Activation Of Mitochondrial Apoptotic Pathway Research Articles

Electrical Stimulation Induces Activation of Mitochondrial Apoptotic Pathway and Down-Regulates Heat Shock Proteins in Pork: An Innovative Strategy for Enhancing the Ripening Process and Quality of Dry-Cured Loin Ham.

A fundamental regulatory framework to elucidate the role of electrical stimulation (ES) in reducing long production cycles, enhancing protein utilization, and boosting product quality of dry-cured ham is essential. However, how mitochondria and enzymes in meat fibers are altered by ES during post-processing, curing, and fermentation procedures remains elusive. This study sought to explore the impact of ES on the regulation of heat shock proteins (HSP27, HSP70), apoptotic pathways, and subsequent influences on dry-cured pork loin quality. The gathered data validated the hypothesis that ES notably escalates mitochondrial oxidative stress and accelerates mitochondrial degradation along the ripening process. The proapoptotic response in ES-treated samples was increased by 120.7%, with a cellular apoptosis rate 5-fold higher than that in control samples. This mitochondrial degradation is marked by increased ratios of Bax/Bcl-2 protein along the time course, indicating that apoptosis could contribute to the dry-cured ham processing. ES was shown to further down-regulate HSP27 and HSP70, establishing a direct correlation with the activation of mitochondrial apoptosis pathways, accompanied by dry-cured ham quality improvements. The findings show that ES plays a crucial role in facilitating the ripening of dry-cured ham by inducing mitochondrial apoptosis to reduce HSP expression. This knowledge not only explains the fundamental mechanisms behind myofibril degradation in dry-cured ham production but also offers a promising approach to enhance quality and consistency.

Tannic Acid Impedes the Proliferation of Bladder Cancer Cells by Elevating Mitochondrial Pathways of Apoptosis.

Bladder cancer stands as a prevailing neoplasm among men globally, distinguished for its pronounced malignancy attributed to invasiveness and metastatic proclivity. Tannic acid (TA), an organic compound in many plants, has garnered recent attention for its discernible anti-mutagenic attributes. This investigation endeavored to scrutinize the repercussions of TA on grade II bladder cancer, with a concerted focus on unraveling its anti-cancer mechanisms. The cytotoxic effects of TA on grade II bladder cancer cells were investigated using multiple techniques, including MTT assay, flow cytometry, TUNEL assay, and western blot. Our findings revealed that elevated concentrations of TA induced cytotoxic effects in grade II bladder cancer cells. Both flow cytometry and the TUNEL assay substantiated the dose-dependent capacity of TA to prompt apoptosis. Western blot analysis corroborated that TA treatment in bladder cancer cells resulted in the upregulation of cleaved caspase-3 expression and PARP. Furthermore, heightened TA dosage elicited an augmentation in the expression of pro-apoptotic proteins, namely Bax and Bak, alongside a reduction in the expression of the anti-apoptotic protein Bcl-2 within bladder cancer cells. This study confirms TA as a potential anticancer agent, demonstrably diminishing the viability of bladder cancer cells. TA exerts cytotoxicity through the activation of mitochondrial apoptotic pathways. Specifically, TA initiates the cleavage of PARP and caspase-3, concurrently augmenting the expression of pro-apoptotic proteins to facilitate apoptosis. Collectively, the present study indicates that TA effectively impedes the proliferation of bladder cancer cells by instigating apoptosis through the intrinsic mitochondrial pathway.

ANKZF1 knockdown inhibits glioblastoma progression by promoting intramitochondrial protein aggregation through mitoRQC

Protein homeostasis is fundamental to the development of tumors. Ribosome-associated quality-control (RQC) is able to add alanine and threonine to the stagnant polypeptide chain C-terminal (CAT-tail) when protein translation is hindered, while Ankyrin repeat and zinc-finger domain-containing-protein 1 (ANKZF1) can counteract the formation of the CAT-tail, preventing the aggregation of polypeptide chains. In particular, ANKZF1 plays an important role in maintaining mitochondrial protein homeostasis by mitochondrial RQC (mitoRQC) after translation stagnation of precursor proteins targeting mitochondria. However, the role of ANKZF1 in glioblastoma is unclear. Therefore, the current study was aimed to investigate the effects of ANKZF1 in glioblastoma cells and a nude mouse glioblastoma xenograft model. Here, we reported that knockdown of ANKZF1 in glioblastoma cells resulted in the accumulation of CAT-tail in mitochondria, leading to the activated mitochondrial unfolded protein response (UPRmt) and inhibits glioblastoma malignant progression. Excessive CAT-tail sequestered mitochondrial chaperones HSP60, mtHSP70 and proteases LONP1 as well as mitochondrial respiratory chain subunits ND1, Cytb, mtCO2 and ATP6, leading to mitochondrial oxidative phosphorylation dysfunction, membrane potential impairment, and mitochondrial apoptotic pathway activation. Our study highlights ANKZF1 as a valuable target for glioblastoma intervention and provides an innovative insight for the treatment of glioblastoma through the regulating of mitochondrial protein homeostasis.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol.

