
肝细胞癌(HCC)是我国最常见的恶性肿瘤之一[1],其预后差病死率高,病死率位居恶性肿瘤第二位[2-3]。研究表明,肿瘤复发和转移是导致HCC患者死亡的重要原因,抑制癌细胞的迁移、侵袭是HCC防治的关键[4-5]。药用植物及其有效活性成分是良好的抗肿瘤药物来源。没食子酸(gallic acid,GA)是一种天然多酚类化合物,存在于山茱萸、五倍子、牡丹皮、掌叶大黄等多种植物[6],具有抗肿瘤、抗炎、抗氧化、抗病毒等药理作用[7]。研究表明,GA对结肠癌、胃癌等肿瘤细胞增殖、迁移、侵袭具有显著的抑制作用,并促进肿瘤细胞凋亡[8-9]。本研究观察GA对HepG2细胞增殖、凋亡、迁移、侵袭的影响,探讨GA抗肝癌的作用分子机制,以期为抗肝癌新药开发提供理论基础和科学依据。
人肝癌HepG2细胞(江苏凯基生物技术股份有限公司,批号KG020);GA(Med Chem Express, 批号HY-N0523);MEM培养基(江苏凯基生物技术股份有限公司,批号KGL1601-500);胎牛血清(普诺赛,批号164210-50);CCK-8(APEXBIO);Annexin V-FITC/PI细胞凋亡检测试剂盒(BD);Matrigel基质凝胶(Corning);兔抗人B细胞淋巴瘤因子2(B-cell lymphoma-2, Bcl-2)、兔抗人Bcl-2关联X蛋白(Bcl2-associated X protein, Bax)、小鼠抗人基质金属蛋白酶2(matrix metallpproteinase-2,MMP-2)、兔抗人MMP-9、兔抗人裂解的半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)抗体均购自英国Abcam公司。Olympus CHX41倒置显微镜;Rayto RT-2100型多功能酶标仪;上海天能凝胶成像系统;Beckman DxELEX流式细胞仪;上海安亭TGL-20B高速台式离心机。
HepG2细胞接种于96孔板(6×103个/孔),培养24 h后分为对照组、空白组(无细胞)和实验组,对照组和空白组加入100 μL完全培养基,实验组加入100 μL不同浓度GA(5、10、20、30、40、50 μg/mL)的培养基,每组设5个复孔。继续培养24、48 h后,每孔加入CCK-8试剂10 μL,4 h后酶标仪450 nm波长处测定吸光度(A)值。细胞存活率(%)=(A实验组-A空白组)/(A对照组-A空白组)×100%,计算半数抑制浓度(IC50)。实验重复3次。
HepG2细胞接种于6孔板(1×103个/孔),培养24 h后分为对照组、5 μg/mL(1/4 IC50) GA组、10 μg/mL(1/2 IC50) GA组和20 μg/mL(IC50) GA组,对照组加入完全培养基,实验组加入终浓度为5、10和20 μg/mL GA。置于37 ℃、5% CO2培养箱培养至肉眼观察到可见的克隆时终止培养,4%多聚甲醛溶液固定,结晶紫染色,晾干后相机拍照,利用Image J软件对细胞集落数进行计数。实验重复3次。
HepG2细胞接种于6孔板(1×106个/孔),培养24 h后用200 μL移液器枪尖垂直六孔板底部划线,磷酸盐缓冲液(PBS)冲洗去除悬浮细胞,实验分组同“1.3”,划痕24 h观察划痕愈合情况并计算各自细胞的相对迁移率。相对迁移率=(0 h划痕宽度-培养24 h后划痕宽度)/0 h划痕宽度×100%。实验重复3次。
50 μL的Matrigel基质凝胶(1∶8稀释)涂布于Transwell小室上室膜面上,置于培养箱中固化4 h,上室接种5×104个GA预处理24 h的HepG2细胞,下室加入600 μL含20%胎牛血清的完全培养基,培养24 h取出上室,棉签擦去聚碳酸酯膜上的上层细胞,将Transwell小室4%多聚甲醛固定30 min,结晶紫染色,显微镜下观察拍照并计数穿过膜细胞数。实验重复3次。
