Effects Of DADS On EMT,Invasion And Drug Resistance Of HUMAN Gastric Cancer SGC7901 Cells By Down-regulating DJ-1 And Its Mechanism

Diallyl Disulfide(DADS),a hydrophobic and effective medicinal component of allyl sulfide in garlic,is a promising anticancer drug.The human DJ-1 gene,also known as RS/PARK7/CAP1,is a member of a family of proteins encoded by the PARK7 gene that belong to peptidase C56.It is involved in many biological processes,such as transcriptional regulation,oxidative stress response,mitochondrial regulation,inflammation and glycosylation injury,which are highly expressed in a variety of tumors,and plays a key role in the regulation of tumor progression,clinical invasions,differentiation,cancer cell morphology and drug sensitivity.In the DADS induced HL-60 cell differentiation differential protein identified earlier,we found that the expression of DJ-1 protein was significantly down-regulated.On the basis the our previous researches,this study further explore that the down-regulation of DJ-1 inhibit EMT,invasion and drug resistance in human gastric cancer cells by DADS via PTEN/Akt pathway,confirmed that DJ-1 is one of the targets of DADS inhibiting EMT,invasion and drug resistance of human gastric cancer cells,provide theoretical basis for clinical gastric cancer molecular targeted therapy.Part ? The expression of DJ-1,PTEN and p-Akt in gastric cancer and its clinicopathological significanceObjective: To confirm the level of DJ-1,PTEN and P-Akt in gastric cancer and its clinicopathological significance.Methods: Immunohistochemistry was used to detect the expression of DJ-1,PTEN and p-Akt in gastric cancer tissue,paracancer tissue and normal gastric mucosa tissue.Results: Both DJ-1 and P-Akt were highly expressed in gastric cancer tissues,and the difference was significant compared with normal gastric mucosa and paracancer tissues(P<0.05).In addition,the expression in paracancer tissues was significantly higher than that in normal gastric mucosa(P<0.05).The expression in lymph node metastasis group was significantly higher than that in no lymph node metastasis group(P<0.05).In ? + ? stage expression significantly below ? + ? stage(P < 0.05).However,there was no significant difference in expression among highly differentiated,moderately differentiated and poorly differentiated gastric adenocarcinoma(P>0.05).The expression of PTEN in normal gastric mucosal tissues was significantly higher than that in adjacent gastric tissues and gastric tissues(P<0.05),and the expression in adjacent gastric tissues was significantly higher than that in gastric tissues(P<0.05).The group with lymph node metastasis was significantly lower than the group without lymph node metastasis(P<0.05).In ? + ? expression is significantly higher than ? + ?period(P < 0.05).The expression of gastric adenocarcinoma with high differentiation,medium differentiation and low differentiation was significantly different(P<0.05).There was no significant difference in the expression of the three proteins between different ages and genders(P>0.05).Kaplan-meier survival analysis showed that the high expression of DJ-1 was negatively correlated with 5-year overall survival in gastric cancer patients(P < 0.05).However,high expression of PTEN was positively correlated with 5-year overall survival rate in gastric cancer patients(P < 0.05).Summary:1.the expression of DJ-1 and p-Akt in human gastric cancer are related to the occurrence of gastric cancer,lymph node metastasis and TNM staging.2.The expression of PTEN in gastric cancer is related to the differentiation,lymph node metastasis and TNM staging of gastric cancer.3.The expression of DJ-1 in gastric cancer was negatively correlated with the survival rate of gastric cancer patients,and the expression of PTEN is positively correlated with it.Part ? DADS negatively regulated the PTEN/Akt pathway and inhibited the