Activation Of Caspase Pathway Research Articles

Decoding the biological toxicity of phenanthrene on intestinal cells of Eisenia fetida: Effects, toxicity pathways and corresponding mechanisms

Phenanthrene is frequently detected and exists extensively in the soil environment, and its residues inevitably impose a significant threat to soil organisms. Exposure to and toxicity of phenanthrene on earthworms has been extensively studied before, however, the possible mechanisms and related pathways associated with phenanthrene-triggered toxicity at the intestinal cell level remain unclear. Herein, primary intestinal cells isolated from Eisenia fetida (Annelida, Oligochaeta) intestine were used as targeted receptors to probe the molecular mechanisms involved in ROS-mediated damaging effects and the potential pathways of phenanthrene-induced toxicity at cellular and sub-cellular levels. Results indicated that phenanthrene exposure induced oxidative stress by activating intracellular ROS (elevated O2−, H2O2, and OH− content) bursts in E. fetida intestinal cells, causing various oxidative damage effects, including lipid peroxidation (increased MDA content), protein oxidation (enhanced PCO levels), and DNA damage (enhanced 8-OHdG levels). The enzymatic and non-enzymatic strategies in earthworm cells were activated to mitigate these detrimental effects by regulating ROS-mediated pathways involving defense regulation. Also, phenanthrene stress destroyed the cell membrane of E. fetida intestinal cells, resulting in cellular calcium homeostasis disruption and cellular energetic alteration, ultimately causing cytotoxicity and cell apoptosis/death. More importantly, the mitochondrial dysfunction in E. fetida cells was induced by phenanthrene-caused mitochondrial membrane depolarization, which in turn caused un-controlled ROS burst and induced apoptosis through mitochondria-mediated caspase-3 activation and ROS-mediated mitochondrial-dependent pathway. Furthermore, exposure to phenanthrene activated an abnormal mRNA expression profile associated with defense regulation (e.g., Hsp70, MT, CRT, SOD, CAT, and GST genes) in E. fetida intestinal cells, resulting in various cellular dysfunctions and pathological conditions, eventually, apoptotic cell death. Taken together, this study offers valuable insights for probing the toxic effects and underlying mechanisms posed by phenanthrene at the intestinal cell level, and is of great significance to estimate the detrimental side effects of phenanthrene on soil ecological health.

CLASRP oncogene as a novel target for colorectal cancer

Clk4-associated serine/arginine-rich protein (CLASRP), an alternative splicing regulator, may be involved in the development and progression of cancer by regulating the activity of the CDC-like kinase (Clk) family. This study explored the biological function of CLASRP in colorectal cancer (CRC). The expression of CLASRP, which is associated with clinicopathological features, was analysed in CRC tissues and paired noncancer tissues by RT–PCR. The roles of CLASRP were investigated in CRC cells transfected with plasmids or shRNA through proliferation, migration and invasion assays in vitro and a xenograft model in vivo. Apoptosis was analysed using CLASRP-overexpressing CRC cells by western blotting. Clk inhibitors were used to perform functional research on CLASRP in CLASRP-overexpressing CRC cells. CLASRP was significantly upregulated in CRC cell lines, while high CLASRP expression was correlated with metastasis in CRC patients. Functionally, overexpression of CLASRP significantly promoted the proliferation, migration and invasion of CRC cells in vitro and tumour growth in vivo. Mechanistically, the proliferation, migration and invasion of CLASRP-overexpressing CRC cells were inhibited by Clk inhibitors, accompanied by low expression of CLASRP at the gene and protein levels. Clk inhibitors induced apoptosis of CLASRP-overexpressing CRC cells, resulting in direct blockade of cell growth. The expression levels of cleaved caspase 3 and cleaved caspase 8 were increased in CLASRP-overexpressing CRC cells treated with Clk inhibitors. CLASRP might serve as a promotional oncogene in CRC cells and be suppressed by Clk inhibitors through activation of caspase pathways.

Neuronal somatic plasmalemmal permeability and dendritic beading caused by head rotational traumatic brain injury in pigs-An exploratory study.

Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY+ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY+ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY+ and LY- cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.

Mitochondria-targeted cyclometalated iridium-β-carboline complexes as potent non-small cell lung cancer therapeutic agents.

Natural products and metals play a crucial role in cancer research and the development of antitumor drugs. We designed and synthesized three new carboline-based cyclometalated iridium complexes [Ir(C-N)2(PPβC)](PF6), where PPβC=N-(1,10-phenanthrolin-5-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxamide, C-N=2-phenylpyridine (ppy, Ir1), 2-(2,4-difluorophenyl) pyridine (dfppy, Ir2), 7,8-benzoquinoline (bzq, Ir3), by combining iridium with β-carboline derivative. These iridium complexes exhibited high potential antitumor effects after being promptly taken up by A549 cells. Accumulating in mitochondria rapidly and preferentially, Ir1-3 caused a series of changes in mitochondrial events, including the loss of mitochondrial membrane potential, the depletion of cellular ATP, and the elevation of reactive oxygen species, leading to significant death of A549 cells. Moreover, the activation of intracellular caspase pathway and apoptosis was further validated to contribute to iridium complexes-induced cytotoxicity. These novel iridium complexes exerted a prominent inhibitory effect on tumor growth in a three-dimensional multicellular tumor spheroid model.

Activation of Nrf-2 Transcription Factor and Caspase Pathway with Royal Jelly Reduces Fluoride Induced Testicular Damage and Infertility in Rats.

This study was carried out to investigate the protective properties of royal jelly on the testicular tissue of rats with testicular damage by giving fluoride. Sperm motility, epididymal sperm density and abnormal sperm ratios were examined and visualized with a light microscope. Expression levels of Caspase-3, Bcl-2, Nrf-2, NF-κB, COX-2, TNF-α and IL1-α proteins in testis tissue were determined by western blot technique. As a result of the study, MDA level, expression level of Bcl-2, NFҡB, COX-2, TNF-α and IL1-α proteins, abnormal sperm rates were found higher in Fluoride-50 and Fluoride100 groups compared to other groups. In addition GSH, Catalase enzyme levels, expression levels of Caspase-3 and Nrf-2 proteins were found to be higher in Fluoride + Royal Jelly groups compared to Fluoride-50 and Fluoride-100 groups. In addition, lower degeneration of testicular tissue was found in the histological evaluation in the Fluoride + Royal Jelly groups compared to the other groups. When the data are evaluated royal jelly provides effective protection against testicular damage. From this point of view, we hope that similar results will be obtained when royal jelly is tested on humans.

Curcumin Nanofibers: A Novel Approach to Enhance the Anticancer Potential and Bioavailability of Curcuminoids.

Development of novel treatment methods for cancer is needed given the limitations of current treatment methods, including side effects and chemotherapeutic resistance, which may provide new hope to cancer patients. Cancer is the second leading cause of global mortality. Curcumin, the active ingredient of turmeric, has been used since ancient times for various therapeutic purposes. Several studies have identified its activity against cancer. Despite the established anticancer activity of curcumin, its low aqueous solubility and bioavailability are barriers to its effectiveness. In an attempt to solve this problem, many studies have formulated curcumin nanofiber preparations using a variety of methods. Electrospinning is a simple and affordable method for the production of nanofibers. Studies have shown increased curcumin bioavailability in nanofibers resulting from their high surface/volume ratio and porosity. We have undertaken a detailed review of studies on the anticancer effects of curcumin nanofibers. Curcumin acts by inhibiting various biological cancer pathways, including NF-κB, mTOR, complex I, cytokines, expression of p-p65, Ki67, and angiogenesis-associated genes. It also induces apoptosis through activation of caspase pathways and ROS production in cancer cells. Curcumin-loaded PLA50/PVP50/Cur15 nanofibers were investigated in breast cancer, one of the most studied cancers, and was shown to have significant effects on the widely used HeLa-cell line. Most of the studies undertaken have been performed in cell lines in vitro, while relatively few animal studies have been reported. More preclinical and clinical studies are needed to evaluate the anticancer activity of curcumin nanofibers. Amongst studies undertaken, a variety of curcumin nanofibers of various formulations have been shown to suppress a variety of cancer types. Overall, curcumin nanofibers have been found to be more efficient than free curcumin. Thus, curcumin nanofibers have been observed to improvise cancer treatment, offering great potential for effective cancer management. Further studies, both in vitro and in vivo, involving curcumin nanofibers have the potential to benefit cancer management.