HepG2细胞接种于6孔板(2×105个/孔),分组同“1.3”,干预48 h后收集各组细胞,严格按照试剂盒染色说明书收集细胞、染色,流式细胞仪检测细胞凋亡率。细胞凋亡率=(早期凋亡细胞数+晚期凋亡细胞数)/细胞总数×100%。实验重复3次。
HepG2细胞分组同“1.3”,干预48 h后收集各组细胞,使用含有磷酸酶和蛋白酶抑制剂的PIPA裂解液提取细胞蛋白。取20 μg蛋白样品进行SDS-PAGE凝胶电泳并转至PVDF膜上,5%脱脂奶粉室温封闭4 h,加入一抗4 ℃孵育过夜。TBST洗膜后加入辣根过氧化物酶标记二抗,室温孵育2 h,ECL发光试剂显色并拍照。目的蛋白的相对表达水平=目的蛋白条带灰度值/内参β-actin条带灰度值。实验重复次3次。
采用SPSS 22.0统计软件对数据进行统计分析,计量资料以
经5、10、20、30、40、50 μg/mL GA作用24、48 h后,HepG2细胞活性明显下降(F值分别为189.489、447.520,P值均<0.001)(图1),GA作用HepG2细胞24、48 h的IC50均值分别为(38.02±2.58)μg/mL和(18.36±1.54)μg/mL;结合预实验结果设置5、10、20 μg/mL GA作用48 h进行后续实验。
对照组和5、10、20 μg/mL GA组HepG2细胞的细胞集落数分别为:(239.00±29.45)个、(210.00±19.00)个、(144.33±16.03)个、(57.00±9.55)个。与对照组比较,10、20 μg/mL GA组细胞集落形成数显著减少(P值均<0.05);与5 μg/mL GA组比较,10、20 μg/mL GA组集落形成数显著减少(P值均<0.05),且呈浓度依赖性(图2)。
与对照组比较,5、10、20 μg/mL GA组HepG2细胞迁移率明显降低(P值均<0.05),且呈剂量依赖性(图3,表1)。
组别 | 迁移率(η/%) | 穿膜细胞数(个) |
---|---|---|
对照组 | 42.62±7.82 | 230.30±15.30 |
5 μg/mL GA组 | 35.34±6.421) | 182.12±12.601) |
10 μg/mL GA组 | 21.85±4.421) | 137.20±7.501) |
20 μg/mL GA组 | 12.57±3.541) | 124.40±6.801) |
F值 | 40.030 | 82.926 |
P值 | <0.001 | <0.001 |
注:与对照组比较,1)P<0.05。 |
与对照组比较,5、10、20 μg/mL GA组HepG2细胞中穿膜细胞数明显减少(P值均<0.05),且呈剂量依赖性(图4,表1)。
对照组和5、10、20 μg/mL GA组HepG2细胞凋亡率分别为:0.67%±0.08%、13.27%±1.07%、20.94%±2.45%、40.74%±2.63%。与对照组比较,不同剂量GA组细胞凋亡率明显升高(P值均<0.05);与5 μg/mL GA组比较,10、20 μg/mL GA组细胞凋亡率显著升高(P值均<0.05)(图5)。
与对照组比较,5、10、20 μg/mL GA组HepG2细胞迁移相关蛋白MMP-2、MMP-9表达水平显著下调(P值均<0.05);10、20 μg/mL GA组凋亡相关蛋白Bcl-2表达水平显著下调(P值均<0.05);5、10、20 μg/mL GA组Bax、Cleaved caspase-3蛋白表达水平显著上调(P值均<0.05)(图6)。
