Development Of Anti-cancer Agents Targeting Both Hedgehog And Apoptosis Signaling Pathways

Background and significance: Hedgehog signaling pathway is abnormally activated in many solid tumors,such as basal cell carcinoma,medulloblastoma,leukemia and so on.Two drugs that targeted the key factor of SMO in the Hedgehog signaling pathway have been approved in recent years.Although these drugs show a promising anti-tumor effect,but the drug resistance of occurs overtime.Induction of tumor cell apoptosis is an important approach to kill tumor cell.Apoptotic cells are engulfed and digested immune cells to achieve the anti-tumor effect.The development of new anti-Smo compounds with pro-apoptotic activity will offer new opportunities for the treatment of cancers associated with aberrant activation of Hedgehog signaling.Objective: This study aims to develop pro-apoptotic Hedgehog inhibitors and explore the mechanisms of their actions and anti-tumor effect in vivo.Methods: In order to identify pro-apoptotic Hedgehog inhibitors,we screened a small molecule library of 300 candidate Hedgehog inhibitors.We found two pro-apoptotic small molecule inhibitors Hh78 and Hh262 that can activate apoptosis.In order to identify the inhibition of these two compounds on the Hedgehog signaling pathway,we used the NIH3T3-GRE-Luc reporter system to monitor their inhibitory effects.We also detected their induction of apoptosis in various tumor cells.By flow cytometry analysis of Annexin V / PI positive rate,we found Annexin V positive cells were observed in the presence of Hh78 or Hh262.Activation of caspase was detected by western blot in tumor cells treated with Hh78 or Hh262.The effect of Hh78 or Hh262 on body weight,blood system and organ was measured in vivo.The tumor model of nude mice xenograft with medulloblastoma cells(MB),which exerted aberrant Hedgehog was constructed to test the anti-tumor effects of Hh262.Results:(1)Several small molecule compounds are found to induce cell death,including Hh78 and Hh262.(2)Compounds show no obvious toxicity in normal cells.(3)Both Hh78 and Hh262 induce caspase-dependent cell death.(4)Tumor cells treated with Hh78 and Hh262 display Annexin V / PI positive.(5)Hh78 and Hh262 are more effective than Vismodegib based on the evaluation of the inhibitory effect on Hedgehog signaling pathway by using NIH3T3-GRE-Luc system.(6)Hh78 and Hh262 inhibit GLI and PTCH at the transcription level.(7)Hh78 and Hh262 can induce cell death in a variety of cancer cells.(8)Hh78 and Hh262 induce the activation of caspase8 and PARP.(9)Caspase8 silencing significantly inhibits apoptosis induced by Hh78 or Hh262.(10)Caspase9 silencing can not block Hh262 induced apoptosis.(11)Hh262 has no toxic in vivo.(12)Hh262 significantly inhibits tumor growth in mouse medulloblastoma-model.Conclusion: We found Hh78 and Hh262 inhibited the Hedgehog pathway,induced caspase8-dependent apoptosis in tumor cells.Our in vivo studies showed that Hh262 had significant antitumor effects in mouse medulloblastoma-model without obvious toxicity.These findings suggest that Hh78 and Hh262 have the potential for use in the development of anticancer therapies for the treatment of cancers associated with aberrant Hedgehog activity.