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The effect of Menoprogen (MPG) on ovarian granulosa cell (GC) apoptosis was investigated in vitro and in vivo in an aged rat model of menopause. Intragastric administration of Menoprogen or estradiol valerate to 14-month-old senile female rats for eight weeks increased plasma E2 levels, as well as the weight of both ovarian and uterine tissues. Flow cytometric (FCM) analysis of isolated GCs from MPG-treated aged rats showed reductions in the G0/G1 ratio and apoptotic peaks. Isolated GCs also exhibited an increase in cell size and the number of cytoplastic organelles and intracellular gap junctions, the reappearance of secretory granules, and a lack of apoptotic bodies as determined by TEM. Results from a TdT-mediated dUTP nick end-labeling (TUNEL) assay revealed a reduction in TUNEL-positive GCs after MPG treatment. Immunohistochemical analysis showed a downregulation of proapoptotic Bax proteins and an upregulation of antiapoptotic Bcl-2 proteins. The addition of MPG-medicated serum to the media of cultured GCs also reduced cadmium chloride-induced apoptosis and downregulated caspase-3 protein expression. This work demonstrates that Menoprogen inhibits GC apoptosis in aged female rats and thereby increases E2 production. This represents a novel mechanism of action for this herbal medicine in the treatment of menopausal symptoms. [ABSTRACT FROM AUTHOR]

Cadmium is one of the most common heavy metal pollutants in the aquatic environment. Mud crab (Scylla paramamosain) is considered a model organism to monitor the impact of heavy metals. However, knowledge about toxicological mechanism of cadmium in crustaceans still remains limited. In this study, mud crabs were exposed to different concentrations of cadmium (0, 1.25, 2.5, 5 and 10 mg/L) for 72 h. Cadmium exposure significantly decreased superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidative capacity (T-AOC), and significantly increased malondialdehyde (MDA) and H2O2 levels. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity significantly increased after cadmium exposure. Moreover, integrated biological responses version 2 (IBRv2) analysis suggested that cadmium exposure exerted stronger toxicity on mud crab. Furthermore, oxidative stress induced by cadmium exposure could decrease total hemocyte count (THC), interrupt Ca2+ homeostasis, and lead to cytological damage. Cadmium exposure induced DNA damage, which activated DNA damage response signaling ATR-CHK1-p53 pathway. Our results also showed that cadmium exposure significantly increased the apoptosis and caspase-3 mRNA levels, which implied that cadmium induced apoptosis through a caspase-3 pathway. • Cadmium exposure induced oxidative stress of mud crab. • Cadmium exposure led to DNA damage and activated DNA damage response pathway of mud crab. • Cadmium exposure induced apoptosis through a caspase-dependent pathway. [ABSTRACT FROM AUTHOR]

Spinal cord ischemia/reperfusion injury (SCII) is a severe complication driven by apoptosis and neuroinflammation. An increase in the expression of c‐Fos, a member of the AP‐1 family, is known as a neuronal activation marker in SCII. The AP‐1 family is composed of Jun, Fos, and is associated with the regulation of cytokines expression and apoptosis. Fra‐1 is a member of the Fos family, however, the contribution of Fra‐1 to SCII is still unclear. In our study, Fra‐1 was highly upregulated especially in neurons and microglia and promoted apoptosis by changing the expression of Bax/Bcl‐2 after SCII. Furthermore, we found that Fra‐1 directly regulated the transcription expression of S100A8. We demonstrated that knockdown of Fra‐1 alleviated S100A8 mediated neuronal apoptosis and inflammatory factor release, thus improved motor function after SCII. Interestingly, we showed that administration of TAK‐242, the TLR4 inhibitor, to the ischemia/reperfusion (I/R) injury induced rats suppressed the activation of the ERK and NF‐κB pathways, and further reduced Fra‐1 expression. In conclusion, we found that Fra‐1‐targeted S100A8 was expressed the upstream of Fra‐1, and the Fra‐1/S100A8 interaction formed a feedback loop in the signaling pathways activated by SCII. [ABSTRACT FROM AUTHOR]

We provide unprecedented genetic and biochemical evidence that the antiapoptotic transcription factor STAT3 serves as a substrate for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK in an ecdysone-inducible mammalian expression system results in STAT3 activation, as documented by tyrosine phosphorylation and nuclear translocation of STAT3, as well as amplified expression of several STAT3 target genes. STAT3 activation after oxidative stress (OS) is strongly diminished in DT40 chicken B-lineage lymphoma cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 domain-mutated, but not a kinase domain-mutated, syk gene into SYK-deficient DT40 cells restores OS-induced enhancement of STAT3 activity. Thus, SYK plays an important and indispensable role in OS-induced STAT3 activation and its catalytic SH1 domain is critical for this previously unknown regulatory function. These results provide evidence for the existence of a novel mode of cytokineindependent cross-talk that operates between SYK and STAT3 pathways and regulates apoptosis during OS. We further provide experimental evidence that SYK is capable of associating with and phosphorylating STAT3 in human B-lineage leukemia/lymphoma cells challenged with OS. In agreement with a prerequisite role of SYI( in OS-induced STAT3 activation, OS does not induce tyrosine phosphorylation of STAT3 in SYK-deficient human proB leukemia cells. Notably, inhibition of SYK with a small molecule drug candidate prevents OS-induced activation of STAT3 and overcomes the resistance of human B-lineage leukemia/lymphoma cells to OSinduced apoptosis. [ABSTRACT FROM AUTHOR]

