Bcl-2 Homologous Antagonist Killer Research Articles

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK.

Yak-Derived CXCL14 Activates the Pro-Inflammatory Response of Macrophages and Inhibits the Proliferation and Migration of HepG2.

CXCL14 (C-X-C motif chemokine ligand 14) is an important chemokine involved in infection and immunity and plays an important role in a variety of immune-related diseases. The 446 bp cDNA sequence of the CXCL14 gene in yaks was obtained. Additionally, the prokaryotic expression vector of the CXCL14 protein with a molecular weight of 27 kDa was successfully constructed and expressed. The proliferation activities and migration abilities of spleen macrophages were significantly inhibited after treatment with the CXCL14 protein at different concentrations (1, 10 and 20 μg/mL) (p < 0.05). Furthermore, the expressions of pro-inflammatory cytokines interleukin 1 beta (IL-1β), interleukin 6 (IL6), interleukin 8 (IL8) and interferon-α (TNF-α) were significantly increased (p < 0.05), but the expression of anti-inflammatory factor interleukin 10 (IL10) was significantly decreased (p < 0.05). The contents of inflammatory factors in the supernatant of cells were detected using ELISA, and it was also found that the contents of TNF-α, IL6 and cytochrome c oxidase subunit II (COX2) were significantly increased under different CXCL14 protein concentrations (p < 0.05). Finally, the exogenous addition of CXCL14 inhibited the activity, clonal formation and migration of hepatoma cells (HepG2). Additionally, after HepG2 cells were treated with 20 μg/mL CXCL14 protein for 12 h, 24 h and 36 h, the expression levels of BCL2 homologous antagonist/killer (BAK) and the BCL2-associated X apoptosis regulator (BAX) were increased to varying degrees, while the expression levels of hypoxia-inducible factor 1 subunit alpha (HIF1A), the mechanistic target of rapamycin kinase (mTOR) and cyclin-dependent kinase 1 (CDK1) genes decreased compared to the control group. In conclusion, the CXCL14 protein can inhibit the proliferation and migration of HepG2 cells by inducing the expression of macrophage pro-inflammatory factors and activating apoptosis-related genes to exert innate immunity. These results are helpful to further study the function of the CXCL14 protein and provide research data for the innate immune mechanism of yaks under harsh plateau environments.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed invivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. Shc was significantly induced in patients with AH (P< .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1β, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. Invivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

Pore-forming proteins as drivers of membrane permeabilization in cell death pathways.

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways:apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein(MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

COMMENTARY: Direct Indexing: A Point Solution for Global Crises

Background: Canine melanoma is the most common type of tumor in dogs. We investigated the effects of canine interferon-beta (cIFN-β)-overexpressing adipose tissue-derived mesenchymal stem cells (cATMSCs) on apoptosis and proliferation of canine melanoma cells. Materials and Methods: Expression of IFN-β in cATMSCs was confirmed using reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assays. Flow cytometry was performed for cell-cycle analysis and apoptotic cell quantification of LMeC (melanoma) cells. Protein expression of cyclin D1, procaspase-3, activated caspase-3, and Bcl-2 homologous antagonist killer (Bak) was evaluated by western blot analysis. Results: Decreased proportions of cells in S- and G₀/G₁ phases were observed in parallel with decreased cyclin D1 expression in LMeC cells treated with cIFN-β-cATMSC-conditioned media. Protein expression of active forms of caspase 3 and Bak increased in response to treatment with cIFN-β-cATMSC-conditioned media. Conclusion: IFN-β overexpression by cATMSCs was associated with pro-apoptotic and growth-inhibitory effects on canine melanoma cells. The antitumor effects of these cells have therapeutic potential for the treatment of canine melanoma.

Tumor Suppressive Role of miR-342-5p and miR-491-5p in Human Osteosarcoma Cells.

