【作者】 杨军国；

【导师】 沈生荣；

【作者基本信息】 浙江大学 ， 茶学， 2009， 博士

【摘要】 前列腺癌在欧美等西方国家是男性最常见的和第2位的癌症致死病因。而近几年，我国的前列腺癌发病率呈明显上升趋势。茶作为世人普遍引用的饮料，对多种癌症具有防治作用，如肺癌、乳腺癌、前列腺癌等。虽然茶与前列腺癌的流行病学调查呈不一致的结论，但在中国、日本等地区进行的病例对照调查表明饮用绿茶能有效的抑制前列腺癌的发病风险。其中，主要的抗癌活性成分为茶儿茶素。EGCG作为绿茶儿茶素的主要组分，具有显著的抗氧化、抗癌、抗突变等生物学活性。近来，EGCG与金属离子的相互作用及其生物学效应开始成为国内外学者研究的热点。锌是人体必需的微量元素。其缺乏与过量都有诱发前列腺癌的危险。因此，研究EGCG与Zn²⁺相互作用对EGCG防治前列腺癌具有重要意义。本研究主要利用激素依赖型前列腺癌细胞株LNCaP培养体系研究了EGCG和Zn²⁺对LNCaP生长状态的影响及其作用机制，以及Zn²⁺存在时EGCG对前列腺癌细胞的作用行为。MTT实验和划痕实验结果表明，EGCG和Zn²⁺都能明显抑制前列腺癌细胞株LNCaP的增殖和迁移，呈浓度依赖效应；光学倒置显微镜观察细胞形态后发现，高浓度的EGCG和Zn²⁺（80～160μM）处理能明显诱导细胞形态发生变化，由正常的梭形均匀分布到变圆簇聚成块；Hoechst 33258试剂盒和罗丹明123染色法检测表明，80μM的EGCG和Zn²⁺能够诱导LNCaP细胞凋亡。这些实验结果表明，EGCG和Zn²⁺能够抑制前列腺癌细胞LNCaP的增殖和迁移，诱导LNCaP细胞凋亡。为明确EGCG和Zn²⁺对前列腺癌细胞LNCaP生长抑制的机理，研究了EGCG和Zn²⁺对前列腺癌细胞LNCaP中的表皮生长因子（EGF）、细胞膜流动性和线粒体功能的影响。研究结果表明，80μM EGCG能明显抑制LNCaP细胞中EGF的表达，降低细胞膜的流动性，改变细胞内锌的分布和调控细胞线粒体功能，包括诱导活性氧（ROS）的产生、抑制ATP的合成、线粒体膜电位的降低、细胞色素C的释放和激活Caspase-3的表达。80μM Zn²⁺之所以能诱导前列腺癌细胞LNCaP的凋亡，主要是通过与细胞线粒体直接的相互作用和引起线粒体Ca²⁺水平积聚，损伤细胞线粒体，释放细胞色素C等凋亡因子，激活Caspase-3，诱导细胞凋亡。同时，本研究还表明了Zn²⁺存在时EGCG对前列腺癌细胞的作用行为。在对细胞膜流动性、活性氧和EGF的研究中，表明Zn²⁺存在时能降低EGCG对前列腺癌细胞的作用行为。相反地，在对线粒体功能调控中，发现Zn²⁺存在时却提高了EGCG对前列腺癌细胞线粒体功能的调控行为。说明EGCG与Zn²⁺共存时，究竟是提高还是降低EGCG的作用行为，与Zn²⁺的作用靶标极其相关：Zn²⁺能直接损伤线粒体功能和促使Ca²⁺在线粒体积聚，且作用效果Zn²⁺＞EGCG，则EGCG+Zn²⁺＞EGCG；Zn²⁺不能诱导线粒体活性氧的产生和抑制细胞中EGF的表达，作用效果Zn²⁺＜EGCG，则EGCG+Zn²⁺＜EGCG。更多还原

【Abstract】 Prostate cancer is the most common malignancy and the second leading cause ofmale death in USA and many European countries. However, the reported incidence ofprostate cancer in China is increasing rapidly in recent years. Tea is the commonlyconsumed beverage in the world that has been investigated for chemopreventive effectson many kinds of cancer such as lung cancer, breast cancer and prostate cancer.Although epidemiological studies on tea and prostate cancer have generatedinconsistent results, the case-control studies from China and Japan reported asignificant inhibition between green tea consumption and prostate cancer risk. EGCG,the main compound of catechins derived from green tea, has exhibited the greatsubstantial properties such as antioxidant, anticancer and antimutation. Recently,special interest has been directed to the interaction of EGCG and metal ions. Zinc isindispensable to human health. Higher and lower zinc ion concentration may increasethe risk of prostate cancer. Hence, the investigations of EGCG and Zn²⁺ play animportant role in EGCG against prostate cancer.In the present work, we aimed to study the effect（s） of EGCG and Zn²⁺ on viabilityof LNCaP cells, and actions elicited by EGCG in the presence of Zn²⁺. MTT assaysand scratch test showed that treatments with EGCG or Zn²⁺ inhibited the growth andmigration of LNCaP cells in a concentration-dependent manner. After treatments withhigh concentrations of EGCG or Zn²⁺ （80～160μM）, morphology of LNCAP cells weremarkedly changed from on the plate uniformly with compressed shape to round inshape and clustered on the plate. Hoechst 33258 and rhodamine 123 stainings indicatedthat 80μM EGCG or Zn²⁺ induced apoptosis of LNCaP cells. These observations gavea hint that EGCG or Zn²⁺ could inhibit the proliferation and migration of LNCaP cells,and induce apoptosis of LNCaP cells. In order to investigate the mechanisms of EGCGor Zn²⁺ on inhibition of growth of LNCaP cells, we futher studied the effects of EGCGand Zn²⁺ on expression of EGF, membrane fluidity and mitochondrial functions. The results showed that 80μM EGCG significantly inhibited the expression of EGF,decreased the membrane fluidity, altered the distribution of cellular zinc and regulatedmitochondrial functions including inducing the generation of ROS, inhibiting thesynthesis of ATP, decline of mitochondrial membrane potential, release of cytochromeC and activation of Caspase-3.80μM Zn²⁺ induced apoptosis of LNCaP cells throughdirect interactions with mitochondria and accumulation of Ca²⁺ in mitochondria, whichresulted in mitochondrial dysfunction and triggered cell apoptotic pathway.We also investigated the bioactivity of EGCG in the presence of Zn²⁺. The resultsindicated that the effect was EGCG+ Zn²⁺ （1:1）＜EGCG in the trials of EGF, ROS andmembrane fluidity. However, the effect was EGCG+ Zn²⁺ （1:1）＞EGCG in theregulation of mitochondrial function. These results showed that Zn²⁺ enhanced theaction of EGCG due to its targets in the mixture of EGCG and Zn²⁺ （1:1）: Treatmentwith Zn²⁺ could directly interact with mitochondria and increased the accumulation ofmitochondrial Ca²⁺, and the effects were Zn²⁺＞EGCG and EGCG+Zn²⁺＞EGCG;Treatment with Zn²⁺ did not significantly stimulate the generation of ROS and inhibitthe expression of EGF, and the effects were Zn²⁺＜EGCG and EGCG+Zn²⁺＜EGCG.更多还原