DJ-1 Is Involved In The Multidrug Resistance Of Gastric Cancer Cells By Upregulating NQO1,HO-1,and GST Expressions Via PTEN/PI3K/Akt/Nrf2 Pathway

Objective:This research aims to explore whether DJ-1 is involved in the multidrug resistance（MDR）of gastric cancer cells by upregulating NQO1,HO-1 and GST expressions via PTEN/PI3K/Akt/Nrf2 pathway at the cellular level.Methods:1.The DJ-1-overexpressed SGC7901/LV-DJ-1 cells were constructed on the basis of parental SGC7901 gastric cancer cells,and DJ-1-knockdowned SGC7901/VCR/sh DJ-1 cells were constructed on the basis of SGC7901/VCR gastric cancer cells of the MDR.Subsequently,the expression of DJ-1 protein was assessed by western blot in SGC7901,SGC7901/VCR,SGC7901/VCR/sh DJ-1,and SGC7901/LV-DJ-1 cells.CCK-8 assay was used to examine the semi-inhibitory concentration(IC₅₀)of these gastric cancer cells after co-incubation with four different chemotherapeutic drugs including vincristine（VCR）,adriamycin（ADR）,5-fluorouracil（5-FU）,and cisplatin（DDP）for 24 h,respectively.In addition,in order to observe the effects of inhibiting PI3K/Akt pathway or Nrf2 pathway on DJ-1-mediated the MDR of gastric cancer cells,we further detected the IC₅₀ values of the above four chemotherapy drugs by CCK-8 assay in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells which were treated with 10μM LY294002 or transfected with Nrf2 si RNA,respectively.2.In SGC7901,SGC7901/VCR,SGC7901/VCR/sh DJ-1,and SGC7901/LV-DJ-1 gastric cancer cells,the following indicators were used to observe the role of DJ-1 in the Nrf2 pathway:（1）The expressions of DJ-1 and p-Nrf2 were analyzed by western blot.（2）Nrf2 expressions in cytoplasm,nucleus,and whole-cell lysates were detected by western blot.（3）Dissociation of Nrf2-Keap1 was assessed by co-immunoprecipitation method.3.To determine the effect of DJ-1 on the PI3K/Akt pathway in DJ-1-mediated the MDR of gastric cancer cells,p-Akt（Ser473）expression was detected by western blot in SGC7901,SGC7901/VCR,SGC7901/VCR/sh DJ-1,and SGC7901/LV-DJ-1gastric cancer cells.Subsequently,to further clarify the relationship among DJ-1,PI3K/Akt,and Nrf2 in DJ-1-mediated the MDR of gastric cancer cells,the expressions of DJ-1,p-Akt（Ser473）,and p-Nrf2 and the nuclear translocation of Nrf2were detected by western blot in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells incubated with 10μM LY294002 or Nrf2 si RNA for 24 h..4.In four different gastric cancer cells of SGC7901,SGC7901/VCR,SGC7901/VCR/sh DJ-1 and SGC7901/LV-DJ-1,the following indicators were used to observe the effects of DJ-1 on DJ-1-PTEN interaction,p-PTEN expression,and PTEN activity:（1）The interaction between DJ-1 and PTEN was detected by co-immunoprecipitation.（2）The expression of p-PTEN protein was analyzed by western blot.（3）PTEN protease activity was assessed by corresponding assay kit.5.To confirm that the effects of DJ-1 on the MDR-related factors regulated by Nrf2 in DJ-1-mediated the MDR of gastric cancer cells,we detected m RNA and protein levels of NQO1,HO-1,GST,MRP1,and UGT in SGC7901,SGC7901/VCR,SGC7901/VCR/sh DJ-1,and SGC7901/LV-DJ-1 gastric cancer cells.In addition,realtime PCR and western blot were used to further detect the m RNA and protein levels of NQO1,HO-1 and GST in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells after 10μM LY294002 pretreatment or Nrf2 si RNA transfection for 24 h.We aim to further elucidate the relationship among PI3K/Akt pathway,Nrf2 pathway,and the expressions of MDR-associated factors like NQO1,HO-1,and GST in DJ-1-mediated the MDR of gastric cancer cells.Results:1.Compared with the parental SGC7901 gastric cancer cells,the expression of DJ-1 was significantly up-regulated in SGC7901/VCR gastric cancer cells,and the IC₅₀ values were all increased in response to VCR,ADR,5-FU or DDP.However,the above effects were inhibited in SGC7901/VCR/sh DJ-1 gastric cancer cells,but were reproduced in SGC7901/LV-DJ-1 gastric cancer cells.Interestingly,the IC₅₀ values of the above four chemotherapeutic drugs were significantly reduced after SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells pretreated with LY294002or Nrf2 si RNA for 24 h.2.Compared with the parental SGC7901 gastric cancer cells,Nrf2phosphorylation was increased,the interaction of Keap1-Nrf2 was attenuated,and Nrf2 nuclear translocation was enhanced in SGC7901/VCR gastric cancer cells.The above effects were inhibited in SGC7901/VCR/sh DJ-1 gastric cancer cells and were reproduced in SGC7901/LV-DJ-1 gastric cancer cells.3.Compared with the parental SGC7901 gastric cancer cells,Akt phosphorylation（Ser473）was significantly increased in SGC7901/VCR gastric cancer cells.The same effect was observed in SGC7901/LV-DJ-1 gastric cancer cells,but was inhibited in SGC7901/VCR/sh DJ-1 gastric cancer cells.Importantly,the expression of DJ-1 in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells was not significantly changed after pretreatment with LY294002.But Akt（Ser473）phosphorylation and Nrf2 phosphorylation were remarkably inhibited,and Nrf2nuclear translocation was reduced.However,in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells,only Nrf2 phosphorylation was inhibited by Nrf2 si RNA.There had no significant effects on DJ-1and p-Akt（Ser473）expressions.4.Compared with the parental SGC7901 gastric cancer cells,the interaction between DJ-1 and PTEN protein was noticeably enhanced in SGC7901/VCR gastric cancer cells,PTEN phosphorylation level was increased and PTEN activity was decreased.The above effects were also inhibited in SGC7901/VCR/sh DJ-1 gastric cancer cells and were reproduced in SGC7901/LV-DJ-1 gastric cancer cells.5.Compared with the parental SGC7901 gastric cancer cells,m RNA and protein expression levels of MDR-related factors（NQO1,HO-1 and GST）were remarkably increased in SGC7901/VCR gastric cancer cells,while MRP1 and UGT expression levels were not significantly changed.Similarly,the above effects were inhibited in SGC7901/VCR/sh DJ-1 gastric cancer cells and mimicked in SGC7901/LV-DJ-1gastric cancer cells.Importantally,the m RNA and protein expression levels of NQO1,HO-1,and GST were decreased in SGC7901/VCR and SGC7901/LV-DJ-1 gastric cancer cells after treatments with LY294002 or Nrf2 si RNA.Conclusion:This work revealed that activating PTEN/PI3K/Akt/Nrf2 pathway and subsequently up-regulating NQO1,HO-1,and GST expressions could be a key mechanism by which DJ-1 mediates the MDR of gastric cancer cells.