Death-associated Protein Research Articles

Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation

Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1–RIPK3–MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.

Regulation of necroptosis by targeting tumor suppressor death-associated protein kinase 1

Abstract Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic induction by different stimuli. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK and AKT, were not regulated by DAPK. In contrast, DAPK selectively promoted MKK3-induced p38 MAPK activation, resulting in enhanced MK2 phosphrylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.

Structural and thermodynamic analyses of interactions between death-associated protein kinase 1 and anthraquinones.

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase that regulates apoptosis and autophagy. DAPK1 is considered to be a therapeutic target for amyloid-β deposition, endometrial adenocarcinomas and acute ischemic stroke. Here, the potent inhibitory activity of the natural anthraquinone purpurin against DAPK1 phosphorylation is shown. Thermodynamic analysis revealed that while the binding affinity of purpurin is similar to that of CPR005231, which is a DAPK1 inhibitor with an imidazopyridazine moiety, the binding of purpurin was more enthalpically favorable. In addition, the inhibition potencies were correlated with the enthalpic changes but not with the binding affinities. Crystallographic analysis of the DAPK1-purpurin complex revealed that the formation of a hydrogen-bond network is likely to contribute to the favorable enthalpic changes and that stabilization of the glycine-rich loop may cause less favorable entropic changes. The present findings indicate that purpurin may be a good lead compound for the discovery of inhibitors of DAPK1, and the observation of enthalpic changes could provide important clues for drug development.

Presynaptic Caytaxin prevents apoptosis via deactivating DAPK1 in the acute phase of cerebral ischemic stroke

Death-associated protein kinase 1 (DAPK1) is a key protein that mediates neuronal death in ischemic stroke. Although the substrates of DAPK1 and molecular signal in stroke have been gradually discovered, the modulation of DAPK1 itself is still unclear. Here we first reveal that Caytaxin, a brain-specific member of BCL2/adenovirus E1B -interacting protein (BNIP-2), increases and interacts with DAPK1 as early as 2 h after middle cerebral artery occlusion (MCAO) in the penumbra area of mouse brain. Furthermore, Caytaxin binds to DAPK1 at the presynaptic site and inhibits DAPK1 catalytic activity. Silencing Caytaxin by Caytaxin shRNA (Sh-Caytaxin) enhances DAPK1 activity, deteriorates neuronal apoptosis and brain injuries both in vivo and in vitro. Thus, elevating presynaptic Caytaxin could prevent neuronal apoptosis by inhibiting DAPK1 activation in the acute stage of ischemic stroke. Caytaxin may physiologically protect neuronal cells and represent a potential prevention and therapeutic target in the early phase of cerebral ischemic stroke.

Oxytocin Alleviates MPTP-Induced Neurotoxicity in Mice by Targeting MicroRNA-26a/Death-Associated Protein Kinase 1 Pathway.

Neurotoxicity is one of the major pathological changes in multiple neurological disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), the second popular neurodegenerative disease in aged people. It is known that the AD and PD share the similar neuropathological hallmarks, such as the oxidative stress, loss of specific neurons, and aggregation of specific proteins. However, there are no effective therapeutic drugs for both AD and PD yet. Oxytocin (OXT) is a small peptide with 9 amino acids that is neuroprotective to many neurological disorders. Whether OXT administration confers neuroprotection to 1-methyl-4-phenyl-1, 2, 3, 6- tetrahydropyridine (MPTP)-induced neurotoxicity in mice are still not known. In this study, we first found that the OXT levels are decreased in MPTP mice. Supplementation with OXT effectively rescues the locomotor disabilities and anxiety-like behaviors in MPTP mice. OXT also alleviates the hyperphosphorylation of α-synuclein at S129 site and the loss of dopaminergic neurons in the substantia nigra pars compacta, as well as the oxidative stress in the MPTP mice, and alleviates both oxidative stress and cell cytotoxicity in vitro. Furthermore, we found that OXT could inhibit the miR-26a/DAPK1 signal pathway in MPTP mice. In summary, our study demonstrates protective effects of OXT in MPTP mice and that miR-26a/DAPK1 signaling pathway may play an important role in mediating the protection ofOXT.

