DJ-1 activates the noncanonical NF-κB pathway via interaction with Cezanne to inhibit the apoptosis and promote the proliferation of Ishikawa cells.

BACKGROUND: Endometrial cancer is generally one of the most evident malignant tumours of the female reproductive system, and the mechanisms underlying its cell proliferation and apoptosis are key to research in gynaecological oncology. In the paper, the in-depth molecular mechanism by which DJ-1 protein regulates the proliferation and apoptosis of Ishikawa cells was investigated. METHODS AND RESULTS: DJ-1 knockdown and overexpressing Ishikawa stable cell lines were established by lentiviral transduction. The levels of DJ-1 and noncanonical NF-κB signaling key proteins were evaluated by Western blotting. Cell counting kit-8 (CCK-8) and flow cytometry were applied to analyze the cell viability and apoptosis. Co-immunoprecipitation experiment was utilized to assess the DJ-1-Cezanne interaction. The results showed that DJ-1 overexpression conferred apoptosis resistance and high proliferation on Ishikawa cells, while DJ-1 knockdown in Ishikawa cells produced the opposite results. These findings again suggested that DJ-1 inhibits the apoptosis and promotes the proliferation of Ishikawa cells. More crucially, further data showed that the noncanonical NF-κB activation was required for the regulation of Ishikawa cell proliferation and apoptosis by DJ-1. Meanwhile, it was found that noncanonical NF-κB pathway may be activated by DJ-1 interacting with and negatively regulating Cezanne in Ishikawa cells. CONCLUSIONS: Overall, this work revealed that DJ-1 associates with and negatively regulates Cezanne and consequently triggers the noncanonical NF-κB activation, thereby regulating Ishikawa cell proliferation and apoptosis.

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Bacillus sp. SMrs28, metabolites from which had significant nematicidal activity, was isolated from the rhizosphere soil of Stellera chamaejasme. To determine the optimal fermentation conditions of the strain and the resin type of preliminary purified active ingredient, fermentation conditions were optimized by single factor experiment, while the macroporous resin types were screened in a static adsorption experiment. The results showed that the optimal fermentation conditions of SMrs28 strain were as follows: glucose and yeast powder were the best carbon source and nitrogen source, fermentation for 48 h, inoculum volume of 10%, temperature at 28 ℃,a rotation speed of 180 r·min-1, liquid volume of 30 mL in 150 mL triangular flask, and with an initial pH of 7.2. The static adsorption experiments showed that the adsorption and desorption of active ingre-dients in the fermentation broth by the macroporous adsorption resin D101 was significantly better than that of XAD-4, HP20 and AB-8, with the nematicidal activity of the desorption liquid being significantly improved. The nematicidal activity of fermentation broth was significantly improved by the optimization of fermentation conditions and the screening of optimal macroporous adsorption resins. These results laid a foundation for the further isolation and purification of active ingredients from SMrs28 strain, and provided theoretical basis for the development and utilization of microbial nematicides.

Mitochondrial Translocation of DJ-1 Is Mediated by Grp75: Implication in Cardioprotection of Resveratrol Against Hypoxia/Reoxygenation-Induced Oxidative Stress.

Resveratrol (Res) was recently reported to ameliorate hypoxia/reoxygenation (H/R)-caused oxidative stress in H9c2 cardiomyocytes through promoting the mitochondrial translocation of DJ-1 protein and subsequently preserving the activity of mitochondrial complex I. However, it is noteworthy that DJ-1 possesses no mitochondria-targeting sequence. Therefore, how Res induces DJ-1 mitochondrial translocation is an important and interesting question for further exploration. Glucose-regulated protein 75 (Grp75), whose N-terminus contains a 51-amino acid long mitochondrial-targeting signal peptide, is a cytoprotective chaperone that partakes in mitochondrial import of several proteins. Here, the contribution of Grp75 to mitochondrial import of DJ-1 by Res was investigated in a cellular model of H/R. Our results showed that Res upregulated the expression of DJ-1 protein, enhanced the interaction of DJ-1 and Grp75, and promoted DJ-1 translocation to mitochondria from cytosol in H9c2 cardiomyocytes undergoing H/R. Importantly, knockdown of Grp75 markedly reduced the interaction of DJ-1 with Grp75 and subsequent DJ-1 mitochondrial translocation induced by Res. Furthermore, Res pretreatment promoted the association of DJ-1 with ND1 and NDUFA4 subunits of complex I, preserved the activity of complex I, decreased mitochondria-derived reactive oxygen species production, and eventually ameliorated H/R-caused oxidative stress damage. Intriguingly, these effects were largely prevented also by small interfering RNA targeting Grp75. Overall, these results suggested that Grp75 interacts with DJ-1 to facilitate its translocation from cytosol to mitochondria, which is required for Res-mediated preservation of mitochondria complex I and cardioprotection from H/R-caused oxidative stress injury.

