Investigation Of The Apoptosis Effects And Mechanism Induced By Echinacoside On MG-63 And SW480 Cells

In recent years, the development of targeted cancer drug is a new way to treat cancer which can avoid side effects on normal cell that has made some progress. Difficulties also emerged, such as oncogenes frequent mutations and heterogeneity of tumor. Particularly, drug resistance compels scientists to search for tumor-specific targets of new drugs.Cancer cells endure significantly higher oxidative stress than normal cells due to prominent metabolism status. 8-oxoG is a most common DNA damage form of the cell that was formed when dGTP was oxidized by oxygen species and the following insertion will cause mutations. 8-oxoG is prone to mismatch adenine which will result in genomic instability that leads to DNA damage response. A large number of single-stranded and double-stranded DNA will form and increase the risk of cancer.MTH1 is a pyrophosphatase, which can dephosphorylate 8-oxoG avoiding it inserted into DNA. Two recent studies reported on MTH1 attracted the attention of scientists, Gad et al., who found that overexpression MTH1 can inhibit oxidative damage caused by reactive oxygen species in cancer cells. Deletion of MTH1 has no impact normal cells but lead to cancer cellular death. And it can be compensated by wild-type MTH1 that reveals requirement of MTH1 for cancer cells’ survival. All of this indicates MTH1 is potent target for cancer treatment.Traditional Chinese Medicine has been used for thousands of years. They are not initially accepted for lacking of clinical research until the effective treatment of disease was revealed. And a growing number of western scholars became interested in Traditional Chinese Medicine. Many lead compounds are chemicals medicine monomers, such as paclitaxel, camptothecin and homoharringtonine alkali that has got better cilincal application. The purpose of this study was to high-throughput screen Traditional Chinese Medicine monomer which was capable of inhibiting the activity of MTH1.In this study, 55 kinds of Traditional Chinese Medicine that may have a role in inhibition of cancer have been screened for inhibition of MTH1. The results show that echinacoside which is a main component of Cistanche stems has a protruding inhibition. Both in vitro and animal research suggest that echinacoside has neuro-protective, hepatoprotective, anti-fatigue and anti-tumor effects. But the mechanism of echinacoside is obscure.Here we firstly find echinacoside can induce apoptosis of MG-63 and SW480 cells which was caused by increase of 8-oxoG. Following, MTH1 and echinacoside docking was achieved by computer simulation also ADMET(drug absorption, distribution, metabolism, excretion and toxicity) was predicted. Echinacoside provide a reference for the study of the structure of MTH1 inhibitors. Echinacoside may be lead compound of cancer treatment.In vitro enzymatic screening showed that echinacoside MTH1 can effectively inhibit the activity of MTH1 with IC50 of 7.01 ± 2.13 μM. MTT assay showed that only MG-63 and SW480 cells are sensitive to echinacoside. Immunofluorescence staining of 8-oxoG in MG-63 and SW480 cells indicated enchinacoside induced 8-oxoG accumulation that should lead to DNA damage response. As the speculation, 53BP1 as DNA repair protein form foci in the two cell lines. The results of western blot showed that cleaved PARP, caspase3 and G1 / S-CDK inhibitor p21 Cip / WAF1 expression increased after echinacoside treatment. Apoptotic bodies and chromatin condensation was observed through DAPI staining. Flow cytometry results showed that echinacosdid induced apoptosis and G1/S cell cycle arrest of MG-63 and SW480 cells which also exhibited in a dose-dependent manner. After 24 h treatment with echinacoside, there is definitely no diploid in MG-63 and SW480 cells which indicated replication-defect in the cells. Colony formation and scratched assay confirmed that echinacoside inhibited the growth of MG-63 and SW480 cells.All the results above indicated echinacoside induced MG-63 and SW480 cells apoptosis and G1/S cell cycle arrest through inhibited MTH1 that resulted in accumulation of 8-oxoG which leaded to DNA replication-defect and apoptosis.At last computer simulation was used to obtain MTH1 and echinacosde docking, ADMET and TOPAT parameters. Echinacoside was water-soluble with a poor intestinal absorption, blood-brain barrier permeability and less than 90% plasma protein binding rate.It may cause a dose-dependent toxic liver damage. TOPKAT results showed that there is no potential developmental toxicity and may be cause male mice carcinogenic.