Analysis of Promoter Hypermethylation of DAPK and BAX Apoptotic Genes in Iranian Gastric Cancer Patients Undergoing Chemotherapy

Background: The apoptotic route is mostly damaged in gastric cancer tumor cells. DNA methylation of promoter associated CpG islands inactivates tumor suppressor genes. The objective of the present study was to analyze the hypermethylation of death-associated protein kinase (DAPK) and Bcl-2-associated X protein (BAX) genes in individuals suffering from gastric cancer and undergoing chemotherapy. Methods: Genomic DNA was extracted from blood samples and the tissue fixed in the paraffin of 30 patients and normal individuals. Hypermethylation investigation of DAPK and BAX genes was conducted via methylation specific PCR technique, the outcomes of which were analyzed through electrophoresis and SPSS software version 20. Results: Methylation of both BAX and DAPK genes with a frequency of (28.3%, 21.7%) in blood and (23.3%, 23.3%) in tissue, respectively, had a significant relationship with gastric cancer (P˂0.01). A significant relationship was also observed between the methylation of BAX gene in tissue and tumor type (12, 35.3% and P˂0.01). No relationship was found between methylation and grade, stage, node, age, sex, and other pathologic and clinical data of the patients (P>0.05). There was a significant association between simultaneous methylation of DAPK and BAX genes in tumor and typical tissues with methylation a frequency of 40% and 95.83%, respectively (P˂ 0.01). Conclusion: Methylation of the BAX and DAPK genes can be used as a biomarker in bloodand an approach in the early detection of malignity and illness management. Methylation inhibitors with the potential for drug targeting of DAPK and BAX can further be employed in pharmacotherapy.

P53 expression in ischemic rat models after the administration of ketamine and ketamine-xylazine

Ketamine and ketamine-xylazine are often used as anesthetic drugs in animal models of ischemia. However, their neuroprotective and neurotoxic effects in ischemic animal models that have undergone tBCCAO are still under debate. The protein p53 is a pro-apoptotic factor involved in the cellular mechanism of ischemia. The interaction between death-associated protein kinase 1 (DAPK 1) and p53 is fundamental in determining whether cells experience necrosis or apoptosis in an ischemic stroke. This study was purposed to identify the presence or absence of differences between the p53 expressions in the brains of tBCCAO-induced ischemic rat models after the administration of ketamine and ketamine-xylazine. It employed a post-test control group design with four groups of adult male Wistar rats as the subject: (1) sham group operated with ketamine, (2) sham group operated with ketamine-xylazine, (3) models of tBCCAO-induced ischemia with ketamine, and (4) models of tBCCAO-induced ischemia with ketamine-xylazine. Ketamine was administered at the dose of 75mg/kg BW, while xylazine was at 8 mg/kg BW. The expression of p53 in rat brains was assessed by semi-quantification, specifically IHC staining with anti-p53 antibodies. P53 expression appeared as brownish stains in the cytoplasm of forebrain pyramidal neurons, and in this study, it was measured using the Allred score. The ANOVA test yielded a p -value of >0.05, implying the absence of difference between the p53 expressions in the brains of tBCCAO-induced ischemic rat models receiving ketamine and ketamine-xylazine.

Potential Prognostic Role for SPOP, DAXX, RARRES1, and LAMP2 as an Autophagy Related Genes in Prostate Cancer.