proliferation,EMT and invasion of SGC-7901 cells by down-regulating DJ-1Objective: To investigate the effects of DADS on proliferation,EMT and invasion of SGC7901 cell line in human gastric cancer SGC7901 cell line with high expression of DJ-1.Methods: SGC-7901 cell lines with stable and high expression of DJ-1were established,and the effects of DADS on proliferation,EMT and invasion of human gastric cancer SGC-7901 cells with high expression of DJ-1 were analyzed by CCK-8,marking experiment,invasion experiment,q RT-PCR,Western blot,and cellular immunofluorescence,respectively.Results: Western blot showed that the expression of DJ-1 in gastric cancer cells treated by DADS 30mg/L for 24 h was significantly lower than that in the control group,among which SGC7901 cells showed the most significant difference.Therefore,SGC7901 was selected as the research object in the follow-up experiment.Further q RT-PCR and Western blot were used to confirm the successful construction of SGC-7901 cell line with stable and high expression of DJ-1.CCK8 detection showed that,before treatment,the proliferative activities of the high expression group of DJ-1 were 0.579±0.082,0.886±0.153,1.138±0.124 and 1.344±0.194 respectively at 24 h,48h,72 h and 96 h,which showed a time-dependent increase compared with the vector group of 0.436±0.045,0.557±0.059,0.667±0.057 and 0.715±0.075(P<0.05),indicating that high expression of DJ-1 could significantly improve the proliferation capacity of SGC7901 cells.After DADS30mg/l treatment for 24 h,28h,72 h,and 96 h,the proliferative activities of the DJ-1 high expression group were 0.560±0.031,0.873±0.017,0.910±0.014,and 0.936±0.019,respectively,which were significantly lower than those before DADS treatment(P<0.05),indicating that DADS partially inhibited the expression of DJ-1 and thereby inhibited the proliferation of SGC7901 cells.The scratch test showed that the high expression of DJ-1 could enhance the migration ability of SGC-7901 cells.However,after DADS treatment of 30mg/l DADS for48 h,the cell migration ability of both the DJ-1 high expression group and the empty carrier group was weakened,indicating that DADS could reduce the effect of DJ-1 high expression on SGC7901 cell migration.Transwell invasion assay showed that the number of cells on the DJ-1high expression group and vector group passing through matrigel matrix gel was 179.7±6.0 and 251.3±4.0(P<0.05),respectively,without DADS treatment group.indicating that the high expression of DJ-1 could significantly promote the invasion of SGC7901 cells,while the number of cells passing through Matrigel matrix gel after DADS 30mg/ L treatment for 24 h was 87.3±7.1 and 130.0±8.5(P<0.05),which was significantly reduced compared with the untreated group.DADS antagonized the effect of high expression of DJ-1 on the invasions ability of SGC7901 cells.Western blot confirmed that compared with the vector group,the expression of DJ-1 and p-AKT protein on high expression of DJ-1 group was significantly increased,and the expression of PTEN was significantly reduced,AKT expression has no obvious difference,EMT related proteins E-cadherin and TIMP3 significantly reduced,the expression of Vimentin,snail and MMP-9 was significantly higher,which prompt the DJ-1 can promote the activation of AKT,inhibit PTEN expression and then start the process of EMT.However,after DADS treatment of 30mg/l for 24 h,DJ-1 expression in the DJ-1 high expression group and the vector group was significantly down-regulated,PTEN expression was significantly increased,p-Akt level was significantly decreased,EMT-related proteins e-cadherin and TIMP3 were significantly increased,and the expressions of Vimentin,Snail and MMP-9 were significantly decreased.It was confirmed that DADS down-regulation of DJ-1 could regulate the PTEN/Akt pathway and inhibit EMT of human gastric cancer cells.Further immunofluorescence test