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Hedgehog/GLI1 signaling pathway regulates the resistance to cisplatin in human osteosarcoma

ABSTRACT: Purpose: This study aimed to investigate the role and mechanism of Hedgehog/GLI1 signaling pathway in regulating the resistance to cisplatin in osteosarcoma (OS). Materials and methods: Immunohistochemistry, western blotting and qRT-PCR assay were performed to analyze and compare the expression of GLI1 in OS tumor tissue and normal bone tissue as well as in cisplatin sensitive and resistant cell lines (SOSP-9607 and SOSP-9607/CR). Meanwhile, the biological role of GLI1 in OS was investigated by using down-regulated expression of GLI1 and functional assays, including CCK-8, colony formation assay, flow cytometry, and wound healing assay. Moreover, the relationship between GLI1 and γ-H2AX (DNA damage protein) in cells treated with GLI1 siRNA and cisplatin was examined using western blot analysis. In addition, GANT61, a inhibitor of Hedgehog pathway was used in xenograft tumor model to further verify the effect and mechanism of GLI1 on cisplatin resistance in OS. Results: We showed that GLI1 expression was up-regulated in OS patients and cisplatin-resistant cells. Silencing GLI1 significantly restored the sensitivity of OS to cisplatin, reduced proliferation, migration and cloning capacity of cisplatin sensitive and resistant cells, and increased the apoptosis rate in vitro. Furthermore, combined administration of GANT61 and cisplatin markedly inhibitted tumor growth in the mouse model. Mechanitic studies found that γ-H2AX is involved in the cisplatin resistance, and blockade of Hedgehog/GLI1 pathway increased the expression of γ-H2AX. Conclusion: Abnormal activation of Hedgehog-GLI1 pathway can regulate the expression of γ-H2AX, thus affecting DNA damage and repair functions, and promoting acquired cisplatin resistance of OS.

SUBMITTER: Chen D

PROVIDER: S-EPMC8518013 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of the Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B, and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunoblotting for microtubule-associated protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining, and transmission electron microscopy. Hh inhibition-induced autophagy was associated with up-regulation of Bnip3, as determined by immunoblotting and real-time polymerase chain reaction (PCR) assay. Knockdown of Bnip3 by RNAi impaired GANT61-induced autophagy. Additionally, Hh inhibition-induced autophagy was associated with Bnip3-mediated displacement of Bcl-2 from Beclin-1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST-1 assays. Pharmacological or genetic inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 small interfering RNA (siRNA) partially suppressed GANT61-induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3-MA.These findings provide novel evidence that Hh inhibition induces autophagy through up-regulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh-targeted therapies.

Project description:BackgroundMicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. Specifically, microRNA-221 (miR-221) is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-221 in human osteosarcoma has not been totally elucidated. In the present study, the effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated.Methodology/principal findingsReal-time quantitative PCR analysis revealed miR-221 was significantly upregulated in osteosarcoma cell lines than in osteoblasts. Both human osteosarcoma cell lines SOSP-9607 and MG63 were transfected with miR-221 mimic or inhibitor to regulate miR-221 expression. The effects of miR-221 were then assessed by cell viability, cell cycle analysis, apoptosis assay, and cisplatin resistance assay. In both cells, upregulation of miR-221 induced cell survival and cisplatin resistance and reduced cell apoptosis. In addition, knockdown of miR-221 inhibited cell growth and cisplatin resistance and induced cell apoptosis. Potential target genes of miR-221 were predicted using bioinformatics. Moreover, luciferase reporter assay and western blot confirmed that PTEN was a direct target of miR-221. Furthermore, introduction of PTEN cDNA lacking 3'-UTR or PI3K inhibitor LY294002 abrogated miR-221-induced cisplatin resistance. Finally, both miR-221 and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR and immunohistochemistry. High miR-221 expression level and inverse correlation between miR-221 and PTEN levels were revealed in osteosarcoma tissues.Conclusions/significanceThese results for the first time demonstrate that upregulation of miR-221 induces the malignant phenotype of human osteosarcoma whereas knockdown of miR-221 reverses this phenotype, suggesting that miR-221 could be a potential target for osteosarcoma treatment.

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Hedgehog/GLI1 signaling pathway regulates the resistance to cisplatin in human osteosarcoma

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