BACKGROUNDEndoplasmic reticulum (ER) stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases. Previous studies have revealed that endoplasmic reticulophagy (ER-phagy) promotes the selective clearance of damaged ER fragments during ER stress, playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis. Family with sequence similarity 134 member B (FAM134B) is a receptor involved in ER-phagy that can form a complex with calnexin (CNX) and microtubule-associated protein 1 light chain 3 (LC3). The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis. However, the precise regulatory mechanisms remain unclear.AIMTo elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A (BRL-3A) rat hepatocytes and the potential regulatory mechanisms.METHODSER stress-related hepatocyte apoptosis was induced using dithiothreitol (DTT). Proteins related to ER stress and autophagy were measured with western blotting. Protein complex interactions with FAM134B were isolated by co-immunoprecipitation. ER-phagy was evaluated in immunofluorescence experiments. Cell cycle distribution and apoptosis were measured by flow cytometry. Mitochondrial Ca2+ levels were evaluated by the co-localization of intracellular Ca2+-tracker and Mito-tracker. The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells.RESULTSER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment. Furthermore, co-immunoprecipitation confirmed an interaction between FAM134B, CNX, FAM134B, and LC3 in BRL-3A cells. Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment, but increased after long-term treatment. Mitochondrial Ca2+ levels and apoptotic rates were dramatically elevated, and more cells were arrested in the G1 stage after short-term DTT treatment; however, these decreased 48 h later. Moreover, FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress.CONCLUSIONFAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway. Our findings provide new evidence highlighting the importance of FAM134B-mediated ER-phagy in attenuating hepatocyte apoptosis.

Protective Effects and Potential Mechanisms of Dietary Resveratrol Supplementation on the Spleen of Broilers Under Heat Stress.

Resveratrol (RSV) is a natural polyphenolic compound with potent antioxidant and anti-inflammatory properties. This study aimed to investigate the protective effects of RSV supplementation on the inflammatory responses of broilers during heat stress. A total of 432 28-d-old white-feathered broilers (817 crossbred chicken) with an average weight of 549 ± 4 g were randomly allotted to 4 equal groups (Half male and half female, 6 replicates/group, 18 chickens/replicate), including normal temperature (NT) group (24 ± 2°C for 24 h/d, basal diet), NT+RSV group (24 ± 2°C for 24 h/d, basal diet + RSV), heat stress (HT) group (37 ± 2°C for 8 h/d, basal diet), and HT+RSV group (37 ± 2°C for 8 h/d, basal diet + RSV). Serum samples were collected on d 7 and 14 of heat stress, and thymus, spleen, jejunum, and bursa of Fabricius samples were collected and analyzed on d14. RSV treatment decreased the feed conversion ratio, partially reversed the negative alternations in body weight, average daily gain, and average daily feed intake caused by heat stress. RSV treatment also decreased the elevated levels of corticosterone on d 14, adrenocorticotropic hormone, and triiodothyronine in serum on d 7 caused by heat stress, and significantly increased the villus height to crypt depth ratio in the jejunum on d 14. Dietary RSV also reduced heat stress-induced splenic pro-inflammatory cytokine concentrations. TUNEL assay showed that RSV significantly reduced heat stress-induced the number of apoptotic cells. Remarkably, RSV down-regulated some splenic related genes for apoptosis genes, including BCL-2, Apaf-1, and MDM2 mRNA levels induced by heat stress. According to GO and KEGG enrichment analyses, the differential genes between HT and HT + RSV groups were mainly associated with immune system process, hematopoietic or lymphoid organ development, and toll-like receptor signaling pathway. The relative mRNA expression of NF-κB, heat shock protein 70 (HSP70), and p38 MAPK were markedly decreased by the combination of RSV and heat stress. These findings showed that RSV might reduce the splenic inflammatory response in heat-stressed white-feather broilers by inhibiting heat stress-induced activation of NF-B, MAPK, and HSP70, as well as inhibiting the activation of mitochondrial apoptotic pathways.

