5-氟尿嘧啶联合白藜芦醇的抗肿瘤作用研究
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摘要
在发展中国家,癌症是仅次于心脏疾病的死亡率最高的疾病,随着生存环境的恶化,人口增长与老龄化,环境污染以及与癌症有关的生活方式的改变(如吸烟、运动减少以及更多地采取西化饮食结构),癌症的发生率逐年增加。目前恶性肿瘤的治疗方法包括手术、放疗、化疗和免疫治疗等手段,以化学治疗为主的综合治疗在其中占有十分重要的地位。化学治疗指的是使用化学药物杀灭肿瘤细胞,理想的化疗药物应该是通过抑制细胞生长或细胞毒作用,只针对肿瘤细胞特异性杀伤而不影响正常细胞。但事实上,临床常用的化疗药物其肿瘤抑制作用大多与剂量相关,在杀伤肿瘤细胞的同时具有细胞毒作用的无选择性,从而对宿主全身会产生毒副作用,成为化疗实施的障碍,从而在很大程度上影响肿瘤治疗的效果。因此,除了寻找高效低毒的新化疗药物以外,还可以通过与其他化合物或药物联合产生相加或协同作用以提高化疗效果。如能改变肿瘤细胞的周期进程,使大量肿瘤细胞暂时性蓄积在某一时相,同时应用细胞周期特异性药物杀伤处于此药物敏感时相的肿瘤细胞将能明显提高治疗效果,同时也可降低化疗药物剂量从而减轻不良反应,这样的研究将具有重要意义。
5-氟尿嘧啶(5-Fluorouracil,5-Fu)是常见的化疗药物,常用于实体瘤的治疗,可以通过将细胞周期阻滞于G1/S期并干扰细胞基本生物合成过程诱导肿瘤细胞的凋亡从而起到抑制肿瘤生长的作用。5-Fu还可以作为胸苷酸合成酶(TS)的抑制剂掺入DNA和RNA结构中使肿瘤细胞无法正常合成DNA,影响其正常功能。鉴于5-Fu的作用机理,它在治疗过程中也会影响正常细胞的功能而产生毒副作用,另外,目前比较常见的5-Fu使用中产生的抗药性也成了阻碍其发挥疗效的障碍。现在已有很多二线药物如紫杉醇、丝裂霉素、四氢叶酸、喃氟啶等开始与5-Fu联合使用,其目的是在保持其抗肿瘤作用最大化的同时减少5-Fu的毒副作用。除了这些药物以外,一些天然植物素也能达到对5-Fu增效减毒的效果。
白藜芦醇(Resveratrol,Res)是一种广泛存在于自然界中的植物抗毒素,是植物对抗外界环境压力如气候变化,暴露于更多的臭氧环境中,日晒和重金属刺激以及病原微生物感染等情况时所产生的。大量研究结果显示白藜芦醇可以对抗多种疾病甚至癌症。研究发现白藜芦醇对肿瘤的起始、促进和发展三个阶段均有抑制作用,可作为天然的肿瘤化学预防剂。其抗肿瘤作用可能与其影响细胞生长、抗炎、诱导细胞凋亡和抗侵袭能力有关。白藜芦醇可以影响体内多种基因表达,其中包括肿瘤抑制因子p53和Rb,细胞周期调节蛋白Cyclin家族以及多种凋亡调节因子。诸多的体内外研究证实白藜芦醇可以作为抗肿瘤药物或与其他化疗药物联合使用治疗一些有抗药性的癌症。
皮肤癌尤其是非黑色素性皮癌(NMSC)是世界上发病数量最多的人类肿瘤,近年来的发病率日益升高,据估计每年新增病例超过一百万。如此高的年发病率使其越发受到公众的关注。食管癌的死亡率很高,在世界恶性肿瘤死亡率中占据第六位,而我国正是是食管癌的高发地区。鉴于这两种肿瘤的高发性,本实验中选取食管鳞状细胞癌株TE-1、人表皮癌A431细胞株作为研究对象来考察5-Fu与白藜芦醇联合在细胞生长、细胞周期和凋亡方面的作用,并对可能与联合作用有关的凋亡通路进行了研究。实验共分为四部分:
第一部分5-氟尿嘧啶联合白藜芦醇对肿瘤细胞A431、TE-1生长的抑制和细胞毒作用
目的:研究5-Fu联合白藜芦醇对肿瘤细胞A431、TE-1生长的抑制作用和致坏死能力
方法:本部分实验中采用MTT法、生长曲线实验、平板克隆实验对5-Fu和白藜芦醇的联合用药进行评价,联合用药的效果采用Chou- Talalay法进行定量评价分析。细胞毒作用采用LDH溢出实验进行评价。
结果: (1) MTT实验显示5-Fu有抑制肿瘤细胞的生长的能力,而且这种作用呈时间与浓度依赖性。而白藜芦醇在较高浓度(不低于10μmol/L)才能够产生抑制作用。二者联合使用时在很大的浓度范围内都可以产生协同作用(白藜芦醇不低于10μmol/L)。联合用药可以大大降低5-Fu或白藜芦醇对A431的中效浓度(5-Fu的IC50从单独使用时的322.89μM降至71.99μM;白藜芦醇的IC50从174.58μM降至37.56μM)。对TE-1联合用药可以分别将5-Fu的IC50从480.83μM降至151.87μM ,而白藜芦醇的IC50可从248.64μM降至69.42μM。该结果显示5-Fu和白藜芦醇联合使用可在很大程度上减少各药物的用量。
采用Chou -Talalay法对5-Fu和白藜芦醇的联合作用48h的效应进行评价,当两药联合使用对肿瘤的的抑制效应达到50%时,A431细胞的联合指数CI为0.438,而对TE-1的联合指数CI为0.595。二者联合在高于0.2的效应范围内CI值均不高于1,说明在一个很大的效应范围内(0.2-0.95)5-Fu联合白藜芦醇都能产生协同作用。(2)生长曲线实验结果显示,当5-Fu和白藜芦醇单独使用时都能对肿瘤细胞的生长起到抑制作用,当二者联合使用时对肿瘤细胞的生长曲线抑制更为明显,与对照组和各单药组相比均存在显著性差异。(3)平板克隆实验结果显示14天后A431和TE-1各自有17.60%和26.75%的克隆形成率,白藜芦醇单独使用可以使两种细胞的克隆形成率分别降至A431 5.42%,TE-1 10.13%。而5-Fu单独使用的克隆形成率分别为A431 1.07% ,TE-1 2.23%。两种细胞的联合用药组则几乎没有克隆形成,各组间在统计学上表现为极显著性差异。(4) LDH活性实验显示了5-Fu和白藜芦醇单独使用所导致的A431和TE-1细胞坏死分别出现在48h和72h后,而联合给药组在24h即出现LDH的溢出,说明联合用药对肿瘤细胞的抑制作用可能与坏死有一定关系,而且其程度均高于各单独用药组。
结论: (1) 5-Fu和白藜芦醇都可以对细胞产生时间依赖和浓度依赖性抑制,联合用药组在大部分效应范围内都表现为协同作用。(2) 5-Fu和白藜芦醇联合或单独使用都会对A431和TE-1的生长曲线产生抑制作用,联合用药与单独用药相比存在显著性差异。(3)细胞坏死在5-Fu单独作用48h后参与到5-Fu对肿瘤细胞的抑制作用中来,而直到72h白藜芦醇才会引起肿瘤细胞的坏死,二者联用则从24h即有细胞坏死现象的存在,并呈现时间依赖性。
第二部分5-Fu联合白藜芦醇对肿瘤细胞A431、TE-1周期和增殖的影响
目的:研究了5-Fu联合白藜芦醇对A431、TE-1细胞周期和周期增殖相关蛋白的影响。
方法:采用流式细胞术对A431和TE-1的细胞周期进行分析。Western blot检测周期相关蛋白和增殖相关蛋白的表达。
结果: (1)白藜芦醇单独作用24~72h均会引起A431和TE-l细胞的S期数量增多与相应的G1期细胞数量减少,这种结果导致了A431和TE-l细胞的S期阻滞现象,与对照组相比,50μM白藜芦醇对A431作用24h导致A431细胞的S-期比例从29.2±1.4%上升到65.3±2.5% (P <0.01)。70μM白藜芦醇对TE-1作用24h导致TE-1细胞的S期比例从26.2±3.2%增长到89.3±3.7% (P <0.01)。5-Fu单独用药组和联合给药组都会引起A431和TE-1的G1期细胞的少量增加,但是这种增加在24h时与对照组相比没有统计学意义,48h后才与对照组相比出现显著性差异,而且5-Fu组和联合用药组之间在不同时间点都没有显著性差异。(2) Western blot对周期蛋白的分析证实了流式的结果,白藜芦醇导致的S期阻滞使CyclinD1和PCNA蛋白的表达量出现减少。
结论: (1) 5-Fu单独给药或与白藜芦醇联合使用会使A431和TE-1的细胞周期产生G0/G1期阻滞,白藜芦醇单独用药会对A431和TE-1的细胞周期产生S期阻滞,鉴于5-Fu的抗肿瘤作用机制是与S期特异性杀伤有关,我们推测可能是白藜芦醇对细胞周期的影响(将肿瘤细胞大量阻滞于S期)使得A431和TE-1细胞对5-Fu的敏感性增加,从而出现了协同作用。(2)对肿瘤细胞增殖的抑制可能是其抗癌作用的机制之一,CyclinD1和PCNA蛋白表达的下降也证实了这个假设。
第三部分5-氟尿嘧啶联合白藜芦醇对A431、TE-1细胞凋亡的影响
目的:研究5-Fu联合白藜芦醇对A431和TE-1细胞凋亡的影响及相关机制。
方法:采用了普通光镜,DAPI染色和DNA片段化凝胶电泳分析等方法对药物诱导A431和TE-1凋亡的现象进行了观察。使用Annexin V-PI流式分析术对细胞的早期凋亡进行分析。测定细胞内Ca2+变化,western blot检测凋亡相关蛋白,对5-Fu与白藜芦醇联合给药的凋亡机制进行研究。
结果: (1)采用普通光镜和DAPI染色法观察了给药后的A431和TE-1,观察到A431和TE-1细胞都出现了膜不对称性和细胞间连接的消失,以及细胞皱缩等现象。DAPI染色可以看到诸如核固缩,染色质凝集等现象,并可观测到凋亡小体。与单独给药组相比,联合药物组可以观察到A431和TE-1细胞更多的形态学变化。5-Fu和白藜芦醇作用48h后,光镜下可以观察到更多凋亡小体和典型的形态学变化。(2)给药后产生凋亡的情况也可以通过检测DNA梯状条带的变化来进行比较,DNA梯状条带的数量可以反映不同药物的作用情况。5-Fu作用48h后,DNA断裂不明显,只有似有似无的现象出现,但是Res作用48h后,可以清晰地辨别出DNA的梯状条带(DNA Ladder),在联合药物组作用48h后,DNA有序片段化更加明显,条带增多。(3)采用Annexin V-FITC和PI(propidium iodide)双染的方法,用流式细胞仪检测给药后A431和TE-1细胞的早期凋亡现象。与对照组相比,5-Fu和白藜芦醇单独给药组都出现了早期凋亡细胞比例的明显增加(P<0.05)。对照组A431细胞出现了少于6%的自发早期凋亡;而5-Fu (70μmol/L )或白藜芦醇(50μmol/L)各自处理24 h后分别诱发了A431细胞19%和9.3%的早期凋亡。二者同时给药时,出现了凋亡率的大幅增加,约达到34.2%的早期凋亡率(与单药组相比P<0.01)。TE-1对照组细胞出现了低于5%的自发凋亡,5-Fu (150μmol/L )或白藜芦醇(70μmol/L)单独作用24h可分别将TE-1的早期凋亡率提升到11%和7.5%.二者联合给药后TE-1的早期凋亡率增加到24.2%,与单独给药组相比有极显著性差异(P<0.01)。(4)钙离子在细胞内处于信号转导通路的上游,其变化与凋亡的发生有关, 5-Fu或白藜芦醇单独用均可使细胞中钙离子的量增加,与单独给药相比,两药联用使细胞中的钙离子强度与有大量增加,与单独给药组呈极显著性差异(P<0.01)。为了研究药物联合使用的机制是否与凋亡相关蛋白的改变有关,我们用western blot检测了凋亡相关蛋白Bcl-2、Bax、caspase-3和PARP等的表达。结果显示无论是5-Fu或白藜芦醇都可以诱发凋亡抑制蛋白Bcl-2表达水平的明显下降,联合药物组的Bcl-2减少更为明显。而凋亡促进蛋白Bax和p53的表达水平却随着给药出现大量增加。另外,联合用药组的凋亡促进蛋白与凋亡抑制蛋白的比值(Bax/Bcl-2)与单独给药组相比大大增加。药物作用48h后细胞中caspase-3和PARP蛋白酶原的表达量减少,相应活化的caspase-3和PARP片段的表达上调,与单独给药组相比,联合用药组表现出了更为明显的下降和上调(P<0.01)。以上结果提示5-Fu和白藜芦醇联合用药产生协同作用可能与上调凋亡促进蛋白Bax和p53的表达,下调凋亡抑制蛋白Bcl-2的表达有关。
结论: (1) 5-Fu和Res都可以诱导肿瘤细胞A431、TE-1发生不同程度的凋亡。(2) 5-Fu和Res联合使用对肿瘤细胞A431、TE-1的凋亡有协同作用。(3) 5-Fu和Res联合使用对肿瘤细胞A431、TE-1凋亡作用机制可能是通过上调促凋亡基因p53, Bax,下调Bcl-2基因表达激活线粒体通路,从而活化caspase-3发生级联反应进而活化底物PARP而诱导了细胞凋亡的发生。
第四部分5-氟尿嘧啶联合白藜芦醇对小鼠两阶段皮肤癌模型的抗肿瘤作用
目的:研究了5-Fu联合白藜芦醇对小鼠两阶段皮肤癌模型的抗肿瘤作用
方法:小鼠皮肤乳头状瘤模型采用经典的DMBA/TPA两阶段促癌法进行诱发。western-blot检测小鼠皮肤肿瘤中p53、Bax和Bcl-2的表达水平。免疫组织化学检测鼠皮中活化caspase-3的表达。
结果: (1)给药后皮肤中p53、Bax的表达水平上升,Bcl-2的表达水平下降,联合给药组组织中促凋亡蛋白与抗凋亡蛋白的比值(Bax /Bcl-2)明显增加。(2) 5-Fu或白藜芦醇单独给药后均可引起小鼠表皮细胞中caspase-3的活化,联合给药组的活化程度与单独给药组相比有明显的增加。
结论:(1)与单药相比,5-Fu和Res联合给药可以对小鼠表皮癌产生良好的治疗效果,并减轻5-Fu对皮肤的强烈刺激。(2) 5-Fu和Res联合给药可以通过调节p53、Bax和Bcl-2基因的表达以及上调Bax/Bcl-2的比例诱导小鼠表皮肿瘤产生凋亡。(3) 5-Fu和Res联合给药还可以使小鼠表皮细胞中的凋亡执行因子caspase-3大量活化,从而诱导细胞产生凋亡。
结论
通过我们的实验研究可以得出以下结论:1、5-Fu和Res联合给药对人表皮癌A431和人食管鳞癌TE-1的生长抑制有协同作用,而且呈现时间和浓度依赖性,与5-Fu和Res单独用药相比,可以更好的抑制A431和TE-1细胞的生长曲线和克隆形成,并在一定程度上通过增加A431和TE-1的坏死程度起到肿瘤抑制作用。2、Res可以影响A431和TE-1细胞的细胞周期,将大量细胞阻滞在对5-Fu敏感的S期,同时降低PCNA的表达使细胞增殖减少,而5-Fu和Res联合给药也可将部分细胞阻滞在DNA合成前期从而减少肿瘤细胞的增殖。二者抗肿瘤的协同作用可能是上述原因综合作用的结果。3、5-Fu和Res联合给药与单独给药相比能更大程度的影响与凋亡有关的生物转导因子Ca2+的胞内表达和凋亡相关蛋白的表达(上调p53、Bax/Bcl-2比例,活化caspase-3,切割其特定底物PARP),产生更强程度的凋亡。具体表现为Ca2+荧光强度大幅度增加,凋亡小体的增多,DNA有序片段化条带增多,膜磷脂外翻程度增加等。4、5-Fu和Res联合给药在两阶段诱发的皮肤癌整体动物模型上也表现出了协同作用,可以对小鼠皮肤癌进行有效的治疗,并影响小鼠皮肤凋亡相关蛋白的表达(上调p53、Bax/Bcl-2比例,增加胞浆内活化caspase-3比例)。
Cancer, next only to heart disease, is the second leading cause of death in developing countries. The burden of cancer is increasing in economically developing countries as a result of environment pollution,population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and‘‘westernized”diets. Cancer can be treated by surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or other methods.
