Peritoneal metastasis is the most frequent cause of death in patients with advanced gastric carcinoma (GC). The phosphatase of regenerating liver-3 (PRL-3) is recognized as an oncogene and plays an important role in GC peritoneal metastasis. However, the mechanism of how PRL-3 regulates GC invasion and metastasis is unknown. In the present study, we found that PRL-3 presented with high expression in GC with peritoneal metastasis, but phosphatase and tensin homologue (PTEN) was weakly expressed. The p-PTEN/PTEN ratio was also higher in GC with peritoneal metastasis than that in the normal gastric tissues. We also found the same phenomenon when comparing the gastric mucosa cell line with the GC cell lines. After constructing a wild-type and a mutant-type plasmid without enzyme activity and transfecting them into GC SGC7901 cells, we showed that only PRL-3 had enzyme activity to downregulate PTEN and cause PTEN phosphorylation. The results also showed that PRL-3 increased the expression levels of MMP-2/MMP-9 and promoted the migration and invasion of the SGC7901 cells. Knockdown of PRL-3 decreased the expression levels of MMP-2/MMP-9 significantly, which further inhibited the migration and invasion of the GC cells. PRL-3 also increased the expression ratio of p-Akt/Akt, which indicated that PRL-3 may mediate the PI3K/Akt pathway to promote GC metastasis. When we transfected the PTEN siRNA plasmid into the PRL-3 stable low expression GC cells, the expression of p-Akt, MMP-2 and MMP-9 was reversed. In conclusion, our results provide a bridge between PRL-3 and PTEN; PRL-3 decreased the expression of PTEN as well as increased the level of PTEN phosphorylation and inactivated it, consequently activating the PI3K/Akt signaling pathway, and upregulating MMP-2/MMP-9 expression to promote GC cell peritoneal metastasis.

We have recently shown that DJ-1 is implicated in the delayed cardioprotective effect of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R) injury as an endogenous protective protein. This study aims to further investigate the underlying mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress. Using a well-characterized cellular model of HPC from rat heart-derived H9c2 cells, we found that HPC promoted nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein-1 (Keap1) dissociation and resulted in increased nuclear translocation, antioxidant response element-binding, and transcriptional activity of Nrf2 24 hours after HPC, with subsequent upregulation of manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO-1), which provided delayed protection against H/R-induced oxidative stress in normal H9c2 cells. However, the aforementioned effects of HPC were abolished in DJ-1-knockdown H9c2 cells, which were restored by restoration of DJ-1 expression. Importantly, we showed that inhibition of the Nrf2 pathway in H9c2 cells mimicked the effects of DJ-1 knockdown and abolished HPC-derived induction of antioxidative enzymes (MnSOD and HO-1) and the delayed cardioprotection. In addition, inhibition of Nrf2 also reversed the effects of restored DJ-1 expression on induction of antioxidative enzymes and delayed cardioprotection by HPC in DJ-1-knockdown H9c2 cells. Taken together, this work revealed that activation of Nrf2 pathway and subsequent upregulation of antioxidative enzymes could be a critical mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress in H9c2 cells.

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Abstract. DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain anti-oxidant enzymes and attenuate hypoxia/re-oxygenation (H/R)-induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2-like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti-oxidant genes via binding to the anti-oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti-oxidative enzymes by DJ-1 and contributes to the protective functions of DJ-1 against H/R-induced oxidative stress in H9c2 cells. The results demonstrated that DJ-1-overexpressing H9c2 cells exhibited anti-oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R-induced oxidative stress compared with native cells, whereas DJ-1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ-1-mediated anti-oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ-1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE-binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ-1-mediated induction of anti-oxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2 cells. IntroductionOxidative stress is known to have an important role in the pathogenesis of certain cardiovascular diseases, including atherosclerosis, myocardial ischemia-reperfusion injury and chronic heart failure (1). Endogenous anti-oxidant systems include a number of proteins or enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as non-enzymatic, low-molecular-weight anti-oxidant compounds, including glutathione and ascorbic acid, which constitute the first line of defence against oxidative stress and maintain the redox environment of the body (2). Mounting evidence indicates that stimulation of endogenous anti-oxidant systems is an important strategy for achieving cardioprotection against ischemia-reperfusion injury, and is also one of essential mechanisms of anti-oxidative stress effect of certain proteins (3,4).DJ-1, a ubiquitously expressed and highly conserved intracellular protein, was originally discovered in 1997 as a novel oncogene product that can transform mouse NIH3T3 cells in combination with H-Ras or c-Myc (5). Subseq...

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DJ‑1 protein, as a multifunctional intracellular protein, has been demonstrated to serve a critical role in regulating cell survival and oxidative stress. To provide in vivo evidence that DJ‑1 is involved in the delayed cardioprotection induced by ischemic preconditioning (IPC) against oxidative stress caused by ischemia/reperfusion (I/R), the present study subjected male Sprague‑Dawley rats to IPC (3 cycles of 5‑min coronary occlusion/5‑min reperfusion) 24 h prior to I/R (30‑min coronary occlusion/120‑min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ‑1 in situ. Lactate dehydrogenase (LDH) and creatine kinase‑MB (CK‑MB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ‑1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ‑1 protein. In addition, IPC reduced LDH and CK‑MB release, attenuated myocardial infarct size, improved cardiac function following I/R, and inhibited the elevation of ROS and MDA and the decrease in activities of the antioxidant enzymes SOD, CAT and GPx. However, in situ knockdown of DJ‑1 attenuated the IPC‑induced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/R‑induced oxidative stress. The present study therefore provided novel evidence that DJ‑1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ‑1 is required for IPC to inhibit I/R‑induced oxidative stress.

Abstract. Sasanquasaponin (SQS) is an active component ofCamellia oleifera Abel. A recent study by our group demonstrated that SQS was able to inhibit ischemia/reperfusion-induced elevation of the intracellular chloride ion concentration ([Cl -] i ) and exerted cardioprotective effects; however, the underlying intracellular signal transduction mechanisms have yet to be elucidated. As protein kinase C ε (PKCε) is able to mediate Cl -homeostasis, the present study investigated its possible involvement in the effects of SQS on cardiomyocytes subjected to ischemia/reperfusion injury. Cardiomyocytes were pre-treated with or without SQS or SQS plus εV1-2, a selective PKCε inhibitor, followed by simulated ischemia/reperfusion (sI/R). The effects on cell viability, PKCε phosphorylation levels, [Cl -] i , mitochondrial membrane potential and reactive oxygen species (ROS) production were assessed using an MTS assay, western blot analysis, colorimetric assays and flow cytometry. The results revealed that treatment with SQS prior to sI/R increased the viability of cardiomyocytes, and efficiently attenuated lactate dehydrogenase and creatine phosphokinase release induced by sI/R. In addition, SQS promoted PKCε phosphorylation and inhibited sI/R-induced elevation of [Cl -] i , paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation. However, when the cardiomyocytes were treated with εV1-2 prior to SQS pre-conditioning, the cardioprotection induced by SQS was reduced and the inhibitory effects of SQS on sI/R-induced elevation of [Cl -] i , production of ROS and loss of mitochondrial membrane potential were also attenuated. These findings indicated that SQS may inhibit sI/R-induced elevation of [Cl -] i through the PKCε signaling pathway to elicit cardioprotection in cultured cardiomyocytes.

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