| BackgroundThere is an urgent need to further decipher the molecular mechanisms,explore new target biomarkers and identify therapeutic strategies to inhibit the progression of advanced GC.Desmocollin 2(DSC2)is the key component of functional desmosomal junction and also the only isoform of DSCs expressed in normal gastric tissue.DSC2 mRNA levels were significantly higher in stage I/II GC tissues than that in stage Ⅲ/Ⅳ samples of GC patients,suggesting DSC2 may play an important role in inhibiting distal metastasis of GC.However,the underlying mechanisms of DSC2 in GC progression require further exploration.We aimed to investigate the mechanism of DSC2 on suppressing the growth and metastasis of GC,and explore its upstream regulatory factors,so as to provide new therapeutic targets and plans for advanced GC.Modified low molecular citrus pectin(MCP)significantly increased DSC2 expression of carotid artery endothelial cells through activating SOD,while ROS inhibited the expression of DSC2.Considering that ROS promoted the development of most tumors,whlie SOD3 was recognized as a tumor suppressor among the three subtypes of SODs,we predicted that MCP might upregulate the expression of DSC2 in GC cells by upregulating the activity of SOD3.Oxaliplatin(OXA)is the first-line chemotherapy drug,while the remission rate of systemic chemotherapy based on OXA was just 40%～67%.In addition,chemotherapy-induced peripheral neuropathy(CIPN)induced by OXA through generating ROS also led to the termination of treatment.CIPN induced by OXA was dose-dependent,and reducing the dose of OXA through combining natural products and combined with compounds with anti-ROS activity were thought to be important plans to reduce the toxicity.Based on the premise that MCP could significantly increased the expression of DSC2 through activating SOD3,we speculated that MCP combined with OXA could enhance the cytotoxicity of OXA for GC cells,suppress the migration and invasion,and alleviate the symptoms of CIPN through anti-ROS.Based on the above,this therapeutic strategy is promising for the treatment of advanced GC.Part 1 Study on the effect and mechanisms of DSC2 on inhibiting the growth and metastasis of GCResearch purposesTo explore the effect of DSC2 in inhibiting the growth and metastasis of GC,and the associated molecular mechanisms.Research methods1)The relationship between the DSC2 expressions and the gastric carcinogenesis were analyzed using TCGA database,47 pairs fresh primary GC specimens from hospital,human gastric mucosal cell GES-1 and GC cells.2)We constructed GC cells that stably overexpression of DSC2 through lentivirus transfection,knocked down the expression of DSC2 through transfecting siRNA.Investigated the effect of DSC2 on the viability,migration and invasion through MTT assay,transwell assay,et al.3)BioGRID data and LC-MS/MS assay were performed to predict the key interacting proteins for DSC2.The regulatory effect of DSC2 on the targets were detected through Co-immunoprecipitation(Co-IP)assay.4)Mouse tumor xenograft model,tumor metastasis model and IVIS imaging were performed to investigate the effect of DSC2 on the growth and metastasis in vivo.5)BRD4 inhibitor JQ1,β-catenin transcription inhibitor ICG-001,PI3K inhibitor LY294002 and PI3K activator IGF1 were used to investigate the effects of DSC2 on key targets and signaling pathways.Experimental results1)The expression level of DSC2 was significantly downregulated in GC tissues and cells,which was negatively correlated with pTNM stage.2)DSC2 suppressed the viability,invasion and migration of GC cells both in cells and in xenografts.3)Overexpression of DSC2 increased the expression of complex ofγ-catenin/DSC2,reduced γ-catenin expression in the nucleus,upregulated the expression of P53 and PTEN,downregulated the level of BCL-2,and suppressed the phosphorylation of PI3K and AKT.4)IGF1 dramatically reversed the inhibitory effect on the survival rate of GC cells that stably overexpressed DSC2,and LY294002 markedly enhanced the inhibitory effect.While,neither IGF1 nor LY294002 affected the expression of DSC2 and the nuclear translocation of γ-catenin.5)Overexpression of DSC2 increased the complex of DSC2/BRD4,inhibited the nuclear translocation of BRD4 and β-catenin,downregulated the expression of Snail,N-cadherin,CD44 and MMP9.6)JQ1 and ICG-001 significantly inhibited the migration and invasion of GC cells(sgRNA-NC/Low-DSC2),but not further inhibited the migration and invasion of GC cells that overexpressing DSC2.SummaryDSC2 was significantly downregulated in GC tissues and GC cells.DSC2 suppressed the growth of GC mainly through inhibiting the nuclear translocation and transcriptional activity of y-catenin.DSC2 suppressed the metastasis of GC mainly through inhibiting the BRD4/Snail signaling pathway and the transcriptional activity of β-catenin,downregulating the expression of Snail,N-cadherin,CD44 and MMP9,and inhibiting epithelial-mesenchymal transition(EMT).Part 2 Study on the effect and mechanism of MCP on