白藜芦醇通过调节抗氧化酶活性而呈现抗肿瘤效应
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摘要
研究背景：恶性肿瘤严重危害人类健康,已成为重要的致死性疾病,上世纪的发病率显著增加。尽管现代医学研究取得了巨大进步,但是大多数肿瘤仍不能被治愈。基于肿瘤的发病机制复杂而使其有效治疗举步维艰。现有的治疗方法如化疗或放疗均具有明显的毒／副作用,医学科学工作者正研究靶向治疗,使其药物仅杀死肿瘤细胞而不伤害健康细胞。恶性肿瘤的发生受环境因素和遗传因素共同影响,环境因素指除遗传因素之外的所有范围,而不仅仅指环境污染。常见的致癌环境因素包括饮食和肥胖(30-35%)、吸烟(25-30%)、感染(15-20%)、辐射(10%)、精神紧张、缺乏锻炼和环境污染物等。大多数肿瘤发生与环境因素相关,其中部分环境因素是可由生活方式控制的。因此,其实癌症是一种有希望通过日常调理、药物和疫苗来预防的疾病。恶性肿瘤的发生实际上是细胞分裂和生长失控,并侵入邻近的身体部位而成癌。
在众多影响肿瘤发生的因素中,氧化应激诱导的DNA损伤及其对细胞信号转导途径的干扰在肿瘤的发生发展中起着重要的作用。活细胞中许多的氧化反应需要酶催化,氧化反应即分子间的氢质子或电子转移,这类代谢反应是生命过程所需能量的主要来源。地球上绝大多数复杂的生命体都依赖氧而生存,但是氧是一种高活性分子,生物体会分解氧气产生包括过氧化氢(H2O2)、次氯酸(HOC1)和自由基如羟基自由基(·OH)、超氧阴离子(02-)以及脂质过氧化物等活性氧簇(reactive oxygen species, ROS),这些代谢产物可以直接或间接地对核酸、脂类和蛋白质造成暂时或永久性损伤,从而对生物体自身造成损害。氧化-抗氧化状态之间的不平衡造成的氧化损伤与众多疾病的发生相关,包括癌症、神经系统疾病、动脉粥样硬化、高血压、糖尿病、急性呼吸窘迫综合症、慢性阻塞性肺疾病和哮喘等。基于自身保护,机体需要多种类型的抗氧化剂以维持氧化代谢的平衡。如谷胱甘肽、维生素C、维生素E以及一些酶类,包括过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)等。抗氧化酶可保护细胞免受氧化应激损伤,抗氧化酶活性和表达的失调与肿瘤发生密切相关,是肿瘤发生发展的重要影响因素。氧化应激能诱导DNA损伤并影响细胞内信号转导途径。ROS以多种形式损伤DNA,包括碱基修饰、自由基损害(嘌呤和嘧啶)、DNA断裂及DNA-蛋白质交互连接等。氧化应激对肿瘤发生与治疗的影响被越来越受到重视。有研究报道,前列腺癌、膀胱癌、乳腺癌、肝癌、多发性骨髓瘤和其他多种肿瘤都伴有抗氧化酶活性降低或其表达下调；也有研究发现某些肿瘤中抗氧化酶水平无变化,或部分抗氧化酶的表达水平甚至升高。近期研究表明,多发性骨髓瘤患者血清中SOD、GPX、 CAT、维生素C和维生素E水平均显著降低；非小细胞肺癌(NSCLC)患者红细胞裂解液中的SOD活性较低而GPX活性升高；前列腺癌患者的GPX、CAT和Cu/Zn-SOD活性均降低；口腔鳞状细胞癌(OSCC)患者的SOD和CAT水平均降低;急性淋巴细胞白血病(ALL)患者全血中CAT和SOD活性降低；膀胱癌组织中CAT和SOD活性均显著低于正常膀胱组织；肺肿瘤组织中总SOD活性升高,CAT活性下降；肺部炎症会产生高水平Mn-SOD,但CAT水平降低,二者共同作用可导致细胞内的H202水平上升,因此肺炎将会为DNA损伤和细胞恶变提供有利条件,在对肺癌组织和肺癌A549细胞株的研究中发现SOD的水平增加、CAT水平降低、而GPX水平则无改变。
恶性肿瘤是一种典型的多因素疾病,遗传因素与环境因素对肿瘤发生的影响相互相存。DNA变异可能引起原癌基因活化或抑瘤基因失活,从而使细胞分化、凋亡失控。基因突变可能是自发突变或者是暴露于辐射或致癌物质所致。抗氧化酶可防止DNA的氧化损伤和延缓或改变细胞周期。某些病毒如人类T淋巴细胞病毒及某些化学物质,特别是含苯或烷基的药物等均是重要的致癌因素。已观察到在造血系统恶性肿瘤中ROS的产生过量和／或抗氧化途径障碍,已发现在一些类型的白血病中抗氧化酶活性缺陷或表达异常。因此,保持一定水平的抗氧化酶活性对于预防恶性肿瘤的发生发展是至关重要的。另一方面,靶向抗氧化酶活性可作为肿瘤药物治疗的重要发展策略,研究发现,单独采用GPX和Mn-SOD基因疗法可以分别减缓肿瘤生长速率达51%和54%,GPX和Mn-SOD基因共转染则可达到81%的肿瘤细胞生长抑制率。此外,研究抗癌药物对抗氧化酶活性和表达水平的影响将加速药物的作用机制研究。
白藜芦醇(resveratrol, RSV)是一种由植物产生的多酚类物质(stilbenoid),化学名称为3,5,4’-三羟基-反式-二苯乙烯(见下图),具有可利用的生物学活性。RSV主要存在于红葡萄皮、豌豆、坚果、蓝莓、桑椹、蔓越莓、姜黄、菠菜、百合等植物组织中,白藜芦醇苷是RSV的前体,属于二苯乙烯类化合物,是葡萄汁中主要的RSV前体物质；白藜芦醇苷经米曲霉催化可以形成RSV。事实上,白藜芦醇苷是自然界中最丰富的RSV储存形式。植物中RSV的合成或转化可通过应激因素调节,如真菌污染或紫外线辐射。植物中的RSV可作为一种植物抗毒素,能抑制某些植物传染病的发生与发展。
研究表明,RSV能显著增加抗氧化酶SOD、CAT、GPX等和非酶类抗氧化物质如还原型谷胱甘肽、维生素C、维生素E和β-胡萝卜素等的抗氧化作用,并降低脂质过氧化水平。RSV具有抗衰老、抗肿瘤活性,能够延缓衰老过程并最大限度地减少与老化相关的并发症的发生；此外,RSV还具有保护心脏、抗糖尿病、抗炎症、抗病毒及保护神经系统等多种作用。已在肿瘤细胞株、动物模型甚至临床研究中发现RSV能够抑制肿瘤的发生发展；并在体内和体外研究中证实RSV对皮肤癌、乳腺癌、肝癌、前列腺癌、大肠癌、胰腺癌等具有治疗或辅助治疗效应。机制研究表明,RSV可通过诱导肿瘤细胞凋亡、改变细胞周期、抑制血管生成、抑制核因子-κB和环氧合酶信号转导途径、抑制致癌物的代谢活化、改变与肿瘤发生相关的miRNA表达模式及抗氧化等而影响肿瘤细胞的增殖、转移或凋亡(见下图)。近期研究表明,RSV可通过干扰pAktl/p70S6K信号途径而抑制人类鼻咽癌(NPC)细胞增殖和促进其凋亡。但目前对RSV发挥作用的确切机制尚未完全了解。本研究即以肿瘤细胞株为试验模型,研究RSV经抗氧化而抑制肿瘤细胞生长的分子机制。
本研究采用3种不同的肿瘤细胞株,即前列腺癌细胞株PC-3、乳腺癌细胞株MCF-7和肝癌细胞株HepG2细胞。现今,这三种肿瘤都被认为是最危险的危及生命的疾病。在全球男性癌症死亡排名六大癌症中,前列腺癌排名第二；乳腺癌是女性最常见的浸润性肿瘤；肝癌则是全球最常见的恶性肿瘤之一,其死亡率仅次于肺癌。人类前列腺癌细胞株PC-3细胞对于研究中晚期前列腺癌细胞的生化变化以及评价其对化疗药物的反应性很有帮助,PC-3细胞株是雄激素非依赖性前列腺癌细胞,建株于1979年,是从一名62岁的白人男性IV级前列腺癌骨转移样品中分离得到的；乳腺癌细胞株MCF-7细胞是于1970年从一名69岁的白人女性患者组织样品中分离得到的,是最常用于研究肿瘤发生机制的乳腺癌细胞；人类肝癌细胞株HepG2细胞来自于一名15岁美国白人男孩的高分化肝癌组织,HepG2细胞常用于研究肝癌的发生发展与治疗机理。为了比较RSV对这些肿瘤细胞的作用及其机制,本研究中我们采用人胚肾细胞株HEK293T细胞作为对照。本研究同时采用了几株人类白血病细胞。人类早幼粒细胞白血病细胞株HL-60细胞来自于美国一位患急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)的36岁女性,是研究髓样分化的分子事件的人源细胞,APL是一种特殊类型的急性髓系白血病(acute myeloid leukemia, AML), HL-60细胞已成为研究AML的良好模型；K-562细胞是建株的第一个具有无限增殖特性的人类白血病细胞系,主要用作研究慢性髓系白血病(chronic myelogenous leukemia, CML)的发生与治疗机理,K-562细胞具有特征性的基于bcr/abl融合基因的费城染色体；KT-1/A3和KT-1/A3R细胞株是本研究采用的另外两类CML细胞,其中KT-1/A3细胞呈现对干扰素的抗癌效应敏感,而KT-1/A3R细胞则呈现干扰素抗性。
