青蒿琥酯对胃癌细胞系HGC27细胞增殖、凋亡的影响及其机制

2014-05-08 16:31吴方红戈伟周学军郑永法文静
中国医药导报 2014年5期
关键词:琥酯青蒿胃癌

吴方红+戈伟+周学军+郑永法+文静

[摘要] 目的 探讨青蒿琥酯对大胃癌细胞系HGC27细胞增殖、凋亡的影响及其机制。 方法 体外培养HGC27细胞,采用不同浓度青蒿琥酯处理24、48、72 h,MTT法测定其对HGC27细胞增殖的影响;倒置显微镜下观察细胞的形态学变化;青蒿琥酯(浓度分别为20、40、80 mg/L)处理HGC27细胞48 h后,分别采用流式细胞仪检测细胞凋亡、分光光度计检测Casepase-3、Caspase-9相对活性、Western blot法检RUNX-3蛋白表达情况。 结果 青蒿琥酯(浓度10~100 mg/L)能抑制HGC27细胞的增殖,呈剂量和时间依赖性;倒置显微镜下可观察到典型的细胞凋亡形态。青蒿琥酯(浓度分别为20、40、80 mg/L)作用HGC27细胞48 h后,细胞凋亡率分别为11.5%、21.4%、36.6%,而对照组凋亡率仅为2.2%;处理后Casepase-3相对活性分别为(0.19±0.02)、(0.25±0.04)和(0.31±0.03),对照组为(0.11±0.02),Casepase-9相对活性分别为(0.18±0.02)、(0.23±0.03)和(0.30±0.04),对照组为(0.10±0.02),与对照组比较,青蒿琥酯处理组Casepase-3、Caspase-9相对活性显著增加,差异均有统计学意义(均P < 0.05);RUNX-3蛋白表达上调,呈剂量依赖性。 结论 青蒿琥酯能抑制HGC27细胞的增殖并诱导其凋亡,其作用机制可能与增加细胞Casepase-3、Caspase-9活性、上调RUNX-3蛋白的表达有关。青蒿琥酯是一种前景广阔的抗肿瘤药物。

[关键词] 青蒿琥酯;HGC27细胞;Caspase-3;Caspase-9;人类相关转录因子3

[中图分类号] R285.5 [文献标识码] A [文章编号] 1673-7210(2014)02(b)-0009-04

Effects of artesunate on cell proliferation and apoptosis in human gastric cancer HGC27 cells and its mechanisms

WU Fanghong GE Wei ZHOU Xuejun ZHENG Yongfa WEN Jing

Department of Oncology, Renmin Hospital of Wuhan University, Hubei Province, Wuhan 430060, China

[Abstract] Objective To investigate the effect of artesunate on cell proliferation and apoptosis in gastric cancer line HGC27 cells and discuss its possible mechanisms. Methods HGC27 cells were cultured in vitro. After treatment by artesunate at different concentrations respectively at 24, 48, 72 h, the cell survival was determined by the MTT method. The changes of cell morphology were observed by inverted microscope. After 48 h treatment by artesunate (20, 40, 80 mg/L), the HGC27 cell apoptosis was detected by flow cytometry, the relative activity of Caspase-3 and Caspase-9 was monitored by spectrophotometer, the change of protein expression of RUNX-3 was detected by western blot. Results From the data of MTT, the cell proliferation of human gastricl cancer HGC27 cells was inhibited by artesunate (10-100 mg/L) in a dose-dependent and time-dependent manner. Typical apoptosis morphology of HGC27 cells was observed by inverted microscope. Flow cytometry assays showed that artesunate significantly induced apoptosis in HGC27 cells. After treated with artesunate (20, 40, 80 mg/L), the apoptosis rate of HGC27 cells was 11.5%, 21.4% and 36.6% respectively, which showed an obvious concentration-effect relationship, while the apoptosis rate of HGC27 cells was 2.2% in the control group. The relative activity of Caspase-3 of artesunate group was (0.19±0.02), (0.25±0.04) and (0.31±0.03) respectively, which was significantly increased than the control group (0.11±0.02) (P < 0.05). And the relative activity of Caspase-9 of artesunate group was (0.18±0.02), (0.23±0.03) and (0.30±0.04), which was significantly increased than the control group (0.10±0.02) (P < 0.05). The data of Western blot showed that artesunate up-regulated RUNX-3 in a dose-dependent manner. Conclusion Artesunate can inhibit the proliferation of HGC27 cells and induce apoptosis, and the mechanism of artesunate on apoptosis may be related to the up-regulation of RUNX-3 expression, as well as the increase of relative activity of Caspase-3 and Caspase-9. Artesunate may be a promising antitumor agent for gastric cancer treatment.

