Investigations Of The Cytotoxicity Of Epigallocatechin-3-gallate And YIGSR Against PC-3 Cells Meidiated By 67LR

Prostate cancer is one of the leading causes of death, killing about 200,000 menannually throughout the world. The major polyphenolic constituent presented in greentea, (-) epigallocatechin-3-gallate (EGCG), is believed to be the compound which ismost responsible for inhibiting prostate cancer cell growth both in cell culture systemsand tumor models.The interaction of EGCG with metal ions changes their bioactivities andmetabolisms, and this interaction may be an important way to prevent or cure prostatediseases. There are various kinds of heavy metals in prostate glands, such as zinc andcadmium, and they are partly secreted into semen during normal metabolism. Innormal prostate glands, the metabolisms of Zn²⁺ and Cd²⁺ are in a balance state, butprostate disease may occur when this balance is disturbed. In the present paper, effectsof EGCG, Zn²⁺, Cd²⁺ and their mixture (EGCG+Zn²⁺, EGCG+Cd²⁺) onandrogen-insensitive prostate cancer cells PC-3 were estimated respectively, and theirmechanisms were thoroughly investigated. The results showed that EGCG, Zn²⁺ andCd²⁺ suppressed viability and migration ability of PC-3 cells, and the suppressioneffect was enhanced when EGCG added with 80μmol/L Zn²⁺ or 20μmol/L Cd²⁺. Ithas been reported that the inhibitory effect of EGCG on tumor cells proliferation isexerted by its binding to the 67kD laminin receptor (67LR), which is a high affinitynon-intergrin laminin receptor. Here we found that Zn²⁺ or Cd²⁺ significantlyenhanced the inhibitory effect of EGCG by up-regulating the 67LR expression, whichcould provide more binding sites of the cell surface for EGCG. Conformationalchanges in membrane proteins were determined using electron paramagneticresonance spin labeling. The results showed that the treatments of EGCG, Zn²⁺ andCd²⁺ disturbed membrane protein conformation, and S/W ratio decreased significantlywhen EGCG added with 80μmol/L Zn²⁺ or 20μmol/L Cd²⁺. In the meanwhile, wealso found that EGCG and Cd²⁺ directly interacted with mitochondrial, and themixture of EGCG and Cd²⁺ (80μmol/L EGCG+20μmol/L Cd²⁺) significantly caused loss of the mitochondrial membrane potential, decrease of the ATP content andactivation of caspase-9 compared with EGCG treated alone.The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide,corresponding to the 929-933 sequence ofβ1 chain, has been found to inhibit tumormetastasis and growth by competing with laminin for its receptor on tumor cells, andblocking the binding sites of these cells to basement membranes. In this paper, wefound that YIGSR inhibited the growth and migration ability of PC-3 cellsdose-dependently. Meanwhile, YIGSR caused the loss of mitochondrial membranepotential, decrease of the ATP content and activation of caspase-9, so PC-3 cellsdamage caused by YIGSR might be through a mitochondria-dependent mechanism.MTT and clonogenecity assay demonstrated that cytotoxicity of EGCG against PC-3cells was weakened in the presence of YIGSR. To elucidate the molecular mechanismby which the YIGSR and EGCG affects tumor growth, we investigated theinteractions of YIGSR, EGCG with 67LR by using MVD. The results showed thatYIGSR and EGCG have similar binding modes with 67LR. They recognized andbinded to the same sites of 67LR in the N-terminal of 3BCHB. YIGSR appears tohave potential interactions with Thr¹⁸⁸, Ser¹⁹⁰, Glu¹⁹², His¹⁹³, Ile¹⁸⁹, while EGCGinteracts with Leu¹⁸³, Arg¹⁸⁴, Thr¹⁸⁸, Ile¹⁸⁹, Ser¹⁹⁰, Glu¹⁹², His¹⁹³.