| At present,cancer is the second leading cause of death in the world,seriously endangering human health.Leukotriene A4 hydrolase(LTA4H)is a bifunctional zinc metalloenzyme with the activities of epoxide hydrolase and aminopeptidase,which has become a new target for anti-tumor drug development due to its role in the development of cancer.Based on the analysis of the inhibition effect of LTA4H structure,26 novel 1,5-disubstituted indole derivatives were designed and synthesized.In this study,26 indoles derivatives were selected as the research objects.Compounds with inhibitory effects on LTA4H were screened first,and then their effects on the proliferation,migration and angiogenesis of tumor cells were studied at the cellular level by using the candidate compounds.Firstly,the recombinant plasmid pET-28a/LTA4H was successfully transformed into e.coli translator induced protein expression with high purity and a concentration of 1.25 mg/mL.By testing its activity,it was determined that the optimal test concentration of the activity inhibition of this enzyme should be set to 33 μg/mL,and a stable LTA4H inhibitor screening platform was successfully established.The test results of 26 compounds showed that compounds 1,2,5,7,8,11 and 12 had a good inhibitory effect on the activity of ammonia peptidase in LTA4H,and the inhibition rate of ammonia peptidase at 10 was about 80%,close to that of positive control Bestatin.These seven candidate compounds also had high inhibition rate of LTA4H hydrolase activity measured by LTB4 ELISA kit.Thus,seven compounds with good inhibitory effect on LTA4H enzyme activity were obtained.Next,the toxicity of these seven compounds to human tumor cells and normal cells was tested by MTT assay.The results showed that compared with other tumor cell lines,HCT116 was the most sensitive to these seven candidate LTA4H inhibitors,and compound LHB-A235-133-1 had the most broad-spectrum and effective inhibition of tumor cell proliferation.The IC50 value of LHB-A235-133-1 acting on HCT116 is 16.53 μM.Finally,the antitumor activity of LHB-A235-133-1 was studied.Western Blot was used to detect the expression level of LTA4H in tumor cells used in the anti-tumor spectrum test,and the results showed that the endogenous LTA4H expression level of HCT116 was the highest,which was consistent with the above experimental results,suggesting that the candidate LTA4H inhibitor inhibits the proliferation of tumor cells was related to the inhibition of the expression of endogenous LTA4H in tumor cells.Western Blot analysis showed that LHB-A235-133-1 could inhibit the expression of LTA4H in colon cancer cells HCT116.Morphological observation,MTT experiment and scratch repair experiment showed that LHB-A235-133-1 could affect the growth of HCT116 cells and inhibit the proliferation and migration of the cells.The results of apoptosis and cycle experiments showed that LHB-A235-133-1 could induce the apoptosis of HCT116 cells and make the cycle stop at G1 phase.In addition,in vitro tube formation experiments have shown that LHB-A235-133-1 can affect the vascularization of human umbilical vein endothelial cell HUVEC and destroy its lumen structure.In summary,the candidate compound LHB-A235-133-1 with inhibition effect of LTA4H was screened in this study and its effect on proliferation,migration and angiogenesis of colon cancer cells HCT116 was studied,which laid an important foundation for further study on the anti-tumor activity of these compounds and the regulation mechanism of LTA4H. |