Osteosarcomas are the most common type of malignant bone tumor. These tumors are characterized by the synthesis of an osteoid matrix. Current treatments are based on surgery and combination chemotherapy. However, for metastatic or recurrent tumors, chemotherapy is generally ineffective, and osteosarcomas are sometimes unresectable. Thus, the use of microRNAs (miRNAs) may represent an attractive alternative for the development of new therapies. Using high-throughput functional screening based on impedancemetry, we previously selected five miRNAs with potential chemosensitizing or antiproliferative effects on chondrosarcoma cells. We validated the tumor-suppressive activity of miR-491-5p and miR-342-5p in three chondrosarcoma cell lines. Here, we carried out individual functional validation of these five miRNAs in three osteosarcoma cell lines used as controls to evaluate their specificity of action on another type of bone sarcoma. The cytotoxic effects of miR-491-5p and miR-342-5p were also confirmed in osteosarcoma cells. Both miRNAs induced apoptosis. They increased Bcl-2 homologous antagonist killer (Bak) protein expression and directly targeted Bcl-2 lymphoma-extra large (Bcl-xL). MiR-342-5p also decreased B-cell lymphoma-2 (Bcl-2) protein expression, and miR-491-5p decreased that of Epidermal Growth Factor Receptor (EGFR). MiR-342-5p and miR-491-5p show tumor-suppressive activity in osteosarcomas. This study also confirms the potential of Bcl-xL as a therapeutic target in osteosarcomas.

Differential Effects of Chronic Binge Alcohol and Ovariectomy on Apoptosis in Omental Adipose Tissue of SIV‐infected Macaques

Adipose tissue maladaptations including cell death, inflammation, adipokine disruption, dysfunction of adipocytes and adipose tissue metabolism are central to metabolic alterations in HIV, at-risk alcohol use, and aging. Though adipose tissue dysregulation is a well-accepted mechanism meditating metabolic comorbidities in HIV, the effects of alcohol use and estrogen loss on adipose tissue cell death are not fully understood. Using a relevant preclinical model of HIV-infection, the aim of our study was to investigate the differential effects of chronic binge alcohol (CBA) administration and ovarian hormone loss, simulated by ovariectomy (OVX), on alterations in omental adipose tissue (OmAT) expression of genes involved in cell death. CBA or isovolumetric water (VEH) was administered through an intragastric catheter at a concentration of 30% (w/v) for 30 minutes, 5 days a week, with blood alcohol concentrations reaching 50-60 mM. Three months after initiation of CBA/VEH administration, macaques were infected with SIVMac251 and 2.5 months later initiated on ART. After 1 month of ART, macaques underwent sham surgery or ovariectomy. Unbiased quantitative proteomic analysis of OmAT revealed that proteins implicated in the apoptotic pathway were differentially regulated by CBA, OVX and the combination of both. Specifically, CBA and OVX independently led to proteome changes consistent with activation of apoptosis and necrosis pathways while the combination of CBA and OVX led to proteome changes consistent with inhibition of both apoptosis and necrosis. To confirm these results, OmAT expression of genes involved in apoptosis was determined using quantitative PCR. B-cell lymphoma 2 (BCL2), an antiapoptotic gene was increased by CBA but decreased in the CBA/OVX group. CBA increased the gene expression of BCL-2 homologous antagonist killer (BAK), a proapoptotic gene and increased the ratio of BCL2 associated X protein (BAX) to BCL2, an indicator of cell susceptibility to apoptosis. Our quantitative PCR results confirm and support the results of the OmAT proteomic analysis, suggesting that CBA and OVX differentially regulate the apoptotic pathway. However, in contrast to the results from the proteomic analysis, the preliminary gene expression data suggest that CBA is a major driver of apoptosis in the OmAT of female rhesus macaques. Our ongoing studies using TUNEL staining and analysis of crown-like structures will determine the effects of CBA and OVX on OmAT cell death. Additionally, the functional effects of CBA and OVX on adipose-derived stem cell death will be determined using flow cytometry.

BH3 Mimetics for the Treatment of B-Cell Malignancies—Insights and Lessons from the Clinic

