白 雪，張 月，李 慧，何 平

山楂酸對(duì)肺癌95D細(xì)胞增殖、凋亡的影響

白 雪，張 月，李 慧，何 平*

目的 探討山楂酸對(duì)肺癌細(xì)胞95D增殖及凋亡產(chǎn)生的影響。方法 將山楂酸作用于人的肺癌細(xì)胞系95D，通過(guò)四甲基偶氮唑藍(lán)(MTT)法檢測(cè)山楂酸對(duì)細(xì)胞增殖的影響；通過(guò)流式細(xì)胞儀Annexin V/PI雙染法檢測(cè)細(xì)胞凋亡；通過(guò)熒光顯微鏡檢測(cè)細(xì)胞核的形態(tài)變化；采用Western blot方法檢測(cè)凋亡相關(guān)蛋白的影響。結(jié)果 隨著山楂酸濃度增大，95D細(xì)胞的增殖率明顯降低，早期及晚期凋亡率逐漸增加，與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義；熒光電子顯微鏡觀察山楂酸作用后的細(xì)胞，可見染色體邊集、核碎裂等典型的凋亡改變，而對(duì)照組沒有相應(yīng)的變化；Western blot顯示，隨著山楂酸濃度的增加，Bcl-2蛋白表達(dá)逐漸降低，Bax蛋白表達(dá)逐漸升高。結(jié)論 山楂酸可以抑制肺癌95D細(xì)胞增殖，且具有濃度依賴性，山楂酸抑制肺癌95D細(xì)胞增殖是通過(guò)誘導(dǎo)細(xì)胞凋亡實(shí)現(xiàn)的。

山楂酸；肺癌；增殖；凋亡

0 引言

肺癌是目前世界上發(fā)病率和死亡率最高的惡性腫瘤之一，盡管近年來(lái)肺癌的診療技術(shù)有了一定程度的提高，肺癌的5年生存率仍較低[1-2]。近年來(lái)，中藥抗腫瘤作用及機(jī)制已成為國(guó)內(nèi)外研究的熱點(diǎn)[3-4]。相關(guān)研究表明，中藥抗腫瘤的作用機(jī)制包括：誘導(dǎo)腫瘤細(xì)胞凋亡、抑制腫瘤血管生成、調(diào)節(jié)細(xì)胞信號(hào)傳導(dǎo)、誘導(dǎo)腫瘤細(xì)胞分化、細(xì)胞毒作用、調(diào)節(jié)機(jī)體免疫功能、逆轉(zhuǎn)腫瘤細(xì)胞多藥耐藥作用等[5-7]。

本研究選取的藥物—山楂酸(Maslinic acid)，是一種三萜類天然有機(jī)化合物，存在于多種天然植物，特別是山楂、紅棗、枇杷葉和油橄欖中，對(duì)人體的安全性較高，具有抗癌、抗氧化、抗艾滋病、抗菌、抗糖尿病等多種生物活性[8-10]。近年研究發(fā)現(xiàn)，山楂酸對(duì)人結(jié)腸癌HT29細(xì)胞[11]、人前列腺癌 DU145細(xì)胞[12]、小鼠黑色素瘤細(xì)胞[13]等有較強(qiáng)的抗腫瘤活性，其中部分作用機(jī)制與誘導(dǎo)腫瘤細(xì)胞凋亡相關(guān)。本實(shí)驗(yàn)主要研究山楂酸對(duì)非小細(xì)胞肺癌95D細(xì)胞的增殖及凋亡的影響。