[Key words] Artesunate; HGC27 cell; Caspase-3; Caspase-9; RUNX-3

胃癌是常见的消化道恶性肿瘤,胃癌死亡率居肿瘤相关死亡率前列[1]。我国胃癌的发病率呈逐年上升趋势,严重威胁着人民群众的生命健康。胃癌治疗方法以手术切除为主,辅以化疗,但常规的化疗药物毒性大且常出现耐药性,因此寻找有效且毒副作用小的抗肿瘤药已经成为国内外研究热点。青蒿素及其衍生物是我国自主知识产权的高效速效抗疟药物。近年来研究发现,青蒿素及其衍生物除了有抗疟作用外,还对人类多种肿瘤细胞具有明显的杀伤或抑制作用[2-4],但具体机制尚不十分清楚。为探讨青蒿素衍生物之一青蒿琥酯的抗癌机制,本研究以人胃癌HGC27细胞为对象,观察青蒿琥酯对人胃癌细胞增殖、凋亡的影响,并检测青蒿琥酯对人胃癌细胞Caspase-3、Caspase-9活性变化及抑癌基因人类相关转录因子3(human runt-related transcription factor 3,RUNX3)表达的影响。

1 材料与方法

1.1 材料

人胃癌细胞HGC27细胞购自中国科学院上海细胞库;青蒿琥酯购于桂林南药股份有限公司;AnnexinV/PI试剂盒购于BENDER公司;胎牛血清购自Invitrogen公司;RPMI1640培养液购于杭州四季青生物材料研究所;胎牛血清系GIBCO产品;MTT试剂盒购于武汉谷歌生物科技有限公司;Caspase-3、Caspase-9活性检测试剂盒购于南京凯基生物科技发展有限公司;RUNX-3单克隆抗体购自Cell Signaling Technology公司;辣根酶标记兔抗山羊IgG购自武汉博士德生物科技有限公司。

1.2 方法

1.2.1 细胞培养 HGC27细胞培养于RPMI1640培养液中(含10%小牛血清,100 U/mL青霉素,100 μg/mL链霉素),置于37℃,饱和湿度,5%CO2培养箱内培养,根据生长情况3~5 d传代一次。

1.2.2 MTT法检测细胞增殖抑制率 取对数生长期的HGC27细胞,制成细胞悬液,以1×104/mL的浓度接种于96孔板,每孔200 μL,待细胞贴壁后分组:青蒿琥酯组加入青蒿琥酯终浓度为10、20、40、80、100 mg/L的培养液,对照组加入等量的培养液,并设立调零孔。每组设5个复孔,分别培养24、48、72 h,实验结束前4 h加入MTT试剂20 μL/每孔,继续孵育4 h,小心吸掉上清,每孔加150 μL DMSO,振荡10 min,使结晶充分溶解,酶标仪上检测每孔在570 nm处的吸光值(A值)。抑制率=[1-(实验组平均A值-调零孔A值)/(对照组平均A值-调零孔A值)]×100%。

1.2.3 显微镜下观察细胞形态 取对数生长期细胞消化传代并培养24 h后,换青蒿琥酯终浓度为10、20、40、80、100 mg/L的培养液连续培养24、48、72 h后置于倒置显微镜下观察细胞生长情况。

1.2.4 流式细胞仪检测细胞凋亡 取对数生长期细胞,以1×106/mL浓度接种于6孔培养板中,贴壁后分为实验组和对照组,实验组分别加入青蒿琥酯终浓度为20、40、80 mg/L的培养液,对照组加入等量培养液,培养48 h后,收集细胞,离洗固定后加入Annexin V-FITC 和PI染色。筛网过滤送流式细胞仪检测细胞凋亡率。

1.2.5 分光光度法检测Caspase-3、Caspase-9活性变化 细胞接种及分组同“1.2.4”项下,培养48 h后,收集细胞,分别加入50 μL冷裂解缓冲液,冰浴裂解15 min,然后4℃,1200 r/min离心15 min,将上清移至预冷的离心管中,置于冰上,取5 μL用BCA法测定蛋白浓度,取50 μL调好蛋白浓度(2~4 g/L),加入20 μL反应缓冲液吹打均匀,加入5 μL Caspase-3、Caspase-9反应底物Ac-DEVD-pNA和Ac-LEHD-pNA,细胞培养箱中避光孵育4 h,酶标仪上405 nm处测定吸光度(OD405)。