Simple SummaryB-cell malignancies, including chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and plasma cell dyscrasias, are significant contributors to cancer morbidity and mortality worldwide. The pathogenesis of many B-cell malignancies involves perturbations in the intrinsic pathway of apoptosis that allow cells to evade cell death. BH3 mimetics represent a class of anti-cancer agents that can restore the ability of cancer cells to undergo apoptosis. Venetoclax, a recently approved BH3 mimetic, has transformed the therapeutic landscape for CLL. Other BH3 mimetics are currently under development. This review summarizes the available data on existing BH3 mimetics and highlights both the rapidly expanding role of BH3 mimetics in the treatment of B-cell malignancies and the clinical challenges of their use.The discovery of the link between defective apoptotic regulation and cancer cell survival engendered the idea of targeting aberrant components of the apoptotic machinery for cancer therapy. The intrinsic pathway of apoptosis is tightly controlled by interactions amongst members of three distinct subgroups of the B-cell lymphoma 2 (BCL2) family of proteins. The pro-survival BCL2 proteins prevent apoptosis by keeping the pro-apoptotic effector proteins BCL2-associated X protein (BAX) and BCL2 homologous antagonist/killer (BAK) in check, while the BH3-only proteins initiate apoptosis by either neutralizing the pro-survival BCL2 proteins or directly activating the pro-apoptotic effector proteins. This tripartite regulatory mechanism is commonly perturbed in B-cell malignancies facilitating cell death evasion. Over the past two decades, structure-based drug discovery has resulted in the development of a series of small molecules that mimic the function of BH3-only proteins called the BH3 mimetics. The most clinically advanced of these is venetoclax, which is a highly selective inhibitor of BCL2 that has transformed the treatment landscape for chronic lymphocytic leukemia (CLL). Other BH3 mimetics, which selectively target myeloid cell leukemia 1 (MCL1) and B-cell lymphoma extra large (BCLxL), are currently under investigation for use in diverse malignancies. Here, we review the current role of BH3 mimetics in the treatment of CLL and other B-cell malignancies and address open questions in this rapidly evolving field.

Small molecule CDS-3078 induces G2/M phase arrest and mitochondria-mediated apoptosis in HeLa cells.

The tumor suppressor p53 serves important roles in cell cycle arrest and apoptosis, and its activation increases the sensitivity of cancer cells to radiotherapy or chemotherapy. In the present study, the small molecule 2-[1-(4-(benzyloxy)phenyl)-3-oxoisoindolin-2-yl)-2-(4-methoxyphenyl)] acetic acid (CDS-3078) significantly increased p53 mRNA expression levels in a dose-dependent manner. Treatment with CDS-3078 increased p53 expression levels and p53-mediated activation of its downstream target genes in HeLa cells. Additionally, p53+/+ HeLa cells treated with CDS-3078 presented with dysfunctional mitochondria, as indicated by the decrease in Bcl-2 levels, the increase in Bcl-2 homologous antagonist killer and the increase in cytochrome c release from the mitochondria to the cytoplasm. The present results suggested that CDS-3078 treatment significantly induced G2/M phase cell cycle arrest. Therefore, CDS-3078 administration induced apoptosis via p53-mediated cell cycle arrest, causing mitochondrial dysfunction and resulting in apoptotic cell death in cervical cancer cells. Collectively, the present results suggested that CDS-3078 may be a potential anticancer agent.

Linker Histone H1.2 Directly Activates BAK through the K/RVVKP Motif on the C-Terminal Domain

H1.2 is a key mediator of apoptosis following DNA double-strand breaks. The link between H1.2 and canonical apoptotic pathways is unclear. One study found that H1.2 stimulates cytochrome c (Cyt c) release; in contrast, apoptosis-inducing factor was found to be released in another study. The C-terminal domain (CTD) of H1.2 has been implicated in the latter pathway, but activation of the proapoptotic protein BCL-2 homologous antagonist/killer (BAK) is a common denominator in both pathways. This study aimed to determine whether the CTD of H1.2 is also responsible for mitochondrial Cyt c release and whether a previously identified K/RVVKP motif in the CTD mediates the response. This study investigated if H1.2 mediates apoptosis induction through direct interaction with BAK. We established that the CTD of H1.2 stimulates mitochondrial Cyt c release in vitro in a mitochondrial permeability transition-independent manner and that the substitution of a single valine with threonine in the K/RVVKP motif abolishes Cyt c release. Additionally, we showed that H1.2 directly interacts with BAK with weak affinity and that the CTD of H1.2 mediates this binding. Using two 20-amino acid peptides derived from the CTD of H1.2 and H1.1 (K/RVVKP motif inclusive), we determined the main residues involved in the direct interaction with BAK. We propose that H1.2 operates through the K/RVVKP motif by directly activating BAK through inter- and intramolecular interactions. These findings expand the view of H1.2 as a signal-transducing molecule that can activate apoptosis in a BAK-dependent manner.

Cucurbita ficifolia Fruit Extract Induces Tp53/Caspase-Mediated Apoptosis in MCF-7 Breast Cancer Cells.