Fumonisin B1 Induces Immunotoxicity and Apoptosis of Chicken Splenic Lymphocytes

Fumonisin B1 (FB1), produced by Fusarium, is among the most abundant and toxic mycotoxin contaminations in feed, causing damages to the health of livestock. However, the mechanisms of FB1 toxicity in chickens are less understood. As splenic lymphocytes play important roles in the immune system, the aim of this study was to investigate the immunotoxic effects and mechanisms of FB1 on chicken splenic lymphocytes. In the present study, the chicken primary splenic lymphocytes were harvested and treated with 0, 2.5, 5, 10, 20 and 40 μg/mL FB1. Then, the cell proliferation, damage, ultrastructure, inflammation and apoptosis were evaluated. Results showed that the proliferation rate of splenic lymphocytes was decreased by FB1 treatments. The activity of lactate dehydrogenase (LDH) was increased by FB1 treatments in a dose-dependent manner, implying the induction of cell damage. Consistently, the ultrastructure of splenic lymphocytes showed that FB1 at all the tested concentrations caused cell structure alterations, including nuclear vacuolation, mitochondrial swelling and mitochondrial crest fracture. Besides, immunosuppressive effects of FB1 were observed by the decreased concentrations of interleukin-2 (IL-2), IL-4, IL-12 and interferon-γ (IFN-γ) in the cell culture supernatant. Furthermore, apoptosis was observed in FB1-treated cells by flow cytometry. The mRNA expressions of apoptosis-related genes showed that the expression of Bcl-2 was decreased, while the expressions of the P53, Bax, Bak-1, and Caspase-3 were increased with FB1 treatment. Similar results were found in the concentrations of apoptosis-related proteins in the cell supernatant by ELISA assay. Moreover, regression analysis indicated that increasing FB1 concentration increased LDH activity, concentrations of Bax, Bak-1 and mRNA expression of Bak-1 linearly, increased M1 area percentage quadratically, decreased concentration of IFN-γ, mRNA expression of Bcl-2 linearly, and decreased concentrations of IL-2 and IL-4 quadratically. Besides, regression analysis also showed reciprocal relationships between IL-12 concentration, Caspase-3 mRNA expression and increasing FB1 concentration. The increasing FB1 concentration could decrease IL-12 concentration and increase Caspase-3 mRNA expression. Altogether, this study reported that FB1 induced the immunotoxicity of chicken splenic lymphocytes and caused splenic lymphocytes apoptosis by the Bcl-2 family-mediated mitochondrial pathway of caspase activation.

Methylmercury induced apoptosis of human neuroblastoma cells through the reactive oxygen species mediated caspase and poly ADP-ribose polymerase/apoptosis-inducing factor dependent pathways.

Methylmercury (MeHg) is an environmental neurotoxic substance, which can easily cross the blood-brain barrier, causing irreversible damage to the human central nervous system. Reactive oxygen species (ROS) are involved in various ways of intracellular physiological or pathological processes including neuronal apoptosis. This study attempted to explore the role of ROS-mediated poly ADP-ribose polymerase (PARP)/apoptosis-inducing factor (AIF) apoptosis signaling pathway in the process of MeHg-induced cell death of human neuroblastoma cells (SH-SY5Y). Here, we found that SH-SY5Y cells underwent apoptosis in response to MeHg, which was accompanied by the increased levels of ROS and calcium ion, and the activation of caspase cascades and PARP. Inhibiting the production of ROS can reduce the apoptosis rate to a certain extent. PARP/AIF apoptotic pathway is independent of caspase dependent signaling pathway and regulates it. In conclusion, these results suggest that ROS mediated activation of caspase pathway and PARP/AIF signaling pathway are involved in MeHg induced apoptosis, and these two pathways interact with each other.