研究表明,RSV主要呈现抗氧化效应。但是,为什么在一定条件下RSV能保护正常细胞而对肿瘤细胞呈现细胞毒性仍然是一个有争议的问题。此外,RSV在不同肿瘤中对抗氧化酶的表达和活性的影响效果仍然有一些不一致的结果。基于此,本研究采用非肿瘤细胞株HEK293T细胞作为对照,针对RSV对部分实体瘤及白血病细胞的作用,揭示RSV对肿瘤细胞的抗氧化酶表达水平及其活性的调节作用,进而探讨RSV的抗氧化活性对肿瘤细胞增殖／凋亡的影响,剖析RSV的抗氧化／抗肿瘤机制。
研究方法：采用不同浓度的RSV处理前列腺癌PC-3细胞、肝癌HepG2细胞、乳腺癌MCF-7细胞、CML细胞株(K-562、KT-1/A3、KT-1/A3R)、AML细胞株HL-60细胞和非肿瘤人胚肾细胞株HEK293T细胞24-72小时,台盼蓝染色法检测细胞生长状态,分光光度法检测抗氧化酶的活性和过氧化氢(H2O2)的产量,Western印迹定量检测抗氧化酶的表达水平,流式细胞仪检测细胞周期分布和凋亡率。
采用DMEM培养基培养肝癌HepG2细胞和乳腺癌MCF-7细胞,采用RPMI1640培养基培养前列腺癌PC-3细胞、白血病细胞和非肿瘤HEK293T细胞,均加入10%的胎牛血清于37℃和5%浓度的CO2条件下培养。
以1.0×105/m1细胞接种于含有lml完全培养基的24孔培养板中,其培养基含有不同浓度(0、10、25、50和100μ M)的RSV；培养24、48和72小时后,富集细胞用于后续实验。
用台盼蓝染色法检测细胞的生长状态,贴壁生长细胞用胰蛋白酶处理,使之与培养皿分离,取等体积的悬浮生长细胞培养物或经胰蛋白酶处理的贴壁生长细胞悬液与台盼蓝溶液混合,显微镜下计数未被染成蓝色的活细胞数目。
以1000g、5分钟离心富集培养细胞,弃上清后用磷酸盐缓冲盐水(phosphate buffered saline, PBS)洗涤离心细胞,然后用200μl放射免疫沉淀分析缓冲液(radioimmunoprecipitation assay buffer, RIPA)和1mM苯甲基磺酰氟(phenylmethylsulfonyl fluoride, PMSF)处理细胞,冰浴30分钟。将该溶液于12000g离心15分钟,收集上清蛋白质溶液,贮于-20℃备用。
采用Bradford法测定蛋白质浓度,按体积比1：1将蛋白质溶液与考马斯亮蓝溶液混合,室温静置5分钟后,以牛血清白蛋白作为蛋白质标准溶液,于595nm处测定待测蛋白质溶液的吸光度并计算其蛋白质浓度。
采用非连续聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)分离蛋白质。首先,制备10%的分离胶,取dH2O7.78ml、40%Acr:Bis4. Oml、 pH8.8#的1.5M Tris-HCl4.0ml、10%SDS160μ1、10%APS80μ1和TEMED4.0μ1混合均匀,倒入PAGE玻璃槽；将胶在室温下静置1小时后,制备4%的浓缩胶含dH2O3.3ml、40%Acr:Bis0.96ml、0.5M Tris-HCl (pH6.8)2.0ml,10%SDS80μ1、10%APS40μ1和TEMED4.0μl,并倒入玻璃槽中覆盖分离胶,将梳板插入凝胶中：成胶后,用16μl蛋白质溶液与4μl上样缓冲液混合,95℃加热10分钟后上样；同样点上已知分子量的蛋白质标准物,60V电泳30分钟后,于80-85V电泳90分钟。
将凝胶上分离的蛋白质经电转移至硝酸纤维素膜,然后将印迹膜于室温浸泡在含5%脱脂牛奶的Tris缓冲生理盐水和吐温20(Tris-buffered saline and Tween20, TBST)缓冲液中2小时。印迹膜与第一抗体孵育12小时后用TBST润洗。然后将印迹膜与辣根过氧化物酶(HRP)第二抗体轻轻震荡孵育2小时,然后用TBST洗涤三次。化学发光反应后,在暗室暴光于X-光片,使用凝胶成像系统("ImageJ"软件)对Western印迹条带进行高密度数字化处理,对蛋白质条带进行定量分析。
以12000g离心5分钟收集细胞,用PBS润洗细胞,用含有1mM EDTA的PBS(pH7.0)匀浆,于40C、以12000g离心5分钟后收集细胞上清液测定过氧化氢的含量和酶活性(U/mg蛋白质)；采用黄嘌呤氧化酶法测定SOD活性；采用钼酸铵法测定CAT活性；利用DTNB法测定GPX活性。
经RSV诱导培养72小时后,12000g离心5分钟收集细胞,用PBS (pH7.4)洗涤并于70%乙醇中固定过夜。然后用流式细胞仪(北京鼎国昌盛生物有限公司)分别分析细胞凋亡和细胞周期的百分比。
对以上实验获取的数据经统计学软件SPSS16.0进行单因素方差分析和比较检验,结果以均数±标准差表示,以P≤0.05显示其统计学意义。研究结果：本研究结果显示,在浓度小于50μM的RSV处理24小时后,细胞生长状态均无明显改变；但是,25μM RSV处理48小时显示PC-3细胞和HepG2细胞的生长明显受到抑制,但MCF-7细胞生长未受抑制；用10μM或者25μM的RSV处理72小时,PC-3细胞和HepG2细胞的生长明显受到抑制,但浓度低于25μ M的RSV对MCF-7细胞生长无抑制效应：50μM的RSV处理48小时或72小时对三种细胞的生长都有明显的抑制作用,同时也抑制非肿瘤细胞株HEK293T细胞的生长；100μM的RSV对每种细胞株的生长都有明显的抑制作用。25μM的RSV能抑制实验中所用肿瘤细胞的生长而不影响非肿瘤细胞株HEK293T细胞的生长,本研究选择25μM的RSV作为最佳实验用剂量。
对照组PC-3细胞中的SOD活性为4.94U/mg蛋白质,分别用10μM,25μM和50u M的RSV处理PC-3细胞72小时,其SOD活性分别增加43%、88%和105%；对照组HepG2细胞中SOD活性为3.41U/mg蛋白质,分别用10μM.25μM和50μM的RSV处理72小时,其SOD活性分别增加55%、63%和87%；对照组MCF-7细胞的SOD活性为2.82U/mg蛋白质,用浓度为25μ M和50μ M的RSV处理MCF-7细胞72小时,其SOD活性分别增加54%和60%；对照组HEK293T细胞中SOD的活性为4.8U/mg蛋白质,低浓度的RSV(10μM或25μM)对于其SOD活性没有影响,但是50u M的RSV可使其活性增加49%。Western印迹分析显示用浓度为25μ M的RSV处理细胞72小时,PC-3、HepG2和MCF-7细胞的SOD2表达量分别增加3倍、2.5倍和1.5倍,而对HEK293T细胞的作用则不明显；细胞的处理组和非处理组中的SOD1表达水平无明显差异。PC-3、HepG2、MCF-7和HEK293T细胞中的CAT活性分别为4.20、4.82、4.08和7.28U/mg蛋白质。RSV处理72小时后各种细胞株的CAT活性都有一定程度的增加,25μM或50μ M的RSV处理HepG2细胞后其CAT活性分别增加了45%和42%；然而RSV处理对于其他细胞的CAT活性无明显影响；Western印迹分析显示用RSV处理的各种细胞株的CAT蛋白质水平均无明显改变。HEK293T、PC-3、HepG2和MCF-7细胞中的GPX活性分别为0.65、0.60、0.74和0.60U/mg蛋白质；用10-50μM的RSV处理细胞72小时,GPX活性及GPX1蛋白质水平均无明显变化。
本研究结果显示,RSV能明显提高肿瘤细胞的H202产生量。用10μM的RSV处理肿瘤细胞72小时,其H202水平均略微增加,而用25μ M的RSV处理PC-3细胞和HepG2细胞72小时,其H202产生量均增加约2.5倍,经同样处理的MCF-7细胞的H202产生量略有增加。与此相反,经10-25u M的RSV处理后,HEK293T细胞的H202产生量明显降低。肿瘤细胞的过氧化氢产生量与其SOD活性呈正相关。
