Andrographolide Induces Cell Cycle Arrest And Apoptosis Of Chondrosarcoma By Targeting TCF-1/SOX9 Axis

ObjectiveTo explore the mechanism of Andro induces cell cycle arrest and apoptosis of chondrosarcoma cells.Methods1.Chondrosarcoma cells were treated with Andro,followed by the cell proliferation,cell cycle,and apoptosis were detected by CCK-8 assay,crystal violet staining,flow cytometry analysis and WB.2.The expression of SOX9 in different pathological grades of chondrosarcoma tissue was examined by IHC.Chondrosarcoma cells were treated with Andro,the total proteins,or the cytoplasmic and nuclear protein extracts were prepared and subjected to WB for the detection of SOX9 expression.3.SW 1353 cells were transfected with siSOX9,siβ-catenin,or siTCF-1,and then relative cell number was calculated,or the downstream proteins were detected by WB.4.To confirmed the role of Wnt/β-catenin signaling in chondrosarcoma,the subcellular localization of β-catenin and TCF-1 in chondrosarcoma cells was observed under confocal microscope and WB.5.In order to test whether Andro may directly affect protein stability or de novo protein synthesis of TCF-1 and SOX9.Chondrosarcoma cells were treated with Andro for indicate time in the presence or absence of MG132 or CHX,and so on,the expression of TCF-1 and SOX9 was examined by WB.6.To investigate the relationship between TCF-1 and SOX9 in chondrosarcoma cells,SW 1353 cells were transfected with siTCF-1,or siSOX9,then the mRNA and protein expression of β-catenin,SOX9,and TCF-1 were detected by RT-PCR and WB.In the end,chondrosarcoma cells were transfected with siTCF-1,followed by the treatment of Andro and MG132,the expressionof TCF-1 and SOX9 was examined by WB.Results1.Andro significantly inhibits chondrosarcoma cells growth,induces pronounced cell cycle arrest at G2/M phase,and promotes apoptosis.2.SOX9 is predominantly expressed in the nucleus,and its expression level is gradually increased in moderate-to poor-differentiated chondrosarcoma tissues.Andro diminishes the protein expression of SOX9 in chondrosarcoma cells,albeit of different cell densities.The down-regulation of SOX9 by Andro is mediated at both nuclear and cytoplasmic levels.3.Knockdown of SOX9,β-catenin or TCF-1,significantly inhibits SW 1353 cell growth,and down-regulates the expression of downstream proteins,respectively.4.Andro remarkably down-regulates TCF-1 expression at both mRNA and protein levels,while having little effect on the expression of β-catenin.Andro significantly diminishes nuclear TCF-1 expression,but do not alter the subcellular localization of β-catenin.5.Our investigation found the reduction of TCF-1 and SOX9 by Andro is completely blocked by the addition of proteasome inhibitor MG132.Moreover,CHX rapidly diminishes the expression of TCF-1 and SOX9.However,treatment of MG132 partially abrogates the down-regulation of TCF-1-mediated by Andro.6.Knockdown of TCF-1 significantly down-regulates the expression of SOX9 at protein level.However,knockdown of SOX9 do increase the protein expression,but not mRNA levels of β-catenin and TCF-1.Andro significantly down-regulates the expression of TCF-1,whereas knockdown of TCF-1 further enhances the down-regulation of SOX9-induced by Andro.ConclusionTaken together,the present study shows that Andro causes G2/M cell cycle arrest and subsequent apoptosis of chondrosarcoma cells.We also found that SOX9 is highly expressed in chondrosarcoma tissues,and that knockdown of SOX9 significantly inhibits cell growth via induction of p21.Further,we disclosed a mechanism that Andro exhibits its anti-cancer activity through targeting the mutual regulation of TCF-1 and SOX9,independent of β-catenin.