Autophagy plays a critical role in PCa development. DAXX has a potent pro-survival effect by enhancing cell growth in PCa via suppression of autophagy. Here, we depicted a network governed by DAXX and SPOP by which the autophagy pathway is suppressed through the ubiquitination and modulation of key cellular signaling pathways mediators including LAMP2 and RARRES1. Through network-based bioinformatics approaches, the expression levels of DAXX, RARRES1, LAMP2, and SPOP genes was assessed in 50 PCa tissues and 50 normal adjacent from the same sample as well as 50 benign prostatic hyperplasia (BPH) tissues by quantitative RT-PCR. The normal adjacent tissues were taken from regions more than 5mm away from the bulk of those tumor tissues with clearly distinct margins. RNA extraction, cDNA synthesis and Real-time Quantitative RT-PCR were done for assessment of gene expression. To evaluate the primary gene network centered on autophagy pathway, according to the Query-dependent weighting algorithm, these two networks were integrated with Cytoscape 3.4 software. We found that in PCa tissues the DAXX expression level was significantly increased (P < 0.001) and the expressions of SPOP, RARRES1, and LAMP2 were significantly down-regulated, when compared to both control groups including normal adjacent and BPH tissues. Moreover, significant correlations were observed between expression levels of all four genes. Additionally, ROC curve analysis revealed that LAMP2 had the most sensitivity and specificity. These findings suggest that the contribution of SPOP, DAXX, RARRES1, and LAMP2 together could be a putative regulatory element acting as a prognostic signature and therapeutic target in PCa.

ROS-mediated programmed cell death (PCD) of Thalassiosira pseudonana under the stress of BDE-47

Polybrominated diphenyl ethers (PBDEs) are a series of highly persistent organic pollutants (POPs) ubiquitously distributed in marine environments. As key primary producers, microalgae are the start of PBDEs bioaccumulations and vulnerable to their toxicities. In order to deeply investigate the toxic mechanism of PBDEs on microalgal cells, the occurrence of programmed cell death (PCD) in a model diatom Thalassiosira pseudonana and its possible mediating mechanism were studied. The results indicated: cell death of T. pseudonana happened under the stress of BDE-47, which was proved to be PCD based on the correlations with three biochemical markers (DNA fragmentation, phosphatidylserine externalization and caspase activity) and three molecular markers [Metacaspase 2 gene (TpMC2), Death-associated protein gene (DAP3) and Death-specific protein 1 gene (TpDSP1)]; Furthermore, the changes of cellular ROS levels were correlated with the PCD markers and the dead cell rates, and the cell membrane and the chloroplast were identified as the major ROS production sites. Therefore, we concluded that PCD might be an important toxic mechanism of PBDEs on microalgal cells, and that chloroplast- and cell membrane-produced ROS was an important signaling molecule to mediate the PCD activation process. Our research firstly indicated microalgal PCD could be induced by PBDEs, and increased our knowledge of the toxic mechanisms by which POPs affect microalgal cells.

The transcription factor REST up-regulates tyrosine hydroxylase and antiapoptotic genes and protects dopaminergic neurons against manganese toxicity

Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein-binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1β (IL-1β), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2-associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration.

Dapk1 improves inflammation, oxidative stress and autophagy in LPS-induced acute lung injury via p38MAPK/NF-κB signaling pathway