Chemotherapy plays a main role in the anti-tumor therapy. Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. Ideally, chemotherapeutic agents should target cancer cells specifically without affecting normal cells, by inducing cytostatic or cytotoxic effects. In fact, the lack of specificity, intrinsic or acquired drug resistance, especially the induction of side effects due to high dosage affects the therapeutic effect of many chemotherapeutics. In addition to finding more effective substances of low toxicity, these problems can also be overcome by combining other compounds or drugs with a lower dose treatment agents to get an additive or synergistic therapeutic effect. Using the cell cycle-specific agent to interfere the cell cycle and block the cancer cells at a certain period, will improve the sensitivity of cancer cells to chemotherapeutics. Meanwhlie,it can also decrease side effect by reducing the dose of chemotherapeutics.
Among the chemotherapy drugs, Fluorouracil (5-Fu) is widely used in the treatment of a range of solid tumors as well as for cutaneous diseases. It works by blocking the cancer cells at G1/S phase and induces apoptosis of cancer cells by inhibiting essential biosynthetic processes, or by being incorporated into RNA and DNA as a thymidylate synthase inhibitor to influence their normal function. In the process, 5-Fu also destroys normal cells, causing several side effects. In addition, 5-Fu resistance during the course of treatment has become common nowadays, which is an important cause of failure for cancer therapies. To reduce the adverse effects and maximize the anti-tumor effects of 5-Fu, efforts have been made in combination of 5-Fu with several second-line agents, such as paclitaxel, mitomycin, leucovorin, tegafur, and so on. In addition, some natural phytochemicals have also shown increased effects and decreasing cytotoxicity in combination with 5-Fu.
Resveratrol (Res) is a phytoalexin produced by the enzyme stilbene synthase in response to environmental stress such as vicissitudes in climate, exposure to ozone, sunlight and heavy metals, and infection by pathogenic microorganisms. Numerous studies show that resveratrol has the ability in protecting against many diseases including cancer. Resveratrol can potentially interfere with all three major stages of carcinogenesis (initiation, promotion and progression). The anti-tumor properties of resveratrol have been associated with its antioxidant activity and ability to affect cell growth, inflammation, apoptosis, angiogenesis, and invasion. It has the ability to affect many gene expression via many pathways, these include tumor suppressors p53 and Rb, cell cycle regulators Cyclins and many apoptotic and survival regulators. These in vitro and in vivo studies provide a rational basis in support of using resveratrol in human cancer chemoprevention, in a combinatorial approach with other chemotherapeutic drugs for the highly efficient treatment of drug refractory tumor cells.
Skin cancer, especially the nonmelanoma skin cancers (NMSC), is the most common form of human cancer. It is estimated that over 1 million new cases occur annually. The annual rates of all forms of skin cancer are increasing each year, representing a growing public concern. Esophageal cancer is the sixth leading cause of cancer mortality. China is among the areas of high incidence of esophageal cancer. Given the high incidence of the two cancers, we chose human epidermal cancer cell lines A431and esophageal squamous cell cancer TE-1 to determine the individual and combined effects of resveratrol and 5-Fu on cell growth, cell cycle, and apoptosis. We also investigated apoptotic pathways that may be involved in the interactions of resveratrol and 5-Fu.
PART 1 The Growth Inhibition and Cytotoxicity of 5-Fu Combined with Resveratrol on the of A431 and TE-1 Cell Lines
Objective: To study the effects of 5-Fu combined with Res on the growth and necrosis of A431 and TE-1 cell lines
Methods: The effects of 5-Fu combined with Res on the viability of A431 and TE-1 cell lines were evaluated by MTT assay, growth curves assay and clonogenic assay. The inhibitory effect of combination of the two drugs was analyzed by the method of Chou and Talalay, which is a quantitative measure of the degree of drug interaction between two agents. Cytotoxicity induced by 5-Fu combined with Res on A431 and TE-1 was measured by LDH releasing assay.
Results: (1) MTT assay showed that 5-Fu decreased cell viability in a concentration-dependent and time-dependent manner. Res at higher concentrations (no less than 10μmol/L) also showed an inhibition on cell viability in a concentration-dependent and time-dependent manner. Combination of 5-Fu with Res was more effective than either alone at most concentrations. Decreased the median inhibitory concentration (IC50) to A431 cells from 322.89μM to 71.99μM (5-Fu) and 174.58μM to 37.56μM (Res), respectively. And decreased IC50 to TE-1 cells from 480.83μM to 151.87μM (5-Fu) and 248.64μM to 69.42μM (Res), respectively. The results indicated a synergistic anti-tumor effect of combining 5-Fu and Res. The inhibitory effect of combination of the two drugs was assessed by the MTT assay. After treated with 5-Fu with or without Res for 48 h, the inhibition rates were analyzed by the method of Chou and Talalay. The CI value was determined at effective concentration (EC) of 5-Fu plus Res that caused minimal inhibition (10%, EC10) and maximal inhibition (95%, EC95). 5-Fu and Res acted synergistically when both agents inhibited cell viability by 50% in A431 (CI=0.438) and TE-1(CI=0.595) .The CI values were <1 when the fractions affected were higher than 0.2, which indicated that combination of 5-Fu and Res had a synergistic inhibitory effect on the viability of A431 and TE-1 cells across the broad range of fraction affected (20-95% cell death). (2) The growth rate of cells was reduced by 5-Fu or Res, compared with the control group. When 5-Fu combined with Res, the growth inhibitory effect became even more pronounced and resulted in almost complete inhibition of cell growth throughout the assay period (day 1~day 9). (3) In vitro clonogenic assays indicated that cells could shape clone at day 14. The clone formation rate of A431 and TE-1 was 17.60% and 26.75% respectively in control group, which was 5.42% and 10.13% in Res-treated group and 1.07% and 2.23% in 5-Fu treated group. There is almost no clone exists when exposed to combination of 5-Fu with Res in both A431 and TE-1 cells. The differences among the treated group were statistically significant. (4) LDH activity assay demonstrated that necrosis pathway involved in the 5-Fu and Res induced A431 and TE -1 cell death after 24h and 48h, respectively. 5-Fu plus Res also showed a stronger effect in necrosis compared with 5-Fu and Res used solely in a time-dependent manner.
Conclusions: (1) 5-Fu and Res time-dependently and concentration-dependently suppressed the viability of A431 and TE-1 cells. Combination of two drugs showed a statistically significant increase in inhibition of A431 and TE-1 cells. (2) 5-Fu and Res can reduce the growth rate and colony formation of A431 and TE-1cells. The difference between combination group and group of sole medication was statistically significant. (3) Necrosis pathway was involved in the 5-Fu induced cell death at that time of 48h, and it was not until 72h that necrosis pathway was involved in the Res induced cell death. When used in combination, 5-Fu and Res began to show a significant stronger effect from 24h in a time-dependent manner.
Part 2 The Effects of 5-Fu Combined with Resveratrol on the Cell cycle and protein related to proliferation of A431 and TE-1Cell Lines
Objective: To study the effects of 5-Fu Combined with Res on the Cell cycle and protein related to proliferation of A431 and TE-1Cell Lines.
Methods: Cell-cycle distribution of A431 and TE-1 was determined by fluorescence -activated cell sorting (FACS). Proteins related to cell cycle and proliferation were identified by Western-blot.
Results: (1) Treatment of A431 and TE-1with Res for 24h~72h led to the accumulation of the S-phase cell population, which was accompanied by the reduction of the ratio of G1-cells compared with the control group (P<0.05) . Compared with control group, the S-Phase fraction of A431 increased from 29.2±1.4% to 65.3±2.5% with 50μM Res (P <0.01). Res had the same effect on TE-1 cell-cycle progression with S-phase fraction being increased from 26.2±3.2% to 89.3±3.7% with 70μM Res (P <0.01). Meanwhile, compared to the control group, some of the G1-phase cells were increased slightly in 5-Fu- treated group and the combination group, but the changes were not statistically significant up to 24h treatment. The cells at G0/G1 phase increased significantly while compared with control group after treatment of A431 and TE-1 cells with 5-Fu with or without Res for 48h, but the difference between 5-Fu group and combination group was not statistically significant. (2) Analysis of the protein level of the key cell cycle regulators substantiated the results of the flow cytometry. The cell cycle arrest in S-phase induced by resveratrol coincided with decrease of the levels of CyclinD1 and PCNA.
Conclusions: (1) Treatment of A431 and TE-1 cells with 5-Fu with or without Res increased the population of cells in G0/G1 phase. Treatment of A431 and TE-1 cells with Res increased the population of cells in S-phase. In the light of the mechanism of 5-Fu,we hypothesize that one reason of the synergistic interaction between resveratrol and 5-Fu is that resveratrol enhances the sensitivity of tumor cells to 5-Fu by increasing numbers of S-phase cells. (2) 5-Fu and Res inhibited the proliferation of A431 and TE-1 cells by stagnating the cells at different phase. Decreasing in expression of the CyclinD1 and PCNA confirmed that possibility.
Part 3 Effects of 5-Fu with or without Resveratrol on the Apoptosis of A431 and TE-1 cell Lines
Objective: To study the effects of 5-Fu with or without Res on apoptosis of A431 and TE-1cell lines and the underlying mechanism.
Methods: Apoptosis of A431 and TE-1 was determined by inverted microscope, DAPI staining, gel electrophoresis of DNA fragment analysis and AnnexinV-PI assay. Intracellular Ca2+ concentration was determined and Western-blot (Proteins related to apoptosis) was used to study the underlying mechanisms of 5-Fu with or without Res on the apoptosis of A431 and TE-1 cell lines.
Results: (1) The morphology of A431 and TE-1 cells was observed by invert microscope and fluorescence microscopy following DAPI staining. After treated with 5-Fu with or without Res, the loss of cell membrane asymmetry and attachment and cell shrinkage were found in A431 and TE-1 cells by invert microscope. Other morphologic changes such as nucleuspyknosis, chromatin condensation and apoptotic body formation were found in the cells incubated continuously with 5-Fu with or without resveratrol for 48 h by DAPI staining. Much more fragmented nuclei and typical morphological changes of apoptosis were observed in the cells treated with 5-Fu and resveratrol in combination than that in the cells treated with the agents alone. (2) The treatment-induced apoptosis was also apparent from the changes detected by DNA Ladder. There appeared to be differences in the amount of DNA laddering formed following different treatment. A distinct banding pattern of fragmented DNA was found easily in cells treated with combination of 5-Fu and resveratrol, while smaller amount of fragmented DNA was seen in the cells treated with resveratrol alone and DNA ladders were indistinct following 5-Fu treatment. (3) After treated with 5-Fu, the percent of apoptotic cells were assessed by Annexin V-FITC and propidium iodide staining, followed by flow cytometric analysis. Either 5-Fu or resveratrol alone caused an apparent increase in the percentage of early apoptotic cells compared to control (P<0.05). Untreated A431 cells show less than 6% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (70μmol/L) or resveratrol (50μmol/L) for 24 h induced 19% and 9.3% apoptosis in A431, respectively. When cells incubated with both 5-Fu (70μmol/L) and resveratrol (50μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 34.2% apoptosis rate) (P<0.01 vs sole medication). Untreated TE-1 cells show less than 5% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (150μmol/L) or resveratrol (70μmol/L) for 24 h induced 11% and 7.5% apoptosis in TE-1, respectively. When cells incubated with both 5-Fu (150μmol/L) and resveratrol (70μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 24.2% apoptosis rate) (P<0.01 vs sole medication). (4) The increase of [Ca2+] was involved in the apoptotic induction and at the upstream of the signal pathway. Both 5-Fu and Res alone increased [Ca2+], and combination of the two drugs induced a significant increase of [Ca2+] compared with either drug alone (P<0.01). (5) To determine whether the treatment-induced apoptosis was associated with expression of apoptosis-regulating proteins, the cells were treated for 48 h with 5-Fu and/or resveratrol, and then were subjected to Western blotting. Since the expression of Bcl-2, Bax, caspases-3 and PARP has been reported to play a crucial role in apoptotic response mediated by many chemopreventive agents, the changes in the expression levels of these proteins were observed by Western blot analysis in our study. The results showed that either 5-Fu or resveratrol caused a marked down-regulation of Bcl-2 protein expression, and the combination was even more effective. Expression of pro-apoptotic proteins Bax and p53 was also significantly increased by 5-Fu and/or resveratrol. In addition, the ratio of pro-apoptotic/anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. The expression of caspase-3 and PARP proteins was down-regulated concurrently with the increase of caspase-3 and cleaved PARP expression after 48h treating with 5-Fu in combination with resveratrol, and the expression of caspase-3 and cleaved PARP was synergistically increased by the combination of the two compounds. These findings suggested that the synergistic effect on apoptosis of 5-Fu and resveratrol might be due to enhancing expression of pro-apoptotic Bax and p53 proteins and reducing expression of anti-apoptotic Bcl-2 protein. Besides, active caspase-3 was generated by either 5-Fu or
resveratrol and combination treatment was more effective than either alone in both A431 and TE-1 (P<0.01).
Conclusions: (1) 5-Fu with or without Rescan induced apoptosis of A431 and TE-1 cells (2) 5-Fu combined with Res showed a synergistic effect for inducing apoptosis of A431 and TE-1 cells (3)The synergistic effect in apoptosis of A431 and TE-1 cells could be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways were involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.
Part 4 Therapeutic effects of 5-Fu and Resveratrol alone or in combination on two-stage mouse skin carcinogenesis
Objective: To study the therapeutic effects of 5-Fu with or without Res on mouse skin papilloma chemically induced by DMBA/TPA.
Methods: The mouse skin papilloma model was established by 7,12- dimethylbenz (a) anthracene (DMBA) and 12-O-tetradecanoylphorbol-13- acetate (TPA),which was a classical two-stage cancer model. The expression of p53, Bax and Bcl-2 in A431and TE-1 cells was identified by western-blot method. Expression of actived- caspase-3 in mouse skin was examined by immunohistochemistry.
Results: (1) Treatment with 5-Fu with or without Res increased the pro-apoptotic proteins Bax and p53, and decreased the anti-apoptotic protein Bcl-2 expression. In addition, the ratio of pro-apoptotic / anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. (2) Active caspase-3 was generated by either 5-Fu or resveratrol and 5-Fu in combination with Res was more effective than either alone.
Conclusions: (1) 5-Fu in combination with Res produced visible effects in curing the epidermal carcinoma of mice and reduced skin irritation of 5-Fu. (2) The synergistic effect in apoptosis of mice epidermal carcinoma may be associated with an increasing of p53 and ratio of Bax to Bcl-2.(3)5-Fu in combination with Res may induce apoptosis of mice epidermal carcinoma through activating much more activation caspase-3 than the group of sole medication.
