Posaconazole-Inhibited Hedgehog/SMO Signaling Induces Autophagy And Cell Cycle Arrest In Rhabdomyosarcoma

Purpose:In this study,we aimed to reveal the anti-tumor mechanism of Posaconazole in Rhabdomyosarcoma and provide a new direction for targeted therapy of Rhabdomyosarcoma.Methods:1.Expression of SMO receptor in human bladder rhabdomyosarcomaImmunohistochemical staining of SMO expression in human bladder Rhabdomyosarcoma was conducted to confirm the status of Hedgehog signaling pathway in bladder rhabdomyosarcoma.2.To determine the inhibitory effect of Posaconazole on cell proliferation of human Rhabdomyosarcoma cellsXTT assay was used to determine the effect of Posaconazole on the cell proliferation of RD,RMS-YM and KYM-1 human Rhabdomyosarcoma cells.3.To investigate the anti-tumor mechanism of Posaconazole on human Rhabdomyosarcoma cells3.1 The changes of Hedgehog signaling pathway,cell cycle and autophagy-related proteins were analyzed by Western Blot to determine whether Posaconazole could induce autophagy and cell cycle arrest in Rhabdomyosarcoma cells through inhibiting Hedgehog signaling pathway.3.2 Flow cytometry FCM was performed to measure the cell cycle distribution in human Rhabdomyosarcoma treated with Posaconazole.3.3 The formation of autophagosomes in human Rhabdomyosarcoma cells treated with Posaconazole was observed by transmission electron microscopy TEM.3.4 The distribution and expression changes of LC3B in treated group were detected by immunofluorescence to further verify the occurrence of autophagy.4.To assess the anti-tumor effect of Posaconazole on RD xenograft tumor mice model4.1 RD xenograft mice model was established to evaluate the effect of Posaconazole in vivo.4.2 Hematoxylin-eosin staining was used to verify the inhibitory effect of Posaconazole on Rhabdomyosarcoma and to determine the degree of liver damage caused by Posaconazole in the mice model.4.3 Detection of serum biochemical markers of mice further proved that Posaconazole had no obvious side effects on mouse liver.Results:1.Posaconazole inhibited the Hedgehog signaling pathway in three Rhabdomyosarcoma cell lines1.1 Western Blot analysis showed that Posaconazole down-regulated the expression levels of SMO and Gli1 proteins in human Rhabdomyosarcoma cells.1.2 Immunohistochemistry of human bladder Rhabdomyosarcoma indicated that high expression of SMO receptor in bladder Rhabdomyosarcoma,compared with that of the adj acent tissue.2.Posaconazole suppressed the cell proliferation in human Rhabdomyosarcoma cellsPosaconazole(0,10,25,50,100 μM)inhibited the cell proliferation in a timeand concentration-dependent manner.3.Posaconazole triggered G0/G1 phase arrest in human Rhabdomyosarcoma cells3.1 According to the results of flow cytommetry,after Posaconazole treatment,more cells stagnated in the G0/G1 phase,and fewer cells progressed into the S phase and G2/M phase,indicating that Posaconazole induced cell cycle arrest in the G0/G1 phase.3.2 Western Blot demonstrated that Posaconazole up-regulated the expression of p21 WAF1/CIP1 while down-regulated the expression of c-myc,CDK4 and CDK6.4.Posaconazole induced cellular autophagy in human Rhabdomyosarcoma cells4.1 The results of transmission electron microscopy showed that autophagosomes were formed in Rhabdomyosarcoma cells treated with Posaconazole for 24 h.4.2Western Blot showed that in Posaconazole group,autophagy related proteins were significantly up-regulated with the concentration increasing.4.3 Immunofluorescence staining showed that LC3B levels in the cytoplasm and nucleus of RD and RMS-YM cells were significantly up-regulated after Posaconazole treatment.5.The anti-tumor effect of Posaconazole on RD cells was proved in vivo5.1 After the treatment of Posaconazole(70mg/kg),the tumor size and volume were significantly lower than those in the control group.5.2 Hematoxylin-eosin staining of xenograft tumor tissue sections showed that tumor cell infiltration in the treatment group was visibly less than that in the control group.5.3 The results of Hematoxylin-eosin staining of mice liver sections and serum biochemical markers of mice indicated that Posaconazole had no significant side effects on mice liver functions.Conclusion:Our research implies that Posaconazole suppresses cell viability and induces G0/G1 cell cycle arrest and autophagy in three Rhabdomyosarcoma cell lines through inhibition of Hedgehog signaling pathway.Moreover,our proposal might also be applicable in bladder RMS.Hence,we expect that Posaconazole to be a potent therapeutic agent targeting at Hedgehog signaling pathway in Rhabdomyosarcoma,even bladder Rhabdomyosarcoma.