流式细胞分析表明,未经RSV处理的PC-3细胞的凋亡率为4.40%,而经RSV处理后,PC-3细胞的凋亡率提高了近7倍达29.11%；在未经RSV处理的PC-3细胞培养液中,有50.72%的细胞处于G1期、39.71%的细胞处于S期、约9%的细胞处于G2期；而经RSV处理后,处于G1期、S期和G2期的PC-3细胞比例分别为41.33%、58.66%和0.01%。经RSV处理的HepG2细胞的凋亡率(22.89%)比未处理的HepG2细胞的凋亡率(3.70%)提高近7倍；未经RSV处理的HepG2细胞处于G1期、S期和G2期的细胞比例分别为53.5%、39.89%和6.61%；然而,经RSV处理的HepG2细胞处于G1期、S期的细胞分别为50.52%和49.48%,无G2期细胞。然而,RSV对MCF-7细胞的凋亡率影响甚微；未经RSV处理的MCF-7细胞中53.02%的细胞处于G1期、42.04%的细胞处于S期、4.94%的细胞处于G2期,而经RSV处理的MCF-7细胞处于G1期、S期的细胞分别为53.73%和46.27%,无G2期细胞。25μ M的RSV处理HEK293T细胞72h小时后,细胞凋亡率和细胞周期分布均无变化(未处理组的细胞凋亡率为14.48%,处理组的细胞凋亡率为14.54%)。
RSV对白血病细胞的生长抑制呈剂量依赖性,其中对HL-60细胞的生长抑制效应最明显。用低于50μ M的RSV处理24小时后,HL-60细胞的生长状态无明显改变,但是25μM的RSV处理HL-60细胞48小时则明显抑制其生长；50μM的RSV处理K-562细胞72小时可抑制其生长；100μM的RSV对于被检测的各类白血病细胞都有明显的生长抑制效应。RSV对白血病细胞的部分抗氧化酶表达水平及其活性具有显著影响。Western印迹分析显示用25μM的RSV处理HL-60细胞72小时,其SOD2表达水平升高36%,RSV处理可使K-562细胞的SOD2表达水平增加15%。然而,25μ M的RSV对HL-60和K-562细胞的SOD1、 CAT和GPX1的表达水平均无明显影响。未经RSV处理的HL-60细胞的SOD活性为3.3U/mg蛋白质,25u M的RSV处理HL-60细胞72小时则显著增加其SOD活性至4.1U/mg蛋白质(增加24%)；25u M的RSV处理K-562细胞可使其SOD活性增加11%；RSV几乎不改变HL-60细胞和K-562细胞的GPX活性。
流式细胞分析显示,RSV可增加白血病细胞株HL-60和K-562细胞的凋亡率,并改变其细胞周期分布。未经RSV处理的HL-60细胞的凋亡百分比为3.89%,25μM的RSV处理72小时使HL-60细胞的凋亡百分比提高5倍(19.76%)；未经RSV处理的HL-60细胞中,有35%的细胞处于G1期、59.2%的细胞处于S期、5.7%的细胞处于G2期,而经RSV处理的HL-60细胞处于G1期、S期和G2期的百分比分别为63.2%、36%和0.05%。RSV处理K-562细胞可使其凋亡率由4.85%升高至7.9%；未经RSV处理的K-562细胞中,50.7%的细胞处于G1期、49%的细胞处于S期、不到1%的细胞处于G2期；而RSV处理的K-562细胞中,39%的细胞处于G1期、60%的细胞处于S期、不到1%的细胞处于G期(G1/G2)期。
RSV明显影响HL-60细胞的H2O2产生量。用25μM或者更高浓度的RSV处理HL-60细胞明显增加其H2O2的生成。综合分析发现,肿瘤细胞产生H2O2依赖于其SOD活性,并与其细胞凋亡率相关。
以上研究结果表明,RSV对肿瘤细胞的生长抑制呈剂量依赖性。RSV在10μM和25μM时就对PC-3和HepG2细胞生长有抑制作用,但不抑制MCF-7细胞生长；50μM的RSV能显著抑制MCF-7细胞生长,但同时也能抑制HEK293T细胞生长。所以25μM的RSV被确定为PC-3和HepG2细胞生长抑制的最有效剂量,且此浓度不影响非肿瘤细胞株HEK293T细胞的生长。RSV能诱导肿瘤细胞尤其是PC-3和HepG2细胞凋亡。RSV抑制白血病细胞生长呈现剂量依赖性,且对HL-60细胞的生长抑制效果强于对K-562、KT-1/A3的和KT-1A3R细胞的生长抑制效应。低浓度RSV可显着增加PC-3、HepG2和MCF-7细胞的SOD活性；低浓度RSV几乎不影响肝癌细胞株HepG2细胞的CAT活性及GPX活性；RSV能诱导肿瘤细胞表达SOD2,但不影响SOD1、CAT及GPX1的表达。低浓度RSV处理能使PC-3和HepG2细胞大量产生H：O2,高浓度RSV处理能使MCF-7和HEK293T细胞大量产生H2O2。
讨论：细胞内抗氧化酶的表达水平和活性比例失调可能使过氧化氢产生量增加,继而诱导如HepG2和PC-3等肿瘤细胞凋亡。另一方面,RSV对非肿瘤细胞如本研究采用的HEK293T细胞的抗氧化酶表达水平和活性仅呈轻微的但成比例的上调,这可能具有防止正常细胞恶变的作用；故认为RSV可能经上调抗氧化酶表达和活性,缓解氧化应激状态,具有预防肿瘤发生的作用。对白血病细胞的研究表明,低浓度的RSV即能抑制HL-60细胞生长,但RSV对CML细胞生长抑制、凋亡及抗氧化酶如SOD2活性的调节效应不明显；提示RSV可能通过上调SOD2表达水平和活性而使线粒体H2O2产生量增加,继而诱导细胞凋亡,故此认为,RSV具有治疗AML的潜能。进一步需深入研究RSV上调肿瘤细胞中抗氧化酶表达水平和活性的分子和信号转导机制,以加速RSV的临床应用研究进程。
抗氧化酶表达水平的下降或者活性降低可能是细胞癌变的重要机制之一。因此,上调抗氧化酶表达水平或者提高其酶活性可作为预防和治疗恶性肿瘤的重要策略。研究发现孕激素能上调过氧化氢酶表达和活性,从而能够有效预防乳腺癌；染料木素作为一种抗癌异黄酮,能够上调前列腺癌细胞PC-3和DU145的SOD2和过氧化氢酶的表达水平；牛磺酸可经增加SOD、GPX和CAT表达水平而有效抑制B16F10黑色素瘤细胞生长。动物实验发现RSV具有抗肿瘤、抗炎症、降血糖和保护心血管的作用,主要生物学效应是抗氧化,并能猝灭ROS,避免其对细胞的氧化损伤,其抗氧化机制类似于维生素E。所以,RSV的抗氧化活性能保护细胞免受氧化应激损伤。研究发现,葡萄中的RSV能作为减少免疫紊乱状态下的氧化损伤和预防慢性退行性疾病的有效膳食补充剂。RSV对抗氧化损伤的高效保护机制表明RSV的抗氧化活性是预防肿瘤发生的基础(见下图)。
白藜芦醇的抗氧化作用及其机制
白藜芦醇使肿瘤细胞中SOD、CAT和GPX表达水平及其活性失衡,使线粒体中H202不断积累并诱导细胞凋亡;非恶变细胞中,白藜芦醇平衡调节其抗氧化酶表达和活性,使细胞免受氧化应激损伤。
研究结论：研究发现,抗氧化酶表达失衡或其酶活性降低是实体瘤及白血病发生的重要病因之一；肿瘤细胞耐药也常伴有抗氧化酶活性降低。故认为,抗氧化酶表达上调或其活性增加是预防和治疗恶性肿瘤的新策略。
Background and Objectives:Resveratrol (RSV) is a natural polyphenol that is known as a potent chemopreventive and chemotherapeutic molecule. RSV affects cancer cells through different mechanisms of action, most importantly, by inducing apoptosis. However the mechanism of RSV action is away from completely understood. Studies revealed that RSV can serve as either an antioxidant or pro-oxidant reagent depending on the specific microenvironment. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells, as well as apoptosis inducted in cancer cells to clarify RSV's actions on antioxidative axis.
