Identification Of Pseudolaric Acid B As A Novel Hedgehog Pathway Inhibitor In Medulloblastoma

ObjectiveAberrant activation of Hedgehog(Hh)pathway is implicated in the pathogenesis and development of multiple cancers,especially Hh-driven medulloblastoma(MB),one of the most common malignant brain tumors in children.Shh subtype MB accounts for about 30%of all MB patients.Surgical resection followed by craniospinal irradiation and adjuvant chemotherapy,the mainstay treatments for MB patients,frequently lead to long-term serious adverse reactions such as cognitive dysfunction.Thus,people turn their attention to molecular targeted therapy.Smoothened(SMO),a key regulatory element in the upstream of Hh pathway,has been identified as a new therapeutic target for Hh-driven MB.To date,a variety of SMO antagonists have entered human MB clinical trials,achieving promising results.However,the therapeutic application of SMO antagonists is hampered by the occurrence of drug resistance caused by SMO mutations or genetic alterations in downstream elements.Hence,identification of novel Hh pathway-targeted drugs that are capable of overcoming drug resistance is urgently needed for MB treatment.Pseudolaric acid B(PAB)is a natural small-molecule compound with broad-spectrum anti-cancer activity.Currently,the effects of PAB on Hh pathway and MB therapy have been rarely reported.Therefore,this study aims to investigate the anti-cancer activity of PAB in MB therapy and examine the potential molecular mechanisms both in vitro and in vivo.MethodsFor the in vitro experiments,first,we screened 26 natural small anticancer compounds by MTT assay and found that PAB exhibited the most potent cytotoxic effect on human medulloblastoma cell line(DAOY).Then,we selected ten tumor cell lines including DAOY,human lung cancer cell lines(A549,H1299),human gastric cancer cell lines(BGC-823,SGC-7901,AGS),human breast cancer cell lines(MDA-MB-231),human cervical cancer cell lines(Hela),and human liver cancer cells Lines(SMMC-7721,MHCC-97H)to detect the Gli1 protein expression by western blotting and determine cell viability by MTT assay in cells treated with indicated concentrations of PAB.Next,DAOY cells,primary granule neuron precursor cells(GNPs),primary MB cells isolated from Ptch1+/-spontaneous MB mice,and 3T3/GLI-luc cells were chosen as the research objects.They were treated with indicated concentrations of PAB or SMO agonist SAG;meanwhile SMO antagonist cyclopamine or vismodegib was used as a positive control,then MTT assay was performed to detect cell viability,clone formation experiment or EdU incorporation experiment were used to detect cell proliferation,flow cytometry was performed to detect cell apoptosis,western blotting was employed to detect apoptosis-related proteins,Gli1,SMO,cyclin D1 and N-myc protein expression.The tumoroids forming assay was applied to determine the ability of PAB to suppress MB tumoroids formation,the luciferase reporter gene detection was adopted to detect the transcriptional activity of GLI,and the qRT-PCR was used to measure the expression of Hh target genes.Meanwhile,MTT assay was used to detect cell viability,and western blotting was used to detect Glil,cyclin D1,N-myc and SMO protein expression in cells whose SMO was knockdown by specific siRNA.In addition,we constructed NIH3T3 cells overexpressing SMO-GFP,293 T cells overexpressing SMO-WT,and 3T3/GLI-luc cells overexpressing SMO-WT,SMO-D477G,and SMO-W539L,respectively.These cells were treated with indicated concentrations of PAB or SAG,in which vismodegib as used as a positive control.Meanwhile,we used molecular docking to predict the binding potential of PAB to SMO,BODIPY-cyclopamine assay to verify that PAB bound to SMO,CETSA assay to detect SMO stability,luciferase reporter assay to determine GLI transcriptional activity,ciliogenesis and SMO trafficking assay were used to detect cilia and SMO ciliary translocation,and western blotting to detect Glil protein expression,respectively.For in vivo experiments,we used primary MB cells isolated from Ptch1+/-spontaneous MB mice to construct a MB allograft model in