CONCLUSIONS
1 Res in combination with 5-Fu inhibited synergistically growth of A431 and TE-1 cells in a concentration-dependent and time-dependent manner. Necrosis pathway was involved in the cell death induced by 5-Fu and Res in combination.
2 Treatment with Res increased the population of cells in S-phase of A431 and TE-1 cells, which enhanced the sensitivity of tumor cells to 5-Fu. Treatment of 5-Fu with or without Res increased the population of cells in G0/G1 phase of A431 and TE-1 cells.
3 Res combined with 5-Fu showed a synergistic effect on apoptosis of A431 and TE-1 cells. The increase of [Ca2+]i induced by 5-Fu combined with Res was involved in the apoptotic induction and at the upstream of the signal pathway. The synergistic effect on apoptosis of A431 and TE-1 cells may be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways are involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.
4 There was an increase of the pro-apoptotic proteins Bax and p53, and decrease of the anti-apoptotic protein Bcl-2 expression after treatment with 5-Fu with or without Res in the tumor-bearing mice model. In addition, the ratio of pro-apoptotic/ anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. Active caspase-3 in the cells of mice skin was generated by either 5-Fu or resveratrol and 5-Fu in combination resveratrol was more effective than either alone.
5-氟尿嘧啶(5-Fluorouracil,5-Fu)是常见的化疗药物,常用于实体瘤的治疗,可以通过将细胞周期阻滞于G1/S期并干扰细胞基本生物合成过程诱导肿瘤细胞的凋亡从而起到抑制肿瘤生长的作用。5-Fu还可以作为胸苷酸合成酶(TS)的抑制剂掺入DNA和RNA结构中使肿瘤细胞无法正常合成DNA,影响其正常功能。鉴于5-Fu的作用机理,它在治疗过程中也会影响正常细胞的功能而产生毒副作用,另外,目前比较常见的5-Fu使用中产生的抗药性也成了阻碍其发挥疗效的障碍。现在已有很多二线药物如紫杉醇、丝裂霉素、四氢叶酸、喃氟啶等开始与5-Fu联合使用,其目的是在保持其抗肿瘤作用最大化的同时减少5-Fu的毒副作用。除了这些药物以外,一些天然植物素也能达到对5-Fu增效减毒的效果。
白藜芦醇(Resveratrol,Res)是一种广泛存在于自然界中的植物抗毒素,是植物对抗外界环境压力如气候变化,暴露于更多的臭氧环境中,日晒和重金属刺激以及病原微生物感染等情况时所产生的。大量研究结果显示白藜芦醇可以对抗多种疾病甚至癌症。研究发现白藜芦醇对肿瘤的起始、促进和发展三个阶段均有抑制作用,可作为天然的肿瘤化学预防剂。其抗肿瘤作用可能与其影响细胞生长、抗炎、诱导细胞凋亡和抗侵袭能力有关。白藜芦醇可以影响体内多种基因表达,其中包括肿瘤抑制因子p53和Rb,细胞周期调节蛋白Cyclin家族以及多种凋亡调节因子。诸多的体内外研究证实白藜芦醇可以作为抗肿瘤药物或与其他化疗药物联合使用治疗一些有抗药性的癌症。
皮肤癌尤其是非黑色素性皮癌(NMSC)是世界上发病数量最多的人类肿瘤,近年来的发病率日益升高,据估计每年新增病例超过一百万。如此高的年发病率使其越发受到公众的关注。食管癌的死亡率很高,在世界恶性肿瘤死亡率中占据第六位,而我国正是是食管癌的高发地区。鉴于这两种肿瘤的高发性,本实验中选取食管鳞状细胞癌株TE-1、人表皮癌A431细胞株作为研究对象来考察5-Fu与白藜芦醇联合在细胞生长、细胞周期和凋亡方面的作用,并对可能与联合作用有关的凋亡通路进行了研究。实验共分为四部分:
第一部分5-氟尿嘧啶联合白藜芦醇对肿瘤细胞A431、TE-1生长的抑制和细胞毒作用
目的:研究5-Fu联合白藜芦醇对肿瘤细胞A431、TE-1生长的抑制作用和致坏死能力
方法:本部分实验中采用MTT法、生长曲线实验、平板克隆实验对5-Fu和白藜芦醇的联合用药进行评价,联合用药的效果采用Chou- Talalay法进行定量评价分析。细胞毒作用采用LDH溢出实验进行评价。
结果: (1) MTT实验显示5-Fu有抑制肿瘤细胞的生长的能力,而且这种作用呈时间与浓度依赖性。而白藜芦醇在较高浓度(不低于10μmol/L)才能够产生抑制作用。二者联合使用时在很大的浓度范围内都可以产生协同作用(白藜芦醇不低于10μmol/L)。联合用药可以大大降低5-Fu或白藜芦醇对A431的中效浓度(5-Fu的IC50从单独使用时的322.89μM降至71.99μM;白藜芦醇的IC50从174.58μM降至37.56μM)。对TE-1联合用药可以分别将5-Fu的IC50从480.83μM降至151.87μM ,而白藜芦醇的IC50可从248.64μM降至69.42μM。该结果显示5-Fu和白藜芦醇联合使用可在很大程度上减少各药物的用量。
采用Chou -Talalay法对5-Fu和白藜芦醇的联合作用48h的效应进行评价,当两药联合使用对肿瘤的的抑制效应达到50%时,A431细胞的联合指数CI为0.438,而对TE-1的联合指数CI为0.595。二者联合在高于0.2的效应范围内CI值均不高于1,说明在一个很大的效应范围内(0.2-0.95)5-Fu联合白藜芦醇都能产生协同作用。(2)生长曲线实验结果显示,当5-Fu和白藜芦醇单独使用时都能对肿瘤细胞的生长起到抑制作用,当二者联合使用时对肿瘤细胞的生长曲线抑制更为明显,与对照组和各单药组相比均存在显著性差异。(3)平板克隆实验结果显示14天后A431和TE-1各自有17.60%和26.75%的克隆形成率,白藜芦醇单独使用可以使两种细胞的克隆形成率分别降至A431 5.42%,TE-1 10.13%。而5-Fu单独使用的克隆形成率分别为A431 1.07% ,TE-1 2.23%。两种细胞的联合用药组则几乎没有克隆形成,各组间在统计学上表现为极显著性差异。(4) LDH活性实验显示了5-Fu和白藜芦醇单独使用所导致的A431和TE-1细胞坏死分别出现在48h和72h后,而联合给药组在24h即出现LDH的溢出,说明联合用药对肿瘤细胞的抑制作用可能与坏死有一定关系,而且其程度均高于各单独用药组。
结论: (1) 5-Fu和白藜芦醇都可以对细胞产生时间依赖和浓度依赖性抑制,联合用药组在大部分效应范围内都表现为协同作用。(2) 5-Fu和白藜芦醇联合或单独使用都会对A431和TE-1的生长曲线产生抑制作用,联合用药与单独用药相比存在显著性差异。(3)细胞坏死在5-Fu单独作用48h后参与到5-Fu对肿瘤细胞的抑制作用中来,而直到72h白藜芦醇才会引起肿瘤细胞的坏死,二者联用则从24h即有细胞坏死现象的存在,并呈现时间依赖性。
第二部分5-Fu联合白藜芦醇对肿瘤细胞A431、TE-1周期和增殖的影响
目的:研究了5-Fu联合白藜芦醇对A431、TE-1细胞周期和周期增殖相关蛋白的影响。
方法:采用流式细胞术对A431和TE-1的细胞周期进行分析。Western blot检测周期相关蛋白和增殖相关蛋白的表达。
结果: (1)白藜芦醇单独作用24~72h均会引起A431和TE-l细胞的S期数量增多与相应的G1期细胞数量减少,这种结果导致了A431和TE-l细胞的S期阻滞现象,与对照组相比,50μM白藜芦醇对A431作用24h导致A431细胞的S-期比例从29.2±1.4%上升到65.3±2.5% (P <0.01)。70μM白藜芦醇对TE-1作用24h导致TE-1细胞的S期比例从26.2±3.2%增长到89.3±3.7% (P <0.01)。5-Fu单独用药组和联合给药组都会引起A431和TE-1的G1期细胞的少量增加,但是这种增加在24h时与对照组相比没有统计学意义,48h后才与对照组相比出现显著性差异,而且5-Fu组和联合用药组之间在不同时间点都没有显著性差异。(2) Western blot对周期蛋白的分析证实了流式的结果,白藜芦醇导致的S期阻滞使CyclinD1和PCNA蛋白的表达量出现减少。
结论: (1) 5-Fu单独给药或与白藜芦醇联合使用会使A431和TE-1的细胞周期产生G0/G1期阻滞,白藜芦醇单独用药会对A431和TE-1的细胞周期产生S期阻滞,鉴于5-Fu的抗肿瘤作用机制是与S期特异性杀伤有关,我们推测可能是白藜芦醇对细胞周期的影响(将肿瘤细胞大量阻滞于S期)使得A431和TE-1细胞对5-Fu的敏感性增加,从而出现了协同作用。(2)对肿瘤细胞增殖的抑制可能是其抗癌作用的机制之一,CyclinD1和PCNA蛋白表达的下降也证实了这个假设。
第三部分5-氟尿嘧啶联合白藜芦醇对A431、TE-1细胞凋亡的影响
目的:研究5-Fu联合白藜芦醇对A431和TE-1细胞凋亡的影响及相关机制。
方法:采用了普通光镜,DAPI染色和DNA片段化凝胶电泳分析等方法对药物诱导A431和TE-1凋亡的现象进行了观察。使用Annexin V-PI流式分析术对细胞的早期凋亡进行分析。测定细胞内Ca2+变化,western blot检测凋亡相关蛋白,对5-Fu与白藜芦醇联合给药的凋亡机制进行研究。
结果: (1)采用普通光镜和DAPI染色法观察了给药后的A431和TE-1,观察到A431和TE-1细胞都出现了膜不对称性和细胞间连接的消失,以及细胞皱缩等现象。DAPI染色可以看到诸如核固缩,染色质凝集等现象,并可观测到凋亡小体。与单独给药组相比,联合药物组可以观察到A431和TE-1细胞更多的形态学变化。5-Fu和白藜芦醇作用48h后,光镜下可以观察到更多凋亡小体和典型的形态学变化。(2)给药后产生凋亡的情况也可以通过检测DNA梯状条带的变化来进行比较,DNA梯状条带的数量可以反映不同药物的作用情况。5-Fu作用48h后,DNA断裂不明显,只有似有似无的现象出现,但是Res作用48h后,可以清晰地辨别出DNA的梯状条带(DNA Ladder),在联合药物组作用48h后,DNA有序片段化更加明显,条带增多。(3)采用Annexin V-FITC和PI(propidium iodide)双染的方法,用流式细胞仪检测给药后A431和TE-1细胞的早期凋亡现象。与对照组相比,5-Fu和白藜芦醇单独给药组都出现了早期凋亡细胞比例的明显增加(P<0.05)。对照组A431细胞出现了少于6%的自发早期凋亡;而5-Fu (70μmol/L )或白藜芦醇(50μmol/L)各自处理24 h后分别诱发了A431细胞19%和9.3%的早期凋亡。二者同时给药时,出现了凋亡率的大幅增加,约达到34.2%的早期凋亡率(与单药组相比P<0.01)。TE-1对照组细胞出现了低于5%的自发凋亡,5-Fu (150μmol/L )或白藜芦醇(70μmol/L)单独作用24h可分别将TE-1的早期凋亡率提升到11%和7.5%.二者联合给药后TE-1的早期凋亡率增加到24.2%,与单独给药组相比有极显著性差异(P<0.01)。(4)钙离子在细胞内处于信号转导通路的上游,其变化与凋亡的发生有关, 5-Fu或白藜芦醇单独用均可使细胞中钙离子的量增加,与单独给药相比,两药联用使细胞中的钙离子强度与有大量增加,与单独给药组呈极显著性差异(P<0.01)。为了研究药物联合使用的机制是否与凋亡相关蛋白的改变有关,我们用western blot检测了凋亡相关蛋白Bcl-2、Bax、caspase-3和PARP等的表达。结果显示无论是5-Fu或白藜芦醇都可以诱发凋亡抑制蛋白Bcl-2表达水平的明显下降,联合药物组的Bcl-2减少更为明显。而凋亡促进蛋白Bax和p53的表达水平却随着给药出现大量增加。另外,联合用药组的凋亡促进蛋白与凋亡抑制蛋白的比值(Bax/Bcl-2)与单独给药组相比大大增加。药物作用48h后细胞中caspase-3和PARP蛋白酶原的表达量减少,相应活化的caspase-3和PARP片段的表达上调,与单独给药组相比,联合用药组表现出了更为明显的下降和上调(P<0.01)。以上结果提示5-Fu和白藜芦醇联合用药产生协同作用可能与上调凋亡促进蛋白Bax和p53的表达,下调凋亡抑制蛋白Bcl-2的表达有关。
结论: (1) 5-Fu和Res都可以诱导肿瘤细胞A431、TE-1发生不同程度的凋亡。(2) 5-Fu和Res联合使用对肿瘤细胞A431、TE-1的凋亡有协同作用。(3) 5-Fu和Res联合使用对肿瘤细胞A431、TE-1凋亡作用机制可能是通过上调促凋亡基因p53, Bax,下调Bcl-2基因表达激活线粒体通路,从而活化caspase-3发生级联反应进而活化底物PARP而诱导了细胞凋亡的发生。
第四部分5-氟尿嘧啶联合白藜芦醇对小鼠两阶段皮肤癌模型的抗肿瘤作用
目的:研究了5-Fu联合白藜芦醇对小鼠两阶段皮肤癌模型的抗肿瘤作用
方法:小鼠皮肤乳头状瘤模型采用经典的DMBA/TPA两阶段促癌法进行诱发。western-blot检测小鼠皮肤肿瘤中p53、Bax和Bcl-2的表达水平。免疫组织化学检测鼠皮中活化caspase-3的表达。
结果: (1)给药后皮肤中p53、Bax的表达水平上升,Bcl-2的表达水平下降,联合给药组组织中促凋亡蛋白与抗凋亡蛋白的比值(Bax /Bcl-2)明显增加。(2) 5-Fu或白藜芦醇单独给药后均可引起小鼠表皮细胞中caspase-3的活化,联合给药组的活化程度与单独给药组相比有明显的增加。
结论:(1)与单药相比,5-Fu和Res联合给药可以对小鼠表皮癌产生良好的治疗效果,并减轻5-Fu对皮肤的强烈刺激。(2) 5-Fu和Res联合给药可以通过调节p53、Bax和Bcl-2基因的表达以及上调Bax/Bcl-2的比例诱导小鼠表皮肿瘤产生凋亡。(3) 5-Fu和Res联合给药还可以使小鼠表皮细胞中的凋亡执行因子caspase-3大量活化,从而诱导细胞产生凋亡。
结论
通过我们的实验研究可以得出以下结论:1、5-Fu和Res联合给药对人表皮癌A431和人食管鳞癌TE-1的生长抑制有协同作用,而且呈现时间和浓度依赖性,与5-Fu和Res单独用药相比,可以更好的抑制A431和TE-1细胞的生长曲线和克隆形成,并在一定程度上通过增加A431和TE-1的坏死程度起到肿瘤抑制作用。2、Res可以影响A431和TE-1细胞的细胞周期,将大量细胞阻滞在对5-Fu敏感的S期,同时降低PCNA的表达使细胞增殖减少,而5-Fu和Res联合给药也可将部分细胞阻滞在DNA合成前期从而减少肿瘤细胞的增殖。二者抗肿瘤的协同作用可能是上述原因综合作用的结果。3、5-Fu和Res联合给药与单独给药相比能更大程度的影响与凋亡有关的生物转导因子Ca2+的胞内表达和凋亡相关蛋白的表达(上调p53、Bax/Bcl-2比例,活化caspase-3,切割其特定底物PARP),产生更强程度的凋亡。具体表现为Ca2+荧光强度大幅度增加,凋亡小体的增多,DNA有序片段化条带增多,膜磷脂外翻程度增加等。4、5-Fu和Res联合给药在两阶段诱发的皮肤癌整体动物模型上也表现出了协同作用,可以对小鼠皮肤癌进行有效的治疗,并影响小鼠皮肤凋亡相关蛋白的表达(上调p53、Bax/Bcl-2比例,增加胞浆内活化caspase-3比例)。
Cancer, next only to heart disease, is the second leading cause of death in developing countries. The burden of cancer is increasing in economically developing countries as a result of environment pollution,population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and‘‘westernized”diets. Cancer can be treated by surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or other methods.