1.2.6 Western blot法检测RUNX-3 蛋白表达变化 细胞接种及分组同“1.2.4”项下,收集细胞,用PBS漂洗,参照细胞浆蛋白抽提试剂盒说明书进行操作,提取细胞总蛋白,并测定蛋白浓度,蛋白样品加入1/5体积的5×上样缓冲液,沸水煮沸5 min后离心,以每孔20 μg上样,行10%SDS-聚丙烯酰胺凝胶电泳,然后电转至PVDF膜上,用5%脱脂奶粉室温封闭1 h,加入1∶1000稀释的兔抗人RUNX-3蛋白,4℃过夜,β-actin作为内参,TBST洗膜3次,加入1∶1000稀释的辣根酶标记的兔抗山羊IgG,室温孵育2 h,同样洗膜3次,ECL显色,观察各条带深浅变化。

1.3 统计学方法

所有资料经SPSS 17.0统计学软件进行数据分析,计量资料数据用均数±标准差(x±s)表示,多组间的比较采用单因素方差分析,组间两两比较采用LSD-t检验,以P < 0.05 为差异有统计学意义。

2 结果

2.1 青蒿琥酯对HGC27细胞增殖的影响

MTT检测数据显示,青蒿琥酯在不同浓度和不同作用时间均能显著抑制HGC27细胞增殖(P < 0.05),且随着青蒿琥酯浓度的增加和作用时间的延长,其抑制作用逐渐增强,呈明显的浓度和时间依赖效应(图1)。

2.2 细胞形态观察

倒置显微镜下可见对照组HGC27细胞生长旺盛,折光率较高,胞体大,形态成梭形或多边形,胞质均匀透明,随培养时间的延长形态无明显变化。青蒿琥酯处理的细胞增殖减慢,且随着青蒿琥酯浓度的升高和作用时间的延长,细胞逐渐变小、变圆,折光率减弱,核浓缩等,部分脱落漂浮于培养瓶中,但细胞膜完整,最后裂解。青蒿琥酯浓度越高,作用时间越长,上述表现越明显,漂浮细胞越多。

2.3 青蒿琥酯对细胞凋亡的影响

流式细胞仪检测结果显示,不同浓度(20、40、80 mg/L)青蒿琥酯处理HGC27细胞48 h后,细胞凋亡率分别为11.5%、21.4%、36.6%,而对照组凋亡率仅为2.2%,表明青蒿琥酯能诱导HGC27细胞凋亡(图2)。

A: 对照组; B: 20 mg/L青蒿琥酯处理组; C: 40 mg/L青蒿琥酯处理组; D: 80 mg/L青蒿琥酯处理组

图2 流式细胞仪检测青蒿琥酯对HGC27细胞凋亡率的影响

2.4 青蒿琥酯对Caspase-3、Caspase-9活性的影响

Caspase-3、Caspase-9活性检测示,20、40、80 mg/L青蒿琥酯作用HGC27细胞48 h后,Caspase-3相对活性明显升高,分别为(0.19±0.02)、(0.25±0.04)和(0.31±0.03),与对照组(0.11±0.02)比较,差异有统计学意义(P < 0.05);Caspase-9活性亦明显升高,分别为(0.18±0.02)、(0.23±0.03)和(0.30±0.04),与对照组(0.10±0.02)比较,差异有统计学意义(P < 0.05),表明青蒿琥酯能活化Caspase-3、Caspase-9。

2.5 青蒿琥酯对RUNX-3蛋白表达的影响

青蒿琥酯处理HGC27细胞48 h后,结果显示随着青蒿琥酯浓度的升高,RUNX-3蛋白表达量也逐渐升高,见图3。

图3 Western blot检测青蒿琥酯对HGC27细胞

RUNX3 蛋白表达的影响

3 讨论

细胞凋亡是多细胞机体维持内环境稳定的自我调节机制[5],细胞凋亡与细胞增殖之间的平衡在胃癌发生发展中起重要作用[6],细胞凋亡是程序化、多基因调控的细胞死亡过程,通过诱导细胞凋亡已经成为抗肿瘤研究的热点。本研究发现,青蒿琥酯在10~100 mg/L浓度范围,对人胃癌HGC27细胞的增殖均有抑制作用,随着药物浓度的升高和作用时间的延长,抑制细胞增殖的作用亦逐步增强,呈现明显的浓度效应及时间效应关系。青蒿琥酯处理HGC27细胞后,在倒置显微镜下均可见到凋亡细胞的形态;流式细胞仪检测其细胞凋亡率随着青蒿琥酯处理浓度的增加而升高,呈明显的剂量依赖效应。表明青蒿琥酯可抑制胃癌HGC27细胞的增殖及诱导其发生凋亡。

