DJ-1 overexpression confers the multidrug resistance phenotype to SGC7901 cells by upregulating P-gp and Bcl-2.

Gastric cancer (GC) is one of the most malignant tumors with high incidence and mortality worldwide, and the multidrug resistance (MDR) often results in chemotherapy failure in GC. DJ-1 has been well indicated to be associated with drug resistance in multiple cancers. However, the role of DJ-1 in the MDR of gastric cancer cells and its possible mechanism remain to be elucidated. Therefore, the current study was investigated whether DJ-1 expression is differential in parental gastric cancer cell SGC7901 and vincristine (VCR)-induced gastric cancer MDR cell SGC7901/VCR, and whether DJ-1 plays a significant role in development of MDR in gastric cancer. The results showed that DJ-1 expression in SGC7901/VCR cells was significantly higher than its sensitive parental SGC7901 cells. Furthermore, DJ-1 overexpressed gastric cancer cell line SGC7901/LV-DJ-1 led to the increase of cell survival rate, the IC50 of chemotherapeutic drugs and number of cell clones as well as decrease of cell cycle G0/G1 phase ratio compared with its parental cells under the treatment of VCR, adriamycin (ADR), 5-Fluorouracil (5-FU) and cisplatin (DDP). However, the DJ-1 knockdown stable cell line SGC7901/VCR/shDJ-1 reversed the above mentioned series of MDR. Moreover, it was found that upregulation of DJ-1 protein expression promoted the pumping rate of GC cells to ADR and reduced the apoptotic index of GC cells treated with chemotherapeutic drugs by upregulating P-gp and Bcl-2. Similarly, knocking down DJ-1, P-gp or Bcl-2 displayed a converse effect. In conclusion, the current study demonstrated that DJ-1 overexpression confers the MDR phenotype to SGC7901 cells and this process is related to DJ-1 promoting active efflux of drugs and enhancing the anti-apoptotic ability of MDR GC cells by upregulating P-gp and Bcl-2.

DJ-1 plays an obligatory role in the cardioprotection of delayed hypoxic preconditioning against hypoxia/reoxygenation-induced oxidative stress through maintaining mitochondrial complex I activity.

DJ-1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)-induced oxidative stress, but its mechanism against H/R-induced oxidative stress during DHP is not fully elucidated. Here, using the well-established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up-regulated DJ-1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R-induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ-1 knockdown with short hairpin RNA but mimicked by DJ-1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ-1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ-1 mediates the cardioprotection of DHP against H/R-induced oxidative stress damage.

DJ-1 preserving mitochondrial complex I activity plays a critical role in resveratrol-mediated cardioprotection against hypoxia/reoxygenation-induced oxidative stress.

Resveratrol has been demonstrated to have cardioprotective effects by attenuating ischemia/reperfusion (I/R)-induced oxidative stress injury, but its in-depth molecular mechanisms against I/R-induced oxidative stress is not fully elaborated. DJ-1 plays a role in maintenance of mitochondrial complex I activity and is closely associated with oxidative stress. Therefore, this study sought to determine the contribution of DJ-1-mediated maintenance of mitochondrial complex I activity to the anti-oxidative stress effect of Resveratrol in the H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that Resveratrol significantly attenuated the H/R-induced viability loss and lactate dehydrogenase leakage, accompanied by decreases in intracellular reactive oxygen species (ROS) and malondialdehyde contents and increases in the reduced glutathione/oxidized glutathione ratio. Furthermore, Resveratrol increased the expression and mitochondrial translocation of DJ-1 and promoted the direct binding of DJ-1 with complex I subunits ND1 and NDUFS4, which in turn improved mitochondrial complex I activity and inhibited mitochondria-derived ROS production after H/R. Intriguingly, the anti-oxidative stress effect of Resveratrol could be partially blocked by DJ-1 siRNA and Complex I inhibitor Rotenone, respectively. Conclusively, these results indicated that DJ-1 is necessary for Resveratrol-mediated cardioprotective effects against H/R-induced oxidative stress damage, at least in part, through preserving mitochondrial complex I activity, and subsequently decreasing mitochondrial ROS generation.

Sasanquasaponin induces increase of Cl­/HCO3­ exchange of anion exchanger 3 and promotes intracellular Cl­ efflux in hypoxia/reoxygenation cardiomyocytes.

Anion exchanger 3 (AE3) is known to serve crucial roles in maintaining intracellular chloride homeostasis by facilitating the reversible electroneutral exchange of Cl­ for HCO3­ across the plasma membrane. Our previous studies reported that sasanquasaponin (SQS) can inhibit hypoxia/reoxygenation (H/R)­induced elevation of intracellular Cl­ concentration ([Cl­]i) and elicit cardioprotection by favoring Cl­/HCO3­ exchange of AE3. However, the molecular basis for SQS­induced increase of Cl­/HCO3­ exchange of AE3 remains unclear. The present study demonstrated that SQS activates protein kinase Cε (PKCε) and stimulates the phosphorylation of AE3 in H9c2 cells. Notably, SQS­induced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV1­2, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCε­dependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV1­2 and S67A mutation of AE3 eradicated the SQS­induced increase of AE3 activity, reversed the inhibitory effect of SQS on H/R­induced elevation of [Cl­]i, Ca2+ overload and generation of reactive oxygen species, and eliminated SQS­induced cardioprotection. In conclusion, PKCε­dependent phosphorylation of serine 67 on AE3 may be responsible for the increase of Cl­/HCO3­ exchange of AE3 and intracellular chloride efflux by SQS, and contributes to the cardioprotection of SQS against H/R in H9c2 cells.

Extracellular Cl--free-induced cardioprotection against hypoxia/reoxygenation is associated with attenuation of mitochondrial permeability transition pore.

The isotonic substitution of extracellular chloride by gluconate (extracellular Cl--free) has been demonstrated to elicit cardioprotection by attenuating ischaemia/reperfusion-induced elevation of intracellular chloride ion concentration ([Cl-]i). However, the downstream mechanism underlying the cardioprotective effect of extracellular Cl--free is not fully established. Here, it was investigated whether extracellular Cl--free attenuates mitochondrial dysfunction after hypoxia/reoxygenation (H/R) and whether mitochondrial permeability transition pore (mPTP) plays a key role in the extracellular Cl--free cardioprotection. H9c2 cells were incubated with or without Cl--free solution, in which Cl- was replaced with equimolar gluconate, during H/R. The involvement of mPTP was determined with atractyloside (Atr), a specific mPTP opener. The results showed that extracellular Cl--free attenuated H/R-induced the elevation of [Cl-]i, accompanied by increase of cell viability and reduction of lactate dehydrogenase release. Moreover, extracellular Cl--free inhibited mPTP opening, and improved mitochondria function, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Intriguingly, pharmacologically opening of the mPTP with Atr attenuated all the protective effects caused by extracellular Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. These results indicated that extracellular Cl--free protects mitochondria from H/R injury in H9c2 cells and inhibition of mPTP opening is a crucial step in mediating the cardioprotection of extracellular Cl--free.

Sasanquasaponin-induced cardioprotection involves inhibition of mPTP opening via attenuating intracellular chloride accumulation.

Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.