Study On Screening And Anti-leukemia Effects Of Indole HDAC6/Tubulin Dual Target Inhibitors

Background and objectivesIndole compounds are naturally present in many plants,especially with cruciferous vegetables such as cauliflower,broccoli,cabbage,etc.,and various epidemiological studies suggest that the consumption of cruciferous vegetables can help fight cancer,resulting in a reduction morbidity of many cancers,such as lung cancer,prostate cancer,colorectal cancer and breast cancer.In recent years,as the pace of life has accelerated,cancer is one of the diseases that seriously threaten human health,of which the incidence has gradually increased.There are 8.2 million deaths because of cancer worldwide each year.According to report of the World Health Organization,by 2030,the number of cancers will reach 23.6 million.Previous studies have shown that indole derivatives show good biological activity in anti-tumor,usually by regulating the inducing apoptosis,cell cycle,inhibiting angiogenesis and cell invasion to play an anti-cancer effect.At present,a variety of indole anti-tumor drugs have been developed for clinical application.In this paper,a series of novel indole compounds were obtained and compounds with good anti-tumor activity was screened.Then we evaluated the pharmacodynamics of 3ab to study its effects on the proliferation of leukemia cells,and on the apoptosis,and cycle,invasion,and autophagy in vitro Action targets of 3ab were screened through molecular docking simulation experiments,and were verified at the cellular and molecular levels,subsequently,elucidating the molecular mechanisms and targets of compound 3ab against leukemia.Methods1.Indole compounds with anticancer activity was screened.Indole compounds with anticancer activity were screened by the MTT assay,taking Cisplatin(DDP)as a positive control drug,so as to detect their inhibitory effects on the proliferation of K562,HT-29 and A549 cells,and screen out the best anti-cancer compounds.2.The type of sensitive cell lines to compound 3ab was determined.The toxicity effects of compounds 3ab and 3ai on many tumor cells was detected by the MTT assay,with DDP as a positive control drug.The inhibitory of the active compounds 3ab and 3ai on a variety of tumor cells(A549,H460,K562,HL-60,HT-29,B16,K562,MDA-MB-232,BGC-823,PC-3)was determined.3.The cytotoxic effect of compound 3ab on normal human cells was detected.The cytotoxic effects of compound 3ab on human normal tissue cells(HUVECs,GES-1)was detected by the MTT assay,with DDP as a positive control drug.4.The morphology of K562 and HL-60 cells treated with compound 3ab was observed.The cell morphology change of K562 and HL-60 cells treated with compound 3ab was observed under inverse microscope.5.Uptake effect of compound 3ab by K562 and HL-60 cells was detected.Uptake effect of compound 3ab by K562 and HL-60 cells was detected by flow cytometry as a result of the autofluorescence of 3ab.6.The effect of compound 3ab on the apoptosis in K562 and HL-60 cells was detected.Hoechst 33342 staining experiment were used to detect the effect of compound 3ab on the apoptosis in K562 and HL-60 cells,and Annexin V-FITC/PI double staining experiment was used to further quantify the apoptosis of K562 and HL-60 cells induced by compound 3ab.Then,the JC-1 fluorescent probe assay was used to detect the mitochondrial membrane potential of K562 andHL-60 cells after treatment with compound 3ab.Finally,Western blotting was used to detect the effect of 3ab on apoptosis-related protein levels in K562 and HL-60 cells.Simultaneously,the DCFH-DA fluorescent probe method was used to detect the effect of compound 3ab on the level of ROS in K562 and HL-60 cells.7.The effect of compound 3ab on cycle distribution in K562/HL-60 cells was detected.PI staining experiment was used to detect the effect of compound 3ab on cycle distribution in K562 and HL-60 cells.Finally,Western blotting was used to detect the effect of 3ab on the expression level of cycle related proteins.8.The effect of compound 3ab on the invasion ability of K562 and HL-60 cells was detected.Transwell assay was used to detecte the effect of compound 3ab on the invasion ability in K562 and HL-60 cells.9.The level of autophagy was detected in K562 and HL-60 cells treated with compound 3 ab.The acridine orange staining assay was used to detect the autophagy level in K562 and HL-60 cells treated with compound 3ab.10.The targets of compound 3ab werescreened by molecules docking simulation experiment.Computer simulation technology was used to simulate the docking,finding the target of compound 3ab and related protein molecules for target screening11.Effect of compound 3ab on the microtubulin was detected in K562 and HL-60 cells.The immunofluorescence experiment was used to detect the microtubulin in K562 and HL-60 cells treated with compound 3ab.Then,the EBI competitive binding experiment was used to detect whether compound 3ab binds to the colchicine site of tubulin.12.The level of HDAC6 protein in K562 and HL-60 cells treated with compound 3ab was detectedWestern blotting was used to