在细胞凋亡的机制研究中,目前认为主要由3条信号途径:线粒体途径、死亡受体途径及内质网途径。Caspase家族的激活在细胞凋亡过程中起着关键作用,被认为是引起凋亡的直接效应物。Caspase-9是线粒体凋亡途径的关键蛋白酶,处于Caspase“瀑布式”激活的顶端,它的活化对线粒体凋亡通路尤为重要,并进一步激活Caspase-3,Caspase-3活化后可裂解DNA修复相关分子、凋亡抑制蛋白、细胞外基质蛋白和骨架蛋白等,促进细胞凋亡。Caspase-3是凋亡过程的主要效应分子,其活化标志着凋亡进入不可逆阶段[7]。本研究发现,青蒿琥酯处理HGC27细胞48 h后,细胞Caspase-3、Caspase-9活性明显升高,并呈明显浓度依赖效应,提示青蒿琥酯可能通过激活Caspase-3、Caspase-9级联的线粒体依赖性途径诱导人胃癌HGC27细胞凋亡。

此外,本研究还发现,青蒿琥酯能上调HGC27细胞中RUNX3基因表达,且表达量随着药物浓度的升高而增加,呈明显浓度效应。大量研究证明,转录生长因子β(transforming growth factor,TGF-β)和Wnt信号通路在肿瘤的发生发展过程中发挥着重要作用[8-9]。RUNX3参与TGF-β信号通路诱导生长抑制的过程[10]。研究证实,RUNX3基因在多种肿瘤如乳腺癌、胃癌、大肠癌、肝癌及肺癌中表达下调甚至缺失,在肿瘤的发生发展中起重要作用[11-12]。有证据表明,恢复RUNX3基因的表达能通过诱导细胞凋亡、调节细胞周期及下调cyclin D1的表达而显著抑制肿瘤细胞增殖及转移[13]。前期研究发现,青蒿琥酯能将肿瘤细胞阻滞于G1期并诱导细胞凋亡[14-15]。且RUNX3基因表达上调趋势与流式细胞仪检测的凋亡率增升高趋势是一致的。因此,提示青蒿琥酯能通过上调RUNX3基因表达影响细胞周期促进细胞凋亡,进而发挥抗肿瘤作用。

总之,本研究发现,青蒿琥酯在体外能抑制大胃癌HGC27细胞增殖并诱导其凋亡,可能的机制是上调抑癌基因RUNX-3表达及增强Caspase-3、Caspase-9活性。肿瘤的凋亡是一个复杂而又精确调控的网络系统,尽管青蒿琥酯抑制细胞增殖及诱导细胞凋亡的机制还有待于进一步探索和研究来阐明,笔者相信青蒿琥酯在抗肿瘤方面具有广阔前景,并期待包括青蒿琥酯在内的青蒿素及其衍生物能成为高效低度的抗肿瘤药物应用于临床。

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(收稿日期:2013-10-26 本文编辑:程 铭)

[3] Wang Y,Han Y,Yang Y,et al. Effect of interaction of magnetic nanoparticles of Fe304 and artesunate on apoptosis of K562 cells [J]. International Journal of Nanomedicine,2011, 10:1185-1192.

[4] 王利娟,杨玉琮,苟文丽.青蒿琥酯抑制人子宫内膜癌HEC-1B细胞增殖及诱导其凋亡的机制[J].西安交通大学学报:医学版,2013,34(1):93-97.

[5] LaCasse EC,Mahoney DJ,Cheung HH,et al. IAP-targeted therapies for cancer [J]. Oncogene,2008,27(28):6252-6275.

[6] Huang WS,Wang JP,Wang T,et al. ShRNA-mediated gene silencing of beta-catenin inhibits growth of human colon cancer cells [J]. World J Gastroenterol,2007,13(48):6581-6587.

[7] Mazumder S,Plesca D,Almasan A. Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis [J]. Methods Mol Biol,2008,414:13-21.

[8] Calone I,Souchelnytskyi S. Inhibition of TGF-β Signaling and its implications in anticancer treatments [J]. Exp Oncol,2012,34(1):9-16.