The second most biggest cancer worldwide is breast cancer. There is an increasing need for safer, effective, and affordable drug candidates from natural sources to treat breast cancer. In the present investigation, the anticancer effect of Cucurbita ficifolia Bouché (C. ficifolia) fruit extract was tested on the human breast cancer cells such as MCF-7. The cells were exposed with different doses of C. ficifolia, for the assessment of IC50 concentrations on the MCF-7 cell lines for 24 hs. The effect of C. ficifolia fruit extract on morphological and apoptotic changes were evaluated by specific fluorescence staining techniques and real-time PCR in a time-dependent manner for 24 hs and 48 hs. The IC50 value for C. ficifolia fruit extract was found to be 90 μg/mL. Morphological alteration and apoptotic distinctiveness aspect like chromatin condensation and nuclear fragmentation were noticed in C. ficifolia extract exposed breast cancer cells. Further, we observed that C. ficifolia extract-induced programmed cell death in the MCF-7 cells were mediated with the elevated expression of the tumor suppressor gene such as p53 and apoptotic markers such as caspase-8, caspase-9, caspase-3, fatty acid synthase (FAS), Fas-associated protein with death domain (FADD), Bcl-2 homologous antagonist/killer (BAK), and Bcl-2-associated X protein (BAX). These observations established that C. ficifolia significantly concealed the cell division and provoked p53/caspase-mediated programmed cell death. Further, we noticed that this cell death in MCF-7 cells is concentration and time dependent. As evaluated through the comet assay, C. ficifolia induced DNA damage; further upon increasing the duration of the treatment, the DNA damage was higher than before. Thus, our study concludes that C. ficifolia could serve as an effective anticancer agent through vital gene modulation.

Regulation of Fibroblast Apoptosis and Proliferation by MicroRNA-125b in Systemic Sclerosis.

To analyze the expression, regulation, and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc). MiR-125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR-125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase-Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real-time assay in dermal fibroblasts. Expression of miR-125b was significantly down-regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35-0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36-0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and α-smooth muscle actin (α-SMA) expression at the messenger RNA (mRNA) and protein levels. RNA-Seq identified BAK1, BMF, and BBC3 as potential targets of miR-125b. Quantitative PCR confirmed that knockdown of miR-125b up-regulated these genes (P < 0.01; n = 12). Bcl-2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR-125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR-125b overexpression decreased apoptosis. Our findings indicate that miR-125b is down-regulated in SSc skin and primary dermal fibroblasts. MiR-125b down-regulation increases apoptosis and decreases proliferation and α-SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.

CDS-1548 induces apoptosis in HeLa cells by activating caspase 3.

Cervical cancer continues to be a threat to female health globally. In the present study, the potential anticancer activity of 2-[2-hydroxyl-1-(4-methoxy phenyl) ethyl]-3-(4-benzyloxy phenyl) isoindolin-1-one (CDS-1548), was evaluated in HeLa cells. CDS-1548 is an organic small-molecule compound characterized by two chiral centers, with the nuclear parent 1H-isoindolin-1-one. CDS-1548 administration significantly elevated the transcriptional activity of p53 and its downstream target genes in a dose-dependent manner. Additionally, CDS-1548 treatment increased the expression levels of p53 and mouse double minute 2 homolog, as well as inducing apoptosis in HeLa cells. Furthermore, CDS-1548 treatment downregulated the expression of B-cell lymphoma 2, upregulated Bcl-2 homologous antagonist killer, promoted the release of cytochrome c from mitochondria to cytoplasm, and activated the production of caspase 3 and 9. Collectively, these results suggested that CDS-1548 inhibited HeLa cell proliferation by promoting G2/M cell cycle phase arrest and inducting of mitochondria-mediated apoptosis.

Fluoxetine Induces Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits ERK/NF-κB-Modulated Anti-Apoptotic and Invasive Potential in Hepatocellular Carcinoma Cells In Vitro.