Coccinia grandis alleviates flavor‐enhancing high‐lipid diet induced hepatocellular inflammation and apoptosis

In the present socioeconomic era, people are consuming ready-made fast-food regularly with minimal physical exercise. Food processors use monosodium glutamate, saturated fatty acids, and hydrogenated fats to prepare flavor-enhancing high-lipid diet (FHD), which cause oxidative damage to different experimental animals and humans through the generation of reactive oxygen species. This study aimed to assess the protective effects of Coccinia grandis against hepatocellular damage caused by FHD. Rats were fed with FHD (prepared with monosodium glutamate in combination with HLD) with or without ethanol extract of Coccinia grandis leaves (EECGL) for 28 days to measure hematological, biochemical, inflammatory, apoptotic biomarkers, cytomorphological changes, and apoptosis of liver, if any. The results indicate that FHD causes hepatic damage by modifying hematological and biochemical parameters, followed by the activation of NF-kB and caspase pathways. Moreover, FHD altered the Bcl2/Bax ratio, nuclear condensation, shrinkage, and fragmentation of hepatocytes, leading to inflammation and apoptosis. On the other hand, EECGL appears to play a significant role in preventing FHD-induced hepatocellular damage via regulating inflammatory and apoptotic factors. In this regard, EECGL might be a useful dietary supplement to reduce the negative impact of a frequent consumption of FHD as part of fast-food. PRACTICAL APPLICATIONS: Fast food is believed to be a flavor-enhanced high-lipid diet, since its delectable taste stimulates the hunger. Eventually, such a diet functions as a silent assassin for many body systems. The current study primarily focused on the negative health effects of commonly used flavoring agents in high-lipid diets, which presents a warning against the choice of meals, particularly food additive mixed diets, and issues an alarming signal to society concerning the use of such combinations in regular diets. Furthermore, this study recommends using Coccinia grandis, which has a variety of bioactive phytoconstituents, as a dietary supplement to counteract the flavor-enhancing high-lipid diet-induced anomalous condition.

GKK1032B from endophytic Penicillium citrinum induces the apoptosis of human osteosarcoma MG63 cells through caspase pathway activation.

Chemical investigation of the culture extract of an endophytic Penicillium citrinum from Dendrobium officinale, afforded nine citrinin derivatives (1-9) and one peptide-polyketide hybrid GKK1032B (10). The structures of these compounds were determined by spectroscopic methods. The absolute configurations of 1 and 2 were determined for the first time by calculation of electronic circular dichroism (ECD) data. Among them, GKK1032B (10) showed significant cytotoxicity against human osteosarcoma cell line MG63 with an IC50 value of 3.49 μmol·L-1, and a primary mechanistic study revealed that it induced the apoptosis of MG63 cellsvia caspase pathway activation.

Antileukemic Natural Product Induced Both Apoptotic and Pyroptotic Programmed Cell Death and Differentiation Effect.

Acute myeloid leukemia (AML) is one of the most common forms of leukemia. Despite advances in the management of such malignancies and the progress of novel therapies, unmet medical needs still exist in AML because of several factors, including poor response to chemotherapy and high relapse rates. Ardisianone, a plant-derived natural component with an alkyl benzoquinone structure, induced apoptosis in leukemic HL-60 cells. The determination of dozens of apoptosis-related proteins showed that ardisianone upregulated death receptors and downregulated the inhibitor of apoptosis protein (IAPs). Western blotting showed that ardisianone induced a dramatic increase in tumor necrosis factor receptor 2 (TNFR2) protein expression. Ardisianone also induced downstream signaling by activating caspase-8 and -3 and degradation in Bid, a caspase-8 substrate. Furthermore, ardisianone induced degradation in DNA fragmentation factor 45 kDa (DFF45), a subunit of inhibitors of caspase-activated DNase (ICAD). Q-VD-OPh (a broad-spectrum caspase inhibitor) significantly diminished ardisianone-induced apoptosis, confirming the involvement of caspase-dependent apoptosis. Moreover, ardisianone induced pyroptosis. Using transmission electron microscopic examination and Western blot analysis, key markers including gasdermin D, high mobility group box1 (HMGB1), and caspase-1 and -5 were detected. Notably, ardisianone induced the differentiation of the remaining survival cells, which were characterized by an increase in the expression of CD11b and CD68, two markers of macrophages and monocytes. Wright–Giemsa staining also showed the differentiation of cells into monocyte and macrophage morphology. In conclusion, the data suggested that ardisianone induced the apoptosis and pyroptosis of leukemic cells through downregulation of IAPs and activation of caspase pathways that caused gasdermin D cleavage and DNA double-stranded breaks and ultimately led to programmed cell death. Ardisianone also induced the differentiation of leukemic cells into monocyte-like and macrophage-like cells. The data suggested the potential of ardisianone for further antileukemic development.

