Mechanisms Of Interaction Of EGCG With Zn²⁺ And Their Biological Behavior In LNCaP Cells

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.