Mechanism Of Anticancer Activity Of Cimicifuga Natural Product KY17 And The DNA Adducts Of Temozolomide Analogs

Malignant neoplasia has currently become one of the major diseases to human health,because of the living environment pollution and life stress.The chemotherapy is kill cancer cells and inhibit their growth,which is the way better efficacy.Among the many chemotherapeutic drugs,antineoplastic agents from plant origin account for a large proportion,and its anti-tumor activity is more significant.But only a small portion of anticancer drugs derived from plants do biological activity screening and pharmacological effects.Therefore,based on the development of tumor occurrence and mechanism,we set up to screen product of cohosh extract isolated compounds for the anti-tumor activity,then further study its mechanismFirstly,we used of mouse cell lines that transfected with mutant p53(p53-/-+v+Ras and p53-/-+s+Ras)and mouse embryonic fibroblasts(MEF)to screen compounds extracted from cohosh rhizome.The results found that one Cycloartanes compound KY17 had the grow inhibition(IC50=13.1?M;13.94?M).Then,using the colon cancer cell lines(HCT116?HT-29?SW480),we screen the compounds anti-tumor activity by MTT assay(For HCT116,HT-29,SW480 IC50 of 9.31 ± 0,98?M,8.84±1.1(?M and 8.75?M).Cell cycle analysis revealed that KY17 made HT-29 cell cycle arrest in G2/M phase by PI staining.This indicated that KY17 inhibited tumor cell growth was achieved by cycle arrest.Then,It was found that 10?M KY17 can activate apoptosis-related proteins in HT-29 cells,such as PARP,Caspase-3,Caspase-8 cleavage by western blotting analysis.At the same time,with Annexin V/PI double staining,we found that 10?M KY17 induced apoptosis in HT-29 cells.Interestingly,the experiments found that 1?M KY17 treated cells will produce vacuole.Through the method of GFP-LC3 protein fusion tracer,Acridinee orange staining and western blotting analysis,we found KY17 also induced HT-29 cells autophagy.Using autophagy inhibitor,Bafilomycin Al,blocking autophagy,we found that apoptosis was strengthened than without inhibitor.It also suggestted that KY17 induced autophagy in HT?29 cells,this effect might protective onet on cells.Fllowing studies showned that KY17 did not induce HT-29 cells to product reactive oxygen species.In addition,we also made a preliminary study about the regulation of aging-related factor and KY17 in HCT116 cells.We found that miRNA-34a was increased,and SIRT1 was inhibited,and leading to activation of p53 protein.In summary,this study found that KY17 that isolated compound from cohosh inhibited the proliferation of HT-29 cells through inducing the apoptosis and cell cycle arrest.Simultaneously,KY17 also induced autophagy,and had a protective effect in cells.TMZ is a new second-generation oral alkylating antineoplastic that belongs to imidazole tetrazine derivatives.TMZ itself doesn't have the antineoplastic activity.As a prodrugs,under physiological conditions,TMZ was decomposed into the pharmaceutically active substance 5-(3-methyl-triazene-1-)imidazo]e-4-carboxamide(MTIC)further hydrolysis of 5-amino-imidazole-4-carboxamide(AIC)without metabolized by the liver.MTIC has a high degree of antitumor activity.Methylhydrazine is considered to alkylate DNA(methylation)and primarily alkylated guanine of O-6 and N-7,forming 5%O-6 meG,75%N-7 meG.The cytotoxicity of TMZ for cells primarily mediated through O6-methyl(O6-MeG)guanine,O6-MeG lead to base mismatch,the formation of the daughter strand nicked DNA and breaken of single-stranded or double-stranded.Eventually that impeded DNA replication.Then inhibition of tumor growth in the process of DNA replication and leads to cell death.However,DNA repair enzymes,O6-alkyl guanine-DNA-alkyl transferase(O6-methylguanine-DNA methyltransferase,MGMT),can catalyticaly transfer the methyl of O6-guanine to its own the 145s cysteine residue in cells and irreversibly repair the damaged DNA,thus weakening the anti-tumor activity of TMZ.Therefore,MGMT expression is the main resistance of TMZ and limits its application in clinic.Professor Malcolm Stevens group have designed and synthesized some TMZ analogs,like compounds 377 to to overcome MGMT resistance of tumor cells,according to the crystal structure of AGT and the mechanism of action of TMZ.Antitumor activity screening results showed that 377 has a broad spectrum of anticancer activity.However,the current study has yet to reveal the formation and characteristics of DNA adducts with new analog 377.Therefore,the purpose of this study is intended to explore the DNA adducts of TMZ derivative 377 with calf thymus DNA(ctDNA)and reveal the mechanism 377 compounds that can escape resistance compared with TMZ.This provides a theoretical basis for the design and development compounds of overcoming MGMT resistance.We using full wavelength UV spectrophotometer scan,the results show that,the maximum absorption wavelength is increase after the sample TMZ,compound 377 with calf thymus DNA adduct reaction.This suggests that TMZ and compound 377 alkylation reaction with calf thymus DNA may have occurred.Detected by HPLC after and various TMZ,We first test sample in vitro by high performance liquid chromatography(HPLC)found stability and the hydrolyzate of TMZ,377 and the enzymatic calf thymus DNA bases.Further using ultra-high pressure liquid chromatography?high resolution mass spectrometry to identify formula of the various components and the hydrolysis.After inferred hydrolyzate formula,we found the compounds 377 open-loop method like TMZ,under physiological conditions(PH?7.4),the intermediate methylhydrazine is responsible for alkylating DNA.At the same time,we also found that the intermediate compound after hydrolytic of 377 were relatively stable.Then by liquid chromatography,and mass spectrometry to determine the 377 and ctDNA binding sites more easily and affinity addition reaction with DNA occurs The adducts include that N3 methyl adenine,N7-methylguanine,O6-methylguanine,N3 methyl thymine,N3 methylcytosine deoxynucleotides,N3 methyl adenine deoxy nucleotides.In summary,the study found that compound 377 can ring-open to produce methylhydrazine intermediate like TMZ under physiological conditions(PH=7.4),and the intermediate is very stable compared with TMZ.377 compound alkylates DNA to produce O6-various DNA adducts.