The c-Abl inhibitor, radotinib induces apoptosis in multiple myeloma cells via mitochondrial-dependent pathway

Multiple myeloma (MM) is a hematological cancer resulting from accumulated abnormal plasma cells. Unfortunately, MM remains an incurable disease, as relapse is very common. Therefore, there is urgent need to develop new treatment options for MM. Radotinib is a novel anti-cancer drug, currently approved in South Korea for the treatment of chronic myeloid leukemia patients. Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and the platelet-derived growth factor receptor. Generally, the mechanism of inhibition of non-receptor tyrosine kinase c-Abl has played an essential role in the inhibition of cancer progression. However, little is known regarding the effects of the c-Abl inhibitor, radotinib on MM cells. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Interestingly, radotinib caused apoptosis in MM cells including RPMI-8226, MM.1S, and IM-9 cells, even in the absence of c-kit expression in 2 of these lines. Radotinib treatment significantly increased the number Annexin V-positive cells and decreased the mitochondrial membrane potential in MM cells. Additionally, we observed that cytochrome C was localized in the cytosol of radotinib-treated MM cells. Moreover, radotinib decreased the expression of Bcl-2 and Bcl-xL, and increased the expression of Bax and Bak in MM cells. Furthermore, radotinib promoted caspase pathway activation by inducing the expression and activity of caspase-3, -7, and -9. Expression of cleaved PARP-1 was also increased by radotinib treatment in various MM cells. In addition, radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells, and killed ex vivo myeloma cells from patients. In conclusion, radotinib may play an important role as a candidate agent or chemosensitizer for the treatment of MM.

Exploring the role and clinical implications of proteasome inhibition in medulloblastoma.

Ubiquitin proteasome-mediated protein degradation has been implicated in posttranslational oncogenesis in medulloblastoma. Current research is evaluating the clinical implications of proteasome inhibition as a therapeutic target. In medulloblastoma cell lines, proteasome inhibitors induce apoptosis and inhibit cell proliferation via multiple pathways involving activation of caspase pathways, NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway inhibition, reduced AKT/mTOR pathway activity, and pro-apoptotic protein expression. Second-generation proteasome inhibitors demonstrate blood-brain barrier penetration while maintaining antitumor effect. This review summarizes the ubiquitin-proteasome system in the pathogenesis of medulloblastoma and the potential clinical implications.

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

The apoptotic effect of Ferulic acid-synthesized gold nanoparticles against human epidermoid carcinoma (A431) cells via activation of caspase-3 pathway

In the present study, ferulic acid (fa), a polyphenol, was employed to prepare stable gold nanoparticles. It acted as a reducing agent when mixed with hydrogen tetrachloroaurate (III) hydrate at ambient temperature. Subsequently, fa also turned as a stabilizing agent and yielded spherical gold nanoparticles (fa-AuNPs). The synthesized gold nanoparticles were thoroughly characterized using UV/Visible spectroscopy, Scanning electron microscopy, High resolution transmission electron microscopy, Dynamic light scattering and Fourier-transform infrared spectroscopy studies. Then, the synthesized fa-AuNPs were tested in human skin cancer cells (A431) and normal kertotinocytes (HaCaT cells). The fa-AuNPs produced cytotoxicity in A431 cells in a dose and time-dependent manner. The angiogenetic efficacy of the fa-AuNPs was substantiated by the results of CAM assay. The programmed cell death occurred via apoptosis as indicated by the sub G1 population. Increased levels of reactive oxygen species and caspase-3 activity resulted in reduced mitochondrial membrane potential. Hence, this study corroborated that fa-AuNPs successfully stimulated apoptosis in A431 cells through mitochondria based pathways and thus may be considered as a potential agent to treat skin cancer.

