,朱美宣,等.紅景天苷藥理作用及其作用機(jī)理研究進(jìn)展[J].中國生化藥物雜志,2015,35(1):171-175.
9.Hu X,Lin S,Yu D,et al.A preliminary study:the antiproliferation effect of Salidroside on different human cancer cell lines[J].Cell Biol Toxicaol,2010,26(6):499-507.
(2015-09-15收稿)
Effect of Salidroside on the function of human breast cancer cell line MDA-MB-435
Gong Shu1,Duan Chenggang1,Tao Zhonghua2,Liu Xiaoyan3,F(xiàn)u Junjiang3,GanLin41Department of Pathophysiology;2Department of Epidemiology and Statistics of School of Public Health;3The Research Center for Preclinical Medicine;4Department of Biochemistry,Southwest Medical University,Luzhou 646000,Sichuan Province,China
Objective:To explore the effects and mechanisms of Salidroside on human breast cancer.Human breast cancer cell line MDA-MB-435 was treated with Salidroside(Sal),and cell proliferation,apoptosis,cell migration and invasion were evaluated.Methods:The MDA-MB-435 cells were cultured in vitro.The half maximal inhibitory concentration(IC50)of Salidroside on MDA-MB-435 cells was determined by CCK-8 assay. MDA-MB-435 cells treated with IC50 of Sal were considered as the Sal group,and cells without Sal treatment were as the control group.Cell morphology was observed using light microscope,cell proliferation was detected by CCK-8 assay,apoptosis and cell cycle were evaluated by flow cytometry,and cell migration and invasion were studied using Transwell assay.Results:The IC50 of Sal on human breast cancer MDA-MB-435 cells was 4 mg/mL.The cells treated with Sal showed the typical morphology of necrosis and apoptosis under lightmicroscropy.Using CCK-8 assay,cells proliferation was significantly inhibited at the IC50 of Sal at 24 h,48 h and 72 h,relative to the control group(P<0.05),and the inhibition showed a time-dependent manner.Flow cytometry using AnnexinⅤ-FITC/PI apoptosis assay kit demonstrated that the percentage of early apoptotic cells was significantly increased(3.31±0.21)%in the Sal group,compared to(1.26±0.13)%in the control group(P<0.05),and the late apoptosis and necrosis was also significantly induced (28.80±2.15)%in the Sal group, compared to (5.56±0.78)%in the control group(P<0.01).The results of FITC demonstrated that the ratio of G0/G1 was increased,while the cells at S phase and the ratio of G2/M were decreased compared to those of the control group.The cell migration and invasion assay revealed that the average number of invaded and migrated cells in the Sal group in migration assay(39.60±3.37),and in invasive assay(4.20±1.55)were significantly decreased compared to the control group in migration assay(142.20±12.99) (P<0.01)and in invasive assay(13.00 ± 3.09)(P < 0.05),respectively.Conclusion:Salidroside treatment significantly inhibited cell proliferation,and promoted apoptosis,arrested cell cycle and repressed the motility and invasion of MDA-MB-435 cells.
Salidroside;Breast cancer;Proliferation;Apoptosis;Invasion
R737.9
A
10.3969/j.issn.1000-2669.2016.02.003
*四川省科技廳應(yīng)用基礎(chǔ)研究計(jì)劃項(xiàng)目(14JC0155)
龔 舒(1982-),女,碩士。
甘 淋(1976-),女,副教授,博士。E-mail:gl-gump@163.com