The aim of the present study was to verify the effects of fluoxetine on dysregulation of apoptosis and invasive potential in human hepatocellular carcinoma (HCC) SK-Hep1 and Hep3B cells. Cells were treated with different concentrations of fluoxetine for different times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used for testing the effects of fluoxetine on cell viability. The regulation of apoptosis signaling, and anti-apoptotic, proliferation, and metastasis-associated proteins after fluoxetine treatment were assayed by flow cytometry and Western blotting assay. The detection of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation after fluoxetine treatment was performed by NF-κB reporter gene assay. The results demonstrated that fluoxetine significantly reduced cell viability, cell migration/invasion, NF-κB, extracellular signal-regulated kinases (ERK) activation, and expression of anti-apoptotic (Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP), Myeloid cell leukemia-1 (MCL-1), X-Linked inhibitor of apoptosis protein (XAIP), and Survivin), proliferation (Cyclin-D1), angiogenesis (vascular endothelial growth factor (VEGF)), and metastasis-associated proteins (matrix metalloproteinase-9 (MMP-9)). Fluoxetine also significantly induced apoptosis, unregulated extrinsic (activation of first apoptosis signal protein and ligand (Fas/FasL), and caspase-8) and intrinsic (loss of mitochondrial membrane potential (ΔΨm) pathways and increased Bcl-2 homologous antagonist killer (BAK) apoptosis signaling. Taken together, these results demonstrated that fluoxetine induced apoptosis through extrinsic/intrinsic pathways and diminished ERK/NF-κB-modulated anti-apoptotic and invasive potential in HCC cells in vitro.

The role of insulin-like growth factor 1 in ALS cell and mouse models: A mitochondrial protector

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disorder, but little is known about the exact causes and pathophysiology of this disease. In transgenic mouse models of ALS, mitochondrial abnormalities develop during the disease and might contribute to the progression of ALS. Gene therapy was recently shown to induce beneficial effects. For example, the delivery of human insulin-like growth factor-1 (hIGF-1) by self-complementary adeno-associated virus (AAV) vectors has been shown to prolong the lifespan of ALS transgenic mice. However, the function of IGF-1 in mitochondria has not been systematically studied in ALS models. In this study, scAAV9-hIGF-1 was intramuscularly injected into transgenic SOD1G93A mice and administered to cell lines expressing the ∼25-kDa C-terminal fragment of transactive response DNA-binding protein (TDP-25). The mitochondrial electrical transmembrane potential was hyperpolarized, and electron microscopy findings revealed that the abnormal mitochondria were transformed. Moreover, the intrinsic mitochondrial apoptotic process was modified through the upregulation of anti-apoptotic proteins (B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)), the downregulation of pro-apoptotic proteins (Bcl-2-associated x protein (Bax) and Bcl-2 homologous antagonist killer (Bak)) and a reduction in mitochondrial cytochrome c release. Mitophagy was also increased after scAAV9-hIGF-1 treatment, as evidenced by a decrease in the p62 level and an increase in the LC3-II level. Furthermore, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was used to delete the IGF-1 gene in SOD1G93A model mice via an intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 to confirm these findings. The protective effect of IGF-1 on the mitochondria decreased after genetic deletion. These novel findings demonstrate that IGF-1 strongly protects mitochondria from apoptosis and upregulates mitophagy in mouse and cell models of ALS. Therefore, therapies that specifically protect mitochondrial function might be promising strategies for treating ALS.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 μM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

BNIP3/Bcl-2-mediated apoptosis induced by cyclic tensile stretch in human cartilage endplate-derived stem cells.

The present study aimed to investigate the molecular mechanisms of cyclic stretch-induced apoptosis in human intervertebral disc cartilage endplate-derived stem cells (CESCs). CESCs were stretched by the Flexercell-4000™ Tension Plus system, the effect on cell viability was measured by a Cell Counting Kit-8 assay, while cell apoptosis was detected by flow cytometry. Western blot analysis was used to evaluate the expression of B-cell lymphoma 2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), Bcl-2, Bcl-2 homologous antagonist killer (Bak), Bcl-2-associated X protein (Bax), Bcl extra large (Bcl-xl) and the activity of caspase-3, while Z-VAD-FMK was used to inhibit caspase-3. Compared with the control group, the cell viability decreased in a time-dependent manner after stretching. Furthermore, cell apoptosis and the activity of caspase-3 were increased in a time-dependent manner. The ratio of pro-death factor BNIP3 to anti-apoptotic protein Bcl-2 was significantly increased. When cells were stretched for 36 h, the apoptosis-associated proteins Bak and Bax were increased, while Bcl-xl was decreased. The viability and apoptotic ratio of stretched cells were significantly restored after caspase-3 was repressed. In conclusion, cyclic tensile stretch induced apoptosis of CESCs, which was probably due to upregulation of the expression of BNIP3.