在中国,肝癌发病率位居恶性肿瘤第4位,病死率居肿瘤致死病因的第2位,2020年我国肝癌新发病例达到41万人,死亡39万人,占全球的45.1%和46.9%,严重威胁人民的生命安全[10]。对于早期肝癌患者,手术治疗是较为有效的治疗手段,但临床数据分析显示,仅5%~15%的患者发现较早有机会行手术切除[11]。肝癌起病隐匿,进展迅速,大多数患者就诊时已失去根治性手术治疗机会,化疗是晚期肝癌治疗的主要手段。治疗肝癌的一线靶向药物主要包括索拉非尼、仑伐替尼,二线药物主要包括瑞戈非尼、卡博替尼及雷莫芦单抗等[12-13]。我国肝癌患者多数具有乙型肝炎及肝硬化背景,就诊时大多数为中晚期,患者肝内肿瘤负荷大且合并门静脉癌栓、肝功能较差,导致疗效有限且不良反应明显,HCC的总生存率仍然很低,寻找高效低毒的抗肝癌药物是研究热点[14]。吴昊等[15]研究表明,GA能够抑制食管癌细胞的增殖、迁移和集落形成,并通过调控细胞内活性氧水平促进食管癌细胞凋亡;Zhang等[9]研究表明,GA通过调控Janus激酶/信号转导与转录激活因子3信号通路增强顺铂的抗非小细胞肺癌作用。目前关于GA对肝癌细胞迁移、侵袭的影响尚未见报道。本研究结果表明,GA在体外实验中可以抑制HepG2细胞增殖、迁移和侵袭,并促进细胞凋亡,可能具有抗肝癌活性。
肿瘤细胞浸润和转移到其他组织增殖形成新的侵袭转移瘤,是恶性肿瘤的一个重要生物学特性。肿瘤细胞转移与患者病死率密切相关,是决定患者预后的因素之一[16]。MMP能够特异性降解细胞间基质,调节细胞间基质代谢的主要限速酶,其中MMP-2和MMP-9能够降解明胶、层黏连蛋白和Ⅳ型胶原等,参与肿瘤的迁移和侵袭[17]。本研究发现,GA在体外显著降低HepG2细胞MMP-2和MMP-9蛋白表达,GA通过降低MMP表达抑制基底膜和细胞外基质的降解,进而抑制HepG2细胞的迁移和侵袭。
诱导细胞凋亡是多数抗肿瘤药物发挥抗肿瘤效应的重要手段,细胞凋亡是多基因调控的细胞程序性死亡,线粒体通路、死亡受体通路和内质网通路是三条主要的介导细胞凋亡信号转导通路[18-19]。Bcl-2家族是线粒体凋亡通路的主要调控因子,通过阻止线粒体细胞色素C的释放发挥抗凋亡作用,Bax是一个关键的促凋亡蛋白,Bax活化后在线粒体膜中形成多聚体孔洞,释放细胞色素C、Smac等凋亡相关因子,进入细胞质,激活下游Caspase家族蛋白,启动细胞凋亡程序[20-22]。本研究结果显示,GA作用HepG2细胞后,与对照组比较,GA组HepG2细胞抑凋亡蛋白Bcl-2表达显著降低,促凋亡蛋白Bax和Cleaved caspase-3表达水平显著升高,GA可能通过调控凋亡蛋白的表达促进HepG2细胞凋亡,凋亡率明显升高。
综上所述,GA在体外对HepG2细胞增殖、迁移和侵袭具有明显的抑制作用,并促进其凋亡,其机制可能与调控细胞MMP-2、MMP-9及凋亡相关蛋白表达有关,具体如何调控,仍需更多的实验证实。研究结果可为GA抗肝癌的应用提供理论基础,但肝癌发生机制复杂,涉及多靶点、多通路,因此GA抗肝癌作用及其机制仍需进一步深入研究。
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|
组别 | 迁移率(η/%) | 穿膜细胞数(个) |
---|---|---|
对照组 | 42.62±7.82 | 230.30±15.30 |
5 μg/mL GA组 | 35.34±6.421) | 182.12±12.601) |
10 μg/mL GA组 | 21.85±4.421) | 137.20±7.501) |
20 μg/mL GA组 | 12.57±3.541) | 124.40±6.801) |
F值 | 40.030 | 82.926 |
P值 | <0.001 | <0.001 |
注:与对照组比较,1)P<0.05。 |