ObjectiveTo investigate the impact of death-associated protein kinase 1 (Dapk1) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) via p38MAPK/NF-κB pathway. MethodsDapk1+/+ and Dapk1−/− mice were randomized into Control, LPS, SB203580 (a p38MAPK pathway inhibitor) + LPS, and PDTC (a NF-κB pathway inhibitor) + LPS groups. Cell counts, lung wet to dry weight ratio (W/D weight ratio), as well as indicators of oxidative stress were determined followed by the detection with HE staining, ELISA, qRT-PCR, Western blotting and Immunofluorescence. Besides, to explore whether the effect of Dapk1 on ALI directly mediated via p38MAPK/NF-κB pathway, mice were injected with TC-DAPK 6 (a Dapk1 inhibitor) with or without SB203580/PDTC before LPS administration. ResultsLPS induced lung injury with increased lung W/D weight ratio, which could be partly reversed by SB203580 and PDTC in LPS-induced mice with activated p38MAPK/NF-κB pathway in lung tissues, especially in Dapk1−/− mice. SB203580 and PDTC reduced total cells and neutrophils in BALF in LPS-induced mice, accompanying with decreased levels of TNF-α, IL-6, MPO, LPO and MDA and the expressions of beclin-1, Atg5 and LC3II, but with the up-regulated activities of SOD and GSH-Px, as well as p62 protein expression. Besides, TC-DAPK 6 aggravated the pathologic injury in LPS-induced ALI with more serious inflammatory response, oxidative stress and autophagy as well as the activated p38MAPK/NF-κB pathway, which were reversed by SB203580 or PDTC. ConclusionDapk1 improved oxidative stress, inhibited autophagy, and reduce inflammatory response of LPS-induced ALI mice by inhibiting p38MAPK/NF-κB pathway.

Fentanyl inhibits the progression of gastric cancer through the suppression of MMP-9 via the PI3K/Akt signaling pathway.

Fentanyl is a drug commonly used for perioperative and postoperative analgesia. Previous studies have confirmed that fentanyl can affect the progression of gastric cancer; however, this effect has not yet been elucidated. The purpose of our study was thus to investigate the role of fentanyl in gastric cancer and clarify its potential mechanisms. A CCK-8 assay was used to determine the proliferation of MGC-803 cells, while Transwell assay and wound healing assay were used to determine the invasion and migration abilities, respectively. Apoptosis and the cell cycle were assessed by flow cytometry, and the ultrastructure of the cells was examined with a transmission electron microscope. The mRNA expression levels of serine-threonine protein kinase 1 (Akt-1), matrix metalloproteinase-9 (MMP-9), and death-associated protein kinase 1 (DAPK1) were evaluated by real-time (RT) quantitative PCR. The protein expression of p-Akt, MMP-9, and caspase-9 was detected by western blot analysis. To study the interaction of fentanyl with the phosphatidylinositol-3-kinase (PI3K)/Akt/MMP-9 pathway, PI3K inhibitor (LY294002) and MMP-9 inhibitor (SB-3CT) were used to treat the MGC-803 cells. Findings indicated that fentanyl inhibits the proliferation, invasion, and migration of MGC-803 cells. Specifically, fentanyl inhibits the expression of MMP-9 and enhances the expression of apoptosis-promoting factors such as caspase-9 and DAPK1 through the PI3K/Akt signaling pathway. Cell cycle arrest was observed in the G0/G1 phase. Furthermore, the inhibition of PI3K/Akt/MMP-9 by LY294002 and SB-3CT enhanced the anticancer effects of fentanyl. Fentanyl inhibits the proliferation, invasion and migration of gastric cancer cells by inhibiting the PI3K/Akt/MMP-9 pathway, which could be very useful for gastric cancer treatment.

Metformin and trimetazidine ameliorate diabetes-induced cognitive impediment in status epileptic rats

Patients with diabetes and epilepsy are more prone to cognitive impairment, dementia, and even Alzheimer's disease. Diabetes-induced inflammatory process is one of the main contributing factors; however, the impact on seizure is not clear. The current study is aimed to examine the role of metformin and trimetazidine in the reduction of neuronal damage caused by inflammatory mediators and apoptotic factors in diabetic epileptic rodent model. Diabetic epileptic rats received orally either metformin (100 mg/kg) or trimetazidine (10 mg/kg) for 3 weeks exhibited reduced cognitive function and ameliorated the disturbed brain neurotransmission. Besides, they improved both the inflammatory status and the histopathologic alterations. Administration of metformin or trimetazidine ameliorated the deterioration in cognitive function in Morris water maze (MWM) and reduced seizure score. Furthermore, brain neurotransmitters glutamate and γ-aminobutyric acid (GABA) were reverted back to their normal values. Both treatments reduced the rise in inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), apoptotic markers nuclear factor-κB (NF-κB) and caspase-3, and improved the pathological photomicrograph of the hippocampus of diabetic epileptic rats. Such effects were closely correlated to the observed increase in the adenosine triphosphate and adenosine diphosphate (ATP/ADP) ratio and reduction of death-associated protein (DAP) and mammalian target of rapamycin (mTOR). In conclusion, the current study shed light on the potential neuroprotective role of metformin and trimetazidine in the amelioration of cognitive function via hindering inflammatory processes in diabetic epileptic rats.