Chemotherapy plays a main role in the anti-tumor therapy. Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. Ideally, chemotherapeutic agents should target cancer cells specifically without affecting normal cells, by inducing cytostatic or cytotoxic effects. In fact, the lack of specificity, intrinsic or acquired drug resistance, especially the induction of side effects due to high dosage affects the therapeutic effect of many chemotherapeutics. In addition to finding more effective substances of low toxicity, these problems can also be overcome by combining other compounds or drugs with a lower dose treatment agents to get an additive or synergistic therapeutic effect. Using the cell cycle-specific agent to interfere the cell cycle and block the cancer cells at a certain period, will improve the sensitivity of cancer cells to chemotherapeutics. Meanwhlie,it can also decrease side effect by reducing the dose of chemotherapeutics.
Among the chemotherapy drugs, Fluorouracil (5-Fu) is widely used in the treatment of a range of solid tumors as well as for cutaneous diseases. It works by blocking the cancer cells at G1/S phase and induces apoptosis of cancer cells by inhibiting essential biosynthetic processes, or by being incorporated into RNA and DNA as a thymidylate synthase inhibitor to influence their normal function. In the process, 5-Fu also destroys normal cells, causing several side effects. In addition, 5-Fu resistance during the course of treatment has become common nowadays, which is an important cause of failure for cancer therapies. To reduce the adverse effects and maximize the anti-tumor effects of 5-Fu, efforts have been made in combination of 5-Fu with several second-line agents, such as paclitaxel, mitomycin, leucovorin, tegafur, and so on. In addition, some natural phytochemicals have also shown increased effects and decreasing cytotoxicity in combination with 5-Fu.
Resveratrol (Res) is a phytoalexin produced by the enzyme stilbene synthase in response to environmental stress such as vicissitudes in climate, exposure to ozone, sunlight and heavy metals, and infection by pathogenic microorganisms. Numerous studies show that resveratrol has the ability in protecting against many diseases including cancer. Resveratrol can potentially interfere with all three major stages of carcinogenesis (initiation, promotion and progression). The anti-tumor properties of resveratrol have been associated with its antioxidant activity and ability to affect cell growth, inflammation, apoptosis, angiogenesis, and invasion. It has the ability to affect many gene expression via many pathways, these include tumor suppressors p53 and Rb, cell cycle regulators Cyclins and many apoptotic and survival regulators. These in vitro and in vivo studies provide a rational basis in support of using resveratrol in human cancer chemoprevention, in a combinatorial approach with other chemotherapeutic drugs for the highly efficient treatment of drug refractory tumor cells.
Skin cancer, especially the nonmelanoma skin cancers (NMSC), is the most common form of human cancer. It is estimated that over 1 million new cases occur annually. The annual rates of all forms of skin cancer are increasing each year, representing a growing public concern. Esophageal cancer is the sixth leading cause of cancer mortality. China is among the areas of high incidence of esophageal cancer. Given the high incidence of the two cancers, we chose human epidermal cancer cell lines A431and esophageal squamous cell cancer TE-1 to determine the individual and combined effects of resveratrol and 5-Fu on cell growth, cell cycle, and apoptosis. We also investigated apoptotic pathways that may be involved in the interactions of resveratrol and 5-Fu.
PART 1 The Growth Inhibition and Cytotoxicity of 5-Fu Combined with Resveratrol on the of A431 and TE-1 Cell Lines
Objective: To study the effects of 5-Fu combined with Res on the growth and necrosis of A431 and TE-1 cell lines
Methods: The effects of 5-Fu combined with Res on the viability of A431 and TE-1 cell lines were evaluated by MTT assay, growth curves assay and clonogenic assay. The inhibitory effect of combination of the two drugs was analyzed by the method of Chou and Talalay, which is a quantitative measure of the degree of drug interaction between two agents. Cytotoxicity induced by 5-Fu combined with Res on A431 and TE-1 was measured by LDH releasing assay.
Results: (1) MTT assay showed that 5-Fu decreased cell viability in a concentration-dependent and time-dependent manner. Res at higher concentrations (no less than 10μmol/L) also showed an inhibition on cell viability in a concentration-dependent and time-dependent manner. Combination of 5-Fu with Res was more effective than either alone at most concentrations. Decreased the median inhibitory concentration (IC50) to A431 cells from 322.89μM to 71.99μM (5-Fu) and 174.58μM to 37.56μM (Res), respectively. And decreased IC50 to TE-1 cells from 480.83μM to 151.87μM (5-Fu) and 248.64μM to 69.42μM (Res), respectively. The results indicated a synergistic anti-tumor effect of combining 5-Fu and Res. The inhibitory effect of combination of the two drugs was assessed by the MTT assay. After treated with 5-Fu with or without Res for 48 h, the inhibition rates were analyzed by the method of Chou and Talalay. The CI value was determined at effective concentration (EC) of 5-Fu plus Res that caused minimal inhibition (10%, EC10) and maximal inhibition (95%, EC95). 5-Fu and Res acted synergistically when both agents inhibited cell viability by 50% in A431 (CI=0.438) and TE-1(CI=0.595) .The CI values were <1 when the fractions affected were higher than 0.2, which indicated that combination of 5-Fu and Res had a synergistic inhibitory effect on the viability of A431 and TE-1 cells across the broad range of fraction affected (20-95% cell death). (2) The growth rate of cells was reduced by 5-Fu or Res, compared with the control group. When 5-Fu combined with Res, the growth inhibitory effect became even more pronounced and resulted in almost complete inhibition of cell growth throughout the assay period (day 1~day 9). (3) In vitro clonogenic assays indicated that cells could shape clone at day 14. The clone formation rate of A431 and TE-1 was 17.60% and 26.75% respectively in control group, which was 5.42% and 10.13% in Res-treated group and 1.07% and 2.23% in 5-Fu treated group. There is almost no clone exists when exposed to combination of 5-Fu with Res in both A431 and TE-1 cells. The differences among the treated group were statistically significant. (4) LDH activity assay demonstrated that necrosis pathway involved in the 5-Fu and Res induced A431 and TE -1 cell death after 24h and 48h, respectively. 5-Fu plus Res also showed a stronger effect in necrosis compared with 5-Fu and Res used solely in a time-dependent manner.
Conclusions: (1) 5-Fu and Res time-dependently and concentration-dependently suppressed the viability of A431 and TE-1 cells. Combination of two drugs showed a statistically significant increase in inhibition of A431 and TE-1 cells. (2) 5-Fu and Res can reduce the growth rate and colony formation of A431 and TE-1cells. The difference between combination group and group of sole medication was statistically significant. (3) Necrosis pathway was involved in the 5-Fu induced cell death at that time of 48h, and it was not until 72h that necrosis pathway was involved in the Res induced cell death. When used in combination, 5-Fu and Res began to show a significant stronger effect from 24h in a time-dependent manner.
Part 2 The Effects of 5-Fu Combined with Resveratrol on the Cell cycle and protein related to proliferation of A431 and TE-1Cell Lines
Objective: To study the effects of 5-Fu Combined with Res on the Cell cycle and protein related to proliferation of A431 and TE-1Cell Lines.
Methods: Cell-cycle distribution of A431 and TE-1 was determined by fluorescence -activated cell sorting (FACS). Proteins related to cell cycle and proliferation were identified by Western-blot.
Results: (1) Treatment of A431 and TE-1with Res for 24h~72h led to the accumulation of the S-phase cell population, which was accompanied by the reduction of the ratio of G1-cells compared with the control group (P<0.05) . Compared with control group, the S-Phase fraction of A431 increased from 29.2±1.4% to 65.3±2.5% with 50μM Res (P <0.01). Res had the same effect on TE-1 cell-cycle progression with S-phase fraction being increased from 26.2±3.2% to 89.3±3.7% with 70μM Res (P <0.01). Meanwhile, compared to the control group, some of the G1-phase cells were increased slightly in 5-Fu- treated group and the combination group, but the changes were not statistically significant up to 24h treatment. The cells at G0/G1 phase increased significantly while compared with control group after treatment of A431 and TE-1 cells with 5-Fu with or without Res for 48h, but the difference between 5-Fu group and combination group was not statistically significant. (2) Analysis of the protein level of the key cell cycle regulators substantiated the results of the flow cytometry. The cell cycle arrest in S-phase induced by resveratrol coincided with decrease of the levels of CyclinD1 and PCNA.
Conclusions: (1) Treatment of A431 and TE-1 cells with 5-Fu with or without Res increased the population of cells in G0/G1 phase. Treatment of A431 and TE-1 cells with Res increased the population of cells in S-phase. In the light of the mechanism of 5-Fu,we hypothesize that one reason of the synergistic interaction between resveratrol and 5-Fu is that resveratrol enhances the sensitivity of tumor cells to 5-Fu by increasing numbers of S-phase cells. (2) 5-Fu and Res inhibited the proliferation of A431 and TE-1 cells by stagnating the cells at different phase. Decreasing in expression of the CyclinD1 and PCNA confirmed that possibility.
Part 3 Effects of 5-Fu with or without Resveratrol on the Apoptosis of A431 and TE-1 cell Lines
Objective: To study the effects of 5-Fu with or without Res on apoptosis of A431 and TE-1cell lines and the underlying mechanism.
Methods: Apoptosis of A431 and TE-1 was determined by inverted microscope, DAPI staining, gel electrophoresis of DNA fragment analysis and AnnexinV-PI assay. Intracellular Ca2+ concentration was determined and Western-blot (Proteins related to apoptosis) was used to study the underlying mechanisms of 5-Fu with or without Res on the apoptosis of A431 and TE-1 cell lines.
Results: (1) The morphology of A431 and TE-1 cells was observed by invert microscope and fluorescence microscopy following DAPI staining. After treated with 5-Fu with or without Res, the loss of cell membrane asymmetry and attachment and cell shrinkage were found in A431 and TE-1 cells by invert microscope. Other morphologic changes such as nucleuspyknosis, chromatin condensation and apoptotic body formation were found in the cells incubated continuously with 5-Fu with or without resveratrol for 48 h by DAPI staining. Much more fragmented nuclei and typical morphological changes of apoptosis were observed in the cells treated with 5-Fu and resveratrol in combination than that in the cells treated with the agents alone. (2) The treatment-induced apoptosis was also apparent from the changes detected by DNA Ladder. There appeared to be differences in the amount of DNA laddering formed following different treatment. A distinct banding pattern of fragmented DNA was found easily in cells treated with combination of 5-Fu and resveratrol, while smaller amount of fragmented DNA was seen in the cells treated with resveratrol alone and DNA ladders were indistinct following 5-Fu treatment. (3) After treated with 5-Fu, the percent of apoptotic cells were assessed by Annexin V-FITC and propidium iodide staining, followed by flow cytometric analysis. Either 5-Fu or resveratrol alone caused an apparent increase in the percentage of early apoptotic cells compared to control (P<0.05). Untreated A431 cells show less than 6% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (70μmol/L) or resveratrol (50μmol/L) for 24 h induced 19% and 9.3% apoptosis in A431, respectively. When cells incubated with both 5-Fu (70μmol/L) and resveratrol (50μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 34.2% apoptosis rate) (P<0.01 vs sole medication). Untreated TE-1 cells show less than 5% spontaneous apoptosis (early apoptotic cells); however, incubation with 5-Fu (150μmol/L) or resveratrol (70μmol/L) for 24 h induced 11% and 7.5% apoptosis in TE-1, respectively. When cells incubated with both 5-Fu (150μmol/L) and resveratrol (70μmol/L), there was a significant increase in the percentage of cells undergoing apoptosis (approximately 24.2% apoptosis rate) (P<0.01 vs sole medication). (4) The increase of [Ca2+] was involved in the apoptotic induction and at the upstream of the signal pathway. Both 5-Fu and Res alone increased [Ca2+], and combination of the two drugs induced a significant increase of [Ca2+] compared with either drug alone (P<0.01). (5) To determine whether the treatment-induced apoptosis was associated with expression of apoptosis-regulating proteins, the cells were treated for 48 h with 5-Fu and/or resveratrol, and then were subjected to Western blotting. Since the expression of Bcl-2, Bax, caspases-3 and PARP has been reported to play a crucial role in apoptotic response mediated by many chemopreventive agents, the changes in the expression levels of these proteins were observed by Western blot analysis in our study. The results showed that either 5-Fu or resveratrol caused a marked down-regulation of Bcl-2 protein expression, and the combination was even more effective. Expression of pro-apoptotic proteins Bax and p53 was also significantly increased by 5-Fu and/or resveratrol. In addition, the ratio of pro-apoptotic/anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. The expression of caspase-3 and PARP proteins was down-regulated concurrently with the increase of caspase-3 and cleaved PARP expression after 48h treating with 5-Fu in combination with resveratrol, and the expression of caspase-3 and cleaved PARP was synergistically increased by the combination of the two compounds. These findings suggested that the synergistic effect on apoptosis of 5-Fu and resveratrol might be due to enhancing expression of pro-apoptotic Bax and p53 proteins and reducing expression of anti-apoptotic Bcl-2 protein. Besides, active caspase-3 was generated by either 5-Fu or
resveratrol and combination treatment was more effective than either alone in both A431 and TE-1 (P<0.01).
Conclusions: (1) 5-Fu with or without Rescan induced apoptosis of A431 and TE-1 cells (2) 5-Fu combined with Res showed a synergistic effect for inducing apoptosis of A431 and TE-1 cells (3)The synergistic effect in apoptosis of A431 and TE-1 cells could be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways were involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.
Part 4 Therapeutic effects of 5-Fu and Resveratrol alone or in combination on two-stage mouse skin carcinogenesis
Objective: To study the therapeutic effects of 5-Fu with or without Res on mouse skin papilloma chemically induced by DMBA/TPA.
Methods: The mouse skin papilloma model was established by 7,12- dimethylbenz (a) anthracene (DMBA) and 12-O-tetradecanoylphorbol-13- acetate (TPA),which was a classical two-stage cancer model. The expression of p53, Bax and Bcl-2 in A431and TE-1 cells was identified by western-blot method. Expression of actived- caspase-3 in mouse skin was examined by immunohistochemistry.
Results: (1) Treatment with 5-Fu with or without Res increased the pro-apoptotic proteins Bax and p53, and decreased the anti-apoptotic protein Bcl-2 expression. In addition, the ratio of pro-apoptotic / anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. (2) Active caspase-3 was generated by either 5-Fu or resveratrol and 5-Fu in combination with Res was more effective than either alone.
Conclusions: (1) 5-Fu in combination with Res produced visible effects in curing the epidermal carcinoma of mice and reduced skin irritation of 5-Fu. (2) The synergistic effect in apoptosis of mice epidermal carcinoma may be associated with an increasing of p53 and ratio of Bax to Bcl-2.(3)5-Fu in combination with Res may induce apoptosis of mice epidermal carcinoma through activating much more activation caspase-3 than the group of sole medication.