BALB/c nude mice;once the tumor volume reached to approximately 100 mm3,the mice were divided into 4 groups randomly including vehicle solvent group,low-dose PAB group(50 mg/kg),high-dose PAB group(100 mg/kg)and vismodegib group(20 mg/kg);tumor volume and body weight of mice were measured every the other day,after continuous administration for 18 days,the tumors were collected to weight,detect apoptosis and proliferation-related proteins and Glil protein by western blotting,examine Hh target gene expression by qRT-PCR,detect Ki-67 expression level by immunohistochemistry,and observe the histopathological morphology by H&E staining,respectively.ResultsFor in vitro experiments,PAB exhibited the most potent cytotoxic effect on DAOY cells.Compared with other tumor cell lines,PAB exhibited a preferable cytotoxic effect on DAOY cells with an IC50 value of 0.83 μM.PAB inhibited DAOY cells clone forming,up-regulated apoptosis-related protein including cleaved-caspase-3 and PARP cleavage,and induced apoptosis;consistently,PAB also suppressed cell viability of primary MB cells,reduced the percentage of EdU-positive cells,and inhibited tumoroids formation.In terms of Hh pathway inhibitory effect,PAB attenuated the GLI luciferase reporter activity and the transcription activation of Hh target genes,including Gli1 cyclin Dl,and N-myc induced by SAG.Meanwhile,PAB also down-regulated the Glil protein levels in DAOY cells with or without SAG induction,and inhibited the transcriptional activity of Glil and cyclin D1;in the same way,PAB can inhibit the transcriptional levels of Hh target genes,including Gli1,cyclin D1,and N-myc in primary MB cells.Consistently,PAB can down-regulate the expression levels of Hh target proteins including cyclin D1 and N-myc in both DAOY cells and primary MB cells.However,the down-regulation effect of PAB in Glil、SMO、cyclin D1 and N-myc protein in primary MB cells whose SMO was knockdown by specific siRNA almost disappeared.Mechanically,although PAB had no effect on total SMO protein in DAOY cells,molecular docking predicted PAB has two binding sites to SMO,one of which was located at the extracellular entrance pocket of SMO transmembrane domain(TMD),while the other was located at the intracellular loops(ICLs)of TMD;PAB was found to compete with BODIPY-cyclopamine to bind to SMO.Furthermore,CETSA assay showed that PAB can improve the stability of SMO protein.PAB also suppressed ciliogenesis,but had no effect on ciliary translocation of SMO.In terms of overcoming drug resistance,PAB showed a similar inhibitory effect on GLI transcriptional activity in 3T3/GLI-luc cells overexpressing SMO-WT,SMO-D477G and SMO-W539L.Consistently,PAB down-regulated Gli1 protein level in these three cell lines.In addition,PAB exhibited comparable inhibitory effects on the GLI transcription activity in 3T3/GLI-luc cells treated with escalating concentrations of SAG.For in vivo experiments,PAB significantly inhibited the growth of MB allograft,and had no effect on the body weight of mice.Moreover,PAB increased the levels of PARP cleavage and cleaved-casepase-3.Moreover,nuclear aggregation was also observed in tumor tissues by H&E staining.In addition,PAB also down-regulated the PCNA and Ki-67 expression levels in tumor tissues;consistent with the in vitro experiments,PAB down-regulated the Glil protein level and the mRNA expression of Gli1,cyclin D1,and N-myc in tumor tissues.conclusionIn this study,we investigated the molecular mechanism by which Pseudolaric acid B suppresses the growth of medulloblastoma.We found that PAB inhibited MB growth both in vitro and in vivo by blocking the activity of Hh pathway.Moreover,PAB can also overcome SMO antagonist resistance induced by SMO mutations.Mechanically,we demonstrated that PAB directly binds to two sites on SMO and inhibits ciliogenesis at the same time,suggesting that PAB targeted the Hh pathway through multiple mechanisms.Therefore,PAB is a potential therapeutic candidate for Hh-driven MB,especially for those encountering resistance to SMO antagonists,which provides a new direction for the development of novel Hh-targeted drugs.