Screening And Mechanism Study Of Novel Imidazo [1,2-?] Pyridine Compounds For Anti-tumor Activity

Objective: 1.Screening of a batch of novel imidazo [1,2-?] pyridines to obtain drugs with antitumor activity.2.To explore the antitumor mechanism of the novel imidazo [1,2-?] pyridines obtained through screening.Methods: 1.60 new imidazo [1,2-?] pyridine compounds were screened for antitumor activity in vitro by MTT assay,and active compounds LA23-30 and C1 obtained from preliminary screening were further tested for anti-proliferation effects.2.The effect of imidazo [1,2-?] pyridines LA23-30 and C1 on the proliferation of He La cells was evaluated by colony formation assay.3.Further observation of the effects of imidazo [1,2-?] pyridines LA23-30 and C1 on the proliferation of He La cells was performed by detecting lactate dehydrogenase activity..4.Caspase inhibitor Z-VAD-FMK was used to observe whether New Imidazo [1,2-?] pyridines LA23-30 or C1 inhibitted cell proliferation through apoptosis.5.Western blotting and cell immunofluorescence were used to assess the effect of imidazo [1,2-?] pyridines LA23-30 or C1 on the expression of autophagy-related protein LC3 B in tumor cells.6.Ad-m Cherry-GFP-LC3 B double-fluorescent adenovirus was used to test the effect of compound LA23-30 or C1 on the autophagy flux.7.Immunofluorescence double-staining of LC3 B and Lamp1 protein was used to observe the effect of compound LA23-30 or C1 on the fusion of autophagosome and lysosome in He La cells.8.The colocalization of Lyso-Tracker Red and the lysosomal marker protein Lamp1 protein was performed by immunofluorescence to observe the effect of compound LA23-30 or C1 on the lysosomal acid environment of He La cells.9.The effect of compound C1 on the expression of autophagy-related protein Rab7 in He La cells was analyzed by western blotting.10.Co-localization analysis of LC3 B and Rab7 proteins was used to observe the effect of compound C1 on the expression of autophagy-related protein Rab7 in He La cells.11.A tumor-bearing model was established by subcutaneously injecting a He La cell suspension into the axillary region of the right forelimb of the back of a nude mouse.12.The effect of compound LA23-30 on tumors in vivo was detected by monitoring the size of tumor and the volume of tumor in nude mice bearing tumors.13.Immunohistochemical detection was used to observe the effect of compound LA23-30 on the expression of autophagy related protein LC3 B in tumor-bearing tissues.14.The effect of compound LA23-30 on the physiological structure of heart,liver,spleen,lung,kidney and stomach of nude mice was observed by using hematoxylin-eosin staining.Result: 1.It was found that the novel imidazo [1,2-?] pyridines LA23-30 and C1 had a significant inhibitory effect on the proliferation of various tumor cells in a concentration dependent manner.2.The imidazo [1,2-?] pyridine compound LA23-30 could significantly inhibit tumor growth in vivo in tumor-bearing nude mice.3.Compared with the group treated with LA23-30 plus caspase inhibitor Z-VAD-FMK,compound LA23-30 had no significant effect on tumor cell apoptosis.Compared with the group treated with compound C1 plus caspase inhibitor Z-VAD-FMK,compound C1 tumor cells had a slight effect on apoptosis of tumor cells.4.The imidazo [1,2-?] pyridine LA23-30 and C1 can induced of increased autophagy in He La cells(characterized by significantly increased autophagosomes and increased LCB? / ? ratio)5.The compound LA23-30 had no effect on the autophagy flux of He La cells.However compound C1 blocked the autophagy flux which resulted into the accumulation of autophagosomes and / or autophagolysosomes.6.Co-localization analysis of autophagy-related protein LC3 B and lysosomal-related protein Lamp1 showed that compound C1 inhibited fusion of autophagosome and lysosome during autophagy flow in He La cells.The compound LA23-30 had no effect on the fusion.7.Co-localization analysis of lysosomal fluorescent probe Lyso-Tracker Red and lysosomal protein Lamp1 showed that neither compounds LA23-30 nor C1 altered the lysosomal acidic environment of He La cells.8.Compound C1 induced downregulation of an autophagosome and lysosomal fusion-related Rab7 protein in He La cells and also prevented Rab7 from being recruited to autophagosomes.9.The expression of LC3 B in tumor-bearing tissues of nude mice treated with compound LA23-30 was significantly up-regulated.10.The compound LA23-30 had no pathological effects in the heart,liver,spleen,lung,kidney,and stomach of nude mice.Conclusion: 1.After screening,the novel imidazo [1,2-?] pyridine compounds LA23-30 and C1 showed significantly inhibitory effects on proliferation of tumor cells.Moreover,compound LA23-30 demonstrated a strong inhibitory effect on tumor growth in vivo.2.The compound LA23-30 induced tumor cell death by promoting cell autophagy;while compound C1 down-regulates the autophagosome-lysosomal fusion-related protein Rab7 and prevented it from being recruited to autophagosome,thereby causing the autophagosome and lysosomal fusion disorder,leading to autophagy flux blocking,and autophagosome accumulation.