CONCLUSIONS
1 Res in combination with 5-Fu inhibited synergistically growth of A431 and TE-1 cells in a concentration-dependent and time-dependent manner. Necrosis pathway was involved in the cell death induced by 5-Fu and Res in combination.
2 Treatment with Res increased the population of cells in S-phase of A431 and TE-1 cells, which enhanced the sensitivity of tumor cells to 5-Fu. Treatment of 5-Fu with or without Res increased the population of cells in G0/G1 phase of A431 and TE-1 cells.
3 Res combined with 5-Fu showed a synergistic effect on apoptosis of A431 and TE-1 cells. The increase of [Ca2+]i induced by 5-Fu combined with Res was involved in the apoptotic induction and at the upstream of the signal pathway. The synergistic effect on apoptosis of A431 and TE-1 cells may be associated with an increasing of p53 and ratio of Bax to Bcl-2, followed by activation of caspase-3 and PARP, suggesting that mitochondrial pathways are involved in 5-Fu/resveratrol-induced apoptosis of cancer cells.
4 There was an increase of the pro-apoptotic proteins Bax and p53, and decrease of the anti-apoptotic protein Bcl-2 expression after treatment with 5-Fu with or without Res in the tumor-bearing mice model. In addition, the ratio of pro-apoptotic/ anti-apoptotic factor (Bax /Bcl-2) was significantly enhanced by combination treatment with 5-Fu and resveratrol. Active caspase-3 in the cells of mice skin was generated by either 5-Fu or resveratrol and 5-Fu in combination resveratrol was more effective than either alone.
引文
1 Mathers, Colin. The Global Burden of Disease: 2004 Update [R]. Geneva: World Health Organization; 2008
2 Ahmedin Jemal,Freddie Bray,Melissa M., et al Global cancer statistics [J].CA: A Cancer Journal for Clinicians, 2011, 61(2): 69-90
3中华人民共和国卫生部. 2010中国卫生统计年鉴[M].北京:中国协和医科大学出版社, 2010: 268, 307
4吕圭源.药理学[M].北京:中国中医药出版社, 2003: 400-404
5 American Cancer Society. Cancer Facts & Figures 2010 [R]. Atlanta: American Cancer Society; 2010: 1
6 Jemal A, Siegel R, Xu TQ, et al Cancer Statistics [J]. CA Cancer J Clin, 2010, 60 (5): 277-300
7 Lablanca R, Pessi A, Facendola G, et al Modulated 5-fluorouracil (5-FU) regimens in advanced colorectal cancer: a critical review of comparative studies [J]. Eur J Cancer, 1996, 32( l5): 7-12
8 Rajendran D, Senthil S, Dhanaraj SA, et al Comparative evaluation of targeting efficiency of charged and neutral liposomes of 5 -FU [J]. Drug development and industrial pharmacy, 1997, 23(11): 1099-1104
9 Levi F, Zidani R, Misset JL, et al Randomised multicentre trial of chemotherapy with oxaliplatin [J]. Drug Dev Ind Pharm, 1997, 23(11): 1099-1102
10 M Lopez-Velez, F Martinez-Martinez, C Del Valle-Ribes. The study of phenolic compounds as natural antioxidants in wine [J]. Crit Rev Food Sci Nutr, 2003, 43(3): 233-244
11 Bertelli A, Gozzini A, Giovannini L. Plasma and tissue resveratrol concentrations and pharmacological activity [J]. Drugs Exp Clin Res, 1998; 24(3): 133-138
12 Cho DI, Koo NY, Chung WJ, et al Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action [J]. Life Sci, 2002, 71(17): 2071-2082
13 Pace-Asciak CR, Hahn S, Diamandis EP, et al The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease [J]. Clin Chim Acta. 1995; 235(2): 207-19
14 Martín AR, Villegas I, La Casa C, et al Resveratrol, a polyphenol found in grapes, suppresses oxidative damage and stimulates apoptosis during early colonic inflammation in rats [J]. Biochem Pharmacol, 2004, 67(7): 399-410
15 Jang M, Cai L, Udeani GO, et al Cancer chemopreventive activity of resveratrol, a natural product derived from grapes [J]. Science, 1997, 275(5297): 218-220
16 Adhami VM, Afaq F, Ahmad N. Involvement of the retinoblastoma (pRb)-E2F/DP pathway during antiproliferative effects of resveratrol in human epidermoid carcinoma (A431) cells [J]. Biochem. Biophys. Res. Commun, 2001, 288(3): 579-585
17 Afaq F, Adhami VM, Ahmad N, et al Botanical antioxidants for chemoprevention of photocarcinogenesis [J]. Front. Biosci, 2002, 7:784-792
18 Daiki M, Yutaka M, Kazumi Y. Resveratrol inhibits hepatoma cell invasion by suppressing gene expression of hepatocyte growth factor via its reactive oxygen species-scavenging property [J]. Clin. Exp. Metastasis , 2004, 21(5): 445-451
19 Athar M, Back JH, Tang XW, et al A review of pre-clinical studies for human cancer prevention [J]. Toxicol Appl Pharmacol, 2007, 224(3): 274-283
20 Srtatton SP. Prvenetion of non-melnaoma skin cancer [J]. Curr Oncol ReP, 2001, 3(4): 295-300
21 Levi F, Zidani, Misset JL. Randomised multicentre trial of chronotherapy with oxaliplatin, fluorouracil and folinic acid inmetastatic colorectal cancer international organization for cancer chemotherapy [J]. Lancet, 1997, 350 (6): 681-686
22 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors [J]. Adv Enzyme Regul,1984, 22: 27-55
23 Kyungmin I, Jongbong P. Resveratrol at high doses acts as an apoptotic inducer in endothelial cells [J]. Cancer Res Treat, 2006, 38(1): 48-53
24 Ou HC, Chou FP, Sheen HM, et al Resveratrol, a polyphenolic compound inred wine, protects against oxidized LDL-induced cytotoxicity in endothelial cells [J]. Clin Chim Acta. 2006, 364(1-2): 196-204
25 Nishikawa T, Kohjimoto Y, Nishihata M, et al Synergistic antitumor effects of fleroxacin with 5-fluorouracil in vitro and in vivo for bladder cancer cell lines [J].Urology, 2009; 74(6): 1370-1376
26 Chougule M, Patel AR, Sachdeva P, et al Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer [J]. Lung Cancer, 2011, 71(3): 271-282
27 Ghosh R, Ganapathy M, Alworth WL, et al Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells [J]. J Steroid Biochem Mol Biol, 2009, 113(1-2): 25-35
1 Thompson CB. Apoptosis in the pathogenesis and treatment of disease [J]. Science, 1995: 267(5203): 1456-1462
2 McDonald ER , El-Deiry WS. Cell cycle control asa basis for cancer drug development [J]. Int J Oncol, 2000, 16(5): 871-886
3 Kathleen C, Tyler J, Nikola P, et al. The cell cycle and cancer. Proc. Natl. Acad. Sci, 1997, 94(7): 2776-2778
4 Michelle D, Garrett. Cell cycle control and cancer [J]. Curr Sci India, 2001; 81(5): 515-522
5 Motokura T, Bloom T, Kim HG, et al A novel Cyclin encoded by a bcl1-linked candidate oncogene [J]. Nature, 1991, 350 (6318): 512-515
6 Lew DJ, Dulic V, Reed SI. Isolation of three novel human Cyclins by rescue of G1 Cyclin (Cln) function in yeast [J]. Cell, 1991, 66 (6): 197-206
7 Li Jiaqing, Shuichi H, Che XM, et al Role of Cyclin E and p53 expression in progression of early gastric cancer [J]. Gastric Cancer 1998, 1(2): 160 -165
8 Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners [J]. J Cell Sci, 2003, 116(15): 3051-3060
9 Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies [J]. Nat Rev Cancer , 2003, 3(5):330-338
10 Hartwell LH, Kastan MB. Cell cycle control and cancer [J]. Science, 1994, 266: 1821-1823
11 Sherr CJ. Cancer cell cycle [J]. Science, 1996, 274: 1672-1677
12 Whittaker SR,Walton MI, Garrett MD,et a1 The Cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) inhibits retinoblastoma protein phosphorylation, causes loss of CyclinD1, and activates the mitogen-activated protein kinase pathway [J]. Cancer Res, 2004, 64(1): 262 -272
13 Darzynkiewiez Z, Gong JP,Juan G, et a1 Cytometry of Cyclinproteins [J]. Cytometry, 1996, 25(1): 1-13
14 Kelman Z. PCNA: structure, functions and interactions [J]. Oncogene, 1997, 14: 629-640
15 Kelman Z, Hurwitz J. Protein-PCNA interactions: a DNA-scanning mechanism [J]. Trends Biochem. Sci, 1998, 23(7): 236-238
16 Baserga R. Growth regulation of the PCNA gene [J]. J Cell Sci, 1991, 98 (4) : 433-436
17 Prelich G, Kostura M, Mashck DR, et al The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro [J]. Nature, 1987, 326 (6112) : 471-475
1 Fink SL, Cookson BT. Apoptosis, Pyroptosis, and Necrosis: Mechanistic Description of Dead and Dying Eukaryotic Cells [J]. Infection and Immunity, 2005, 73(4): 1907-1916
2 Scott H.Kaufman. Apoptosis: pharmacological implications and therapeutic opptunities [M]. Academic Pr. 1997, 3-6
3 C. S. Potten, James W. Wilson. Apoptosis: the life and death of cells [M]. Cambridge Univ Pr. 2004; 37-56
4 Lawen A. Apoptosis- an introduction [J]. BioEssays , 2003, 25(9), 888-896
5 Hu W, Kavanagh J. Anticancer therapy targeting the apoptotic pathway [J]. Lancet Oncol, 2003, 4(12): 721-729
6 R. Ramírez Chamond, J. Carracedo A?ón, C. Moreno Aguilar y F. Guerra Pasadas. Apoptosis and disease [J]. Alergol Inmunol Clin, 1999, 14(6): 367-374
7 Rudin CM, Thompson CB. Apoptosis and disease: regulation and clinical relevance of programmed cell death [J]. Annu Rev Med, 1997, 48: 267-281
8 Berridge MJ, Bootman MD, Lipp P. Calcium-a life and death signal [J]. Science, 1998, 395(6703): 645-648
9 Jiang S, Chow SC, Nicotera P, et al Intracellular Ca2+ signals activateapoptosis in Thymocytes: studies using the Ca2+-ATPase inhibitor thapsigargin [J]. Exp Cell Res, 1994, 212(1): 84-92
10 Kaise N, Edelman IS. Calcium dependence of glucocorticoid-induced lymphocytolysis [J]. Proc Natl Acad Sci USA, 1977, 74(2): 638-642
11 Mconkey DJ, Nicotera P, Hartzell P, et al Glucocorticoids activate a suide process in thymocytes through an elevation of cytosolic Ca2+ concentration [J]. Arch Biochem Biophys, 1989, 269: 365-370
12 Ghosh R, Ganapathy M, Alworth WL, et al Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis ind uction synergi stically in androgen independent prostate cancer cells [J]. J Steroid Biochem Mol Biol, 2009, 113(1-2): 25-35
13 Herrmann M, Lorenz HM , Voll R, et al A rapid and simple method for the isolation of apoptotic DNA fragments [J]. Nucleic Acids Research, 1994, 22(24): 5506-5507
14 Liu G, Xia T, Chen XB. The Activation Domains, the Proline-rich Domain, and the C-terminal Basic Domain in p53 Are Necessary for Acetylation of Histones on the Proximal p21 Promoter and Interaction with p300/CREB binding Protein [J]. Biological Chemistry, 2003, 278 (19): 17557-17565
15 Vande WB, Tijdens IB, Verbrugge A, et al Cleavage of the actin-capping protein alpha-adducin at AsP-AsP-Ser-AsP633-Ala by caspase-3 is Preceded by its PhosPhorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells [J]. Biol Chem, 2000, 275(33): 25805-25813
16 MacFarlane M,Williams AC. Apoptosis and disease: a life or death decision [J]. EMBO reports, 2004, 5(7): 674-678
17 Sugars KL. Bcl-Xl expression is inhibited by p53 [J]. Nucleic Acidic Research, 2001, 29(22): 4530-4540
18 Jerry E. Chipukdirect activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis [J]. Science, 2004, 303: 1010- 1014
19 Vousden KH, Lane DP. p53 in health and disease. Nature reviews molecularcell biology [J]. 2007, 8(4): 275-283
20 Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival [J]. Science, 1998, 281(5381): 1322-1326
21 Wolter KG, Hsu YT, Smith CL, et al Movement of Bax from the cytosol to mitochondria during apoptosis [J]. Cell Biol , 1997, 139(5): 1281-1292
22 Green DR, Reed JC. Mitochondria and apoptosis [J]. Science, 1998, 281(5381): 1309-1312
23 Mayer B, Oberbauer R. Mitochondrial Regulation of Apoptosis [J]. News in Physiological Sciences, 2003, 18( 3): 89-94
24 Tafani M, Schneider TG, Pastorino JG, et al Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition [J]. Am J Pathol, 2000, 156(6): 2111-2121
25 Wang XD. The expanding role of mitochondria in apoptosis [J]. Genes Dev , 2001, 15(22): 2922-2933
26 Dejean LM, Sonia MC, Stephen M, et al Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins [J]. Biochim Biophys Acta, 2006, 1762 (2): 191-201
27 Dejean LM, Sonia MC, Guo L, et al Oligomeric Bax Is a Component of the Putative Cytochrome c Release Channel MAC, Mitochondrial Apoptosis-induced Channel [J]. Mol Biol Cell, 2005, 16(5): 2424-2432
28 Fan TJ , Han LH, Cong RS,et al Caspase family proteases and apoptosis [J]. Acta Biochimica et Biophysica Sinica, 2005, 37(11): 719-727
29 Yang B,Elnahas AM,Fisher M,et al Inhibitors directed towards caspase-1 and -3 are less effective than pan caspase inhlbition in preventing renal Proximal tubular cell apoptosis [J]. Nephron Exp NePhrol, 2004, 96(2): 39-51
30 Hunot S, Flavell RA. Apoptosis. Death of a monopoly [J]. Science, 2001, 292(5518):865-866
31 Nicholson DW, Thornberry NA. Apoptosis. Life and death decisions [J]. Science 2003, 299(5604): 214-215
32 Cory S, Adams JM. The Bcl-2 family: regulators of the cellular life-or-death switch [J]. Nat Rev Cancer 2002, 2:647-656
1 Moorselaar RJA, Schalken JA, Oosterhof GON, et al Use of animal models in diagnosis and treatment of renal cell carcinoma [J]. World J Urol , 1991, 9(4): 192-197
2 Marek P, Rod B. Insights from Animal Models on the Origins and Progression of Retinoblastoma [J]. Curr Mol Med, 2006, 6(7): 759-781
3 Suzanne OR. Animal models of tumor immunity, immunotherapy and cancer vaccines [J]. Curr Opin Immunol, 2004, 16(2): 143-150
4 SinghA, Shukla Y. Antitumor activity of diallyl sulfide in two-stage mouse skin model of carcinogenesis [J]. Biomedical and Environmental Science, 1998, 11(3): 258-263
5韩锐.肿瘤化学预防及药物治疗[M].北京医科大学中国协和医科大学联合出版社, 1991, 115-116
6 Kozumbo WJ, Cerutti PA. Antioxidants as antitumor promoters [J]. Basic Life Sci, 1986, 39: 491-506
7 Pryor WA . Cancer and free radicals [J]. Basic Life Sci, 1986, 39: 45-59
8 Flemming R. Treatment of Basal Cell Carcinoma of the Skin with 5-Fluorouracil Ointment: A 10-Year Follow-Up Study [J]. Dermatologica, 1979, 158(5): 368-372
1 Gupta SC, Kannappan R, Reuter S, et al Chemosensitization of tumors by resveratrol [J]. Ann N Y Acad Sci. 2011, 1215:150-60
2 Delmas D, Lancon A, Colin D, et al Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer [J]. Curr Drug Targets, 2006, 7(4): 423-442
3 Bavaresco L, Role of viticultural factors on stilbene concentrations of grapes and wine [J]. Drugs Exp Clin Res, 2003, 29(5-6): 181-187
4 Baliga MS, Meleth S, Katiyar SK. Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary carcinoma 4T1 cells in vitro and in vivo systems [J]. Clin Cancer Res. 2005, 11(5): 1918-1927
5 Regev-Shoshani G, Shoseyov O, Bilkis I, et al Glycosylation of resveratrol protects it from enzymic oxidation [J]. Biochem J, 2003, 374(1): 157-163
6 Walle T, Hsieh F, DeLegge MH, et al High absorption but very low bioavailability of oral resveratrol in humans [J]. Drug Metab. Dispos. 2004,32(12): 1377-1382
7 Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev, Drug Discov, 2006, 5: 493-506
8 Soleas GJ, Yan J, Goldberg DM. Ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection [J]. Journal of Chromatography B: Biomedical Sciences and Applications 2001,757(1): 161-172
9 Baur JA, Sinclair DA, Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev Drug Discov, 2006, 5: 493-506
10 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol Med, 2002, 33(3): 387-398
11 Crowell JA, Korytko PJ, Morrissey RL, et al Resveratrol-associated renal toxicity [J]. Toxicol Sci, 2004, 82(2): 614-619
12 Vitrac X, Desmouliere A, Brouillaud B, et al Distribution of [14C]- trans- resveratrol,a cancer chemopreventive polyphenol, in mouse tissues after oral administration [J]. Life Sci, 2003, 72(20): 2219-2233
13 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol Med, 2002, 33(3): 387-398
14 Afaq F, Adhami VM, Ahmad N, et al Botanical antioxidantsfor chemoprevention of photocarcinogenesis [J]. Front Biosci, 2002, 7: 784-792
15 Afaq F, Adhami VM, Ahmad N, Prevention of short-term ultraviolet B radiation-mediated damages by resveratrol in SKH-1 hairless mice [J].Toxicol Appl Pharmacol, 2003, 186: 28-37
16 Parekh P, Motiwale L, Naik N, et al Downregulation of cyclin D1 is associated with decreased levels of p38 MAP kinases, Akt/PKB and Pak1 during chemopreventive effects of resveratrol in liver cancer cells [J]. Exp Toxicol Pathol. 2011, 63(1-2):167-73.