Toll like receptor 4 signaling pathway participated in Salmonella lipopolysaccharide-induced spleen injury in young chicks

Spleen is one of the crucial sites for cellular and humoral immunity but it easily damaged during pathogenic infections resulting in immunosuppression. The current study was therefore performed to explore the mechanism of acute spleen injury induced by salmonella lipopolysaccharide (LPS) in young chicks. Healthy one-day-old Cobb strain broiler chicks were intra-peritoneally injected with saline or LPS. LPS treatment caused significant decreases in body and spleen weights at 36 and 72 h. Histological analysis showed the changes of ellipsoid structures with beginning of nuclear pyknosis and karyolysis similar to steatosis at 12 h, maximum histopathological lesions were seen at 36 h, however these were disappeared at 72 h post LPS stimulation. Cell proliferation was decreased (low PCNA positivity) and apoptosis increased (high ssDNA positivity) in the spleen at 12 and 36 h after LPS treatment. The expression levels of mRNA for caspase-3, caspase-8, B-cell lymphoma 2 (BCL-2), tumor protein p53 or p53 and Bcl-2 homologous antagonist killer (BAK) showed slight increase at some time points following LPS stimulation. LPS treatment also induced significant up-regulation in toll like receptor 4 (TLR4) at 36 h post LPS stimulation and slight increase in expressions of its downstream molecules (MyD88 and NF-κB) at 12 h post LPS treatment. The keystone cytokines (TNF-α and IL-6) exhibited significant up-regulation at 12 h following LPS stimulation. Our findings provided novel information about the histopathological as well as apoptotic and proliferative alterations in spleen mediated by TLR4 signaling induced by Salmonella LPS in avian species.

Suppression of Oral Carcinoma Oncogenic Activity by microRNA-203 via Down-regulation of SEMA6A.

The purpose of this study was to elucidate the molecular mechanism underlying regulation of semaphorin-6A (SEMA6A) involving microRNA-203 (miR-203) as a tumor suppressor in YD-38 human oral cancer cells. miRNA arrays, polymerase chain reaction analyses, MTT assays, immunoblotting, and luciferase assays were carried out in YD-38 cells. MiRNA microarray results showed that expression of miR-203 was significantly down-regulated in YD-38 cells compared to normal human oral keratinocytes. The viability of YD-38 cells was reduced by miR-203 in time- and dose-dependent manners. Overexpression of miR-203 increased the nuclear condensation of YD-38 cells and activated the apoptotic signaling pathway by up-regulating pro-apoptotic factors, such as BCL-2-associated X protein (BAX) and BCL-2 homologous antagonist killer (BAK), and the active forms of caspase-9, caspase-3, and poly-(ADP-ribose)-polymerase (PARP). Furthermore, target gene array analyses revealed that the expression of class 6 semaphorin A (SEMA6A) was down-regulated by miR-203 in YD-38 cells. Both the mRNA and protein levels of SEMA6A were reduced in YD-38 cells transfected with miR-203. Luciferase activity assay confirmed that miR-203 directly targets the SEMA6A 3'-untranslated region to suppress gene expression. Our results indicate that miR-203 induces the apoptosis of YD-38 human oral cancer cells by directly targeting SEMA6A, suggesting its potential application in anticancer therapeutics.

Combination of cecropinXJ and LY294002 induces synergistic cytotoxicity, and apoptosis in human gastric cancer cells via inhibition of the PI3K/Akt signaling pathway.

The aim of the present study was to investigate the cytotoxic and apoptotic effects of cecropinXJ against human gastric cancer BGC823 cells, either alone, or in combination with a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. Cell viability and the apoptosis rate were measured using flow cytometry with Annexin-V staining. Additionally, the expression levels of several RAC-α serine/threonine kinase (Akt) phosphorylation-associated proteins and apoptosis-regulating proteins were evaluated by western blot analysis. It was observed that the combination of cecropinXJ and LY294002 resulted in significant synergistic cytotoxic and apoptosis effects, as compared with any single agent alone, in a dose-dependent manner. Corresponding to enhanced apoptosis, the expression levels of certain apoptosis-regulating proteins were changed, the most notable being the upregulation of caspase-3, B-cell lymphoma-2 (Bcl-2)-associated death promotor, Bcl-2 homologous antagonist killer, Bcl-2 interacting killer, Bcl-2-like protein 11, Bcl-2-like protein 4 and cytochrome c, and the downregulation of phosphorylated-Bad and Bcl-2 proteins. The present study provided a novel therapeutic regimen for the use of the cecropinXJ in combination with LY294002 for the treatment of gastric cancer.