Identification And Functional Research Of Hedgehog Signaling Pathway Inhibitors

Background : Hedgehog(Hh)signaling pathway is one of the classical signal pathways that regulate insect and embryonic development.Hh signaling pathway is highly activated in a variety of human cancers,including basal cell carcinoma,,medulloblastoma,colon cancer,pancreatic cancer and so on..Emerging evidence suggests that inhibition of Hh signaling pathway suppresses Hh-dependent cancer cell proliferation and in vivo tumor growth.Vismodegib is the first Hh inhibitor approved for anti-cancer therapy by targeting Smoothened(SMO),a critical regulator of the Hh pathway.However,acquisition of drug resistance to vismodegib occurs overtime.Apoptosis is a prevalent form of programmed cell death that is executed by Caspases.Induction of tumor cell apoptosis represents an attractive therapeutic strategy to eliminate tumor cells.Therefore,identification of novel Hh inhibitors with apoptosis-inducing activity will offer new opportunities for the treatment of cancers.Methods: In order to identify Hh antagonists with apoptosis-inducing activity,we screened a set of ~300 potential SMO antagonists with novel scaffold structures..Hh003 was found to induce cell death.in multiple cancer cells.To further determine whether Hh003 induced cell death is apoptosis,we stained Hh003-treated cells with trypan blue and performed Annexin V/PI staining by flow cytometry technique.To identify the inhibitory effect of small molecular compound Hh003 on Hedgehog signaling pathway,we used the NIH3T3-GRE-Luc reporter system to detect their inhibitory effects.To further elucidate the molecular mechanism of apoptosis induced by Hh003,we examined apoptosis activation by monitoring the cleavage of caspase8,caspase9 and PARP by Western blot..In addition,we examined the effect of Hh003 on the formation of tumor colonies in vitro.Finally,we constructed a nude mouse xenograft model of human colon cancer cell line HCT116 to explore the anti-tumor effect of Hh003 in vivo.Results:(1)Hh003 induced cell death;(3)Hh003-treated cancer cells showedpositive Trypan blue staining;(4)Hh003-treated cancer cells showed positive Annexin V / PI staining;(5)Inhibition of caspases activity reduced Hh003-induced cell death(6)Hh003 inhibited Hh signaling pathway by directly targeting SMO;(7)Hh003induced activation of PARP and caspase8;(8)The apoptosis induced by Hh003 was prevented by Caspase8 silence,but not by Caspase9 silence;(9)Hh003 did not affect the autocrine of TNF-? in tumor cells(10)Hh003 significantly inhibited the formation of tumor colonies;(11)Hh003 repressed colorectal tumor growth in vivo.Conclusion:We identified Hh003 as a potent Hh inhibitor and effective apoptosis inducer.Hh003 activates caspase8-dependent extrinsic apoptosis pathway.Remarkably,Hh003 exhibits enhanced inhibition of tumor colonies in vitro and tumor growth in vivo compared to Vismodegib.These results are instructive for the development of new Hedgehog inhibitors and provide a new direction for the cure of cancer.