17 Aziz MH, Afaq F, Ahmad N, Prevention of ultraviolet-B radiation damage by resveratrol in mouse skin is mediated via modulation in survivin. Photochem [J]. Photobiol, 2005, 81(1), 25-31
18 Jang M, Cai L, Udeani GO, et al Cancer chemopreventive activity of resveratrol, a natural product derived from grapes [J]. Science, 1997, 275(5297): 218-220
19 Soleas GJ, Grass L, Josephy PD, et al A comparison of the anticarcinogenic properties of four red wine polyphenols [J]. Clin Biochem, 2002, 35(2):119-24
20 Fukui M, Choi HJ, Zhu BT. Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death [J]. Free Radic Biol Med. 2010, 49(5):800-813.
21 Fu ZD, Cao Y, Wang KF, et al Chemopreventive effect of resveratrol to cancer [J]. Aizheng (Chin. J. Cancer), 2004, 23(8): 869-873
22 Benitez DA, Pozo-Guisado E, Alvarez-Barrientos A, et al Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest in prostate cancer-derived cell lines [J]. J Androl. 2007, 28(2):282-93.
23 Ahmad N, Adhami VM, Afaq F, et al Resveratrol causes WAF-1/p21-mediated G1-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 [J]. Cells, 2001, 7(5): 1466- 1473
24 Kim AL, Zhu Y, Zhu H.et al Resveratrol inhibits proliferation of human epidermoid carcinoma A431 cells by modulating MEK1 and AP-1 signalling pathways [J]. Exp. Dermatol, 2006, 15(7): 538-546
25 Sheu SJ, Wu TT. Resveratrol protects against ultraviolet A-mediated inhibition of the phagocytic function of human retinal pigment epithelial cells via large-conductance calcium-activated potassium channels [J]. Kaohsiung J Med Sci. 2009, 25(7):381-388
26 Seve M, Chimienti F, Devergnas S, et al Resveratrol enhances UVA-induced DNA damage in HaCaT human keratinocytes [J]. Med. Chem, 2005, 1(6): 629-633
27 Gatouillat G, Balasse E, Joseph-Pietras D, et al Resveratrol induces cell-cycle disruption and apoptosis in chemoresistant B16 melanoma [J]. J Cell Biochem. 2010, 110(4):893-902.
28 Bhattacharya S, Darjatmoko SR, Polans AS. Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB [J]. Melanoma Res. 2011, 21(3):180-187.
29 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol.Med, 2002, 33(33):387-398
30 Caltagirone S, Rossi C, Poggi A, et al Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential [J]. Int. J. Cancer, 2000, 87(4): 595-600
31 Niles RM, Cook CP, Meadows GG, et al Resveratrol is rapidly metabolized in athymic (Nu/Nu) mice and does not inhibit human melanoma xenograft tumor growth [J]. J Nutr, 2006, 136(10): 2542-2546
32 Atten MJ, Godoy-Romero E, Attar BM et al Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells [J]. Invest New Drugs, 2005, 23(2): 111-119
33 Martini S, D'Addario C, Braconi D, et al Antibacterial activity of grape extracts on cagA-positive and -negative Helicobacter pylori clinical isolates [J]. J Chemother. 2009, 21(5):507-513.
34 Kim MY, Trudel LJ, Wogan GN. Apoptosis induced by capsaicin and resveratrol in colon carcinoma cells requires nitric oxide production and caspase activation [J]. Anticancer Res. 2009, 29(10):3733-3740
35 Atten MJ, Godoy-Romero E, Attar BM, et al Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells [J]. Invest. New Drugs, 2005, 23(2): 111-119
36 Riles WL, Erickson J Nayyar S, Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells [J]. World J Gastroenterol, 2006, 12(35): 5628-5634
37 Kim MY, Trudel LJ, Wogan GN. Apoptosis induced by capsaicin and resveratrol in colon carcinoma cells requires nitric oxide production and caspase activation [J]. Anticancer Res. 2009, 29(10):3733-40.
38 Cosan DT, Soyocak A, Basaran A, et al Effects of various agents on DNA fragmentation and telomerase enzyme activities in adenocarcinoma cell lines. Mol Biol Rep [J]. 2011, 38(4):2463-2469.
39 Corpet DE, Pierre F. Point: from animal models to prevention of colon cancer. Systematic Review of Chemoprevention in Min Mice and Choice of the Model System [J]. Cancer Epidemiol Biomarkers Prev, 2003, 12(5): 391-400.
40 Tessitore L, Davit A, Sarotto I, et al Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting Bax and p21 (CIP) expression [J]. Carcinogenesis, 2000, 21(8): 1619-1622
41 Schneider Y, Duranton B, Gosse F, et al Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis [J]. Nutr Cancer, 2001, 39(1): 102-107
42 Zhou HB, Chen JJ, Wang WX, et al Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice [J]. World J Gastroenterol, 2005, 11(2): 280-284
43 Sale S, Tunstall RG, Ruparelia KC, et al Comparison of the effects of the chemopreventive agent resveratrol and its synthetic analog trans 3,4,5,4′-tetramethoxystilbene (DMU-212) on adenoma development in the Apc (Min+) mouse and cyclooxygenase-2 in human-derived colon cancer cells [J]. Int J Cancer, 2005, 115(2): 194-201
44 Sengottuvelan M, Viswanathan P, Nalini N, Chemopreventive effect of trans-resveratrol-a phytoalexin against colonic aberrant crypt foci and cell proliferation in 1, 2-dimethylhydrazine induced colon carcinogenesis [J]. Carcinogenesis, 2006, 27(5): 1038-1046
45 Sengottuvelan M, Senthilkumar R, Nalini N. Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazineinduced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in rat colon carcinogenesis [J]. Biochim Biophys Acta. 2006, 1760(8):1175-83
46 Ziegler CC, Rainwater L, Whelan J, et al Dietary resveratrol does not affect intestinal tumorigenesis in Apc (Min/+) mice [J]. J Nutr, 2004, 134(1): 5-10
47 Pasqualini JR. Estrogen sulfotransferases in breast and endometrial cancers [J]. Ann N Y Acad Sci. 2009, 1155:88-98.
48 Sakamoto T, Horiguchi H, Oguma E, et al Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells [J]. J Nutr Biochem. 2010, 21(9):856-864.
49 Clément MV, Hirpara JL, Chawdhury SH, et al Chemopreventive Agent Resveratrol, a Natural Product Derived From Grapes, Triggers CD95 Signaling-Dependent Apoptosis in Human Tumor Cells [J]. Blood, 1998, 92(3): 996-1002.
50 Hsieh TC, Wu JM. Suppression of cell proliferation and gene expression by combinatorial synergy of EGCG, resveratrol and gamma-tocotrienol in estrogen receptor-positive MCF-7 breast cancer cells [J]. Int J Oncol. 2008, 33(4):851-859
51 Tang FY, Su YC, Chen NC, et al Resveratrol inhibits migration and invasion of human breast-cancer cells [J]. Mol Nutr Food Res. 2008, 52(6):683-691.
52 Bhat KPL, Pezzuto JM. Cancer chemopreventive activity of resveratrol [J]. Ann. NY Acad Sci, 2002, 957: 210-229
53 Serrero G, Lu R. Effect of resveratrol on the expression of autocrine growth modulators in human breast cancer cells [J]. Antioxid. Redox Signal, 2001, 3(6): 969-979
54 Magee PJ, Rowland IR. Phyto-estrogens, their mechanism of action: current evidence for a role in breast and prostate cancer [J]. The Br J Nutr, 2004, 91(4): 513-531
55 Pozo-Guisado E, Lorenzo-Benayas MJ, Fernández-Salguero PM. Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen receptor alpha-dependent mechanism: relevance in cell proliferation [J]. Int J Cancer , 2004, 109(2): 167-173
56 El-Mowafy A M, Alkhalaf M. Resveratrol activates adenylyl-cyclase in human breast cancer cells: a novel, estrogen receptor-independent cytostatic mechanism [J]. Carcinogenesis , 2003, 24(5): 869-873
57 Scarlatti F, Sala G, Somenzi G, et al Resveratrol induces growth inhibition and apoptosis in metastatic breast cancer cells via de novo ceramide signaling [J]. FASEB J, 2003, 17(15): 2339-2341
58 Shi Y, Yang S, Troup S, et al. Resveratrol induces apoptosis in breast cancer cells by E2F1-mediated up-regulation of ASPP1 [J]. Oncol Rep., 2011, 25(6):1713-1719.
59 Pozo-Guisado E, Merino JM, Mulero-Navarro S, et al Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-kappaB [J]. Int. J. Cancer, 2005, 115(1): 74-84
60 Kotha A, Sekharam M, Cilenti L, et al Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein [J]. Mol Cancer Ther, 2006, 5(3): 621-629
61 Castillo-Pichardo L, Martínez-Montemayor MM, Martínez JE, et al. Inhibition of mammary tumor growth and metastases to bone and liver by dietary grape polyphenols [J]. Clin Exp Metastasis. 2009, 26(6):505-16.
62 Whitsett T, Carpenter M, Lamartiniere CA. Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats [J]. J Carcinog. 2006, 5:15-26.
63 Banerjee S, Bueso-Ramos C, Aggarwal BB. Suppression of 7,
12-dimethylbenz (a) anthracene-induced mammary carcinogenesis in rats by resveratrol: role of nuclear factor-{kappa} B, Cyclooxygenase 2, and Matrix Metalloprotease 9 [J]. Cancer Res, 2002, 62(17): 4945-4954
64 Tang FY, Chiang EP, Sun YC. Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells [J]. J Nutr Biochem. 2008, 19(5):287-294
65 Provinciali M, Re F, Donnini A, et al Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice [J]. Int. J. Cancer, 2005, 115(1): 36-45
66 Garvin S, Ollinger K, Dabrosin C. Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts in vivo [J]. Cancer Lett , 2006, 231(1): 113-122
67 Bove K, Lincoln DW, Tsan MF. Effect of resveratrol on growth of 4T1 breast cancer cells in vitro and in vivo [J]. Biochem Biophys Res Commun, 2002, 291(4): 1001-1005
68 Sato M, Pei RJ, Yuri T, et al Prepubertal resveratrol exposure accelerates N-methyl-N-nitrosourea-induced mammary carcinoma in female Sprague–Dawley rats [J]. Cancer Lett, 2003, 202(2): 137-145
69 Russo J, Russo IH, Biological and molecular bases of mammary carcinogenesis [J]. Lab. Invest. J. Tech. Methods Pathol. 1987, 57(2): 112-137
70 Juan ME, Vinardell MP, Planas JM. The daily oral administration of high doses of trans-resveratrol to rats for 28 days is not harmful [J]. J. Nutr. 2002, 132(2): 257-260
71 Tsuji PA, Walle T. Benzo[a]pyrene-induced cytochrome P450 1A and DNA binding in cultured trout hepatocytes - inhibition by plant polyphenols [J]. Chem Biol Interact. 2007, 169(1):25-31.
72 Hansen T, Seidel A, Borlak J. The environmental carcinogen
3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells. Toxicol Appl Pharmacol [J]. 2007, 221(2):222-34.
73 Revel A, Raanani H, Younglai E, et al Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects lung from DNA damage andapoptosis caused by benzo[a]pyrene [J]. J Appl Toxicol, 2003, 23(4): 255-261
74 Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice [J]. J Nutr, 2001, 131(6): 1844-1849
75 Berge G, Ovrebo S, Eilertsen E, et al Analysis of resveratrol as a lung cancer chemopreventive agent in A/J mice exposed to benzo[a]pyrene [J]. Br. J. Cancer, 2004, 91(7): 1380-1383
76 Hecht SS, Kenney PM, Wang M, et al Evaluation of butylated hydroxyanisole, myo-inositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone-induced lung tumorigenesis in A/ J mice [J]. Cancer Lett, 1999, 137(2): 123-130
77 Zhou HB, Yan Y, Sun YN, et al Resveratrol induces apoptosis in human esophageal carcinoma cells [J]. World J. Gastroenterol, 2003, 9(3): 408-411
78 Szumi?o J. Natural compounds in chemoprevention of esophageal squamous cell tumors--experimental studies [J]. Pol Merkur Lekarski. 2009, 26(152):156-161.
79 Mousa SS, Mousa SS, Mousa SA. Effect of resveratrol on angiogenesis and platelet/fibrin-accelerated tumor growth in the chick chorioallantoic membrane model [J]. Nutr. Cancer, 2005, 52(1): 59-65
80 Brakenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes [J]. FASEB J, 2001, 15(10): 1798-1800
81程海燕,周家华,杨德同.白藜芦醇诱导胰腺癌细胞凋亡通路的实验研究[J].现代医学, 2010, 38(5): 515-517
82 Mouria M, Gukovskaya AS, Jung Y, et al Food-derived polyphenols inhibit pancreatic cancer growth through mitochondrial cytochrome c release and apoptosis [J]. Int J Cancer , 2002, 98(5): 761-769
83 Ding XZ, Adrian TE. Resveratrol inhibits proliferation and inducesapoptosis in human pancreatic cancer cells [J]. Pancreas, 2002, 25(4): 71-76
84 Ganapathy S, Chen Q, Singh KP, et al. Resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor [J]. PLoS One. 2010, 5(12):e15627.