1 材料與方法

1.1 材料、試劑與儀器 人肺癌細(xì)胞系95D購(gòu)于美國(guó)典型培養(yǎng)物保藏中心(American Type Culture Collection，ATCC)。山楂酸購(gòu)于上海純優(yōu)生物科技有限公司，RPMI-1640培養(yǎng)基購(gòu)于Gibco公司，胎牛血清購(gòu)于Hyclone公司，胰蛋白酶購(gòu)于以色列Biological Industries公司，四甲基偶氮唑藍(lán)(MTT)、Hoechst 33342購(gòu)于美國(guó)Sigma公司，AnnexinV/PI雙染試劑盒購(gòu)于BD公司，超凈工作臺(tái)(SW-CJ-1FD)購(gòu)于蚌埠凈化設(shè)備廠，酶標(biāo)儀(BIOTEK-ELX808)購(gòu)于美國(guó)賽默飛世爾公司，流式細(xì)胞儀(FACSCalibur)購(gòu)于美國(guó)BD公司，倒置顯微鏡(Olympus CKX31)購(gòu)于日本奧林巴斯公司，熒光電子顯微鏡(Nikon C1)購(gòu)于日本尼康公司，離心機(jī)購(gòu)于德國(guó)Eppendorf公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng) 人肺癌95D細(xì)胞培養(yǎng)于含10%胎牛血清的RPMI-1640培養(yǎng)基中，置于37 ℃、飽和濕度、5%二氧化碳的培養(yǎng)箱中培養(yǎng)。細(xì)胞呈單層貼壁生長(zhǎng)，接種細(xì)胞過(guò)夜進(jìn)入對(duì)數(shù)生長(zhǎng)期后，將細(xì)胞濃度調(diào)整為5×105/mL，開始進(jìn)行藥物干預(yù)。

1.2.2 MTT實(shí)驗(yàn) 山楂酸對(duì)95D細(xì)胞增殖的抑制作用：消化收集對(duì)數(shù)增殖期細(xì)胞，將細(xì)胞懸液濃度調(diào)整為1×105/mL后接種于96孔板，每孔100 μL，每個(gè)濃度設(shè)3個(gè)復(fù)孔，同時(shí)設(shè)立含RPMI-1640全培養(yǎng)基的空白對(duì)照和未予藥物作用的陰性對(duì)照。實(shí)驗(yàn)組加入終濃度依次為0、2、4、6、8、10 μg/mL的山楂酸溶液，將96孔板置于37 ℃、飽和濕度、5%二氧化碳的培養(yǎng)箱中培養(yǎng)24 h，每孔加入MTT 20 μL，繼續(xù)培養(yǎng)4 h，培養(yǎng)結(jié)束后吸去上清，每孔加入DMSO 150 μL，振蕩混勻10 min。酶標(biāo)儀檢測(cè)波長(zhǎng)570 nm的每孔光密度值(OD值)。增殖抑制率按如下公式計(jì)算：抑制率=(1-加藥組OD值/對(duì)照組OD值)×100%。實(shí)驗(yàn)重復(fù)3次。

1.2.3 流式細(xì)胞儀(FCM)AnnexinV/PI雙染法檢測(cè)山楂酸誘導(dǎo)95D細(xì)胞凋亡 95D細(xì)胞以5×105/mL接種于6孔板中，分別加入終濃度為0、2、4、6、8、10 μg/mL的山楂酸溶液，并設(shè)空白對(duì)照組(加等量RPMI-1640培養(yǎng)基)，于37 ℃、飽和濕度、5%二氧化碳的培養(yǎng)箱中培養(yǎng)24 h后胰酶消化收獲細(xì)胞。4 ℃、1 500 r/min離心5 min后收集細(xì)胞，棄去培養(yǎng)基，冷PBS洗滌、離心細(xì)胞2次。用500 μL Binding Buffer懸浮細(xì)胞，濃度約為1×106/mL，依次加入5 μL AnnexinV-FITC、5 μL PI混勻，2～8 ℃避光條件下孵育15 min，流式細(xì)胞儀檢測(cè)，分析凋亡的百分比。每組實(shí)驗(yàn)重復(fù)3次。