Changes in the Expression of TBP-2 in Response to Histone Deacetylase Inhibitor Treatment in Human Endometrial Cells.

Histone deacetylase inhibitors (HDACi) induce apoptosis preferentially in cancer cells by caspase pathway activation and reactive oxygen species (ROS) accumulation. Suberoylanilide hydroxamic acid (SAHA), a HDACi, increases apoptosis via altering intracellular oxidative stress through thioredoxin (TRX) and TRX binding protein-2 (TBP-2). Because ROS accumulation, as well as the redox status determined by TBP-2 and TRX, are suggested as possible mechanisms for endometriosis, we queried whether SAHA induces apoptosis of human endometrial cells via the TRX–TBP-2 system in endometriosis. Eutopic endometrium from participants without endometriosis, and ectopic endometrium from patients with endometriosis, was obtained surgically. Human endometrial stromal cells (HESCs) and Ishikawa cells were treated with SAHA and cell proliferation was assessed using the CCK-8 assay. Real-time PCR and Western blotting were used to quantify TRX and TBP-2 mRNA and protein expression. After inducing oxidative stress, SAHA was applied. Short-interfering TRX (SiTRX) transfection was performed to see the changes after TRX inhibition. The mRNA and protein expression of TBP-2 was increased with SAHA concentrations in HESCs significantly. The mRNA TBP-2 expression was decreased after oxidative stress, upregulated by adding 2.5 μM of SAHA. The TRX/TBP-2 ratio decreased, apoptosis increased significantly, and SiTRX transfection decreased with SAHA. In conclusion, SAHA induces apoptosis by modulating the TRX/TBP-2 system, suggesting its potential as a therapeutic agent for endometriosis.

Evaluation of Molecular and Cellular Alterations Induced by Neuropathic Pain in Rat Brain Glial cells.

Neuropathic pain originates from illness or damage of the nervous system and affects the somatosensory system. Recently, many efforts have been made to illuminate the influences of neuropathic pain in different parts of central nervous system (CNS). However, the toxic consequences of neuropathic pain in glial cells, which involve in the control of pain is poorly understood. Therefore, the present study aimed to assess the molecular and cellular effects of neuropathic pain in the glial cells of rat brain. Induction of neuropathic pain in rats was associated with oxidative stress as evident by elevated reactive oxygen species (ROS) formation as well as reversible glutathione (GSH) depletion in the glial cells. Moreover, neuropathic pain caused mitochondrial membrane potential collapse (∆Ψm%), lysosomal membrane rapture, and proteolysis, probably due to ROS-induced MPT pore opening. These toxic events could cause cytochrome c release from intermembrane space into the cytosole and trigger caspase activation pathway. Our finding confirmed that the activity of caspase-3 was significantly increased in the glial cells as a core component of the apoptotic machinery. In conclusion, the neuropathic pain induces ROS generation as the major cause of GSH depletion along with mutual mitochondrial/lysosomal potentiation (cross-talk) of oxidative stress in the glial cells. Subsequently, this toxic cross-talk can induce proteolysis and trigger apoptosis by caspase-3 activation in the glial cells of rat brain.

Novel Polymeric Hybrid Nanocarrier for Curcumin and Survivin shRNA Co-delivery Augments Tumor Penetration and Promotes Synergistic Tumor Suppression.