Atmospheric ammonia causes histopathological lesions, cell cycle blockage, and apoptosis of spleen in chickens

This experiment was conducted to investigate the effect of atmospheric ammonia (NH3) on histological changes, cell cycle distribution, and apoptosis of the spleen in chickens. Two hundred forty chickens were randomly allocated to the control group (without NH3 challenge) and NH3 group (70 ± 5 ppm NH3). The experiment lasted for 8 days. The results showed that NH3 exposure caused decreased relative weight ( P < 0.05), dysplasia of white pulps, obvious ultrastructural lesions, upregulation of G0G1 phase cells, excessive apoptosis, and oxidative stress ( P < 0.05) in the spleen. The mechanisms of cell cycle blockage were closely related to the upregulation of the P53 and P21 genes ( P < 0.05), the downregulation of the cyclin D1 and CDK6 genes ( P < 0.05), and the decrease of proliferating cell nuclear antigen (PCNA) protein ( P < 0.05). The activated apoptosis resulted from the increased gene and protein expression of Bax and caspase-3 ( P < 0.05), and the decreased gene and protein expression of Bcl-2 ( P < 0.05). The results suggested that 70 ± 5 ppm NH3 caused the spleen dysplasia, which was closely related to the cell cycle arrest and mitochondrial apoptotic pathway activation.

Connexin 43 hyper-phosphorylation at serine 282 triggers apoptosis in rat cardiomyocytes via activation of mitochondrial apoptotic pathway.

Cx43 is the major connexin in ventricular gap junctions, and plays a pivotal role in control of electrical and metabolic communication among adjacent cardiomyocytes. We previously found that Cx43 dephosphorylation at serine 282 (pS282) caused cardiomyocyte apoptosis, which is involved in cardiac ischemia/reperfusion injury. In this study we investigated whether Cx43-S282 hyper-phosphorylation could protect cardiomyocytes against apoptosis. Adenovirus carrying rat full length Cx43 gene (Cx43-wt) or a mutant gene at S282 substituted with aspartic acid (S282D) were transfected into neonatal rat ventricular myocytes (NRVMs) or injected into rat ventricular wall. Rat abdominal aorta constriction model (AAC) was used to assess Cx43-S282 phosphorylation status. We showed that Cx43 phosphorylation at S282 was increased over 2-times compared to Cx43-wt cells at 24 h after transfection, while pS262 and pS368 were unaltered. S282D-transfected cells displayed enhanced gap junctional communication, and increased basal intracellular Ca2+ concentration and spontaneous Ca2+ transients compared to Cx43-wt cells. However, spontaneous apoptosis appeared in NRVMs transfected with S282D for 34 h. Rat ventricular myocardium transfected with S282D in vivo also exhibited apoptotic responses, including increased Bax/Bcl-xL ratio, cytochrome c release as well as caspase-3 and caspase-9 activities, while factor-associated suicide (Fas)/Fas-associated death domain expression and caspase-8 activity remained unaltered. In addition, AAC-induced hypertrophic ventricles had apoptotic injury with Cx43-S282 hyper-phosphorylation compared with Sham ventricles. In conclusion, Cx43 hyper-phosphorylation at S282, as dephosphorylation, also triggers cardiomyocyte apoptosis, but through activation of mitochondrial apoptosis pathway, providing a fine-tuned Cx43-S282 phosphorylation range required for the maintenance of cardiomyocyte function and survival.

Antitumor Effects of Scorpion Peptide Smp43 through Mitochondrial Dysfunction and Membrane Disruption on Hepatocellular Carcinoma.

Smp43, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows cytotoxicity toward hepatoma cell line HepG2 by membrane disruption. However, its underlying detailed mechanisms still remain to be further clarified. In the present study, we evaluated the cellular internalization of Smp43 and explored its effects on cell viability, cell cycle, apoptosis, autophagy, necrosis, and factor expression related to these cellular processes in human HepG2. Smp43 was found to suppress the growth of HepG2, Huh7, and human primary hepatocellular carcinoma cells while showing low toxicity to normal LO2 cells. Furthermore, Smp43 could interact with the cell membrane and be internalized into HepG2 cells via endocytosis and pore formation, which caused a ROS production increase, mitochondrial membrane potential decline, cytoskeleton disorganization, dysregulation of cyclin expression, mitochondrial apoptotic pathway activation, and alteration of MAPK as well as PI3K/Akt/mTOR signaling pathways. Finally, Smp43 showed effective antitumor protection in the HepG2 xenograft mice model. Overall, these findings indicate that Smp43 significantly exerts antitumor effects via induction of apoptosis, autophagy, necrosis, and cell cycle arrest due to its induction of mitochondrial dysfunction and membrane disruption. This discovery will extend the antitumor mechanisms of antimicrobial peptides and contribute to the development of antitumor agents against hepatocellular carcinoma.