regulating the expression of DSC2 in GC cells through upregulating the expression of SOD3Research purposesWe aimed to evaluate the effect and mechanism of MCP on regulating the expression of DSC2.Research methods1)NBT and qRT-PCR assay were performed to analyze the SOD acitivity and the expression of SOD1,SOD2 and SOD3 mRNA after treating with MCP.2)qRT-PCR and Western blot assay were performed to analyze the DSC2 expression in GC cells after transfecting with siSOD1,siSOD2 and siSOD3,respectively.3)Western blot assay were performed to analyze the expression of SOD3,EGFR,PKP3 and DSC2 after treating with MCP.4)SOD inhibitor DETC and EGFR inhibitor AG1478 were used to investigate the effects of MCP on key targets and signaling pathways.Experimental results1)MCP upregualted the activity of SOD both in cells and in culture.MCP upregualted the expression of SOD3 mRNA,but not affect the expression of SOD1 or SOD2.2)SOD3 upregulated the expression of DSC2 in GC cells,while SOD1 and SOD2 didn’t regulate the expression of DSC2.3)MCP upregualted the expression of SOD3 in GC cells.Both MCP and SOD3 upregualted the expression of PKP3,DSC2,and inhibited the phosphorylation of EGFR.DETC reversed the upregulation of DSC2 and PKP3 expression induced by MCP,and promoting the phosphorylation of EGFR.AG 1478 upregulated the expression of PKP3 and DSC2 of GC cells,but had no effect on the expression of SOD3.SummaryMCP upregulated the expression of DSC2 through activating SOD3/EGFR/PKP3/DSC2 signaling pathway.Part 3 Study on the effect and mechanisms of MCP on enhancing the cytotoxicity and reducing the toxicity of OXA for GC through upregulating the expression of SOD3 and DSC2Research purposesWe aimed to evaluate the effect and mechanism of MCP on the migration and invasion of GC cells,the cytotoxicity of OXA and/or MCP for GC cells,the remission of CIPN induced by OXA and explore the mechanisms in vitro and in vivo.Research methods1)MTT assay and Sperm DNA fragmentation assay were performed to evaluate the effect of OXA and/or MCP on the viability of GC cells(Lenti-NC/High-DSC2).2)DCFH-DA probes assay was performed to analyze the ROS levels after treating with OXA and/or MCP.Mouse tumor xenograft model and I VIS imaging,Von Frey Test,Thermal Nociception to Heat assay and Cold Plate Test were used to investigate the effect of MCP on inhibiting the growth of transplanted tumor,and alleviating the CIPN induced by OXA in vivo.3)We knocked down the expression of DSC2 in GC cells(Lenti-NC/High-SOD3)through transfecting with siRNA.Then the viability,migration and invasion were investigated through MTT assay,scratch wound-healing assay,transwell assay.4)SOD inhibitor DETC and EGFR inhibitor AG1478 were used to investigate the regulatory effects of MCP on key targets and signaling pathways.Experimental results1)Compared with the group treated with OXA only,the IC50 values of OXA for GC cells in the group co-administrated with MCP markedly decreased.Overexpression of DSC2 enhanced the cytotoxicity of OXA on GC cells,but not significantly further enhanced the cytotoxicity of the co-combination group.MCP enhanced the inhibition effect of OXA on tumor growth in vivo.2)MCP suppressed the ROS generation induced by OXA in vitro.Compared to the group treated with OXA(15 mg/kg)only,that co-administrated with MCP increased the paw withdrawl threshold stimulating by Von Frey filament and paw withdrawl latency after heat exposure,but reduced the paw shaking/licking events on the cold plate in vivo.3)SOD3 and MCP both suppressed the viability,migration and invasion of GC cells.DETC reversed the inhibition of MCP on the viability,migration and invasion in GC cells.Knocking down the expression of DSC2 reversed the inhibition of SOD3 on the viability,migration and invasion.AG 1478 suppressed the viability,migration and invasion of GC cells(sgRNA-NC/Low-DSC2).SummaryMCP significantly enhanced the cytotoxicity of OXA on GC cells in vitro and in vivo,and suppressing the migration and invasion of GC cells through activating SOD3/EGFR/PKP3/DSC2 signaling pathway.Co-administrated with MCP significantly alleviated the symptoms of CIPN through upregulating the level of SOD3 and reducing the activity of ROS.Conclusion1)The expression level of DSC2 in GC tissues and cells was significantly downregulated.DSC2 inhibited the growth of GC mainly through inhibiting the nuclear translocation and transcriptional activity of y-catenin.DSC2 suppressed the migration and invasion of GC mainly through inhibiting the BRD4/Snail signaling pathway and the transcriptional activity of β-catenin,and inhibiting EMT.2)MCP upregulated the expression of DSC2 through activating SOD3/EGFR/PKP3 signaling pathway.3)MCP significantly enhanced the cytotoxicity of OXA on GC cells in vitro and in vivo,and suppressed the migration and invaison of GC cells through upregulating the expression of DSC2.MCP alleviated the symptoms of CIPN induced by OXA through upregulating the level of SOD3. |
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