Study On The Effects And Mechanisms Of A Novel Smoothened Protein Antagonist TPB14 On Colon Cancer

BackgroundHedgehog(Hh)signaling pathway plays an important role in the normal development of mammalian embryos and the maintenance of homeostasis.Multiple studies have shown that the Hh signaling pathway is abnormally activated in a variety of tumors.Therefore,the research and development of anti-tumor drugs targeting the Hh pathway is a current research and development hotspot.Among them,the research and development of Hh signaling pathway component Smoothened(SMO)is at the forefront.The SMO protein inhibitors Vismodegib(Vis),Sonidegib and Glasdegib have been approved to market for the treatment of Basal cell carcinoma(BCC)and Acute myeloid leukemia(AML).However,the major side effects and drug resistance of the listed drugs were found in the clinical stage.New SMO protein inhibitors with high efficiency and low toxicity should be developed to overcome this problem.In the previous study,our group designed,synthesized and screened a series of novel SMO targeted inhibitors,among which TPB14 is closely bound to SMO protein,can specifically inhibit the activation of Hh-gli pathway,and has a wide range of anti-tumor cell proliferation activity.ObjectivesThe purpose of this study was to investigate the effect of TPB14 on the proliferation of colon cancer cells in vitro and in vivo,and to explore its mechanism.Methods1.The effect of compound TPB14 on the proliferation of colon cancer cells was detected by MTT EdU cell proliferation assay and plate cloning assay.The effect of compound TPB14 on senescence of colon cancer cells was analyzed bygalactosidase staining.2.The effect of TPB14 on cell migration in colon cancer was detected by scratch test and Transwell test.3.The effect of TPB14 on cell cycle and apoptosis of colon cancer cells was detected by flow cytometry.4.The confocal assay examined the effect of TPB14 on the localization and expression of SMO protein and Glil in cells.5.Acute toxicity test to evaluate the toxicity of TPB14 in vivo.Pharmacokinetic experiments were conducted to investigate the bioavailability and pharmacokinetic characteristics of TPB14 in animals.6.The xenotransplantation model of colon cancer was constructed in nude mice to study the anti-colon cancer cell growth effect of TPB14.The toxicity of TPB14 was analyzed by HE staining of main tissue samples.7.Western-blot and QRT-PCR were used to evaluate the effects of TPB14 on the levels of proteins and mRNA of the main components of the Hh signaling pathway in animal tumor tissues.8.Phosphorylated protein microarray and high-throughput transcriptome sequencing were used to investigate the preliminary mechanism of compound TPB14 in the anti-proliferation of colon cancer cells,and the results were further verified by Western blot.Results1.MTT experimental results showed that while TPB14 inhibited the proliferation of tumor cells,it had almost no cytotoxicity to normal breast epithelial cells(IC50>100μM).At the same time,TPB14 can also inhibit the formation of colon cancer cell cloning,but does not induce cell aging.2.The results of the scratch test and the Tramswell test showed that TPB14 could inhibit the migration of colon cancer cells(p<0.01).3.Flow results showed that TPB14 could block colon cancer cells in G0/G1 phase(p<0.05)and induce apoptosis(p<0.01).4.Confocal test results showed that no SMO protein expression was observed in the primary cilium after TPB14 administration,and Glil expression was decreased in cytoplasm and nucleus.5.Acute toxicity test results showed that the LD50 value of TPB14(>5000 mg/kg)was greater than the positive control drug Vis(2008.17 mg/kg),but the oral bioavailability in rats was lower than the positive control drug Vis.6.In the tumor-bearing experiment of nude mice,the anti-tumor effect of TPB14 at the gavage dose of 100 mg/kg was better than that of the positive drug Vis at the same gavage dose,and the experimental results showed a statistically significant difference(p<0.01).At the same time,the body weight of the experimental animals did not change significantly during the 21-day administration.The results of HE staining showed that TPB14 did not produce obvious toxicity to the main organs of nude mice and had obvious damage to tumor cells within the dose range.7.Western blot and QRT-PCR experiments confirmed that TPB14 treatment can reduce the levels of Hh signaling components Gli1 and Gli2 proteins and mRNA,and increase the levels of Sufu proteins and mRNA,with no significant effect on the expression of PTCH2 and SMO proteins.8.The phosphorylated protein microarray identified 122 different phosphorylated proteins.A total of 18,514 differential genes were identified by highthroughput sequencing.Top 50 differentially phosphorylated proteins and differentially expressed genes were significantly enriched to PI3k-Akt,Ras and cell cycle-related signaling pathways.ConclusionsThis study confirmed that the compound TPB14 showed good anti-colon tumor activity in vivo and in vitro by inhibiting the Hh signaling pathway.