THE MCL-1 INHIBITOR AZD5991 DISPLAYS A PROMISING TARGETED THERAPY FOR ACUTE MYELOID LEUKEMIA

MCL-1 protein is an anti-apoptotic member of BCL-2 protein family. In hematological neoplasms, increased MCL-1 gene expression or protein activity is related to disease relapse, poor prognosis and resistance to chemotherapy, especially to Venetoclax, an BCL-2 inhibitor. The present study aimed at evaluating the MCL-1 gene expression in AML patients as well as investigating the effects of MCL-1 protein inhibition in myeloid cells. Analysis of MCL-1 gene expression was performed in a cohort of 286 AML patients (OHSU/Nature 2018 study at cbioportal.org). Leukemia myeloid cell lines (U937, KG1a, OCI-AML3) were treated with increasing concentrations of the MCL-1 inhibitor compound AZD5991 (AstraZeneca) for 48, 72 and 96h for determination of the 50% inhibiting concentration (IC 50 ). Apoptosis (annexin-V stain), reactive oxygen species (ROS) production (DFCHA dye) and autophagy (acridine orange dye) were assessed by flow cytometry, after 48h of treatment. Coculture assays were performed by adding leukemia cells onto a monolayer of HS5 mesenchymal stromal cells, followed by treatment with AZD5991 for 48h. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal Wallis tests. p-value < .05 was considered significant. Increased MCL-1 gene expression in AML patients with adverse cytogenetic risk was observed when compared to patients with favorable or intermediate cytogenetic risk (p < .001). All leukemia cell lines showed dose and time-dependent sensitivity with AZD5991. IC 50 values of 4.3, 3.4 and 9.2μM in U937; 20.0, 15.2 and 14.9μM in KG1a; and 550, 380 and 420nM in OCI-AML3, for 48, 72 and 96h, respectively. Treatment with AZD5991 (0.5-20μM) in OCI-AML3, U937 and KG1a cells significantly reduced cell viability (69.2%, 50.3%, 40.6%) and increased apoptosis (68.2%, 90.1%, 92.2%) rate in a time and dose-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to reduce ROS production at 15.1% and 17.2% in U937, 10.3% and 5.6% in KG1a, 59.4% and 45.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Similarly, there was a reduction in the formation of acidic vesicles (autophagic process) at 43.0% and 19.0% in U937, 40.5% and 19.0% in KG1a, 62.6% and 18.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Increased cell percentage in G0/G1 phases of cell cycle was also observed. Western blot analyses showed decreased MCL-1, Bax and Bak and increased Bim. MCL-1 overexpression is related with AML progression, and associated with poor prognosis (Li et al. Onco Targets Ther. 2019;12:3295-3304). Our analysis from OHSU data bank showed that patients with adverse cytogenetic risk presented higher MCL-1 gene expression that could induce resistance to pharmacological therapies. Our in vitro studies demonstrated that the MCL1 inhibitor AZD5991 induced apoptosis and reduced viability of leukemia cells, possibly by MCL-1-Bak complex disruption and subsequent Bak-dependent mitochondrial apoptotic pathway activation. The diminished MCL-1 availability in mitochondria, probably caused lower ROS production and impaired autophagy (Mukhopadhyay et al. Apoptosis. 2014;19:555-66). Therefore, these findings provide important tools for further investigations and highlight that MCL-1 inhibitor could be a promising compound for AML treatment due to its potent antitumor activity and may represent a potential therapeutic strategy. FAPESP e CNPq.

Luteolin combined with low-dose paclitaxel synergistically inhibits epithelial-mesenchymal transition and induces cell apoptosis on esophageal carcinoma in vitro and in vivo.