A major barrier for co-delivery of gene medicine with small molecular chemotherapeutic drugs in solid tumors is the inadequate tumor penetration and transfection. In this study, a novel polymeric nanocarrier with integrated properties of tumor penetration, nuclear targeting, and pH-responsive features was designed, and further used to achieve the synergistic anti-tumor effect of curcumin (CUR) and survivin shRNA (pSUR). The polymeric hybrid nanocarrier was constructed from the FDA-approved polymer PLGA and a novel conjugated triblock polymer W5R4K-PEG2K-PHIS (WPH). CUR and pSUR were simultaneously encapsulated in the dual-drug-loaded nanoparticles (CUR/pSUR-NPs) by a modified double-emulsion solvent evaporation (W/O/W) method. The obtained nanoparticles exhibited better pharmaceutical properties with a uniform spherical morphology and sustained release manners of CUR and pSUR. Excellent features including preferable cellular uptake, efficient endosomal escape, enhanced tumor penetration, and elevated transfection efficiency were further proven. Additionally, a markedly enhanced anti-tumor efficacy for CUR/shRNA-NPs was achieved on SKOV-3 and Hela cells. The synergistic anti-tumor effect involved the inhibition of tumor cell proliferation, induction of cell apoptosis, and the activation of caspase-3 pathways. This work sets up an innovative co-delivery nanosystem to suppress tumor growth, contributing to the development of a comprehensive nanoparticulate strategy for future clinical applications.

Microglia Activation in Retinal Ischemia Triggers Cytokine and Toll-Like Receptor Response

Mechanisms and progression of ischemic injuries in the retina are still incompletely clarified. Therefore, the time course of microglia activation as well as resulting cytokine expression and downstream signaling were investigated. Ischemia was induced in one eye by transiently elevated intraocular pressure (60 min) followed by reperfusion; the other eye served as a control. Eyes were processed for RT-qPCR and immunohistochemistry analyses at 2, 6, 12, and 24 h as well as at 3 and 7 days. Already 2 h after ischemia, more microglia/macrophages were in an active state in the ischemia group. This was accompanied by an upregulation of pro-inflammatory cytokines, like IL-1β, IL-6, TNFα, and TGFβ. Activation of TLR3, TLR2, and the adaptor molecule Myd88 was also observed after 2 h. NFκB revealed a wave-like activation pattern. In addition, an extrinsic caspase pathway activation was noted at early time points, while enhanced numbers of cleaved caspase 3+ cells could be observed in ischemic retinae throughout the study. Retinal ischemia induced an early and strong microglia/macrophage response as well as cytokine and apoptotic activation processes. Moreover, in early and late ischemic damaging processes, TLR expression and downstream signaling were involved, suggesting an involvement in neuronal death in ischemic retinae.Graphical

ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia

BackgroundIn acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with a dismal outcome. Although uncoordinated 51-like kinase 1 (ULK1), which plays a central role in the autophagy pathway, has emerged as a novel therapeutic target for various cancers, its role in FLT3-ITD AML remains elusive. In this study, we evaluated the effects of ULK1 inhibition on leukemia cell death in FLT3-ITD AML.MethodWe evaluated ULK1 expression and the levels of apoptosis and autophagy following ULK1 inhibition in FLT3-ITD AML cell lines and investigated the mechanism underlying apoptosis induced by ULK1 inhibition. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc).ResultsFLT3-ITD AML cells showed significantly higher ULK1 expression than FLT3-wild-type (WT) AML cells. Two ULK1 inhibitors, MRT 68921 and SBI-0206965, induced apoptosis in FLT3-ITD AML cells, with relatively minimal effects on FLT3-WT AML cells and normal CD34-positive cells. Apoptosis induction by ULK1 inhibition was associated with caspase pathway activation. Interestingly, ULK1 inhibition paradoxically also induced autophagy, showing synergistic interaction with autophagy inhibitors. Hence, autophagy may act as a prosurvival mechanism in FLT3-ITD AML cells. FLT3-ITD protein degradation and inhibition of the ERK, AKT, and STAT5 pathways were also observed in FLT3-ITD AML cells following treatment with ULK1 inhibitors.ConclusionULK1 is a viable drug target and ULK1 inhibition may represent a promising therapeutic strategy against FLT3-ITD AML.