【作者】 敦洁宁；

【导师】 张永健；

【作者基本信息】 河北医科大学 ， 药理学， 2011， 博士

【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)。更多还原

【Abstract】 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 LinesObjective: To study the effects of 5-Fu combined with Res on the growth and necrosis of A431 and TE-1 cell linesMethods: 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 LinesObjective: 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 LinesObjective: 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 carcinogenesisObjective: 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.CONCLUSIONS1 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.更多还原