Although paclitaxel is a promising frontline chemotherapy agent for various malignancies, the clinical applications have been restricted by side effects, drug resistance, and cancer metastasis. The combination of paclitaxel and other agents could be the promising strategies against malignant tumor, which enhances the antitumor effect through synergistic effects, reduces required drug concentrations, and also suppresses tumorigenesis in multiple ways. In this study, we found that luteolin, a natural flavonoid compound, combined with low-dose paclitaxel synergistically regulated the proliferation, migration, epithelial-mesenchymal transition (EMT), and apoptosis of esophageal cancer cells in vitro, as well as synergistically inhibited tumor growth without obvious toxicity in vivo. The molecular mechanism of inhibiting cell migration and EMT processes may be related to the inhibition of SIRT1, and the mechanism of apoptosis induction is associated with the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) pathway-mediated activation of mitochondrial apoptotic pathway.

Mechanism of piperine in affecting apoptosis and proliferation of gastric cancer cells via ROS-mitochondria-associated signalling pathway.

Piperine (PIP), the main active ingredient in pepper, belongs to the cinnamamide alkaloid. PIP has been found to have functions, including anti‐oxidation, immune regulation, anti‐tumour and promotion of drug metabolism. The present study was mainly designed to reveal the anti‐tumour effect of PIP against gastric cancer and the relevant mechanism. In brief, the undifferentiated human gastric cancer cell HGC‐27 was used, which was treated with different concentrations of PIP. As a result, PIP could inhibit proliferation and induce apoptosis of HGC‐27 cells in a dose‐dependent manner. The mechanism of PIP was associated with ROS increase and mitochondrial damage, simultaneously, the expression of key proteins of apoptosis was affected, including Bcl‐2, Bax, Cyt‐c, Caspase‐9 and Caspase‐3. Pre‐treatment of ROS scavenger NAC HGC‐27 cells could significantly reduce PIP‐induced apoptosis and inhibit the activation of apoptotic signals. Consistently, PIP could induce ROS to increase and activate apoptotic signals in the animal model. Therefore, the present study showed that PIP can induce the generation of ROS, thereby promoting the activation of mitochondrial apoptotic pathway and exerting anti‐tumour effects.

Induction of mitochondrial apoptosis pathway mediated through caspase-8 and c-Jun N-terminal kinase by cadmium-activated Fas in rat cortical neurons.

Cadmium (Cd) is a toxic metal and an environmental pollutant and can cause neurotoxicity by inducing apoptosis. Fas (CD95/Apo-1) is a cell-surface receptor that triggers apoptosis upon ligand binding, mediated through the mitochondrial apoptotic pathway. However, the role and regulatory mechanism of Fas in Cd-induced neuronal apoptosis remain understudied. Here, we demonstrate that activation of caspase-8 and the c-Jun N-terminal kinase (JNK) pathway are mechanisms underlying Cd-induced Fas-mediated activation of the mitochondrial apoptotic pathway in rat cerebral cortical neurons. In vitro, Cd induced apoptosis in primary cortical neurons by activating caspase-8, JNK, and the mitochondrial apoptotic pathway. Fas knockdown enhanced cell viability in the presence of Cd and inhibited apoptosis by blocking Cd-activated Fas, caspase-8, and JNK. Fas knockdown also inhibited the decrease of mitochondrial membrane potential, cleavage of caspase-9/3 and poly (ADP-ribose) polymerase 1, and impaired nuclear translocation of apoptosis-inducing factor and endonuclease G. In vivo, Fas knockdown alleviated Cd-induced neuronal injury and inhibited apoptosis, activation of caspase-8, JNK, and mitochondrial apoptotic pathways in rat cerebral cortical neurons. In summary, our results demonstrate that Cd-activated Fas relays apoptotic signals from the cell surface to the mitochondria via caspase-8 and JNK activation in rat cerebral cortical neurons, leading to aggravation of the neuronal injury.

The effect of ochratoxin A on cytotoxicity and glucose metabolism in human esophageal epithelium Het-1A cells

