A Study On The Synergistic Effect Of Melatonin And (-)-Epigallocatechin-3-Gallate(EGCG) On Cancer Inhibition

We have demonstrated that melatonin could efficiently attenuated hepatotoxicity triggered by high-dose(-)-epigallocatechin-3-gallate(EGCG)in mice.This suggests that EGCG and melatonin are good partners.A large number of studies show that high-dose levels of melatonin and EGCG have clear roles in anticancer.But High doses mean high risk.It is worth studying whether the combination of EGCG and melatonin have a better cancerinhibitory effect thereby decreasing the dose of EGCG and melatonin.No study have been done about the combination of EGCG and melatonin to inhibit cancer cells till now.The current work investigated whether EGCG improves cancer-inhibitory effect of melatonin use of MTT assay,Western blotting,deuteration assay,RT-PCR and Q-PCR.Melatonin(N-acetyl-5-methoxytryptamine)is an indoleamine compound synthesized mainly produced in pineal gland.It is a hormone with many physiological functions,inclouding biological rhythm regulation,immune modulation,antioxidant and antitumor.By western blotting method and MTT assay,we found melatonin dose-dependently increased cell cycle regulator p21 and cytotoxicity in TCA cells.This shows that the upregulation of p21 produced by melatonin is an important cause of cytotoxicity in TCA cells.The upregulation of p21 was due to p53 transcriptional activation caused by the increase of nuclear Trx1 and TrxR1 after melatonin treatment of TCA cells.In HepG2 cells,melatonin in turn suppressed p21,an action like sorafenib that is a mainstay drug used for treating hepatocarcinoma.It has been known that p21 suppression could sensitize cytotoxicity of numerous anti-cancer agents.In conclusion,the response of p21 is an important index reflecting melatonin cytotoxicity.(-)-Epigallocatechin-3-gallate(EGCG),extracted from green tea,exhibits a variety of activities such as antitumor,antiviral and protective nervous system.Through MTT experiments,we found that EGCG can produce cytotoxicity in a dose-dependent manner in TCA cells.By measuring the level of quinoprotein after treatment with EGCG,it was found that EGCG could promote the production of quinoprotein in TCA cells in a dose-dependent manner,and the level of quinoprotein basically reached the plateau phase after 6 hours of EGCG treatment.However,the combination of 2 mM NAC and 50 U/ml CAT antioxidants inhibited the cytotoxicity and quinoprotein levels priduced by EGCG.It can be concluded that quinoproteins levels were positively associated with cytotoxicity of EGCG.To investigate whether EGCG and melatonin can inhibit cancer in synergy,we compared the cytotoxic effect between the co-treatment of EGCG and melatonin and the single EGCG or melatonin in TCA and HepG2 cells.In the co-treatment of EGCG and melatonin,melatonin did not affect the dramatic increase of quinoproteins induced by EGCG and EGCG did not impair p21 upregulation caused by melatonin in TCA cells.Thus the cotreatment generating enhanced cytotoxicity as evidenced by the assays of cell survival,scratch test and colony formation.In HepG2 cells,we found that the co-treatment of melatonin also did not hamper the effect of EGCG on the formation of quinoproteins and greatly enhanced cytotoxic actions of EGCG as reflected by the assays of cell survival,scratch test and colony formation.Taken together,the present study demonstrated that EGCG and melatonin can inhibit cancer in synergy and explain its molecular mechanism.