Generation of a DAPK1 knockout first (conditional ready) human embryonic stem cell line (ZSSYe001-A) by CRISPR-Cas9 technology

Death-associated protein kinase 1 (DAPK1) is a Ca2+/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.

Autophagy and Apoptosis Specific Knowledgebases-guided Systems Pharmacology Drug Research.

Autophagy and apoptosis are the basic physiological processes in cells that clean up aged and mutant cellular components or even the entire cells. Both autophagy and apoptosis are disrupted in most major diseases such as cancer and neurological disorders. Recently, increasing attention has been paid to understand the crosstalk between autophagy and apoptosis due to their tightly synergetic or opposite functions in several pathological processes. This study aims to assist autophagy and apoptosis-related drug research, clarify the intense and complicated connections between two processes, and provide a guide for novel drug development. We established two chemical-genomic databases which are specifically designed for autophagy and apoptosis, including autophagy- and apoptosis-related proteins, pathways and compounds. We then performed network analysis on the apoptosis- and autophagy-related proteins and investigated the full protein-protein interaction (PPI) network of these two closely connected processes for the first time. The overlapping targets we discovered show a more intense connection with each other than other targets in the full network, indicating a better efficacy potential for drug modulation. We also found that Death-associated protein kinase 1 (DAPK1) is a critical point linking autophagy- and apoptosis-related pathways beyond the overlapping part, and this finding may reveal some delicate signaling mechanism of the process. Finally, we demonstrated how to utilize our integrated computational chemogenomics tools on in silico target identification for small molecules capable of modulating autophagy- and apoptosis-related pathways. The knowledge-bases for apoptosis and autophagy and the integrated tools will accelerate our work in autophagy and apoptosis-related research and can be useful sources for information searching, target prediction, and new chemical discovery.

Silencing DAPK3 blocks the autophagosome-lysosome fusion by mediating SNAP29 in trophoblast cells under high glucose treatment

Autophagy plays an essential role in gestational diabetes mellitus (GDM). Death-associated protein kinase-3 (DAPK3) regulates a variety of cellular functions; however, the relationship between DAPK3 and autophagy is unknown. In this study, we aim to investigate whether DAPK3 is associated with autophagy in GDM, and we found that DAPK3 was upregulated in the placenta of GDM patients and extravillous trophoblast cells under high-glucose conditions. Silencing DAPK3 decreased the assembly of the STX17-SNAP29-VAMP8 complex, leading to the blockade of autophagosome-lysosome fusion by mediating synaptosomal-associated protein 29 (SNAP29). Moreover, knockdown of DAPK3 ameliorates cell invasion and mediates autophagy in high glucose, and does not alter the expression of autophagy-related genes in normal glucose. Our study demonstrates the significance of DAPK3 in autophagy and GDM, which may provide new insights into the molecular mechanisms regulating trophoblast invasion.

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells

To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells. HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP). Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L): 12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)% vs. (69.40±0.75)%, ATP (×106 RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin: 0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)% vs. (34.42±0.64)%], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001±0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin: 0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05). There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.

DAPK1 loss triggers tumor invasion in colorectal tumor cells

Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Thymosin β4 promotes autophagy and repair via HIF-1α stabilization in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin β4 (Tβ4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tβ4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tβ4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tβ4.