Ochratoxin A (OTA) is a widespread mycotoxin worldwide that causes major health risks. The esophageal epithelium is unavoidably exposed to food contaminated OTA after ingestion. Yet, few studies have involved in the putative effects of OTA on the cytotoxicity and glucose metabolism responses on esophageal epithelial cells. In this in vitro study, we aimed to investigate the effects of OTA on esophageal epithelial cell intracellular apoptosis, oxidative stress, DNA damage, mitochondrial function and glucose metabolism. Human esophageal epithelial Het-1A cells were exposed to 2.5, 5 or 10 μM OTA for 24 h. The results showed that OTA decreased cell viability and concomitantly increased apoptosis-related indices, reactive oxygen species generation, oxidative DNA damage, mitochondrial dysfunction and mitochondrial apoptotic pathway activation. In addition, OTA switched the glucose metabolism of Het-1A cells from oxidative phosphorylation towards glycolysis by decreasing the expression of tricarboxylic acid cycle-associated enzymes such as α-ketoglutarate dehydrogenase and isocitrate dehydrogenase 1 and by increasing pyruvate dehydrogenase kinase 1 expression. The data indicated that cell apoptosis, oxidative damage, mitochondrial dysfunction and glucose metabolism perturbation might play pivotal roles in the mechanism of OTA-induced esophageal toxicity.

Rutin-protected BisGMA-induced cytotoxicity, genotoxicity, and apoptosis in macrophages through the reduction of the mitochondrial apoptotic pathway and induction of antioxidant enzymes.

Bisphenol-A-glycidyldimethacrylate (BisGMA) is a resin monomer frequently used in dentin restorative treatments. The leakage of BisGMA monomer from BisGMA-based polymeric resins can lead to cytotoxicity in macrophages. Rutin has various beneficial bioeffects, including antioxidation and antiinflammation. In this study, we found that pretreatment of RAW264.7 macrophages with rutin-inhibited cytotoxicity induced by BisGMA in a concentration-dependent manner. BisGMA-induced apoptosis, which was detected by levels of phosphatidylserine from the internal to the external membrane and formation of sub-G1, and genotoxicity, which was detected by cytokinesis-blocked micronucleus and single-cell gel electrophoresis assays, were inhibited by rutin in a concentration-dependent manner. Rutin suppressed the BisGMA-induced activation of caspase-3 and -9 rather than caspase-8. Rutin inhibited the activation of the mitochondrial apoptotic pathway, including cytochrome C release and mitochondria disruption, after macrophages were treated with BisGMA. Finally, BisGMA-induced reactive oxygen species (ROS) generation and antioxidant enzyme (AOE) deactivation could be reversed by rutin. Parallel trends were observed in the elevation of AOE activation and inhibition of ROS generation, caspase-3 activity, mitochondrial apoptotic pathway activation, and genotoxicity. These results suggested that rutin suppressed BisGMA-induced cytotoxicity through genotoxicity, the mitochondrial apoptotic pathway, and relatively upstream factors, including reduction of ROS generation and induction of AOE.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

RAB9A Plays an Oncogenic Role in Human Liver Cancer Cells.

Background RAB9, as a member of the Rab GTPase family, is required for the transport of the mannose-6-phosphate receptor (MPR) from late endosomes to trans-Golgi network (TGN). However, the role of RAB9A in tumors, including liver cancer, is still unknown. Methods We used pcDNA3.1 plasmid to upregulate the expression of RAB9A in Hep3b cells and used specific shRNA to downregulate the expression of RAB9A in HepG2 cells. Biological functions of RAB9A were performed by CCK-8 assay, colony formation assay, apoptosis analysis, transwell assays, and wound healing assays. Finally, an in-depth mechanism study was performed by western blot. Results RAB9A promoted the proliferation and clonality of Hep3b and HepG2 cells. RAB9A also inhibited apoptosis and the activation of mitochondrial apoptotic pathway. In addition, RAB9A promoted the invasion and migration of Hep3b and HepG2 cells. Importantly, RAB9A activated the AKT/mTOR signaling pathway in human liver cancer cells. A double-effect inhibitor (BEZ235) significantly hindered the effect of RAB9A overexpression on the proliferation and invasion of Hep3b cells. Conclusion Our data suggest that RAB9A plays a carcinogenic role in human liver cancer progression partially through AKT signaling pathways, suggesting that RAB9A may serve as a potential therapeutic target for liver cancer therapy.

Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

Hepatitis E virus (HEV) infection has been associated with extrahepatic manifestations, particularly neurological disorders. Although it has been reported that HEV infection induced hepatocyte apoptosis associated with mitochondria injury, activation of mitochondrial apoptotic pathway in the central nervous system during HEV infection was not clearly understood. In this study, the induction of mitochondrial apoptosis-associated proteins and pro-inflammatory cytokines were detected in HEV infected Mongolian gerbil model and primary human brain microvascular endothelial cells (HBMVECs). Mitochondrial exhibited fragments with loss of cristae and matrix in HEV infected brain tissue by transmission electron microscope (TEM). In vitro studies showed that expression of NADPH oxidase 4 (NOX4) was significantly increased in HEV infected HBMVECs (p < 0.05), while ATP5A1 was significantly decreased (p < 0.01). Expressions of pro-apoptotic proteins were further evaluated. Bax was significantly increased in both HEV infected brain tissues and HBMVECs (p < 0.01). In vivo studies showed that caspase-9 and caspase-3 were activated after HEV inoculation (p < 0.01), associated with PCNA overexpression as response to apoptosis. Cytokines were measured to evaluate tissue inflammatory levels. Results showed that the release of TNFα and IL-1β were significantly increased after HEV infection (p < 0.01), which might be attributed to microglia activation characterized by high level of IBA1 expression (p < 0.01). Taken together, these data support that HEV infection induces high levels of pro-inflammatory cytokines, associated with mitochondria-mediated apoptosis. The results provide new insight into mechanisms of extra-hepatic injury of HEV infection, especially in the central nervous system.

Mechanisms of ROS-induced mitochondria-dependent apoptosis underlying liquid storage of goat spermatozoa.

Liquid storage of spermatozoa is important for artificial insemination and herd genetic breeding. However, the extended time of storage inducing the rapid decline in spermatozoa quality limits the development of this technology. The molecular mechanisms underlying liquid storage of spermatozoa remain largely unexplored. In this study, the effects of liquid storage on functional quality of spermatozoa were assessed in goat (Capra hircus). The time-dependent decline in spermatozoa motility showed a strong correlation with the significant increase in apoptosis. Moreover, apoptosis-related ultrastructural changes were observed, especially the defects in mitochondria. A significant decrease in mitochondrial membrane potential and changes in the expression of mitochondrial apoptosis-related proteins indicated mitochondrial dysfunction and mitochondrial apoptotic pathway activation. Notably, the abnormally high level of reactive oxygen species (ROS) caused by liquid storage resulted in oxidative damage to mitochondria and accelerated mitochondria-dependent apoptosis, as demonstrated by the addition of ROS scavenger N-acetylcysteine. Furthermore, critical differentially expressed proteins involved in mitochondria-dependent apoptosis and antioxidant defense were identified and profiled by quantitative proteomic analysis, facilitating the understanding of molecular regulation of ROS-induced mitochondria-dependent apoptosis. These outcomes provide insights into the mechanisms underlying liquid storage of goat spermatozoa and enhance the progress of semen storage technology.

S834 PEGCRISANTASPASE AND VENETOCLAX COMBINATION IS HIGHLY EFFECTIVE AGAINST ACUTE MYELOID LEUKEMIA (AML), MECHANISTICALLY DRIVEN SYNERGISM BETWEEN GLUTAMINE DEPLETION AND BCL‐2 INHIBITION