Materials and Methods:Prostate cancer PC-3cells, hepatic cancer HepG2cells, breast cancer MCF-7cells and non-cancerous HEK293T cells were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. In another set of experiments, three Chronic myelogenous (CML) cell lines K-562, KT-1/A3, KT-1/A3R and one acute myelogenous (AML) cell line HL-60were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. Cell growth was estimated by trypan blue staining assay; activities of the antioxidant enzymes were measured spectrophotometrically; expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots; and the percentage of apoptotic cells was determined by flow cytometry. The content of hydrogen peroxide (H2O2) inside the cells was measured by spectrophotometric method.
Results:RSV showed dose-and time-dependent growth inhibition in tumor cells. It was significantly effective against PC-3and HepG2, but not against MCF-7, even at10μM and25μM concentration. However,50μM of RSV showed significant growth inhibitory effect in MCF-7, as well as HEK293T cells.25μM of RSV was identified as the most effective dosage against PC-3, HepG2and MCF-7cells, without affecting HEK293T cells.
Treatment of RSV at low concentration significantly increased the activity of antioxidant enzyme superoxide dismutase (SOD) in PC-3, HepG2and MCF-7cells, but not in HEK293T cells. Another antioxidant enzyme catalase (CAT) activity was increased in HepG2, but no effect was found on the activity of antioxidant enzyme glutathione peroxidase (GPX) by RSV treatment. RSV-induced SOD2expression was observed in cancer cells, whereas the expressions of SOD1, CAT and GPX1were not affected. The production of H2O2was significantly increased in PC-3and HepG2at lower concentrations of RSV, and induced production of H2O2was seen in MCF-7and HEK293T when treated with higher concentrations of RSV. The apoptosis was increased by RSV treatment in cancer cells, especially PC-3and HepG2.
In all tested leukemic cells, RSV showed dose dependent growth inhibition, and HL-60was found more sensitive to the RSV than K-562, KT-1/A3and KT-1/A3R. After24hours of treatment of RSV at a concentration of less than50μM, the cell growth was not significantly changed. However,48hours treatment of25μM RSV showed significant growth inhibitory effect in HL-60cells, but not in the other cells. The growth of HL-60cells was inhibited by10μM RSV when treated for72hours. RSV showed growth inhibitory effect in K-562cells only when treated with50μM for72hours. In KT-1/A3or KT-1/A3R cells, RSV showed no significant effect when treated with≤50μM. However,100μM of RSV was found lethal to all the tested leukemic cell lines.
The SOD activity in HL-60cells was significantly increased (by24%) after RSV treatment for72hours at25μM concentration. When the K-562treated with25μM of RSV, the SOD activity was not significantly increased (only by11%). The CAT activity was slightly increased in K-562cells and slightly decreased in HL-60cells, but none of the effects was significant. In addition, the GPX activity was not significantly changed by RSV in either HL-60or K-562cells. Western blot analysis showed the expression of SOD2in HL-60increased (by36%) significantly when treated with25μM RSV for 72hours, but the effect on K-562was non-significant when the cells were treated with the same conditions. However, in HL-60or K-562cells, the effect of25μM RSV on SOD1, CAT and GPX1was not significant. The flow cytometric analysis revealed that the percentage of apoptotic cells was increased more potently in HL-60cells (6-fold) than in K-562cells (1.5fold) when treated with RSV. RSV also showed significant effect on the production of H2O2in HL-60cells. Treatment of10μM of RSV for72hours decreased the H2O2content slightly in cancer cells, but25μM or higher concentrations of RSV significantly increased H2O2production in HL-60cells.
