The Anticancer Effects And Mechanism Of Methyl Haematommate

Objective:To explore the isolation and structure of the anticancer compound,which is a bioactive compound of the Usnea.Then investigate the inhibitory effects and it's durg combination studies with cisplatin(DDP)on human cancer cell lines in vitro.To evaluate a further observation on its anticancer activities in vivo by the model of human carcinoma transplanted in nude mice.Developing its anti-tumor mechnism by the technology of flow cytometry and gene microarray.Methods:1.The compound of Usnea was isolated and purified by the methods of solvent extraction,gel column chromatography and re-crystallization technology and it's structures was characterized by1H-NMR and 13C-NMR.2.The MTT assay was used to determine the Antiproliferation effect of the compound on A549,HepG2,XWLC-05,MCF-7,NCI-H157,NCI-H460,T24 and CNE cancer cell lines at different levels of concentrations(i.e.at 2?g/ml?4pg/ml?8?g/ml?16?g/ml?32?g/ml respectively).3.Antiproliferation effect of combination therapy with the compound in different concentration and Cis-DDP in vitro were determined by MTT method,such as XWLC-05 and T24.And the Jin's formula was used to analyse the effect of drug combination.4.Long and short diameters of the tumors were measured to calcuate the tumor volume by the model of human carcinoma transplanted in nude mice.The mice were sacrificed after 16 times treatment,their body and main organs were weighted accurately,the organ coefficient was calculated,then hematological parameters and hematological biological parameters were measured.5.The Usnea Bioactive Compound's regulation of cell cycle in HepG2 was detected by flow cytometry.Cells were stained by propidium iodid,then DNA content in different cellular cycle was detected by flow cytometry at 488nm,thus the mechanism of the compound on cell cycle of HepG2 was analyzed.6.Microarray analysis was used to identify differentially expressed gene profiles in human Breast carcinoma cell line(MCF-7)after the compound treatment.Human Transcriptome Array 2.0 was performed to detect IncRNA and mRNA expression profiles.Bioinformatic analyses(gene ontology,pathway,and net-work analysis)were applied for further study of these differentially expressed mRNAs.Results:1.The compound was isolated and purified from Usnea,which molecular structural formula could be determined as 3-Formyl-2,4-dihydroxy-6-methyl-benzonic acid methyl ester(Methyl haematommate).2.The results of MTT assay has been showed that proliferation of the A549.HepG2?XWLC-05?MCF-7?NCI-H157?H460?T24 and CNE cancer cell lines was inhibited by Methyl Haematommate in a concentration-dependent manner after 72h.3.IC50 values of the Methyl Haematommate were 13.388 ?g/ml?11.784 ?g/ml?8.771?g/ml?13.188 ?g/ml?25.165 ?g/ml?28.719 ?g/ml?7.598 ?g/ml and 18.475 ?g/ml in A549?HepG2?XWLC-05?MCF-7?NCI-H157?H460?T24 and CNE cell lines.The sensitivity was obviously divergent in different cancer cell lines,which proved that the antitumor effects of Methyl haematommate in vitro was targeted.4.The inhibitory rate of Methyl Haematommate at different levels of concentrations(i.e.at 2pg/ml?4?g/ml?8?g/ml?16pg/ml?32?g/ml respectively)in A549 were 3.09%,4.40%,20.03%,52.45%,76.89%at 24h;2.17,6.75%,19.73%,60.9%,86.72%at 48h;5.99%,8.30%,14.18%,56.63%,91.45%at 72h.The in vitro anticancer effects had no time-course relationship.5.The combination effects of Methyl haematommate with cisplatin in the human cancer cell lines were determines by q value.The q value were 0.56,0.55,0.708 on XWLC-05 and were 0.717,1.046,1.01 on T24.According to the judgment standard,the in vitro combinative anticancer were antagonism effect on XWLC-05 and additive effect on T24.6.In the vivo anticancer studies,methyl haematommate(5mg/ml)group presented significant inhibition effect on tumor growth in a time-dependent manner(p<0.05).The relative proliferation rate of tumor in nude mice were 69.3%?71.9%?54.4%?52.4%?49.5%?46.90%?49.5%?46.6%.7.Methyl haematommate has influences on hematological biochemical parameters(TP/ALB/GLB)and blood routine index(RBC).The results show that the compound has some damage to the liver,but it can improve the immunity of the organism in vivo.8.The FCM assay indicated that Methyl haematommate altered the cell cycle when treated for 24 h,the percentages of cells in the G1 phase transition were increased and the percentages of cells in the S phase or G2/M phase transition were decreased,suggesting a possible cell cycle arrest at G0/G1 phase in HepG2 cells induced by the compound.9.37,000 lncRNAs and 34,000 mRNAs were siginificantly dysregulated(fold change>1.5)between methyl haematommate treated and control samples.The PLCB4 gene has the maximal interaction with the other genes and is a potential key regulatory gene in the network.We found 393 of gene notology and 93 of pathway for differentially expressed mRNAs.Conclusion:Methyl haematommate is isolated from Usnea for the first time.Both in vivo and in vitro,It has the inhibitory effects in a concentration-dependent manner.The underlying mechanisms are supposed to be related to the regulation of the cell cycle and gene expression.