Background:We reported that Erwinaze could effectively deplete plasma glutamine (Gln) to undetectable levels in patients (pts) with relapsed/refractory (R/R) AML and these pts had higher probability of clinical response. We hypothesized that Gln depletion (pegcrisantaspase, a recombinant pegylated Erwinia crisantaspase, PegC [Jazz Pharmaceuticals]) leads to activation of mitochondrial apoptotic pathways and synergizes with Bcl‐2 inhibition (venetoclax, Ven). In vitro, we demonstrated that anti‐AML activity of Ven was potentiated 10–44 fold by PegC; presence of PegC (0.01 IU/mL) decreased Ven's IC50 (5.3→0.12 μM in MOLM‐14 &amp; 7.5→0.76 μM in MonoMac‐6 cells). Ven‐PegC demonstrated synergy in killing AML cells. Here, we report the mechanism of action (MOA), and in vivo safety and efficacy of Ven‐PegC in AML.Aims:(1) To determine toxicity profile of Ven‐PegC in mice (2) To analyze effect of Ven‐PegC on plasma Gln in vivo (3) To determine efficacy of Ven‐PegC in orthotopic pt‐derived xenograft (PDX), and (4) To assess changes in the transcriptome/translatome of AML cells treated with Ven‐PegC.Methods:Toxicity profile of Ven‐PegC was investigated in immune competent (CD1) and incompetent (NRG/NSG) mice. To measure pharmacodynamic (PD) effect of PegC, NSG mice were dosed once IV at 3 different doses [125, 250 and 500 IU/kg (n = 4 mice/dose)]. We euthanized 2 mice on days 3 and 10 and measured plasma amino acids by LCMS. Efficacy of Ven‐PegC was assessed in an AML pt‐derived xenograft model (AML‐45) that expresses luciferase. We injected 1x106 AML‐45‐luc cells IV into NRG mice, and after engraftment mice were treated with vehicle, Ven (75 mg/kg, PO, 5x/week), PegC (200 IU/kg, IV, 1x/week), or Ven‐PegC for 2 weeks (wks), 1 wk off, then 2 more wks. Mice were imaged weekly and survival monitored. For MOA, MOLM‐14 cells were treated with each agent alone and combined followed by RNA isolation, RNA‐seq and transcriptome analysis. For translatome, cells were treated similarly and subjected to western blotting, cap enrichment and polysomal fractionation.Results:For safety, Ven‐PegC resulted in mild weight loss with full recovery at PD active doses. Amylase, lipase, creatinine, and transaminases were unchanged after 3 wks of Ven‐PegC treatment. Ven‐PegC induced myelosuppression as expected (7.4 ± 1.0x103/μL control vs 1.9 ± 0.3x103/μL combination, p&lt;0.003). For PD, asparagine was not detectable on days 3 &amp; 10 post dosing. No plasma Gln was detected on day 3 and on day 10 was undetectable at the highest dose. For efficacy, Ven‐PegC significantly lowered leukemia burden compared to monotherapies (p &lt; 0.03). On Day 36, AML was virtually undetectable in mice treated with Ven‐PegC (Fig. 1A‐1B). For MOA, immunoblot analysis showed decreased phosphorylation of mTOR substrates (p70S6K &amp; 4E‐BP1) with Ven‐PegC. A significant decrease in cap driven translation complexes and in eIF4E phosphorylation at Ser‐209 followed by enhanced interaction with 4E‐BP1 were observed with Ven‐PegC (Fig. 1C). Transcriptome analysis showed differential gene expression pattern between groups (RT‐qPCR validated; Fig. 1D). p90RSK, a key gene in ribosomal biogenesis and cell proliferation, was reduced upon Ven‐PegC treatment in MOLM‐14 transcriptome profiling (RNA‐qPCR/immunoblot analysis confirmed). Analysis of altered translational profile is ongoing.Summary/Conclusion:Ven‐PegC is a mechanistically‐driven, highly active treatment against AML in the PDX model. Ven‐PegC appears to broadly regulate the ribosomal biosynthetic pathway, a major driver for chemo‐resistance. The Phase 1 clinical trial in pts with R/R AML is planned.image

Excessive backfat of sows at mating promotes oxidative stress and up-regulates mitochondrial-mediated apoptotic pathway in the full-term placenta

The objective of this investigation was to evaluate the influence of back-fat thickness (BF), at mating of sows, on the placental oxidative stress, mitochondrial injury and apoptosis. For that purpose, we performed iTRAQ labeling based proteomic analysis on term placentas obtained by vaginal delivery from BFI (15–20 mm) and BFII (21–27 mm) sows formed according to BF at mating. Proteomic analysis revealed 413 proteins to be significantly different in placenta from BFII sows by ≥1.2-fold. Gene ontology (GO) analysis identified proteins related to oxidative stress response and apoptotic process to be altered in placenta from BFII sows. Indicative of increased oxidative stress and impaired mitochondrial function, higher placental MDA, H2O2 and O2− levels and reduced CAT and SOD activities were linked to decreased mitochondrial membrane potential and reduced mitochondrial DNA copy number and increased MPTP opening activity in placenta from BFII group (P< 0.05). Furthermore, a TUNEL apoptosis assay showed that increased back-fat triggered a 33% increase of apoptotic cells in placental villi; and apoptosis was confirmed by the decreased protein contents of Bcl-2, as well as the increased expression of Bax, cytochrome c, Apaf-1, caspase 9 and cleaved caspase 3 in placenta from BFII sows that was associated with higher caspase 3 activity (P< 0.05), suggesting the activation of mitochondrial apoptotic pathway. Excessive back-fat was also associated with increased activation of Mark4 and JNK1 and reduced activation of p38 MAPK in pig term placenta, suggesting the potential mechanisms underlying increased oxidative stress and apoptosis in placenta from BFII sows. Together, these findings indicate that excessive back-fat of sows at mating leads to increased oxidative stress that is associated with mitochondrial dysfunction and augmented apoptosis in the full-term placenta.