found that compared with the vector group,The fluorescence intensity of d J-1,Vimentin,TIMP3 and MMP-9proteins in the high EXPRESSION group of DJ-1 was significantly enhanced.It showed that high expression of DJ-1 promoted the expression of EMT-related proteins Vimentin,TIMP3 and MMP-9.The fluorescence intensity of PTEN and E-cadherin in the high EXPRESSION group of DJ-1 was significantly decreased,indicating that high expression of DJ-1 could inhibit the expression of PTEN and E-cadherin.After DADS treatment of 30mg/l for 24 h,the cell staining of DJ-1,Vimentin,MMP-9 and TIMP3 in each group was decreased,while that of PTEN and E-cadherin was increased,compared with the untreated group,indicating that DADS inhibited the expression of DJ-1and thus affected the expression of PTEN and EMT-related proteins.Summary:DADS inhibits proliferation,EMT and invasion of SGC-7901 cells by downregulating the PTEN/Akt pathway negatively by DJ-1.Part ? DADS downregulated DJ-1 to inhibit the resistance of SGC7901 cells to 5-FuObjective: To investigate the down-regulation of DJ-1 by DADS on proliferation,apoptosis and 5-Fu sensitivity of SGC7901 cells.Methods: MTT assay and flow cytometry were used to determine the effects of DADS on the proliferation and apoptosis of SGC7901 cells.The changes of Bcl-2,XIAP,caspase-3,MDR-1 and P-gp expression levels in cells of SGC7901 with DADS were detected by q RT-PCR,Western blot and immune-free fluorescence respectively.Results: After treatment with different concentrations of 5-Fu for 24 h,the proliferation and inhibition rate of DADS 30mg/l cells in each group was significantly higher than that before treatment(P<0.05).However,the inhibition of SGC7901/DJ-1 cell proliferation was not as obvious as SGC7901 and SGC7901/ VCR cells.DADS was shown to inhibit the proliferation of SGC7901 and SGC7901/VCR cells and improve the sensitivity to 5-Fu.Flow cytometry showed that the apoptosis of SGC7901/DJ-1 cells was lower than that of SGC7901 and SGC7901/VCR(P<0.05),and the apoptosis rate of SGC7901 cells was higher than that of SGC7901/VCR(P<0.05).After treatment with DADS,the levels of apoptosis in each group were significantly higher than those in the control group(P<0.05).QRT-PCR and Western blot showed that MDR-1,P-gp,Bcl-2 and XIAP of SGC7901/DJ-1 cells were significantly higher than those of SGC7901 and SGC7901/VCR cells(P<0.05),and caspase3 was significantly inhibited(P<0.05).After DADS treatment,MDR-1,P-gp,Bcl-2 and XIAP were significantly decreased in SGC7901,SGC7901/VCR and SGC 7901/DJ-1 cells(P<0.05),while caspase-3 was significantly up-regulated(P<0.05).Immunofluorescence showed that the fluorescence intensity of SGC7901/DJ-1 cells P-gp,Bcl-2 and XIAP was significantly increased compared with SGC7901 and SGC7901/VCR cells,while the fluorescence intensity of Caspase-3 was significantly decreased.After DADS treatment,the cell staining of P-gp,Bcl-2 and XIAP proteins in the three groups was decreased compared with that in the untreated group,while caspase-3 staining was enhanced.The results were consistent with those of q RT-PCR and Western blot.Summary:The down-regulation of DJ-1 by DADS could promote the sensitivity of SGC7901 cells to 5-Fu,which was related to the inhibition of P-gp,MDR-1,Bcl-2,XIAP and the promotion of Caspase-3.Conclusion:1.The expression of DJ-1,PTEN and p-Akt proteins in gastric cancer tissues is related to the occurrence,lymph node metastasis and TNM stage of gastric cancer.2.DADS inhibited proliferation,EMT and invasion of SGC-7901 cells by down-regulating DJ-1 and negatively regulating PTEN/Akt pathway.3.DADS down-regulation of DJ-1 promoted the sensitivity of SGC7901 cells to 5-Fu,which was related to the inhibition of P-GP,MDR-1,Bcl-2 and XIAP and the promotion of Caspase-3.4.DJ-1 may be one of the targets of DADS inhibiting EMT,invasion and drug resistance of human gastric cancer cells.