| Background and objectivesWith increasing morbidity and mortality,the global cancer burden is increasing and has become the second leading cause of death worldwide.At present,chemotherapy is one of the main cancer treatment methods.Chemotherapy drugs currently used often have large side effects and are prone to drug resistance,resulting in limited chemotherapy effects.Therefore,the development and application of compounds with effective anti-tumor activity is of great significance for cancer treatment.Indole and its derivatives have shown great biological activity in anti-tumor and have attracted widespread attention.Indole compounds show broad-spectrum biological activity in anti-tumor,anti-inflammatory,insecticidal and bactericidal.Previous studies have shown that indole derivatives can induce apoptosis and inhibit a variety of cellular signaling pathways,including the PI3K/Akt/mTOR signaling pathway.Several indole anti-tumor drugs have been developed,such as vinca alkaloids,indirubin,etc.In view of the potent anti-cancer activity of indoles,exploring novel indole derivatives would contribute to the development of anti-cancer drugs.In this paper,we screened a series of novel indole derivatives and obtained derivatives with good anti-tumor activity.Then we observed the effects of the active derivative 3p on proliferation,cycle distribution,apoptosis,autophagy,microtubules,ROS levels,DNA damage and metastasis of human liver cancer PLC/PRF/5 and BEL7402 cells,and explored the possible mechanism of action of 3p.Methods1.Screening of active derivatives with good anti-tumor activity from novel oxidized indole derivatives.We first detect the cytotoxicity of a series of derivatives on A549,K562 and HT-29 cells via MTT assay and using cisplatin(DDP)as a positive control drug,so as to obtain active derivatives that could effectively inhibit tumor cells.2.The screening of sensitive cell lines by detecting the inhibitory effect of most active derivative 3p and 3r on a variety of tumor cells.The cytotoxicity of the active derivative 3p and 3r on various tumor cells(A549,H1299,MDA-MB-231,PLC/PRF/5,BEL7402,SMMC7721,HCT116,HT-29,BGC-823 and PC-3)was determined by MTT assay and using DDP as a positive drug.3.The cytotoxicity of 3p on normal human cells(HUVECs,GES-1,HL-7702)was investigated by MTT assay.4.The uptake of the derivative 3p by PLC/PRF/5 and BEL7402 cells was detected using flow cytometer.5.Anti-proliferative activity of derivative 3p on PLC/PRF/5 and BEL7402 cells.The effect of 3p on cell population dependence and proliferation ability of derivative 3p on PLC/PRF/5 and BEL7402 cells was observed by cell colony formation assay.6.The effect of derivative 3p on cell cycle distribution of PLC/PRF/5 and BEL7402 cells was examined using PI staining assay by flow cytometiy.The expression levels of the cycle regulation-related proteins were observed through Western blotting assay.7.The inhibitory effect of derivative 3p on microtubules of PLC/PRF/5 and BEL7402 cells was observed using immunofluorescence assay by laser scanning confocal microscope.The molecular docking simulation of 3p and tubulin was performed by computer simulation technology.Furthermore,whether 3p works by binding to the colchicine binding site of tubulin was determined by EBI competitive binding assay.8.The effect of derivative 3p on the apoptosis of PLC/PRF/5 and BEL7402 cells was determined via Hoechst 33342 nucleic acid staining and Annexin V-FITC/PI double-staining assays.And the change of mitochondrial membrane potential of PLC/PRF/5 and BEL7402 cells after treated with 3p was detected by JC-1 fluorescent probe assay.Furthermore,Western blotting assay was used to observe changes of the apoptosis related proteins' expression level.9.The effect of derivative 3p on intracellular ROS levels in PLC/PRF/5 and BEL7402 cell was detected by DCFH-DA fluorescent probe assay.10.The effect of derivative 3p on DNA molecule damage of PLC/PRF/5 and BEL7402 cells.The expression level of DNA molecule damage marker protein(y-H2AX)was observed via Western blotting assay.11.The effect