1.2.4 熒光顯微鏡觀察凋亡 將預(yù)處理過(guò)的蓋玻片置于六孔板內(nèi)，95D細(xì)胞以5×105/mL接種于蓋玻片中培養(yǎng)過(guò)夜，加入終濃度為8 μg/mL的山楂酸溶液，設(shè)空白對(duì)照組(加等量RPMI-1640培養(yǎng)基)，于37 ℃、飽和濕度、5%二氧化碳的培養(yǎng)箱中培養(yǎng)24 h。PBS洗2次，吹干，固定15 min，加入Hoechst 33342熒光染料(5 mg/L) 1 mL，37℃避光孵育15 min，含10%甘油的PBS封片，PBS洗2次，于熒光顯微鏡下觀察、照相。每組實(shí)驗(yàn)重復(fù)3次。

1.2.5 Western blot檢測(cè)Bcl-2、Bax的表達(dá) 95D細(xì)胞培養(yǎng)過(guò)夜，加入終濃度為4、6、8 μg/mL的山楂酸溶液，并設(shè)空白對(duì)照組(加等量RPMI-1640培養(yǎng)基)，于37 ℃、飽和濕度、5%二氧化碳的培養(yǎng)箱中培養(yǎng)24 h后胰酶消化，收獲細(xì)胞。裂解，低溫離心10 min (12 000 r/min，4 ℃)取上清液，加入上樣緩沖液，煮沸5 min，冷卻。加樣，Marker，行SDS-PAGE電泳，濃縮膠80 V電泳約30 min，分離膠100 V、90 min電泳至底部。轉(zhuǎn)移、封閉后，加入一抗，本實(shí)驗(yàn)所用一抗為Bcl-2、Bax、GAPDH (1∶200；Santa Cruz；USA)，4 ℃過(guò)夜孵育。應(yīng)用羊抗鼠IgG-HRP(1∶3 000)標(biāo)記二抗，孵育2 h。PVDF膜上均勻滴加適量發(fā)光液，行ECL底物發(fā)光。

2 結(jié)果

2.1 山楂酸對(duì)95D細(xì)胞生長(zhǎng)抑制作用 MTT法檢測(cè)細(xì)胞增殖表明，隨著山楂酸作用濃度的增加，對(duì)95D細(xì)胞的增殖有明顯的抑制作用，呈現(xiàn)劑量效應(yīng)關(guān)系，與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見圖1。

2.2 山楂酸對(duì)95D細(xì)胞凋亡的誘導(dǎo)作用 流式細(xì)胞儀AnnexinV/PI雙染法檢測(cè)表明，山楂酸作用24 h，隨山楂酸濃度的增高，早期凋亡和晚期凋亡細(xì)胞的比例均逐漸增加，與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見圖2。

圖1 山楂酸(MA)對(duì)肺癌95D細(xì)胞增殖的抑制作用注：*與MA 0 μg/mL比較，P<0.05

圖2 流式細(xì)胞儀AnnexinV/PI雙染法檢測(cè)MA作用于肺癌 95D細(xì)胞的凋亡率注：*與MA 0 μg/mL比較，P<0.05

2.3 Hoechst33342染色熒光顯微鏡觀察細(xì)胞核形態(tài) 8 μg/mL山楂酸作用于95D細(xì)胞24 h后，細(xì)胞形態(tài)出現(xiàn)顯著變化，呈現(xiàn)染色質(zhì)邊集、核碎裂等改變。隨著藥物濃度增加，細(xì)胞數(shù)量明顯減少，凋亡的細(xì)胞明顯增加，凋亡改變明顯。見圖3。

圖3 山楂酸作用于95D細(xì)胞后引起細(xì)胞核形態(tài)改變(400×)

2.4 山楂酸對(duì)凋亡相關(guān)蛋白Bcl-2、Bax表達(dá)的影響 Western blot方法檢測(cè)結(jié)果顯示，隨著山楂酸作用濃度的增加，Bcl-2蛋白表達(dá)逐漸降低，Bax蛋白表達(dá)逐漸增高。見圖4。