Conclusions:This study demonstrates:
(1) RSV inhibits PC-3, HpG2, MCF-7cancer cell growth with minimal effect on non-cancerous cells.
(2) RSV is more growth inhibitory against HL-60AML cells than CML cells.
(3) Mechanistically, the disproportional up-regulation of SOD, CAT and GPX expression and their enzymatic activity in PC-3, HepG2, MCF-7tumor cells, and HL-60leukemic cells result in mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis. On the other hand, slight but proportional up-regulation of anti-oxidative enzymes and reducing oxidative stress could explain RSV's protective effect on non-cancerous cells.
在众多影响肿瘤发生的因素中,氧化应激诱导的DNA损伤及其对细胞信号转导途径的干扰在肿瘤的发生发展中起着重要的作用。活细胞中许多的氧化反应需要酶催化,氧化反应即分子间的氢质子或电子转移,这类代谢反应是生命过程所需能量的主要来源。地球上绝大多数复杂的生命体都依赖氧而生存,但是氧是一种高活性分子,生物体会分解氧气产生包括过氧化氢(H2O2)、次氯酸(HOC1)和自由基如羟基自由基(·OH)、超氧阴离子(02-)以及脂质过氧化物等活性氧簇(reactive oxygen species, ROS),这些代谢产物可以直接或间接地对核酸、脂类和蛋白质造成暂时或永久性损伤,从而对生物体自身造成损害。氧化-抗氧化状态之间的不平衡造成的氧化损伤与众多疾病的发生相关,包括癌症、神经系统疾病、动脉粥样硬化、高血压、糖尿病、急性呼吸窘迫综合症、慢性阻塞性肺疾病和哮喘等。基于自身保护,机体需要多种类型的抗氧化剂以维持氧化代谢的平衡。如谷胱甘肽、维生素C、维生素E以及一些酶类,包括过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)等。抗氧化酶可保护细胞免受氧化应激损伤,抗氧化酶活性和表达的失调与肿瘤发生密切相关,是肿瘤发生发展的重要影响因素。氧化应激能诱导DNA损伤并影响细胞内信号转导途径。ROS以多种形式损伤DNA,包括碱基修饰、自由基损害(嘌呤和嘧啶)、DNA断裂及DNA-蛋白质交互连接等。氧化应激对肿瘤发生与治疗的影响被越来越受到重视。有研究报道,前列腺癌、膀胱癌、乳腺癌、肝癌、多发性骨髓瘤和其他多种肿瘤都伴有抗氧化酶活性降低或其表达下调；也有研究发现某些肿瘤中抗氧化酶水平无变化,或部分抗氧化酶的表达水平甚至升高。近期研究表明,多发性骨髓瘤患者血清中SOD、GPX、 CAT、维生素C和维生素E水平均显著降低；非小细胞肺癌(NSCLC)患者红细胞裂解液中的SOD活性较低而GPX活性升高；前列腺癌患者的GPX、CAT和Cu/Zn-SOD活性均降低；口腔鳞状细胞癌(OSCC)患者的SOD和CAT水平均降低;急性淋巴细胞白血病(ALL)患者全血中CAT和SOD活性降低；膀胱癌组织中CAT和SOD活性均显著低于正常膀胱组织；肺肿瘤组织中总SOD活性升高,CAT活性下降；肺部炎症会产生高水平Mn-SOD,但CAT水平降低,二者共同作用可导致细胞内的H202水平上升,因此肺炎将会为DNA损伤和细胞恶变提供有利条件,在对肺癌组织和肺癌A549细胞株的研究中发现SOD的水平增加、CAT水平降低、而GPX水平则无改变。
恶性肿瘤是一种典型的多因素疾病,遗传因素与环境因素对肿瘤发生的影响相互相存。DNA变异可能引起原癌基因活化或抑瘤基因失活,从而使细胞分化、凋亡失控。基因突变可能是自发突变或者是暴露于辐射或致癌物质所致。抗氧化酶可防止DNA的氧化损伤和延缓或改变细胞周期。某些病毒如人类T淋巴细胞病毒及某些化学物质,特别是含苯或烷基的药物等均是重要的致癌因素。已观察到在造血系统恶性肿瘤中ROS的产生过量和／或抗氧化途径障碍,已发现在一些类型的白血病中抗氧化酶活性缺陷或表达异常。因此,保持一定水平的抗氧化酶活性对于预防恶性肿瘤的发生发展是至关重要的。另一方面,靶向抗氧化酶活性可作为肿瘤药物治疗的重要发展策略,研究发现,单独采用GPX和Mn-SOD基因疗法可以分别减缓肿瘤生长速率达51%和54%,GPX和Mn-SOD基因共转染则可达到81%的肿瘤细胞生长抑制率。此外,研究抗癌药物对抗氧化酶活性和表达水平的影响将加速药物的作用机制研究。
白藜芦醇(resveratrol, RSV)是一种由植物产生的多酚类物质(stilbenoid),化学名称为3,5,4’-三羟基-反式-二苯乙烯(见下图),具有可利用的生物学活性。RSV主要存在于红葡萄皮、豌豆、坚果、蓝莓、桑椹、蔓越莓、姜黄、菠菜、百合等植物组织中,白藜芦醇苷是RSV的前体,属于二苯乙烯类化合物,是葡萄汁中主要的RSV前体物质；白藜芦醇苷经米曲霉催化可以形成RSV。事实上,白藜芦醇苷是自然界中最丰富的RSV储存形式。植物中RSV的合成或转化可通过应激因素调节,如真菌污染或紫外线辐射。植物中的RSV可作为一种植物抗毒素,能抑制某些植物传染病的发生与发展。
研究表明,RSV能显著增加抗氧化酶SOD、CAT、GPX等和非酶类抗氧化物质如还原型谷胱甘肽、维生素C、维生素E和β-胡萝卜素等的抗氧化作用,并降低脂质过氧化水平。RSV具有抗衰老、抗肿瘤活性,能够延缓衰老过程并最大限度地减少与老化相关的并发症的发生；此外,RSV还具有保护心脏、抗糖尿病、抗炎症、抗病毒及保护神经系统等多种作用。已在肿瘤细胞株、动物模型甚至临床研究中发现RSV能够抑制肿瘤的发生发展；并在体内和体外研究中证实RSV对皮肤癌、乳腺癌、肝癌、前列腺癌、大肠癌、胰腺癌等具有治疗或辅助治疗效应。机制研究表明,RSV可通过诱导肿瘤细胞凋亡、改变细胞周期、抑制血管生成、抑制核因子-κB和环氧合酶信号转导途径、抑制致癌物的代谢活化、改变与肿瘤发生相关的miRNA表达模式及抗氧化等而影响肿瘤细胞的增殖、转移或凋亡(见下图)。近期研究表明,RSV可通过干扰pAktl/p70S6K信号途径而抑制人类鼻咽癌(NPC)细胞增殖和促进其凋亡。但目前对RSV发挥作用的确切机制尚未完全了解。本研究即以肿瘤细胞株为试验模型,研究RSV经抗氧化而抑制肿瘤细胞生长的分子机制。
本研究采用3种不同的肿瘤细胞株,即前列腺癌细胞株PC-3、乳腺癌细胞株MCF-7和肝癌细胞株HepG2细胞。现今,这三种肿瘤都被认为是最危险的危及生命的疾病。在全球男性癌症死亡排名六大癌症中,前列腺癌排名第二；乳腺癌是女性最常见的浸润性肿瘤；肝癌则是全球最常见的恶性肿瘤之一,其死亡率仅次于肺癌。人类前列腺癌细胞株PC-3细胞对于研究中晚期前列腺癌细胞的生化变化以及评价其对化疗药物的反应性很有帮助,PC-3细胞株是雄激素非依赖性前列腺癌细胞,建株于1979年,是从一名62岁的白人男性IV级前列腺癌骨转移样品中分离得到的；乳腺癌细胞株MCF-7细胞是于1970年从一名69岁的白人女性患者组织样品中分离得到的,是最常用于研究肿瘤发生机制的乳腺癌细胞；人类肝癌细胞株HepG2细胞来自于一名15岁美国白人男孩的高分化肝癌组织,HepG2细胞常用于研究肝癌的发生发展与治疗机理。为了比较RSV对这些肿瘤细胞的作用及其机制,本研究中我们采用人胚肾细胞株HEK293T细胞作为对照。本研究同时采用了几株人类白血病细胞。人类早幼粒细胞白血病细胞株HL-60细胞来自于美国一位患急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)的36岁女性,是研究髓样分化的分子事件的人源细胞,APL是一种特殊类型的急性髓系白血病(acute myeloid leukemia, AML), HL-60细胞已成为研究AML的良好模型；K-562细胞是建株的第一个具有无限增殖特性的人类白血病细胞系,主要用作研究慢性髓系白血病(chronic myelogenous leukemia, CML)的发生与治疗机理,K-562细胞具有特征性的基于bcr/abl融合基因的费城染色体；KT-1/A3和KT-1/A3R细胞株是本研究采用的另外两类CML细胞,其中KT-1/A3细胞呈现对干扰素的抗癌效应敏感,而KT-1/A3R细胞则呈现干扰素抗性。