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of β-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a β-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates β-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer. [ABSTRACT FROM AUTHOR]

Purposes: Long intergenic non-protein coding RNA 525 (LINC00525), a long noncoding RNA, has been implicated in the carcinogenesis and progression of many human cancer types. However, the detailed roles of LINC00525 in chordoma and the underlying mechanisms are not fully understood. Here, we aimed to determine whether LINC00525 could modulate the oncogenicity of chordoma cells and to elucidate in detail the molecular events underlying these tumor-promoting activities. Methods: Reverse-transcription quantitative polymerase chain reactions were performed to assess LINC00525 expression in chordoma. The effects of LINC00525 silencing on chordoma cell proliferation, apoptosis, migration, and invasiveness in vitro and tumor growth in vivo were respectively tested using CCK-8 assay, flow cytometry, migration and invasion assays, and xenograft experiments. Results: High LINC00525 expression levels were detected in chordoma tissues. The proliferative, migratory, and invasive abilities of chordoma cells in vitro and their tumor growth in vivo were suppressed by the LINC00525 knockdown, whereas apoptosis was induced by it. Mechanistically, LINC00525 acted as a molecular sponge of microRNA-505-3p (miR-505-3p) and upregulated the expression of high mobility group box 1 (HMGB1), which is directly targeted by miR-505-3p. Rescue assays indicated that increasing the output of miR-505-3p–HMGB1 axis attenuated the effects of LINC00525 depletion on chordoma cells. Conclusion: LINC00525, a pro-oncogenic long noncoding RNA, promotes chordoma progression by regulating the miR-505-3p–HMGB1 axis. The LINC00525–miR-505-3p–HMGB1 pathway may be a novel therapeutic target in chordoma. [ABSTRACT FROM AUTHOR]

The present study investigated autophagy changes and the expression of HMGB1 in human glioblastoma cells, responding to carbon-ion beam irradiation (35 keV/μm, 80.55 MeV/u). U251 cells were irradiated with carbon-ion beams and cell proliferation was measured by counting the number of living cells. The expression of Light Chain 3 beta (LC3B), Beclin 1, high-mobility-group box 1 (HMGB1), pro-form caspase-3 and Cellular FLICE-like inhibitory protein (c-FLIP) was analyzed by western blotting. Caspase enzyme activity was determined via a caspase cleavage based florescent substrate commercial Kit. Living cell counting demonstrated a time- and dose-dependent cell death in U251 cells. The expression of LC3B and Beclin 1 revealed that, a high level of autophagy was induced 24 h after irradiation with 1 Gy carbon ions and then decreased in a time- and dose-dependent manner. The expression of the whole HMGB1 showed a well correlation with the dynamic autophagic level. Cytoplasmic HMGB1 maintained autophagy was concluded. Enzyme-Linked Immuno Sorbent Assay (ELISA) measurement found that, HMGB1 was released into the extracellular space in a time- and dose-dependent manner. Lower intracellular HMGB1 levels correlated with decreased autophagy as measured by the expression of LC3B. Decreased expression of pro-form caspase-3 and c-FLIP as well as the increased caspase enzyme activity indicated that apoptosis was induced by carbon-ion beam irradiation. Inhibition of HMGB1 release from the area of intracellular to that of extracellular significantly increased cell survival. In summary, carbon-ion beam irradiation could elevate autophagy and HMGB1 expression efficiently, which would protect the cells from programmed cell death via inducing autophagy. Apoptosis as measured by expression of caspase activities increased as the dose increased, which was accompanied with decreased levels of LC3B and HMGB1. • Carbon-ion irradiation could induce autophagy efficiently. • Apoptosis would be promoted accompanied by reduced autophagy. • Change of HMGB1 shows a good correlation to the autophagic level. • Accumulated HMGB1 protects cell from carbon-ion beam irradiation induced cell death. [ABSTRACT FROM AUTHOR]