85 Fulda S, Debatin KM. Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol [J]. Cancer Res, 2004, 64(1): 337-346
86 Kotha A, Sekharam M, Cilenti L, et al Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein [J]. Mol. Cancer Ther, 2006, 5(3): 621-629
87 Kuroiwa Y, Nishikawa A, Kitamura Y, et al Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters [J]. Cancer Lett, 2006, 241(2): 275-280.
88 Manson MM, Farmer PB, Gescher A, et al Innovative agents in cancer prevention. Recent results in cancer research [J]. Fortschr Krebsforsch, 2005, 166: 257-275
89 Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev Drug Discov, 2006, 5(6): 493-506
2 Ahmedin Jemal,Freddie Bray,Melissa M., et al Global cancer statistics [J].CA: A Cancer Journal for Clinicians, 2011, 61(2): 69-90
3中华人民共和国卫生部. 2010中国卫生统计年鉴[M].北京:中国协和医科大学出版社, 2010: 268, 307
4吕圭源.药理学[M].北京:中国中医药出版社, 2003: 400-404
5 American Cancer Society. Cancer Facts & Figures 2010 [R]. Atlanta: American Cancer Society; 2010: 1
6 Jemal A, Siegel R, Xu TQ, et al Cancer Statistics [J]. CA Cancer J Clin, 2010, 60 (5): 277-300
7 Lablanca R, Pessi A, Facendola G, et al Modulated 5-fluorouracil (5-FU) regimens in advanced colorectal cancer: a critical review of comparative studies [J]. Eur J Cancer, 1996, 32( l5): 7-12
8 Rajendran D, Senthil S, Dhanaraj SA, et al Comparative evaluation of targeting efficiency of charged and neutral liposomes of 5 -FU [J]. Drug development and industrial pharmacy, 1997, 23(11): 1099-1104
9 Levi F, Zidani R, Misset JL, et al Randomised multicentre trial of chemotherapy with oxaliplatin [J]. Drug Dev Ind Pharm, 1997, 23(11): 1099-1102
10 M Lopez-Velez, F Martinez-Martinez, C Del Valle-Ribes. The study of phenolic compounds as natural antioxidants in wine [J]. Crit Rev Food Sci Nutr, 2003, 43(3): 233-244
11 Bertelli A, Gozzini A, Giovannini L. Plasma and tissue resveratrol concentrations and pharmacological activity [J]. Drugs Exp Clin Res, 1998; 24(3): 133-138
12 Cho DI, Koo NY, Chung WJ, et al Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action [J]. Life Sci, 2002, 71(17): 2071-2082
13 Pace-Asciak CR, Hahn S, Diamandis EP, et al The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: implications for protection against coronary heart disease [J]. Clin Chim Acta. 1995; 235(2): 207-19
14 Martín AR, Villegas I, La Casa C, et al Resveratrol, a polyphenol found in grapes, suppresses oxidative damage and stimulates apoptosis during early colonic inflammation in rats [J]. Biochem Pharmacol, 2004, 67(7): 399-410
15 Jang M, Cai L, Udeani GO, et al Cancer chemopreventive activity of resveratrol, a natural product derived from grapes [J]. Science, 1997, 275(5297): 218-220
16 Adhami VM, Afaq F, Ahmad N. Involvement of the retinoblastoma (pRb)-E2F/DP pathway during antiproliferative effects of resveratrol in human epidermoid carcinoma (A431) cells [J]. Biochem. Biophys. Res. Commun, 2001, 288(3): 579-585
17 Afaq F, Adhami VM, Ahmad N, et al Botanical antioxidants for chemoprevention of photocarcinogenesis [J]. Front. Biosci, 2002, 7:784-792
18 Daiki M, Yutaka M, Kazumi Y. Resveratrol inhibits hepatoma cell invasion by suppressing gene expression of hepatocyte growth factor via its reactive oxygen species-scavenging property [J]. Clin. Exp. Metastasis , 2004, 21(5): 445-451
19 Athar M, Back JH, Tang XW, et al A review of pre-clinical studies for human cancer prevention [J]. Toxicol Appl Pharmacol, 2007, 224(3): 274-283
20 Srtatton SP. Prvenetion of non-melnaoma skin cancer [J]. Curr Oncol ReP, 2001, 3(4): 295-300
21 Levi F, Zidani, Misset JL. Randomised multicentre trial of chronotherapy with oxaliplatin, fluorouracil and folinic acid inmetastatic colorectal cancer international organization for cancer chemotherapy [J]. Lancet, 1997, 350 (6): 681-686
22 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors [J]. Adv Enzyme Regul,1984, 22: 27-55
23 Kyungmin I, Jongbong P. Resveratrol at high doses acts as an apoptotic inducer in endothelial cells [J]. Cancer Res Treat, 2006, 38(1): 48-53
24 Ou HC, Chou FP, Sheen HM, et al Resveratrol, a polyphenolic compound inred wine, protects against oxidized LDL-induced cytotoxicity in endothelial cells [J]. Clin Chim Acta. 2006, 364(1-2): 196-204
25 Nishikawa T, Kohjimoto Y, Nishihata M, et al Synergistic antitumor effects of fleroxacin with 5-fluorouracil in vitro and in vivo for bladder cancer cell lines [J].Urology, 2009; 74(6): 1370-1376
26 Chougule M, Patel AR, Sachdeva P, et al Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer [J]. Lung Cancer, 2011, 71(3): 271-282
27 Ghosh R, Ganapathy M, Alworth WL, et al Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells [J]. J Steroid Biochem Mol Biol, 2009, 113(1-2): 25-35
1 Thompson CB. Apoptosis in the pathogenesis and treatment of disease [J]. Science, 1995: 267(5203): 1456-1462
2 McDonald ER , El-Deiry WS. Cell cycle control asa basis for cancer drug development [J]. Int J Oncol, 2000, 16(5): 871-886
3 Kathleen C, Tyler J, Nikola P, et al. The cell cycle and cancer. Proc. Natl. Acad. Sci, 1997, 94(7): 2776-2778
4 Michelle D, Garrett. Cell cycle control and cancer [J]. Curr Sci India, 2001; 81(5): 515-522
5 Motokura T, Bloom T, Kim HG, et al A novel Cyclin encoded by a bcl1-linked candidate oncogene [J]. Nature, 1991, 350 (6318): 512-515
6 Lew DJ, Dulic V, Reed SI. Isolation of three novel human Cyclins by rescue of G1 Cyclin (Cln) function in yeast [J]. Cell, 1991, 66 (6): 197-206
7 Li Jiaqing, Shuichi H, Che XM, et al Role of Cyclin E and p53 expression in progression of early gastric cancer [J]. Gastric Cancer 1998, 1(2): 160 -165
8 Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners [J]. J Cell Sci, 2003, 116(15): 3051-3060
9 Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies [J]. Nat Rev Cancer , 2003, 3(5):330-338
10 Hartwell LH, Kastan MB. Cell cycle control and cancer [J]. Science, 1994, 266: 1821-1823
11 Sherr CJ. Cancer cell cycle [J]. Science, 1996, 274: 1672-1677
12 Whittaker SR,Walton MI, Garrett MD,et a1 The Cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) inhibits retinoblastoma protein phosphorylation, causes loss of CyclinD1, and activates the mitogen-activated protein kinase pathway [J]. Cancer Res, 2004, 64(1): 262 -272
13 Darzynkiewiez Z, Gong JP,Juan G, et a1 Cytometry of Cyclinproteins [J]. Cytometry, 1996, 25(1): 1-13
14 Kelman Z. PCNA: structure, functions and interactions [J]. Oncogene, 1997, 14: 629-640
15 Kelman Z, Hurwitz J. Protein-PCNA interactions: a DNA-scanning mechanism [J]. Trends Biochem. Sci, 1998, 23(7): 236-238
16 Baserga R. Growth regulation of the PCNA gene [J]. J Cell Sci, 1991, 98 (4) : 433-436
17 Prelich G, Kostura M, Mashck DR, et al The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro [J]. Nature, 1987, 326 (6112) : 471-475
1 Fink SL, Cookson BT. Apoptosis, Pyroptosis, and Necrosis: Mechanistic Description of Dead and Dying Eukaryotic Cells [J]. Infection and Immunity, 2005, 73(4): 1907-1916
2 Scott H.Kaufman. Apoptosis: pharmacological implications and therapeutic opptunities [M]. Academic Pr. 1997, 3-6
3 C. S. Potten, James W. Wilson. Apoptosis: the life and death of cells [M]. Cambridge Univ Pr. 2004; 37-56
4 Lawen A. Apoptosis- an introduction [J]. BioEssays , 2003, 25(9), 888-896
5 Hu W, Kavanagh J. Anticancer therapy targeting the apoptotic pathway [J]. Lancet Oncol, 2003, 4(12): 721-729
6 R. Ramírez Chamond, J. Carracedo A?ón, C. Moreno Aguilar y F. Guerra Pasadas. Apoptosis and disease [J]. Alergol Inmunol Clin, 1999, 14(6): 367-374
7 Rudin CM, Thompson CB. Apoptosis and disease: regulation and clinical relevance of programmed cell death [J]. Annu Rev Med, 1997, 48: 267-281
8 Berridge MJ, Bootman MD, Lipp P. Calcium-a life and death signal [J]. Science, 1998, 395(6703): 645-648
9 Jiang S, Chow SC, Nicotera P, et al Intracellular Ca2+ signals activateapoptosis in Thymocytes: studies using the Ca2+-ATPase inhibitor thapsigargin [J]. Exp Cell Res, 1994, 212(1): 84-92
10 Kaise N, Edelman IS. Calcium dependence of glucocorticoid-induced lymphocytolysis [J]. Proc Natl Acad Sci USA, 1977, 74(2): 638-642
11 Mconkey DJ, Nicotera P, Hartzell P, et al Glucocorticoids activate a suide process in thymocytes through an elevation of cytosolic Ca2+ concentration [J]. Arch Biochem Biophys, 1989, 269: 365-370
12 Ghosh R, Ganapathy M, Alworth WL, et al Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis ind uction synergi stically in androgen independent prostate cancer cells [J]. J Steroid Biochem Mol Biol, 2009, 113(1-2): 25-35
13 Herrmann M, Lorenz HM , Voll R, et al A rapid and simple method for the isolation of apoptotic DNA fragments [J]. Nucleic Acids Research, 1994, 22(24): 5506-5507
14 Liu G, Xia T, Chen XB. The Activation Domains, the Proline-rich Domain, and the C-terminal Basic Domain in p53 Are Necessary for Acetylation of Histones on the Proximal p21 Promoter and Interaction with p300/CREB binding Protein [J]. Biological Chemistry, 2003, 278 (19): 17557-17565
15 Vande WB, Tijdens IB, Verbrugge A, et al Cleavage of the actin-capping protein alpha-adducin at AsP-AsP-Ser-AsP633-Ala by caspase-3 is Preceded by its PhosPhorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells [J]. Biol Chem, 2000, 275(33): 25805-25813
16 MacFarlane M,Williams AC. Apoptosis and disease: a life or death decision [J]. EMBO reports, 2004, 5(7): 674-678
17 Sugars KL. Bcl-Xl expression is inhibited by p53 [J]. Nucleic Acidic Research, 2001, 29(22): 4530-4540
18 Jerry E. Chipukdirect activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis [J]. Science, 2004, 303: 1010- 1014
19 Vousden KH, Lane DP. p53 in health and disease. Nature reviews molecularcell biology [J]. 2007, 8(4): 275-283
20 Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival [J]. Science, 1998, 281(5381): 1322-1326
21 Wolter KG, Hsu YT, Smith CL, et al Movement of Bax from the cytosol to mitochondria during apoptosis [J]. Cell Biol , 1997, 139(5): 1281-1292
22 Green DR, Reed JC. Mitochondria and apoptosis [J]. Science, 1998, 281(5381): 1309-1312
23 Mayer B, Oberbauer R. Mitochondrial Regulation of Apoptosis [J]. News in Physiological Sciences, 2003, 18( 3): 89-94
24 Tafani M, Schneider TG, Pastorino JG, et al Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition [J]. Am J Pathol, 2000, 156(6): 2111-2121
25 Wang XD. The expanding role of mitochondria in apoptosis [J]. Genes Dev , 2001, 15(22): 2922-2933
26 Dejean LM, Sonia MC, Stephen M, et al Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins [J]. Biochim Biophys Acta, 2006, 1762 (2): 191-201
27 Dejean LM, Sonia MC, Guo L, et al Oligomeric Bax Is a Component of the Putative Cytochrome c Release Channel MAC, Mitochondrial Apoptosis-induced Channel [J]. Mol Biol Cell, 2005, 16(5): 2424-2432
28 Fan TJ , Han LH, Cong RS,et al Caspase family proteases and apoptosis [J]. Acta Biochimica et Biophysica Sinica, 2005, 37(11): 719-727
29 Yang B,Elnahas AM,Fisher M,et al Inhibitors directed towards caspase-1 and -3 are less effective than pan caspase inhlbition in preventing renal Proximal tubular cell apoptosis [J]. Nephron Exp NePhrol, 2004, 96(2): 39-51
30 Hunot S, Flavell RA. Apoptosis. Death of a monopoly [J]. Science, 2001, 292(5518):865-866
31 Nicholson DW, Thornberry NA. Apoptosis. Life and death decisions [J]. Science 2003, 299(5604): 214-215
32 Cory S, Adams JM. The Bcl-2 family: regulators of the cellular life-or-death switch [J]. Nat Rev Cancer 2002, 2:647-656
1 Moorselaar RJA, Schalken JA, Oosterhof GON, et al Use of animal models in diagnosis and treatment of renal cell carcinoma [J]. World J Urol , 1991, 9(4): 192-197
2 Marek P, Rod B. Insights from Animal Models on the Origins and Progression of Retinoblastoma [J]. Curr Mol Med, 2006, 6(7): 759-781
3 Suzanne OR. Animal models of tumor immunity, immunotherapy and cancer vaccines [J]. Curr Opin Immunol, 2004, 16(2): 143-150
4 SinghA, Shukla Y. Antitumor activity of diallyl sulfide in two-stage mouse skin model of carcinogenesis [J]. Biomedical and Environmental Science, 1998, 11(3): 258-263
5韩锐.肿瘤化学预防及药物治疗[M].北京医科大学中国协和医科大学联合出版社, 1991, 115-116
6 Kozumbo WJ, Cerutti PA. Antioxidants as antitumor promoters [J]. Basic Life Sci, 1986, 39: 491-506
7 Pryor WA . Cancer and free radicals [J]. Basic Life Sci, 1986, 39: 45-59
8 Flemming R. Treatment of Basal Cell Carcinoma of the Skin with 5-Fluorouracil Ointment: A 10-Year Follow-Up Study [J]. Dermatologica, 1979, 158(5): 368-372
1 Gupta SC, Kannappan R, Reuter S, et al Chemosensitization of tumors by resveratrol [J]. Ann N Y Acad Sci. 2011, 1215:150-60
2 Delmas D, Lancon A, Colin D, et al Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer [J]. Curr Drug Targets, 2006, 7(4): 423-442
3 Bavaresco L, Role of viticultural factors on stilbene concentrations of grapes and wine [J]. Drugs Exp Clin Res, 2003, 29(5-6): 181-187
4 Baliga MS, Meleth S, Katiyar SK. Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary carcinoma 4T1 cells in vitro and in vivo systems [J]. Clin Cancer Res. 2005, 11(5): 1918-1927
5 Regev-Shoshani G, Shoseyov O, Bilkis I, et al Glycosylation of resveratrol protects it from enzymic oxidation [J]. Biochem J, 2003, 374(1): 157-163
6 Walle T, Hsieh F, DeLegge MH, et al High absorption but very low bioavailability of oral resveratrol in humans [J]. Drug Metab. Dispos. 2004,32(12): 1377-1382
7 Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev, Drug Discov, 2006, 5: 493-506
8 Soleas GJ, Yan J, Goldberg DM. Ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection [J]. Journal of Chromatography B: Biomedical Sciences and Applications 2001,757(1): 161-172
9 Baur JA, Sinclair DA, Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev Drug Discov, 2006, 5: 493-506
10 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol Med, 2002, 33(3): 387-398
11 Crowell JA, Korytko PJ, Morrissey RL, et al Resveratrol-associated renal toxicity [J]. Toxicol Sci, 2004, 82(2): 614-619
12 Vitrac X, Desmouliere A, Brouillaud B, et al Distribution of [14C]- trans- resveratrol,a cancer chemopreventive polyphenol, in mouse tissues after oral administration [J]. Life Sci, 2003, 72(20): 2219-2233
13 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol Med, 2002, 33(3): 387-398
14 Afaq F, Adhami VM, Ahmad N, et al Botanical antioxidantsfor chemoprevention of photocarcinogenesis [J]. Front Biosci, 2002, 7: 784-792
15 Afaq F, Adhami VM, Ahmad N, Prevention of short-term ultraviolet B radiation-mediated damages by resveratrol in SKH-1 hairless mice [J].Toxicol Appl Pharmacol, 2003, 186: 28-37
16 Parekh P, Motiwale L, Naik N, et al Downregulation of cyclin D1 is associated with decreased levels of p38 MAP kinases, Akt/PKB and Pak1 during chemopreventive effects of resveratrol in liver cancer cells [J]. Exp Toxicol Pathol. 2011, 63(1-2):167-73.