圖4 MA作用于肺癌95D細(xì)胞后對(duì)Bcl-2、Bax表達(dá)的影響

3 討論

目前，許多抗腫瘤藥物通過(guò)促進(jìn)腫瘤細(xì)胞發(fā)生凋亡來(lái)實(shí)現(xiàn)抗腫瘤作用[14-17]。Bcl-2家族是細(xì)胞凋亡的關(guān)鍵調(diào)節(jié)因子，在線粒體凋亡途徑的調(diào)控中起重要作用，其中，Bcl-2、Bax在目前研究最為廣泛。當(dāng)細(xì)胞接受死亡信號(hào)刺激，Bax被激活，從細(xì)胞漿轉(zhuǎn)移到線粒體膜上，進(jìn)而形成同源二聚體，促進(jìn)細(xì)胞凋亡，而Bcl-2則在Bax誘發(fā)凋亡過(guò)程中對(duì)其功能進(jìn)行限制。當(dāng)細(xì)胞內(nèi)Bcl-2表達(dá)增多時(shí)，細(xì)胞凋亡受到抑制，Bax表達(dá)增多時(shí)，細(xì)胞凋亡受到促進(jìn)，Bcl-2/Bax比例對(duì)細(xì)胞凋亡的發(fā)生意義重大[18]。

近年來(lái)，中藥及其有效成分因其抗腫瘤作用確切，在腫瘤治療中的地位日趨受到重視。研究發(fā)現(xiàn)，從中藥中提取的一些萜類化合物，尤其是三萜類化合物，具有潛在的抗腫瘤作用，其抗腫瘤機(jī)制與促進(jìn)腫瘤細(xì)胞凋亡有關(guān)[19-24]。

本研究表明，山楂酸具有較強(qiáng)的抗氧化、抗炎、抗病毒、抗腫瘤等多種活性作用，研究顯示，山楂酸對(duì)肝癌、乳腺癌、胰腺癌、結(jié)腸癌細(xì)胞有很強(qiáng)的抑制作用[25-29]。Fernando等[30]通過(guò)山楂酸對(duì)人結(jié)腸癌癌細(xì)胞HT29的實(shí)驗(yàn)證明，山楂酸通過(guò)直接激活線粒體凋亡通路導(dǎo)致人結(jié)腸癌癌HT29細(xì)胞死亡。

本實(shí)驗(yàn)MTT結(jié)果顯示，山楂酸對(duì)95D細(xì)胞具有明顯的增殖抑制作用，并且存在劑量依賴關(guān)系。AnnexinV/PI雙染法結(jié)果顯示，山楂酸對(duì)95D細(xì)胞的增殖抑制作用是由誘導(dǎo)細(xì)胞凋亡來(lái)實(shí)現(xiàn)的，且凋亡率隨著藥物濃度的增加而升高，呈劑量依賴性。通過(guò)熒光顯微鏡，從形態(tài)學(xué)上證實(shí)，山楂酸可以誘導(dǎo)95D細(xì)胞凋亡。Western blot檢測(cè)結(jié)果顯示，隨著山楂酸作用濃度的增加，Bcl-2蛋白表達(dá)逐漸降低，Bax蛋白表達(dá)逐漸增高。

綜上所述，山楂酸可以有效地抑制人肺癌95D細(xì)胞的增殖，且呈濃度依賴性，山楂酸抑制肺癌95D細(xì)胞的增殖是通過(guò)誘導(dǎo)其凋亡來(lái)實(shí)現(xiàn)的，凋亡通路之一可能是線粒體凋亡通路。

[1] Blumenthal GM,Karuri SW,Zhang H,et al.Overall response rate,progression-free survival,and overall survival with targeted and standard therapies in advanced non-small-cell lung cancer:US Food and Drug Administration trial-level and patient-level analyses[J].J Clin Oncol,2015,33(9):1008-1014.

[2] Li Y,Jiang L,Liu XG,et al.Efficacy and survival rate analysis of lung cancer with spinal metastases[J].Beijing Da Xue Xue Bao,2014,46(1):138-143.