研究表明,RSV主要呈现抗氧化效应。但是,为什么在一定条件下RSV能保护正常细胞而对肿瘤细胞呈现细胞毒性仍然是一个有争议的问题。此外,RSV在不同肿瘤中对抗氧化酶的表达和活性的影响效果仍然有一些不一致的结果。基于此,本研究采用非肿瘤细胞株HEK293T细胞作为对照,针对RSV对部分实体瘤及白血病细胞的作用,揭示RSV对肿瘤细胞的抗氧化酶表达水平及其活性的调节作用,进而探讨RSV的抗氧化活性对肿瘤细胞增殖／凋亡的影响,剖析RSV的抗氧化／抗肿瘤机制。
研究方法：采用不同浓度的RSV处理前列腺癌PC-3细胞、肝癌HepG2细胞、乳腺癌MCF-7细胞、CML细胞株(K-562、KT-1/A3、KT-1/A3R)、AML细胞株HL-60细胞和非肿瘤人胚肾细胞株HEK293T细胞24-72小时,台盼蓝染色法检测细胞生长状态,分光光度法检测抗氧化酶的活性和过氧化氢(H2O2)的产量,Western印迹定量检测抗氧化酶的表达水平,流式细胞仪检测细胞周期分布和凋亡率。
采用DMEM培养基培养肝癌HepG2细胞和乳腺癌MCF-7细胞,采用RPMI1640培养基培养前列腺癌PC-3细胞、白血病细胞和非肿瘤HEK293T细胞,均加入10%的胎牛血清于37℃和5%浓度的CO2条件下培养。
以1.0×105/m1细胞接种于含有lml完全培养基的24孔培养板中,其培养基含有不同浓度(0、10、25、50和100μ M)的RSV；培养24、48和72小时后,富集细胞用于后续实验。
用台盼蓝染色法检测细胞的生长状态,贴壁生长细胞用胰蛋白酶处理,使之与培养皿分离,取等体积的悬浮生长细胞培养物或经胰蛋白酶处理的贴壁生长细胞悬液与台盼蓝溶液混合,显微镜下计数未被染成蓝色的活细胞数目。
以1000g、5分钟离心富集培养细胞,弃上清后用磷酸盐缓冲盐水(phosphate buffered saline, PBS)洗涤离心细胞,然后用200μl放射免疫沉淀分析缓冲液(radioimmunoprecipitation assay buffer, RIPA)和1mM苯甲基磺酰氟(phenylmethylsulfonyl fluoride, PMSF)处理细胞,冰浴30分钟。将该溶液于12000g离心15分钟,收集上清蛋白质溶液,贮于-20℃备用。
采用Bradford法测定蛋白质浓度,按体积比1：1将蛋白质溶液与考马斯亮蓝溶液混合,室温静置5分钟后,以牛血清白蛋白作为蛋白质标准溶液,于595nm处测定待测蛋白质溶液的吸光度并计算其蛋白质浓度。
采用非连续聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)分离蛋白质。首先,制备10%的分离胶,取dH2O7.78ml、40%Acr:Bis4. Oml、 pH8.8#的1.5M Tris-HCl4.0ml、10%SDS160μ1、10%APS80μ1和TEMED4.0μ1混合均匀,倒入PAGE玻璃槽；将胶在室温下静置1小时后,制备4%的浓缩胶含dH2O3.3ml、40%Acr:Bis0.96ml、0.5M Tris-HCl (pH6.8)2.0ml,10%SDS80μ1、10%APS40μ1和TEMED4.0μl,并倒入玻璃槽中覆盖分离胶,将梳板插入凝胶中：成胶后,用16μl蛋白质溶液与4μl上样缓冲液混合,95℃加热10分钟后上样；同样点上已知分子量的蛋白质标准物,60V电泳30分钟后,于80-85V电泳90分钟。
将凝胶上分离的蛋白质经电转移至硝酸纤维素膜,然后将印迹膜于室温浸泡在含5%脱脂牛奶的Tris缓冲生理盐水和吐温20(Tris-buffered saline and Tween20, TBST)缓冲液中2小时。印迹膜与第一抗体孵育12小时后用TBST润洗。然后将印迹膜与辣根过氧化物酶(HRP)第二抗体轻轻震荡孵育2小时,然后用TBST洗涤三次。化学发光反应后,在暗室暴光于X-光片,使用凝胶成像系统("ImageJ"软件)对Western印迹条带进行高密度数字化处理,对蛋白质条带进行定量分析。
以12000g离心5分钟收集细胞,用PBS润洗细胞,用含有1mM EDTA的PBS(pH7.0)匀浆,于40C、以12000g离心5分钟后收集细胞上清液测定过氧化氢的含量和酶活性(U/mg蛋白质)；采用黄嘌呤氧化酶法测定SOD活性；采用钼酸铵法测定CAT活性；利用DTNB法测定GPX活性。
经RSV诱导培养72小时后,12000g离心5分钟收集细胞,用PBS (pH7.4)洗涤并于70%乙醇中固定过夜。然后用流式细胞仪(北京鼎国昌盛生物有限公司)分别分析细胞凋亡和细胞周期的百分比。
对以上实验获取的数据经统计学软件SPSS16.0进行单因素方差分析和比较检验,结果以均数±标准差表示,以P≤0.05显示其统计学意义。研究结果：本研究结果显示,在浓度小于50μM的RSV处理24小时后,细胞生长状态均无明显改变；但是,25μM RSV处理48小时显示PC-3细胞和HepG2细胞的生长明显受到抑制,但MCF-7细胞生长未受抑制；用10μM或者25μM的RSV处理72小时,PC-3细胞和HepG2细胞的生长明显受到抑制,但浓度低于25μ M的RSV对MCF-7细胞生长无抑制效应：50μM的RSV处理48小时或72小时对三种细胞的生长都有明显的抑制作用,同时也抑制非肿瘤细胞株HEK293T细胞的生长；100μM的RSV对每种细胞株的生长都有明显的抑制作用。25μM的RSV能抑制实验中所用肿瘤细胞的生长而不影响非肿瘤细胞株HEK293T细胞的生长,本研究选择25μM的RSV作为最佳实验用剂量。
对照组PC-3细胞中的SOD活性为4.94U/mg蛋白质,分别用10μM,25μM和50u M的RSV处理PC-3细胞72小时,其SOD活性分别增加43%、88%和105%；对照组HepG2细胞中SOD活性为3.41U/mg蛋白质,分别用10μM.25μM和50μM的RSV处理72小时,其SOD活性分别增加55%、63%和87%；对照组MCF-7细胞的SOD活性为2.82U/mg蛋白质,用浓度为25μ M和50μ M的RSV处理MCF-7细胞72小时,其SOD活性分别增加54%和60%；对照组HEK293T细胞中SOD的活性为4.8U/mg蛋白质,低浓度的RSV(10μM或25μM)对于其SOD活性没有影响,但是50u M的RSV可使其活性增加49%。Western印迹分析显示用浓度为25μ M的RSV处理细胞72小时,PC-3、HepG2和MCF-7细胞的SOD2表达量分别增加3倍、2.5倍和1.5倍,而对HEK293T细胞的作用则不明显；细胞的处理组和非处理组中的SOD1表达水平无明显差异。PC-3、HepG2、MCF-7和HEK293T细胞中的CAT活性分别为4.20、4.82、4.08和7.28U/mg蛋白质。RSV处理72小时后各种细胞株的CAT活性都有一定程度的增加,25μM或50μ M的RSV处理HepG2细胞后其CAT活性分别增加了45%和42%；然而RSV处理对于其他细胞的CAT活性无明显影响；Western印迹分析显示用RSV处理的各种细胞株的CAT蛋白质水平均无明显改变。HEK293T、PC-3、HepG2和MCF-7细胞中的GPX活性分别为0.65、0.60、0.74和0.60U/mg蛋白质；用10-50μM的RSV处理细胞72小时,GPX活性及GPX1蛋白质水平均无明显变化。
本研究结果显示,RSV能明显提高肿瘤细胞的H202产生量。用10μM的RSV处理肿瘤细胞72小时,其H202水平均略微增加,而用25μ M的RSV处理PC-3细胞和HepG2细胞72小时,其H202产生量均增加约2.5倍,经同样处理的MCF-7细胞的H202产生量略有增加。与此相反,经10-25u M的RSV处理后,HEK293T细胞的H202产生量明显降低。肿瘤细胞的过氧化氢产生量与其SOD活性呈正相关。
流式细胞分析表明,未经RSV处理的PC-3细胞的凋亡率为4.40%,而经RSV处理后,PC-3细胞的凋亡率提高了近7倍达29.11%；在未经RSV处理的PC-3细胞培养液中,有50.72%的细胞处于G1期、39.71%的细胞处于S期、约9%的细胞处于G2期；而经RSV处理后,处于G1期、S期和G2期的PC-3细胞比例分别为41.33%、58.66%和0.01%。经RSV处理的HepG2细胞的凋亡率(22.89%)比未处理的HepG2细胞的凋亡率(3.70%)提高近7倍；未经RSV处理的HepG2细胞处于G1期、S期和G2期的细胞比例分别为53.5%、39.89%和6.61%；然而,经RSV处理的HepG2细胞处于G1期、S期的细胞分别为50.52%和49.48%,无G2期细胞。然而,RSV对MCF-7细胞的凋亡率影响甚微；未经RSV处理的MCF-7细胞中53.02%的细胞处于G1期、42.04%的细胞处于S期、4.94%的细胞处于G2期,而经RSV处理的MCF-7细胞处于G1期、S期的细胞分别为53.73%和46.27%,无G2期细胞。25μ M的RSV处理HEK293T细胞72h小时后,细胞凋亡率和细胞周期分布均无变化(未处理组的细胞凋亡率为14.48%,处理组的细胞凋亡率为14.54%)。