17 Aziz MH, Afaq F, Ahmad N, Prevention of ultraviolet-B radiation damage by resveratrol in mouse skin is mediated via modulation in survivin. Photochem [J]. Photobiol, 2005, 81(1), 25-31
18 Jang M, Cai L, Udeani GO, et al Cancer chemopreventive activity of resveratrol, a natural product derived from grapes [J]. Science, 1997, 275(5297): 218-220
19 Soleas GJ, Grass L, Josephy PD, et al A comparison of the anticarcinogenic properties of four red wine polyphenols [J]. Clin Biochem, 2002, 35(2):119-24
20 Fukui M, Choi HJ, Zhu BT. Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death [J]. Free Radic Biol Med. 2010, 49(5):800-813.
21 Fu ZD, Cao Y, Wang KF, et al Chemopreventive effect of resveratrol to cancer [J]. Aizheng (Chin. J. Cancer), 2004, 23(8): 869-873
22 Benitez DA, Pozo-Guisado E, Alvarez-Barrientos A, et al Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest in prostate cancer-derived cell lines [J]. J Androl. 2007, 28(2):282-93.
23 Ahmad N, Adhami VM, Afaq F, et al Resveratrol causes WAF-1/p21-mediated G1-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 [J]. Cells, 2001, 7(5): 1466- 1473
24 Kim AL, Zhu Y, Zhu H.et al Resveratrol inhibits proliferation of human epidermoid carcinoma A431 cells by modulating MEK1 and AP-1 signalling pathways [J]. Exp. Dermatol, 2006, 15(7): 538-546
25 Sheu SJ, Wu TT. Resveratrol protects against ultraviolet A-mediated inhibition of the phagocytic function of human retinal pigment epithelial cells via large-conductance calcium-activated potassium channels [J]. Kaohsiung J Med Sci. 2009, 25(7):381-388
26 Seve M, Chimienti F, Devergnas S, et al Resveratrol enhances UVA-induced DNA damage in HaCaT human keratinocytes [J]. Med. Chem, 2005, 1(6): 629-633
27 Gatouillat G, Balasse E, Joseph-Pietras D, et al Resveratrol induces cell-cycle disruption and apoptosis in chemoresistant B16 melanoma [J]. J Cell Biochem. 2010, 110(4):893-902.
28 Bhattacharya S, Darjatmoko SR, Polans AS. Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB [J]. Melanoma Res. 2011, 21(3):180-187.
29 Asensi M, Medina I, Ortega A, et al Inhibition of cancer growth by resveratrol is related to its low bioavailability [J]. Free Radical Biol.Med, 2002, 33(33):387-398
30 Caltagirone S, Rossi C, Poggi A, et al Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential [J]. Int. J. Cancer, 2000, 87(4): 595-600
31 Niles RM, Cook CP, Meadows GG, et al Resveratrol is rapidly metabolized in athymic (Nu/Nu) mice and does not inhibit human melanoma xenograft tumor growth [J]. J Nutr, 2006, 136(10): 2542-2546
32 Atten MJ, Godoy-Romero E, Attar BM et al Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells [J]. Invest New Drugs, 2005, 23(2): 111-119
33 Martini S, D'Addario C, Braconi D, et al Antibacterial activity of grape extracts on cagA-positive and -negative Helicobacter pylori clinical isolates [J]. J Chemother. 2009, 21(5):507-513.
34 Kim MY, Trudel LJ, Wogan GN. Apoptosis induced by capsaicin and resveratrol in colon carcinoma cells requires nitric oxide production and caspase activation [J]. Anticancer Res. 2009, 29(10):3733-3740
35 Atten MJ, Godoy-Romero E, Attar BM, et al Resveratrol regulates cellular PKC alpha and delta to inhibit growth and induce apoptosis in gastric cancer cells [J]. Invest. New Drugs, 2005, 23(2): 111-119
36 Riles WL, Erickson J Nayyar S, Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells [J]. World J Gastroenterol, 2006, 12(35): 5628-5634
37 Kim MY, Trudel LJ, Wogan GN. Apoptosis induced by capsaicin and resveratrol in colon carcinoma cells requires nitric oxide production and caspase activation [J]. Anticancer Res. 2009, 29(10):3733-40.
38 Cosan DT, Soyocak A, Basaran A, et al Effects of various agents on DNA fragmentation and telomerase enzyme activities in adenocarcinoma cell lines. Mol Biol Rep [J]. 2011, 38(4):2463-2469.
39 Corpet DE, Pierre F. Point: from animal models to prevention of colon cancer. Systematic Review of Chemoprevention in Min Mice and Choice of the Model System [J]. Cancer Epidemiol Biomarkers Prev, 2003, 12(5): 391-400.
40 Tessitore L, Davit A, Sarotto I, et al Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting Bax and p21 (CIP) expression [J]. Carcinogenesis, 2000, 21(8): 1619-1622
41 Schneider Y, Duranton B, Gosse F, et al Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis [J]. Nutr Cancer, 2001, 39(1): 102-107
42 Zhou HB, Chen JJ, Wang WX, et al Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice [J]. World J Gastroenterol, 2005, 11(2): 280-284
43 Sale S, Tunstall RG, Ruparelia KC, et al Comparison of the effects of the chemopreventive agent resveratrol and its synthetic analog trans 3,4,5,4′-tetramethoxystilbene (DMU-212) on adenoma development in the Apc (Min+) mouse and cyclooxygenase-2 in human-derived colon cancer cells [J]. Int J Cancer, 2005, 115(2): 194-201
44 Sengottuvelan M, Viswanathan P, Nalini N, Chemopreventive effect of trans-resveratrol-a phytoalexin against colonic aberrant crypt foci and cell proliferation in 1, 2-dimethylhydrazine induced colon carcinogenesis [J]. Carcinogenesis, 2006, 27(5): 1038-1046
45 Sengottuvelan M, Senthilkumar R, Nalini N. Modulatory influence of dietary resveratrol during different phases of 1,2-dimethylhydrazineinduced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in rat colon carcinogenesis [J]. Biochim Biophys Acta. 2006, 1760(8):1175-83
46 Ziegler CC, Rainwater L, Whelan J, et al Dietary resveratrol does not affect intestinal tumorigenesis in Apc (Min/+) mice [J]. J Nutr, 2004, 134(1): 5-10
47 Pasqualini JR. Estrogen sulfotransferases in breast and endometrial cancers [J]. Ann N Y Acad Sci. 2009, 1155:88-98.
48 Sakamoto T, Horiguchi H, Oguma E, et al Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells [J]. J Nutr Biochem. 2010, 21(9):856-864.
49 Clément MV, Hirpara JL, Chawdhury SH, et al Chemopreventive Agent Resveratrol, a Natural Product Derived From Grapes, Triggers CD95 Signaling-Dependent Apoptosis in Human Tumor Cells [J]. Blood, 1998, 92(3): 996-1002.
50 Hsieh TC, Wu JM. Suppression of cell proliferation and gene expression by combinatorial synergy of EGCG, resveratrol and gamma-tocotrienol in estrogen receptor-positive MCF-7 breast cancer cells [J]. Int J Oncol. 2008, 33(4):851-859
51 Tang FY, Su YC, Chen NC, et al Resveratrol inhibits migration and invasion of human breast-cancer cells [J]. Mol Nutr Food Res. 2008, 52(6):683-691.
52 Bhat KPL, Pezzuto JM. Cancer chemopreventive activity of resveratrol [J]. Ann. NY Acad Sci, 2002, 957: 210-229
53 Serrero G, Lu R. Effect of resveratrol on the expression of autocrine growth modulators in human breast cancer cells [J]. Antioxid. Redox Signal, 2001, 3(6): 969-979
54 Magee PJ, Rowland IR. Phyto-estrogens, their mechanism of action: current evidence for a role in breast and prostate cancer [J]. The Br J Nutr, 2004, 91(4): 513-531
55 Pozo-Guisado E, Lorenzo-Benayas MJ, Fernández-Salguero PM. Resveratrol modulates the phosphoinositide 3-kinase pathway through an estrogen receptor alpha-dependent mechanism: relevance in cell proliferation [J]. Int J Cancer , 2004, 109(2): 167-173
56 El-Mowafy A M, Alkhalaf M. Resveratrol activates adenylyl-cyclase in human breast cancer cells: a novel, estrogen receptor-independent cytostatic mechanism [J]. Carcinogenesis , 2003, 24(5): 869-873
57 Scarlatti F, Sala G, Somenzi G, et al Resveratrol induces growth inhibition and apoptosis in metastatic breast cancer cells via de novo ceramide signaling [J]. FASEB J, 2003, 17(15): 2339-2341
58 Shi Y, Yang S, Troup S, et al. Resveratrol induces apoptosis in breast cancer cells by E2F1-mediated up-regulation of ASPP1 [J]. Oncol Rep., 2011, 25(6):1713-1719.
59 Pozo-Guisado E, Merino JM, Mulero-Navarro S, et al Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-kappaB [J]. Int. J. Cancer, 2005, 115(1): 74-84
60 Kotha A, Sekharam M, Cilenti L, et al Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein [J]. Mol Cancer Ther, 2006, 5(3): 621-629
61 Castillo-Pichardo L, Martínez-Montemayor MM, Martínez JE, et al. Inhibition of mammary tumor growth and metastases to bone and liver by dietary grape polyphenols [J]. Clin Exp Metastasis. 2009, 26(6):505-16.
62 Whitsett T, Carpenter M, Lamartiniere CA. Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats [J]. J Carcinog. 2006, 5:15-26.
63 Banerjee S, Bueso-Ramos C, Aggarwal BB. Suppression of 7,
12-dimethylbenz (a) anthracene-induced mammary carcinogenesis in rats by resveratrol: role of nuclear factor-{kappa} B, Cyclooxygenase 2, and Matrix Metalloprotease 9 [J]. Cancer Res, 2002, 62(17): 4945-4954
64 Tang FY, Chiang EP, Sun YC. Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells [J]. J Nutr Biochem. 2008, 19(5):287-294
65 Provinciali M, Re F, Donnini A, et al Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice [J]. Int. J. Cancer, 2005, 115(1): 36-45
66 Garvin S, Ollinger K, Dabrosin C. Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts in vivo [J]. Cancer Lett , 2006, 231(1): 113-122
67 Bove K, Lincoln DW, Tsan MF. Effect of resveratrol on growth of 4T1 breast cancer cells in vitro and in vivo [J]. Biochem Biophys Res Commun, 2002, 291(4): 1001-1005
68 Sato M, Pei RJ, Yuri T, et al Prepubertal resveratrol exposure accelerates N-methyl-N-nitrosourea-induced mammary carcinoma in female Sprague–Dawley rats [J]. Cancer Lett, 2003, 202(2): 137-145
69 Russo J, Russo IH, Biological and molecular bases of mammary carcinogenesis [J]. Lab. Invest. J. Tech. Methods Pathol. 1987, 57(2): 112-137
70 Juan ME, Vinardell MP, Planas JM. The daily oral administration of high doses of trans-resveratrol to rats for 28 days is not harmful [J]. J. Nutr. 2002, 132(2): 257-260
71 Tsuji PA, Walle T. Benzo[a]pyrene-induced cytochrome P450 1A and DNA binding in cultured trout hepatocytes - inhibition by plant polyphenols [J]. Chem Biol Interact. 2007, 169(1):25-31.
72 Hansen T, Seidel A, Borlak J. The environmental carcinogen
3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells. Toxicol Appl Pharmacol [J]. 2007, 221(2):222-34.
73 Revel A, Raanani H, Younglai E, et al Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects lung from DNA damage andapoptosis caused by benzo[a]pyrene [J]. J Appl Toxicol, 2003, 23(4): 255-261
74 Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice [J]. J Nutr, 2001, 131(6): 1844-1849
75 Berge G, Ovrebo S, Eilertsen E, et al Analysis of resveratrol as a lung cancer chemopreventive agent in A/J mice exposed to benzo[a]pyrene [J]. Br. J. Cancer, 2004, 91(7): 1380-1383
76 Hecht SS, Kenney PM, Wang M, et al Evaluation of butylated hydroxyanisole, myo-inositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone-induced lung tumorigenesis in A/ J mice [J]. Cancer Lett, 1999, 137(2): 123-130
77 Zhou HB, Yan Y, Sun YN, et al Resveratrol induces apoptosis in human esophageal carcinoma cells [J]. World J. Gastroenterol, 2003, 9(3): 408-411
78 Szumi?o J. Natural compounds in chemoprevention of esophageal squamous cell tumors--experimental studies [J]. Pol Merkur Lekarski. 2009, 26(152):156-161.
79 Mousa SS, Mousa SS, Mousa SA. Effect of resveratrol on angiogenesis and platelet/fibrin-accelerated tumor growth in the chick chorioallantoic membrane model [J]. Nutr. Cancer, 2005, 52(1): 59-65
80 Brakenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes [J]. FASEB J, 2001, 15(10): 1798-1800
81程海燕,周家华,杨德同.白藜芦醇诱导胰腺癌细胞凋亡通路的实验研究[J].现代医学, 2010, 38(5): 515-517
82 Mouria M, Gukovskaya AS, Jung Y, et al Food-derived polyphenols inhibit pancreatic cancer growth through mitochondrial cytochrome c release and apoptosis [J]. Int J Cancer , 2002, 98(5): 761-769
83 Ding XZ, Adrian TE. Resveratrol inhibits proliferation and inducesapoptosis in human pancreatic cancer cells [J]. Pancreas, 2002, 25(4): 71-76
84 Ganapathy S, Chen Q, Singh KP, et al. Resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor [J]. PLoS One. 2010, 5(12):e15627.
85 Fulda S, Debatin KM. Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol [J]. Cancer Res, 2004, 64(1): 337-346
86 Kotha A, Sekharam M, Cilenti L, et al Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein [J]. Mol. Cancer Ther, 2006, 5(3): 621-629
87 Kuroiwa Y, Nishikawa A, Kitamura Y, et al Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters [J]. Cancer Lett, 2006, 241(2): 275-280.
88 Manson MM, Farmer PB, Gescher A, et al Innovative agents in cancer prevention. Recent results in cancer research [J]. Fortschr Krebsforsch, 2005, 166: 257-275
89 Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivo evidence [J]. Nat Rev Drug Discov, 2006, 5(6): 493-506