Molecular Mechanism Underlying Hypoxic Preconditioning-Promoted Mitochondrial Translocation of DJ-1 in Hypoxia/Reoxygenation H9c2 Cells.

DJ-1 was recently reported to be involved in the cardioprotection of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R)-induced oxidative stress damage, by preserving mitochondrial complex I activity and, subsequently, inhibiting mitochondrial reactive oxygen species (ROS) generation. However, the molecular mechanism by which HPC enables mitochondrial translocation of DJ-1, which has no mitochondria-targeting sequence, to preserve mitochondrial complex I, is largely unknown. In this study, co-immunoprecipitation data showed that DJ-1 was associated with glucose-regulated protein 75 (Grp75), and this association was significantly enhanced after HPC. Immunofluorescence imaging and Western blot analysis showed that HPC substantially enhanced the translocation of DJ-1 from cytosol to mitochondria in H9c2 cells subjected to H/R, which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transfection. Importantly, knockdown of Grp75 markedly reduced the mitochondrial translocation of DJ-1 induced by HPC and pFlag-DJ-1 transfection. Moreover, HPC promoted the association of DJ-1 with mitochondrial complex I subunits ND1 and NDUFA4, improved complex I activity, and inhibited mitochondria-derived ROS production and subsequent oxidative stress damage after H/R, which was also mimicked by pFlag-DJ-1 transfection. Intriguingly, these effects of HPC and pFlag-DJ-1 transfection were also prevented by Grp75 knockdown. In conclusion, these results indicated that HPC promotes the translocation of DJ-1 from cytosol to mitochondria in a Grp75-dependent manner and Grp75 is required for DJ-1-mediated protection of HPC on H/R-induced mitochondrial complex I defect and subsequent oxidative stress damage.

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.

Resveratrol attenuates myocardial hypoxia/reoxygenation-induced cell apoptosis through DJ-1-mediated SIRT1-p53 pathway.

Resveratrol, a multi-functional phytoalexin, has been well indicated to exert cardioprotective effects by weakening ischemia/reperfusion (I/R) injury, and cell apoptosis is a vital way in I/R injury. SIRT1-p53 pathway has strong significance in regulating cell apoptosis. DJ-1 can directly bind to SIRT1 and stimulate the activity of SIRT1-p53. Therefore, the current study was determined whether Resveratrol attenuates hypoxia/reoxygenation (H/R)-induced cell apoptosis, and whether DJ-1-mediated SIRT1 activation involves in the cardioprotective effects of Resveratrol. The results showed that remarkable decrease in the number of apoptotic cells along with reduction of lactate dehydrogenase release and restoration of cell viability emerged when Resveratrol was applied in the H9c2 cells exposed to H/R. Moreover, Resveratrol increased DJ-1 expression and promoted the interaction of DJ-1 with SIRT1, which further contributed to subsequent restoration of SIRT1 activity and decrease of acetylation level of p53. However, above cardioprotective effects of Resveratrol were abrogated by DJ-1 siRNA and SIRT1 specific inhibitor Sirtinol. In conclusion, the current study demonstrated that Resveratrol suppressed H/R-induced cell apoptosis, which may be conducted by up-regulating DJ-1, and later activating SIRT1 activity and subsequently inhibiting p53 acetylation level in the H9c2 cells.