[3] Xia L,Plachynta M,Liu T,et al.Pro-inflammatory effect of a traditional Chinese medicine formula with potent anti-cancer activity in vitro impedes tumor inhibitory potential in vivo[J].Mol Clin Oncol,2016,5(6):717-723.

[4] Xu J,Song Z,Guo Q,et al.Synergistic effect and molecular mechanisms of traditional chinese medicine on regulating tumor microenvironment and cancer cells[J].Biomed Res Int,2016,2016:1490738.

[5] Guo Q,Li J,Lin H.Effect and molecular mechanisms of traditional chinese medicine on regulating tumor immunosuppressive microenvironment[J].Biomed Res Int,2015,2015:261620.

[6] Liu M,Jia LQ.Preliminary clinical experience of Prof.Li Pei-wen in treating complications of tumor by external treatment of traditional Chinese medicine[J].Zhongguo Zhong Xi Yi Jie He Za Zhi,2014,34(11):1390-1391.

[7] Zhang YH,Wang Y,Yusufali AH,et al.Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo[J].J Integr Med,2014,12(6):483-494.

[8] Sommerwerk S,Heller L,Kuhfs J,et al.Urea derivates of ursolic,oleanolic and maslinic acid induce apoptosis and are selective cytotoxic for several human tumor cell lines[J].Eur J Med Chem,2016,119:1-16.

[9] Siewert B,Pianowski E,Csuk R.Esters and amides of maslinic acid trigger apoptosis in human tumor cells and alter their mode of action with respect to the substitution pattern at C-28[J].Eur J Med Chem,2013,70:259-272.

[10]Li C,Yang Z,Zhai C,et al.Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor alpha by inhibiting NF-kappaB signaling pathway[J].Mol Cancer,2010,9:73.

[11]Peragón J,Rufino-Palomares EE,Muoz-Espada I,et al.A New HPLC-MS Method for Measuring Maslinic Acid and Oleanolic Acid in HT29 and HepG2 Human Cancer Cells[J].Int J Mol Sci,2015,16(9):21681-21694.

[12]Park SY,Nho CW,Kwon DY,et al.Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells:possible mediation via hypoxia-inducible factor-1alpha signalling[J].Br J Nutr,2013,109(2):210-222.

[13]Mokhtari K,Rufino-Palomares EE,Pérez-Jiménez A,et al.Maslinic acid,a triterpene from olive,affects the antioxidant and mitochondrial status of B16F10 melanoma cells grown under stressful conditions[J].Evid Based Complement Alternat Med,2015,2015:272457.

[14]Zheng X,Jin X,Li F,et al.Inhibiting autophagy with chloroquine enhances the anti-tumor effect of high-LET carbon ions via ER stress-related apoptosis[J].Med Oncol,2017,34(2):25.

[15]Sun D,Mou Z,Li N,et al.Anti-tumor activity and mechanism of apoptosis of A549 induced by ruthenium complex[J].J Biol Inorg Chem,2016,21(8):945-956.

[16]Li W,Tian YH,Liu Y,et al.Platycodin D exerts anti-tumor efficacy in H22 tumor-bearing mice via improving immune function and inducing apoptosis[J].J Toxicol Sci,2016,41(3):417-428.

[17]Wang L,Zhang J,Yuan Q,et al.Separation and purification of an anti-tumor peptide from rapeseed (Brassica campestris L.) and the effect on cell apoptosis[J].Food Funct,2016,7(5):2239-2248.

[18]Brenner C,Lemoine A.Mitochondrial proteins (e.g.,VDAC,Bcl-2,HK,ANT) as major control points in oncology[J].Front Oncol,2014,15(4):365-366.

[19]Bai ST,Zhu GL,Peng XR,et al.Cytotoxicity of Triterpenoid Alkaloids from Buxus microphylla against Human Tumor Cell Lines[J].Molecules,2016,21(9).E1125.