detect the level of HDAC6 protein and related pathway proteins in K562 and HL-60 cells treated with compound 3ab13.The inhibitory effect of compound 3ab on the growth of K562 xenograft tumor in nude mice was detected.The mouse transplanted tumor model of K562 cells was established and the mouses were administered 3ab continuously for 18 days.The effect of compound 3ab on the growth of transplanted tumors in vivo was observed.At the same time,the changes in mouse body weight and transplanted tumor volume were recorded.After the experiment,the transplanted tumor was moved,weighed,and variation in the levels of tubulin and HDAC6 content were detected by immunohistochemistry and Western blotting.Results1.The IC50 valuesof compounds 3ab and 3ai for K562 cells were 4.8 μM and 13.3 μM,respectively.The IC50 of 3ab for K562 cells was lower than that of 3ai,and its inhibitory effect on K562 was better than that of DDP.Therefore,the compounds 3ab and 3ai were selected for the next step of screening for sensitive tumor cell lines.2.Compounds 3ab and 3ai were effective against breast cancer cells(MDA-MB-231),liver cancer cells(K562),colon cancer cells(HT-29),gastric cancer cells(BGC-823),prostate cancer cells(PC-3),melanoma cells(B16)and leukemia cells(K562 and HL-60),with IC50 values ranging from 4.63 to 35.17 μM and 8.87 to 39.42 μM,respectively.Because the IC50 of 3ab was lower than 3ai,and the stability of 3ai was poor.Therefore,3ab was finally selected as the research object to explore its anti-tumor effect on leukemia cells and its molecular mechanism.3.The IC50 of 3ab against human umbilical vein endothelial cells(HUVECs)was 14.73 μM,and against human normal gastric mucosal cells(GES-1)was 17.14μM,both higher than DDP.Therefore,compound 3ab had low toxicity on human normal cells.4.Compound 3ab changed the morphology of K562 and HL-60 cells.The size of the cell morphology varied and became smaller,and the cell debris increased significantly.5.Compound 3ab could be uptaken by K562 and HL-60 cells and accumulate in a concentration-dependent manner.6.Compound 3ab could induce the apoptosis of K562 and HL-60 cells,and cause changes in apoptosis-related proteins,specifically up-regulation of Cleaved-Caspase3 and Cleaved-PARP as well as down-regulation of Bcl-2.This kind of apoptosis occured through the mitochondrial pathway,and 3ab could increase the ROS level in K562 and HL-60 cells in a concentration-dependent manner7.After treatment with diverse concentrations of compound 3ab on K562 and HL-60 cells,the number of cells arrested in G2/M phase will increase and related proteins,Cyclin B41 and CDK1,were upregulated specifically.8.Compound 3ab could increase the ROS level in K562 and HL-60 cells in a concentration-dependent manner.9.Compound 3ab did not affect the autophagy of K562 cells,but could promote the autophagy of HL-60 cells in a concentration-dependent manner10.The action target of compound 3ab may be the tubulin colchicine site and the CD2 domian of HDAC611.Compound 3ab could shrink the microtubule network in K562 and HL-60 cells and gather around the nucleus in a concentration-dependent manner.12.After treatment with compound 3ab,the expression of HDAC6,EGFR,p-AKT and NF-κB in K562 and HL-60 cells was down-regulated in a concentration-dependent manner.13.Compound 3ab could significantly slow down the growth rate of K562 transplanted tumors in nude mice.At the same time,the cell tubulin shranked and the red fluorescence intensity of tubulin decreased in the transplanted tumors of compound 3ab group which was concentration-dependent.The expression of HDAC6 in the transplanted tumors of compound 3ab group also decreased with the increase of the dose of compound 3 ab,indicating that compound 3 ab may be a tubulin/HDAC 6 dual target inhibitor.ConclusionCompound 3ab had a good inhibitory effect on the proliferation of leukemia K562 and HL-60 cells in vitro and in vivo,and could induce G2/M phase arrest and cell apoptosis,and also had a certain degree of influence on theautophagy and invasion.The anti-leukemia effect of compound 3ab was related to inhibit the function of microtubules by binding to colchicine site of tubulin,leading to the G2/M phase arrest.At the same time,by binding to CD2 domain of HDAC6 and inhibiting its expression,compound 3ab induced cell mitochondrial apoptosis via down-regulating the EGFR/AKT/NF-κB pathway.Therefore,3ab is a tubulin/HDAC6 dual target inhibitor with good anti-tumor activity.SignificanceThe indole compound 3ab screened in this article had a good anti-leukemia effect.In view of its good anti-proliferative effect in vitro and in vivo,we continued to explore its role in promoting apoptosis and anti-invasion,and further elucidate its action target and molecular mechanism.To provide new ideas for the development of anti-leukemia,to use more effective chemical drugs in clinical treatment to bring new hopes,and to explore the various possibilities of indole compounds.Compound 3ab as an effective tubulin/HDAC6 dual target inhibitor may be used in cancer treatment,but further experimental verification is required.