RSV对白血病细胞的生长抑制呈剂量依赖性,其中对HL-60细胞的生长抑制效应最明显。用低于50μ M的RSV处理24小时后,HL-60细胞的生长状态无明显改变,但是25μM的RSV处理HL-60细胞48小时则明显抑制其生长；50μM的RSV处理K-562细胞72小时可抑制其生长；100μM的RSV对于被检测的各类白血病细胞都有明显的生长抑制效应。RSV对白血病细胞的部分抗氧化酶表达水平及其活性具有显著影响。Western印迹分析显示用25μM的RSV处理HL-60细胞72小时,其SOD2表达水平升高36%,RSV处理可使K-562细胞的SOD2表达水平增加15%。然而,25μ M的RSV对HL-60和K-562细胞的SOD1、 CAT和GPX1的表达水平均无明显影响。未经RSV处理的HL-60细胞的SOD活性为3.3U/mg蛋白质,25u M的RSV处理HL-60细胞72小时则显著增加其SOD活性至4.1U/mg蛋白质(增加24%)；25u M的RSV处理K-562细胞可使其SOD活性增加11%；RSV几乎不改变HL-60细胞和K-562细胞的GPX活性。
流式细胞分析显示,RSV可增加白血病细胞株HL-60和K-562细胞的凋亡率,并改变其细胞周期分布。未经RSV处理的HL-60细胞的凋亡百分比为3.89%,25μM的RSV处理72小时使HL-60细胞的凋亡百分比提高5倍(19.76%)；未经RSV处理的HL-60细胞中,有35%的细胞处于G1期、59.2%的细胞处于S期、5.7%的细胞处于G2期,而经RSV处理的HL-60细胞处于G1期、S期和G2期的百分比分别为63.2%、36%和0.05%。RSV处理K-562细胞可使其凋亡率由4.85%升高至7.9%；未经RSV处理的K-562细胞中,50.7%的细胞处于G1期、49%的细胞处于S期、不到1%的细胞处于G2期；而RSV处理的K-562细胞中,39%的细胞处于G1期、60%的细胞处于S期、不到1%的细胞处于G期(G1/G2)期。
RSV明显影响HL-60细胞的H2O2产生量。用25μM或者更高浓度的RSV处理HL-60细胞明显增加其H2O2的生成。综合分析发现,肿瘤细胞产生H2O2依赖于其SOD活性,并与其细胞凋亡率相关。
以上研究结果表明,RSV对肿瘤细胞的生长抑制呈剂量依赖性。RSV在10μM和25μM时就对PC-3和HepG2细胞生长有抑制作用,但不抑制MCF-7细胞生长；50μM的RSV能显著抑制MCF-7细胞生长,但同时也能抑制HEK293T细胞生长。所以25μM的RSV被确定为PC-3和HepG2细胞生长抑制的最有效剂量,且此浓度不影响非肿瘤细胞株HEK293T细胞的生长。RSV能诱导肿瘤细胞尤其是PC-3和HepG2细胞凋亡。RSV抑制白血病细胞生长呈现剂量依赖性,且对HL-60细胞的生长抑制效果强于对K-562、KT-1/A3的和KT-1A3R细胞的生长抑制效应。低浓度RSV可显着增加PC-3、HepG2和MCF-7细胞的SOD活性；低浓度RSV几乎不影响肝癌细胞株HepG2细胞的CAT活性及GPX活性；RSV能诱导肿瘤细胞表达SOD2,但不影响SOD1、CAT及GPX1的表达。低浓度RSV处理能使PC-3和HepG2细胞大量产生H：O2,高浓度RSV处理能使MCF-7和HEK293T细胞大量产生H2O2。
讨论：细胞内抗氧化酶的表达水平和活性比例失调可能使过氧化氢产生量增加,继而诱导如HepG2和PC-3等肿瘤细胞凋亡。另一方面,RSV对非肿瘤细胞如本研究采用的HEK293T细胞的抗氧化酶表达水平和活性仅呈轻微的但成比例的上调,这可能具有防止正常细胞恶变的作用；故认为RSV可能经上调抗氧化酶表达和活性,缓解氧化应激状态,具有预防肿瘤发生的作用。对白血病细胞的研究表明,低浓度的RSV即能抑制HL-60细胞生长,但RSV对CML细胞生长抑制、凋亡及抗氧化酶如SOD2活性的调节效应不明显；提示RSV可能通过上调SOD2表达水平和活性而使线粒体H2O2产生量增加,继而诱导细胞凋亡,故此认为,RSV具有治疗AML的潜能。进一步需深入研究RSV上调肿瘤细胞中抗氧化酶表达水平和活性的分子和信号转导机制,以加速RSV的临床应用研究进程。
抗氧化酶表达水平的下降或者活性降低可能是细胞癌变的重要机制之一。因此,上调抗氧化酶表达水平或者提高其酶活性可作为预防和治疗恶性肿瘤的重要策略。研究发现孕激素能上调过氧化氢酶表达和活性,从而能够有效预防乳腺癌；染料木素作为一种抗癌异黄酮,能够上调前列腺癌细胞PC-3和DU145的SOD2和过氧化氢酶的表达水平；牛磺酸可经增加SOD、GPX和CAT表达水平而有效抑制B16F10黑色素瘤细胞生长。动物实验发现RSV具有抗肿瘤、抗炎症、降血糖和保护心血管的作用,主要生物学效应是抗氧化,并能猝灭ROS,避免其对细胞的氧化损伤,其抗氧化机制类似于维生素E。所以,RSV的抗氧化活性能保护细胞免受氧化应激损伤。研究发现,葡萄中的RSV能作为减少免疫紊乱状态下的氧化损伤和预防慢性退行性疾病的有效膳食补充剂。RSV对抗氧化损伤的高效保护机制表明RSV的抗氧化活性是预防肿瘤发生的基础(见下图)。
白藜芦醇的抗氧化作用及其机制
白藜芦醇使肿瘤细胞中SOD、CAT和GPX表达水平及其活性失衡,使线粒体中H202不断积累并诱导细胞凋亡;非恶变细胞中,白藜芦醇平衡调节其抗氧化酶表达和活性,使细胞免受氧化应激损伤。
研究结论：研究发现,抗氧化酶表达失衡或其酶活性降低是实体瘤及白血病发生的重要病因之一；肿瘤细胞耐药也常伴有抗氧化酶活性降低。故认为,抗氧化酶表达上调或其活性增加是预防和治疗恶性肿瘤的新策略。
Background and Objectives:Resveratrol (RSV) is a natural polyphenol that is known as a potent chemopreventive and chemotherapeutic molecule. RSV affects cancer cells through different mechanisms of action, most importantly, by inducing apoptosis. However the mechanism of RSV action is away from completely understood. Studies revealed that RSV can serve as either an antioxidant or pro-oxidant reagent depending on the specific microenvironment. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells, as well as apoptosis inducted in cancer cells to clarify RSV's actions on antioxidative axis.
Materials and Methods:Prostate cancer PC-3cells, hepatic cancer HepG2cells, breast cancer MCF-7cells and non-cancerous HEK293T cells were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. In another set of experiments, three Chronic myelogenous (CML) cell lines K-562, KT-1/A3, KT-1/A3R and one acute myelogenous (AML) cell line HL-60were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. Cell growth was estimated by trypan blue staining assay; activities of the antioxidant enzymes were measured spectrophotometrically; expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots; and the percentage of apoptotic cells was determined by flow cytometry. The content of hydrogen peroxide (H2O2) inside the cells was measured by spectrophotometric method.