[20]Zong J,Wang R,Bao G,et al.Novel triterpenoid saponins from residual seed cake of Camellia oleifera Abel.show anti-proliferative activity against tumor cells[J].Fitoterapia,2015,104:7-13.

[21]Dasgupta A,Sawant MA,Lavhale MS,et al.AECHL-1,a novel triterpenoid,targets tumor neo-vasculature and impairs the endothelial cell cytoskeleton[J].Angiogenesis,2015,18(3):283-299.

[22]Xia Q,Mao W.Anti-tumor effects of traditional Chinese medicine give a promising perspective[J].J Cancer Res Ther,2014,10(Suppl 1):1-2.

[23]Zhou J,Zhou T,Jiang M,et al.Research progress on synergistic anti-tumor mechanisms of compounds in traditional Chinese medicine[J].J Tradit Chin Med,2014,34(1):100-105.

[24]Ling CQ,Yue XQ,Ling C.Three advantages of using traditional Chinese medicine to prevent and treat tumor[J].J Integr Med,2014,12(4):331-335.

[25]Fukumitsu S,Villareal MO,Fujitsuka T,et al.Anti-inflammatory and anti-arthritic effects of pentacyclic triterpenoids maslinic acid through NF-kappaB inactivation[J].Mol Nutr Food Res,2016,60(2):399-409.

[26]Hung YC,Yang HT,Yin MC.Asiatic acid and maslinic acid protected heart via anti-glycative and anti-coagulatory activities in diabetic mice[J].Food Funct,2015,6(9):2967-2974.

[27]Kapoor S.Maslinic acid and its in vitro anti-neoplastic effects[J].Nat Prod Res,2013,27(23):2210-2211.

[28]Rufino-Palomares EE,Reyes-Zurita FJ,García-Salguero L,et al.Maslinic acid,a triterpenic anti-tumoural agent,interferes with cytoskeleton protein expression in HT29 human colon-cancer cells[J].J Proteomics,2013,83:15-25.

[29]Huang L,Guan T,Qian Y,et al.Anti-inflammatory effects of maslinic acid,a natural triterpene,in cultured cortical astrocytes via suppression of nuclear factor-kappa B[J].Eur J Pharmacol,2011,672(1-3):169-174.

[30]Reyes-Zurita FJ,Pachón-Pea G,Lizárraga D,et al.The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism[J].BMC Cancer,2011,11:154.

Effect of maslinic acid on the proliferation and apoptosis of lung cancer 95D cells

BAI Xue,ZHANG Yue,LI Hui,HE Ping*

(Shengjing Hospital of China Medical University,Shenyang 110004,China)

Objective To research the effect of maslinic acid on the proliferation and apoptosis of lung cancer 95D cells.Methods The effects of maslinic acid on proliferation of 95D cells was determined by MTT assay,and the apoptosis was analyzed by Annexin V/PI flow cytometric analysis.The morphology of apoptotic cell was detected by fluorescent microscopy.The effect of apoptosis-related proteins was detected by Western blot.Results Maslinic acid inhibited the proliferation of 95D cells in a dose-dependent manner significantly,and the proportion of Annexin V positive cells in drug-treated cells were increased in a dose-dependent manner,there were significant differences compared with control group (P<0.05).The observation of fluorescent microscopy showed that treatment with maslinic acid for 24 h could significantly induce the apoptotic cell death with chromatin condensation and nuclear fragmentation,while no corresponding change was found in control group.Western blot showed that the expression of Bcl-2 protein decreased gradually and the expression of Bax protein increased gradually with the increase of maslinic acid′s concentration.Conclusion Maslinic acid could inhibit the proliferation of 95D cell lines in a dose-dependent manner,which is achieved by induction of apoptosis.

Maslinic acid;Lung cancer;Proliferation;Apoptosis

2017-02-11

中國(guó)醫(yī)科大學(xué)附屬盛京醫(yī)院，沈陽(yáng) 110004

*通信作者

10.14053/j.cnki.ppcr.201707002