[9] Liu LC,Tsao TC,Hsu SR,et al. EGCG inhibits Transforming Growth Factor-β-mediated Epithelial-to-Mesenchymal Transition via inhibition of Smads and Erk1/2 Signaling Pathways in Non-small Cell Lung Cancer Cells [J]. J Agric Food Chem,2012,60(39):9863-9873.

[10] Watanabe K,Sugai M,Nambu Y,et al. Requirement for RUNX proteins in IgA class awitching acting downstream of TGF-beta 1 and retinoic acid signaling [J]. J Immunol,2010,184(6):2785-2792.

[11] Jeong P,Min BD,Ha YS,et al. Runx3 methylation in normal surrounding urothelium of patients with non-muscle-invasive bladder cancer:Potential role in the prediction of tumor progression [J]. Eur J Surg Oncol,2012,38(11):1095-1100.

[12] Shiraha H,Nishina S,Yamamoto K. Loss of runt-related transcription factor 3 causes development and progression of hepatocellular carcinoma [J]. J Cell Biochem,2011,112(3):745-749.

[13] Chi XZ,Yang JO,Lee KY,et al. RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1)expression in cooperation with transforming growth factor {beta}-activated SMAD [J]. Mol Cell Biol,2005,25(18):8097-107.

[14] Li Y,Shan F,Wu JM,et al. Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle [J]. Bioorg Med Chem Lett,2001,11(1):5-8.

[15] Wu JM,Shan F,Wu G,et al. Synthesis and cytotoxicity of artemisinin derivatives containing cyanoarylmethyl group [J]. Eur J Med Chem,2001,36(5):467-479.

(收稿日期:2013-10-26 本文编辑:程 铭)

[3] Wang Y,Han Y,Yang Y,et al. Effect of interaction of magnetic nanoparticles of Fe304 and artesunate on apoptosis of K562 cells [J]. International Journal of Nanomedicine,2011, 10:1185-1192.

[4] 王利娟,杨玉琮,苟文丽.青蒿琥酯抑制人子宫内膜癌HEC-1B细胞增殖及诱导其凋亡的机制[J].西安交通大学学报:医学版,2013,34(1):93-97.

[5] LaCasse EC,Mahoney DJ,Cheung HH,et al. IAP-targeted therapies for cancer [J]. Oncogene,2008,27(28):6252-6275.

[6] Huang WS,Wang JP,Wang T,et al. ShRNA-mediated gene silencing of beta-catenin inhibits growth of human colon cancer cells [J]. World J Gastroenterol,2007,13(48):6581-6587.

[7] Mazumder S,Plesca D,Almasan A. Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis [J]. Methods Mol Biol,2008,414:13-21.

[8] Calone I,Souchelnytskyi S. Inhibition of TGF-β Signaling and its implications in anticancer treatments [J]. Exp Oncol,2012,34(1):9-16.

[9] Liu LC,Tsao TC,Hsu SR,et al. EGCG inhibits Transforming Growth Factor-β-mediated Epithelial-to-Mesenchymal Transition via inhibition of Smads and Erk1/2 Signaling Pathways in Non-small Cell Lung Cancer Cells [J]. J Agric Food Chem,2012,60(39):9863-9873.

[10] Watanabe K,Sugai M,Nambu Y,et al. Requirement for RUNX proteins in IgA class awitching acting downstream of TGF-beta 1 and retinoic acid signaling [J]. J Immunol,2010,184(6):2785-2792.

[11] Jeong P,Min BD,Ha YS,et al. Runx3 methylation in normal surrounding urothelium of patients with non-muscle-invasive bladder cancer:Potential role in the prediction of tumor progression [J]. Eur J Surg Oncol,2012,38(11):1095-1100.

[12] Shiraha H,Nishina S,Yamamoto K. Loss of runt-related transcription factor 3 causes development and progression of hepatocellular carcinoma [J]. J Cell Biochem,2011,112(3):745-749.

[13] Chi XZ,Yang JO,Lee KY,et al. RUNX3 suppresses gastric epithelial cell growth by inducing p21(WAF1/Cip1)expression in cooperation with transforming growth factor {beta}-activated SMAD [J]. Mol Cell Biol,2005,25(18):8097-107.

[14] Li Y,Shan F,Wu JM,et al. Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle [J]. Bioorg Med Chem Lett,2001,11(1):5-8.

[15] Wu JM,Shan F,Wu G,et al. Synthesis and cytotoxicity of artemisinin derivatives containing cyanoarylmethyl group [J]. Eur J Med Chem,2001,36(5):467-479.

(收稿日期:2013-10-26 本文编辑:程 铭)

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