Anticancer Effects Of Lichen Bioactive Compound Ethyl Haematommate And Its Mechanisms

Objectives:Isolated and appraise lichen bioactive compound.To investigate the in vitro anticancer effects on human cancer cell line and observe cell targeted and selectivity as well as the dose-effect and time-effect relationship.To know the form and strength of vitro combination anticancer effects on bioactive compound and clinical anticancer drugs DDP.Animals anti-cancer experiment was used to study the anti-cancer effect of bioactive compounds in nude mice transplanted human cervical cancer Hela,observe whether the bioactive compounds have toxic effects on animals at the same time.Gene chip and flow cytometry detection was used from the level of gene and cell level to study cancer gene expression and the influence of the cancer cell cycle distribution of bioactive compounds,and explore the active compounds of anticancer mechanism.Methods:1.The alcohol extraction,alcohol precipitation and chloroform extraction was used to separate anti-cancer active ingredients AMH-D.2.Column chromatography separation was used to separate anti-cancer active ingredients,combined with antitumor activity in vitro to gain bioactive compound G.3.Bioactive compound G was tested for purity and mass spectrometry,hydrogen spectrum and the carbon and dept detection,chemical structure analysis shall be conducted by experts.4.MTT method was used to detect in vitro anticancer effects of bioactive compound G on human cancer cell line HepG2,XWLC-05,A-549,MCF-7,T-24,NCI-H157,NCI-H460,CNE and BEAS-2B cells,observe dose-effect,time-effect and sensibility.To know cell targeted and selectivity on the in vitro anticancer effects of bioactive compound G on normal and cancer cell lines.Time-effect relationship of A-549 test was also observed.5.Kim formula was used to detect in vitro combination anticancer effects on bioactive compound G and DDP,and the dose-effect relationship was observed.6.Animals anti-cancer experiment was used to study the anti-cancer effect of bioactive compounds in nude mice transplanted human cervical cancer Hela,observe the influence compound G generate on Hela.7.Flow cytometry detection was used to detect the influence of the cancer cell cycle distribution on cancer cells by bioactive compound G,the influence of bioactive compound G were discussed on cellular level inhibition mechanisms of cancer cells8.Gene chip was used from the level of gene and cell level to study cancer gene expression and the influence of the cancer cell cycle distribution of bioactive-compounds,and explore the active compounds of anticancer mechanismResults:1.Lichen bioactive compound G was isolated.2.The purity of bioactive compound G was 97.448%.3.The formula weight of bioactive compound G was 224.1,themolecular formula 8,was C11H12O5.The English name was ethyl haematommate.4.In the in vitro anticancer effects experiment,the inhibitory ratios measured by MTT were 3.89%,11.09%,21.66%,46.27%,66.69%in HepG2;-3.78%,2.88%,21.85%,62.18%,87.53 in XWLC-05;9.31%,13.53%,12.41%,42.86%,93.80%in A-549:9.09%,11.37%,26.95%,58.10%,94.20%in MCF-7;5.13%,1.86%,47.74%,93.59%,99.30%in T-24;7.52%,8.16%,1.50%,36.86%,65.17%in NCI-H157:13.05%,13.60%,5.98%,35.07%,79.36%in NCI-H460;2.02%,1.97,%,7.10%,40.45%,70.64%in CNE when cancer cells were treated by ethyl haematommate after 72h.The obvious dose-effect relationships were showed in the in vitro anticancer effects experiment.5.The inhibitory ratios measured by MTT were 0.49%,-1.54%,-3.15%,8.36%,90.27%in BEAS-2B,the in vitro anticancer effects was very weak at low dose and selective anticancer effects was existed.6.The IC50 were 18.88 μg/ml,4.22μg/ml,12.41 jig/ml,10.24 μg/ml,7.68 μg/ml,37.11μg/ml,22.55μg/ml,22.84μg/ml in HepG2,XWLC-05,A-549,MCF-7,T-24,NCI-H157,NCI-H460 and CNE.The in vitro anticancer effects of ethyl haematommate had targeted.7.In the in vitro combinative anticancer study,the CDI were 0.57,0.56,0.68 in XWLC-05;0.58,0.48,0.53 in HepG2.The in vitro combinative anticancer effects were antagonism effect on XWLC-05 and HepG2.8.In the in vitro combinative anticancer study,the inhibitory ratios were40.24%,41.69%,55.84%in XWLC-05;52.70%,44.66%,49.78%in HepG2.9.In the vivo anticancer studies,the 50mg/kg ethyl haematommate were injected in Hela xenograft after 4,8,12,16,20 and 24,the relative tumor volume of ethyl haematommate group were 1.28,1.62,2.09,2.54,2.90 and 3.39;the relative proliferation ratios were 63.10%,60.27%,56.07%,46.89%,40.57%and 38.94%.Ethy haematommate had significant in vivo anticancer in Hela xenograft mice,the inhibition increased with time extend.10.The group of ethyl haematommate HepG2 cells were 79.68%in G0/G1 phase,14.95%in S phase and 5.37%in G2/M prase.Cell cycle distribution of cancer cell were changed by ethyl haematommate,the number of cells in G0/G1 phase were increased;the number of cells in S and G2/M prase were reduced.The cancer cells were retardant in G0/G1 phase,cell cycle distribution was intervened,cells proliferation were decreased.11.Compared with control group,301 mRNA and 325 IncRNA was changed outstanding in ethyl haematommate group,18 signal path were influenced.142 mRNA were up-regulated which with the largest fold change was 2.5202,and 158 mRNA were up-regulated which with the largest fold change was-2.2173.181 IncRNA were up-regulated which with the largest fold change was 3.2283,and 143 mRNA were up-regulated which with the largest fold change was-2.8199.Conclusions:1.Lichen bioactive compoundethyl haematommate was isolated.2.Ethyl haematommate had significant in vitro anticancer effects in HepG2,XWLC-05,A-549,MCF-7,T-24,NCI-H157,NCI-H460 and CNE.3.The selective anticancer effects was existed in ethyl haematommate.4.The in vitro anticancer effects had dosedependent relationship.5.Different cancer cells had different sensitivity:XWLC-05>T-24>MCF-7>A-549>HepG2>NCI-H460>BEAS-2B>CNE>NCI-H157;This results showed that ethyl haematommate has targeted anticancer effect.6.The in vitro combined anticancer of ethyl haematommate and DDP had synergistic in XWLC-05 and HepG2.The combined anticancer had time dependent relationship.7.Ethyl haematommate had significant anticancer effects in Hela xenograft mice in the in vivo anticancer experiment,the in vivo anticancer effects had time course relationship.8.Ethyl haematommate had no obvious toxicity on proliferation of nude mice weight and organs weight.9.Ethyl haematommate changed the distribution of cell cycle,the cancer cells were retardant in G0/G1 phase,cell cycle distribution was intervened,cells proliferation were decreased.10.The gene expression and pathways of cancer cells were changed by ethyl haematommate.