Dataset Information

GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells.

ABSTRACT: Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line was the most sensitive (IC50: 36 µM) to the tested compound. The compound's effects on the expression of HH pathways components were analyzed by qPCR and Western blot; cell viability was analyzed by trypan blue assay and flow cytometry were used to investigate cell cycle phase, morphology, and death patterns in HSC3 cells. A significant reduction in mRNA levels of the GLI1 transcription factor was found after 12 h of treatment withGANT61. Protein expression levels of other HH pathway components (PTCH1, SHH, and Gli1) and HSC3 cell viability also decreased after 24 h of treatment. Cell cycle analysis and death pattern evaluations revealed significantly increased nuclear fragmentation in sub-G1 phase, as well as cell death due to apoptosis. In conclusion, the significantly reduced GLI1 gene expression seen in response to the GLI inhibitor indicates diminished downstream activation in HH pathway components. GANT61 significantly reduced cell viability in the metastatic cell line of OSCC and promoted a significant increase in nuclear fragmentation and cell death by apoptosis.

SUBMITTER: Bacelar Sacramento de Araujo T

PROVIDER: S-EPMC7503713 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells.

Bacelar Sacramento de Araújo Taís T de Oliveira Siquara da Rocha Leonardo L Torres Andion Vidal Manuela M Cerqueira Coelho Paulo Lucas PL Galvão Dos Reis Mitermayer M Solano de Freitas Souza Bruno B Botelho Pereira Soares Milena M Almeida Pereira Thiago T Della Coletta Ricardo R Pereira Bezerra Daniel D Borges Dias Rosane R Araújo Gurgel Rocha Clarissa C

International journal of molecular sciences 20200824 17

Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line ...[more]

PMID: 32846867

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Project description:ObjectivesTo investigate the potential role of Ribosomal protein L35 (RPL35) in regulating chondrocyte catabolic metabolism and to examine whether osteoarthritis (OA) progression can be delayed by overexpressing RPL35 in a mouse compression loading model.MethodsRNA sequencing analysis was performed on chondrocytes treated with or without 20 % elongation strain loading for 24 h. Experimental OA in mice was induced by destabilization of the medial meniscus and compression loading. Mice were randomly assigned to a sham group, an intra-articular adenovirus-mediated overexpression of the negative group, and an intra-articular adenovirus-mediated overexpression of the RPL35 operated group. The Osteoarthritis Research Society International score was used to evaluate cartilage degeneration. Immunostaining and western blot analyses were conducted to detect relative protein levels. Primary mouse chondrocytes were treated with 20 % elongation strain loading for 24 h to investigate the role of RPL35 in modulating chondrocyte catabolic metabolism and regulating cellular senescence in chondrocytes.ResultsThe protein expression of RPL35 in mouse chondrocytes was significantly reduced when excessive mechanical loading was applied, while elevated protein levels of RPL35 protected articular chondrocytes from degeneration. In addition, the RPL35 knockdown alone induced chondrocyte senescence, decreased the expression of anabolic markers, and increased the expression of catabolic markers in vitro in part through the hedgehog (Hh) pathway.ConclusionsThese findings demonstrated a functional pathway important for OA development and identified intra-articular injection of RPL35 as a potential therapy for OA prevention and treatment.The translational potential of this articleIt is necessary to develop new targeted drugs for OA due to the limitations of conventional pharmacotherapy. Our study explores and demonstrates the protective effect of RPL35 against excessive mechanical stress in OA models in vivo and in vitro in animals. These findings might provide novel insights into OA pathogenesis and show its translational potential for OA therapy.

Project description:Background and aimsThe cluster of differentiation 47 (CD47)-signal regulatory protein alpha (SIRPα) signaling pathway plays important roles in immune homeostasis and tissue inflammatory response. Activation of the Hedgehog/smoothened (SMO)/GLI family zinc finger 1 (Gli1) pathway regulates cell growth, differentiation, and immune function. However, it remains unknown whether and how the CD47-SIRPα interaction may regulate Hedgehog/SMO/Gli1 signaling in mesenchymal stem cell (MSC)-mediated immune regulation during sterile inflammatory liver injury.Approach and resultsIn a mouse model of ischemia/reperfusion (IR)-induced sterile inflammatory liver injury, we found that adoptive transfer of MSCs increased CD47 expression and ameliorated liver IR injury. However, deletion of CD47 in MSCs exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators. MSC treatment augmented SIRPα, Hedgehog/SMO/Gli1, and Notch1 intracellular domain (NICD), whereas CD47-deficient MSC treatment reduced these gene expressions in IR-stressed livers. Moreover, disruption of myeloid SMO or Notch1 increased IR-triggered liver inflammation with diminished Gli1 and NICD, but enhanced NIMA related kinase 7 (NEK7) and NLR family pyrin domain containing 3 (NLRP3) activation in MSC-transferred mice. Using a MSC/macrophage co-culture system, we found that MSC CD47 and macrophage SIRPα expression were increased after LPS stimulation. The CD47-SIRPα interaction increased macrophage Gli1 and NICD nuclear translocation, whereby NICD interacted with Gli1 and regulated its target gene Dvl2 (dishevelled segment polarity protein 2), which in turn inhibited NEK7/NLRP3 activity.ConclusionsThe CD47-SIRPα signaling activates the Hedgehog/SMO/Gli1 pathway, which controls NEK7/NLRP3 activity through a direct interaction between Gli1 and NICD. NICD is a coactivator of Gli1, and the target gene Dvl2 regulated by the NICD-Gli1 complex is crucial for the modulation of NLRP3-driven inflammatory response in MSC-mediated immune regulation. Our findings provide potential therapeutic targets in MSC-mediated immunotherapy of sterile inflammatory liver injury.

Dataset Information

GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells.

Publications

GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells.

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