Results:RSV showed dose-and time-dependent growth inhibition in tumor cells. It was significantly effective against PC-3and HepG2, but not against MCF-7, even at10μM and25μM concentration. However,50μM of RSV showed significant growth inhibitory effect in MCF-7, as well as HEK293T cells.25μM of RSV was identified as the most effective dosage against PC-3, HepG2and MCF-7cells, without affecting HEK293T cells.
Treatment of RSV at low concentration significantly increased the activity of antioxidant enzyme superoxide dismutase (SOD) in PC-3, HepG2and MCF-7cells, but not in HEK293T cells. Another antioxidant enzyme catalase (CAT) activity was increased in HepG2, but no effect was found on the activity of antioxidant enzyme glutathione peroxidase (GPX) by RSV treatment. RSV-induced SOD2expression was observed in cancer cells, whereas the expressions of SOD1, CAT and GPX1were not affected. The production of H2O2was significantly increased in PC-3and HepG2at lower concentrations of RSV, and induced production of H2O2was seen in MCF-7and HEK293T when treated with higher concentrations of RSV. The apoptosis was increased by RSV treatment in cancer cells, especially PC-3and HepG2.
In all tested leukemic cells, RSV showed dose dependent growth inhibition, and HL-60was found more sensitive to the RSV than K-562, KT-1/A3and KT-1/A3R. After24hours of treatment of RSV at a concentration of less than50μM, the cell growth was not significantly changed. However,48hours treatment of25μM RSV showed significant growth inhibitory effect in HL-60cells, but not in the other cells. The growth of HL-60cells was inhibited by10μM RSV when treated for72hours. RSV showed growth inhibitory effect in K-562cells only when treated with50μM for72hours. In KT-1/A3or KT-1/A3R cells, RSV showed no significant effect when treated with≤50μM. However,100μM of RSV was found lethal to all the tested leukemic cell lines.
The SOD activity in HL-60cells was significantly increased (by24%) after RSV treatment for72hours at25μM concentration. When the K-562treated with25μM of RSV, the SOD activity was not significantly increased (only by11%). The CAT activity was slightly increased in K-562cells and slightly decreased in HL-60cells, but none of the effects was significant. In addition, the GPX activity was not significantly changed by RSV in either HL-60or K-562cells. Western blot analysis showed the expression of SOD2in HL-60increased (by36%) significantly when treated with25μM RSV for 72hours, but the effect on K-562was non-significant when the cells were treated with the same conditions. However, in HL-60or K-562cells, the effect of25μM RSV on SOD1, CAT and GPX1was not significant. The flow cytometric analysis revealed that the percentage of apoptotic cells was increased more potently in HL-60cells (6-fold) than in K-562cells (1.5fold) when treated with RSV. RSV also showed significant effect on the production of H2O2in HL-60cells. Treatment of10μM of RSV for72hours decreased the H2O2content slightly in cancer cells, but25μM or higher concentrations of RSV significantly increased H2O2production in HL-60cells.
Conclusions:This study demonstrates:
(1) RSV inhibits PC-3, HpG2, MCF-7cancer cell growth with minimal effect on non-cancerous cells.
(2) RSV is more growth inhibitory against HL-60AML cells than CML cells.
(3) Mechanistically, the disproportional up-regulation of SOD, CAT and GPX expression and their enzymatic activity in PC-3, HepG2, MCF-7tumor cells, and HL-60leukemic cells result in mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis. On the other hand, slight but proportional up-regulation of anti-oxidative enzymes and reducing oxidative stress could explain RSV's protective effect on non-cancerous cells.
引文
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