Inhibition of NADPH oxidase alleviates germ cell apoptosis and ER stress during testicular ischemia reperfusion injury.

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.

PERK Inhibition Mitigates Restenosis and Thrombosis: A Potential Low-Thrombogenic Antirestenotic Paradigm.

Developing endothelial-protective, nonthrombogenic antirestenotic treatments has been a challenge. A major hurdle to this has been the identification of a common molecular target in both smooth muscle cells and endothelial cells, inhibition of which blocks dysfunction of both cell types. The authors' findings suggest that the PERK kinase could be such a target. Importantly, PERK inhibition mitigated both restenosis and thrombosis in preclinical models, implicating a low-thrombogenic antirestenotic paradigm.

JAK2 regulation by licochalcone H inhibits the cell growth and induces apoptosis in oral squamous cell carcinoma.

BACKGROUND: Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations. PURPOSE: The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms. STUDY DESIGN: We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway. METHODS: To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis. RESULTS: According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade. CONCLUSION: LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Acrolein Is a Pathogenic Mediator of Alcoholic Liver Disease and the Scavenger Hydralazine Is Protective in Mice.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) remains a major cause of morbidity and mortality, with no Food and Drug Administration-approved therapy. Chronic alcohol consumption causes a pro-oxidant environment and increases hepatic lipid peroxidation, with acrolein being the most reactive/toxic by-product. This study investigated the pathogenic role of acrolein in hepatic endoplasmic reticulum (ER) stress, steatosis, and injury in experimental ALD, and tested acrolein elimination/scavenging (using hydralazine) as a potential therapy in ALD. METHODS: In vitro (rat hepatoma H4IIEC cells) and in vivo (chronic+binge alcohol feeding in C57Bl/6 mice) models were used to examine alcohol-induced acrolein accumulation and consequent hepatic ER stress, apoptosis, and injury. In addition, the potential protective effects of the acrolein scavenger, hydralazine, were examined both in vitro and in vivo. RESULTS: Alcohol consumption/metabolism resulted in hepatic accumulation of acrolein-protein adducts, by up-regulation of cytochrome P4502E1 and alcohol dehydrogenase, and down-regulation of glutathione-s-transferase-P, which metabolizes/detoxifies acrolein. Alcohol-induced acrolein adduct accumulation led to hepatic ER stress, proapoptotic signaling, steatosis, apoptosis, and liver injury; however, ER-protective/adaptive responses were not induced. Notably, direct exposure to acrolein in vitro mimicked the in vivo effects of alcohol, indicating that acrolein mediates the adverse effects of alcohol. Importantly, hydralazine, a known acrolein scavenger, protected against alcohol-induced ER stress and liver injury, both in vitro and in mice. CONCLUSIONS: Our study shows the following: (1) alcohol consumption triggers pathologic ER stress without ER adaptation/protection; (2) alcohol-induced acrolein is a potential therapeutic target and pathogenic mediator of hepatic ER stress, cell death, and injury; and (3) removal/clearance of acrolein by scavengers may have therapeutic potential in ALD.

Silica nanoparticles induce endoplasmic reticulum stress response, oxidative stress and activate the mitogen-activated protein kinase (MAPK) signaling pathway.

Application of silica nanoparticles (SiO2-NPs) may result in human exposure. Here we investigate unexplored modes of action by which SiO2-NPs with average size of 225 nm act on human hepatoma cells (Huh7). We focused on the endoplasmic (ER) stress response and on mitogen-activated protein kinase (MAPK) signaling pathways. Both pathways were induced. ER stress and the associated three unfolded protein response (UPR) pathways were activated as demonstrated by significant inductions of BiP and XBP-1s and a moderate but significant induction of ATF-4 at 0.05 and 0.5 mg/ml. In addition to activation of NFкB interferon stimulated genes IP-10, IRF-9, and ISG-15 were up-regulated. As a consequence of ER stress, the pro-inflammatory cytokine TNFα and PP2Ac were induced following exposure to 0.05 mg/ml SiO2-NPs. Additionally, this occurred at 0.005 mg/ml SiO2-NPs for TNFα at 24 h. This in turn led to a strong transcriptional induction of MAP-kinases and its target genes cJun, cMyc and CREB. A strong transcriptional down-regulation of the proapoptotic gene p53 occurred at 0.05 and 0.5 mg/ml SiO2-NP. Exposure of Huh7 cells to the anti-oxidant N-acetyl cysteine reduced transcriptional induction of ER stress markers demonstrating a link between the induction of oxidative stress and ER stress. Our study demonstrates that SiO2-NPs lead to strong ER stress and UPR induction, oxidative stress, activation of MAPK signaling and down-regulation of p53. All of these activated pathways, which are analyzed here for the first time in detail, inhibit apoptosis and induce cell proliferation, which may contribute to a hepatotoxic, inflammatory and tumorigenic action of SiO2-NPs.