of derivative 3p on migration ability of PLC/PRF/5 and BEL7402 cells was observed through wound scratch assay.12.The effect of derivative 3p on PLC/PRF/5 and BEL7402 autophagy.The effect of 3p on the acidic autophagic vesicles in PLC/PRF/5 and BEL7402 cells was detected by acridine orange staining assay.Results1.Inhibitory effects of oxidized indole derivatives on the proliferation of tumor cells.The IC50 values of derivatives 3p and 3r for K562,A549 and HT-29 cells were 40 ?M,18.9 ?M,14.29 pM and 28 ?M,16.24 ?M,19.54 ?M,respectively.Therefore,the derivatives 3p and 3r were selected for the screening of sensitive tumor cells.Derivatives 3p and 3r had great inhibitory effect to lung cancer(A549,H299),breast cancer(MDA-MB-231),liver cancer(PLC/PRF/5,BEL7402,SMMC7721),colon cancer(HCT-116,HT-29),gastric cancer(BGC-823)and prostate cancer(PC-3)cells,with IC50 values ranging from 1.04 to 22.75 ?M and 3.84 to 25.05 ?M,respectively.And the effect of 3p was slightly better than 3r.Based on the current research status of liver cancer,there is a lack of chemotherapeutic drugs with great therapeutic effect and sensitivity.Therefore,the derivative 3p and liver cancer were selected as the research objects.2.Derivative 3p had low cytotoxicity on normal human cells.By detecting the cytotoxicity of 3p on human umbilical vein endothelial cells(HVECs),human normal gastric mucosal cells(GES-1)and human normal liver cells(HL-7702),it was found that the cytotoxicity of 3p to HL-7702 cells(ICso=29.21±2.47 ?M)was much weaker than DDP(IC50=1.72±0.14 ?M),and the cytotoxicity to HUVECs was also significantly lower than DDP.But the cytotoxicity of 3p to GES-1 cells was higher than DDP at higher concentrations.3.Derivative 3p could accumulate in PLC/PRF/5 and BEL7402 cells in a concentration-dependent manner.4.Derivative 3p could significantly inhibit the colony forming ability of PLC/PRF/5 and BEL7402 cells.5.Derivative 3p induced M phase arrest in PLC/PRF/5 and BEL7402 cells,which might be associated with the up-regulation of Cyclin B1,CDK1,p-Histone H3 and P53.6.Derivative 3p could bind to the tubulin of PLC/PRF/5 and BEL7402 cells,thereby inhibiting the function of microtubules and destroying cell structure.However.we only found that its binding site to tubulin was not the colchicine binding site.7.Derivative 3p induced mitochondrial dysfunction-related apoptosis in PLC/PRF/5 and BEL7402 cells by up-regulating the expression level of cleaved-PARP,cleaved-Caspase 3,Cyt C,Bad and Bax,as well as down-regulating the expression of Bcl-2.8.Derivative 3p could increase the ROS level of PLC/PRF/5 and BEL7402 cells in a concentration-dependent manner.9.Derivative 3p up-regulated the expression level of y-H2AX of PLC/PRF/5 and BEL7402 cells,indicating that 3p could induce DNA molecule damage.10.Derivative 3p could inhibit the migration of PLC/PRF/5 and BEL7402 cells.11.Derivative 3p had no effect on the autophagy of PLC/PRF/5 and BEL7402 cells.ConclusionDerivative 3p could inhibit cell growth and proliferation,induce G2/M phase arrest in PLC/PRF/5 and BEL7402 cells,as well as inhibit cell migration.Its molecular mechanism may be related to the inhibitory effect of 3 p on function of microtubules by binding to tubulin,subsequently arresting cells in the M phase and inducing the mitochondrial pathway mediated-apoptosis.At the same time,the inhibition of tubulin function could induce the increase of intracellular ROS levels and DNA molecular damage,thereby further promoting cell apoptosis.The interaction between the derivative 3p and tubulin is not mediated by binding to the colchicine binding site,and the mechanism of interaction between 3p and tubulin needs further study.SignificanceThe development and research of novel indole derivatives is expected to provide new ideas for the design of anti-tumor active derivatives and new directions for the development of tumor therapeutic drugs.Further exploration and optimization of derivative 3p may be used as an effective microtubule targeting agent for cancer treatment. |
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