Abstract Bladder cancer (BC) has been regarded as the most common malignancy of the urinary system worldwide. With lack of investigations for molecular pathogenesis underlying that develop BC, the therapeutic efficacy of several therapeutic approaches existing is still unsatisfactory. Here, our study aimed to explore the potentially biological function of MAN1B1 on BC. In this study, MAN1B1 expression level in BC tissues and normal tissues was analyzed based on The Cancer Genome Atlas (TCGA) data and correlation between its expression and prognosis was determined using Kaplan-Meier analysis. Knockout of MAN1B1 was performed using silencing RNA and the efficacy of MAN1B1 knockout was identified using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The BC cells proliferation was assessed by Cell Counting Kit-8 (CCK8) assay, and then the cells apoptosis was detected by Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI) staining and flow cytometry following MAN1B1 knocked down by small interfering RNA. Protein kinase B (AKT) signaling was evaluated by detecting related markers, namely AKT, p-AKT, 4E-BP-1 and Bax using western blot assay. As a result, the MAN1B1 expression was higher in BC tissues than those in normal tissues, besides, its overexpression was associated with poor prognosis. Moreover, MAN1B1 reduction by silencing RNA approach resulted in BC cells proliferation suppression and BC cells apoptosis promotion. Finally, AKT signaling activity was inhibited by MAN1B1 silencing. Taken together, these results unraveled that MAN1B1 may act on an oncogenic action in BC, which improved the likelihood of MAN1B1 taking on a promising prognostic biomarker and a potential target for treating BC. Highlights • A significant biomarker for bladder cancer progression and prognosis is proposed. • This work indicates the functional role of MAN1B1 in bladder cancer cell proliferation and apoptosis. • Potential molecular mechanism involved in biological effect of MAN1B1 in bladder cancer is developed. • Control strategies targeting the MAN1B1 are predicted to be promising therapies in bladder cancer. [ABSTRACT FROM AUTHOR]

Background: Bone marrow mesenchymal stem cells (MSCs) have been found to produce beneficial effects on ischemia-reperfusion injury. However, most of the MSCs died when transplanted into the ischemic tissue, which severely limit their therapeutic potential. Methods: Using an in vitro model of hypoxia and serum deprivation (H/SD), we investigated the hypothesis that sevoflurane preconditioning could protect MSCs against H/SD-induced apoptosis and improve their migration, proliferation, and therapeutic potential. The H/SD of MSCs and neuron-like PC12 cells were incubated in a serum-free medium and an oxygen concentration below 0.1% for 24 h. Sevoflurane preconditioning was performed through a 2-h incubation of MSCs in an airtight chamber filled with 2 vol% sevoflurane. Apoptosis of MSCs or neuron-like PC12 cells was assessed using Annexin V-FITC/propidium iodide (PI). Furthermore, the mitochondrial membrane potential was assessed using lipophilic cationic probe. The proliferation rate was evaluated through cell cycle analysis. Finally, HIF-1α, HIF-2α, VEGF and p-Akt/Akt levels were measured by western blot. Results: Sevoflurane preconditioning minimized the MSCs apoptosis and loss of mitochondrial membrane potential. Furthermore, it increased the migration and expression of HIF-1α, HIF-2α, VEGF, and p-Akt/Akt, reduced by H/SD. In addition, neuron-like PC12 cells were more resistant to H/SD-induced apoptosis when they were co-cultured with sevoflurane preconditioning MSCs. Conclusion: These findings suggest that sevoflurane preconditioning produces protective effects on survival and migration of MSCs against H/SD, as well as improving the therapeutic potential of MSCs. These beneficial effects might be mediated at least in part by upregulating HIF-1α, HIF-2α, VEGF, and p-Akt/Akt. [ABSTRACT FROM AUTHOR]

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne's disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne's disease in bovines. [ABSTRACT FROM AUTHOR]

Background: Ischaemia reperfusion (IR) induces multiple pathophysiological changes. In addition to its classical role in regulating tumourigenesis, the feedback loop formed by p53 and its driven target p53-upregulated modulator of apoptosis (PUMA) was recently demonstrated to be the common node tightly controlling various cellular responses during myocardial IR. However, the roles of the p53-PUMA feedback loop in the spinal cord remain unclear. This study aimed to elucidate the roles of p53-PUMA feedback interactions in the spinal cord after IR, specifically investigating their regulation of caspase 3-mediated apoptosis and nuclear factor (NF)-κB-mediated cytokine release.Methods: SD rats subjected to 12 min of aortic arch occlusion served as IR models. Neurological assessment as well as p53 and PUMA mRNA and protein expression analyses were performed at 12-h intervals during a 48-h reperfusion period. The cellular distributions of p53 and PUMA were determined via double immunofluorescence staining. The effects of the p53-PUMA feedback loop on modulating hind-limb function; the number of TUNEL-positive cells; and protein levels of caspase 3, NF-κB and cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, were evaluated by intrathecal treatment with PUMA-specific or scramble siRNA and pifithrin (PFT)-α. Blood-spinal cord barrier (BSCB) breakdown was examined by Evans blue (EB) extravasation and water content analyses.Results: IR induced significant behavioural deficits as demonstrated by deceased Tarlov scores, which displayed trends opposite those of PUMA and p53 protein and mRNA expression. Upregulated PUMA and p53 fluorescent labels were widely distributed in neurons, astrocytes and microglia. Injecting si-PUMA and PFT-α exerted significant anti-apoptosis effects as shown by the reduced number of TUNEL-positive cells, nuclear abnormalities and cleaved caspase 3 levels at 48 h post-IR. Additionally, p53 colocalized with NF-κB within the cell. Similarly, injecting si-PUMA and PFT-α exerted anti-inflammatory effects as shown by the decreased NF-κB translocation and release of IL-1β and TNF-α. Additionally, injecting si-PUMA and PFT-α preserved the BSCB integrity as determined by decreased EB extravasation and spinal water content. However, injecting si-Con did not induce any of the abovementioned effects.Conclusions: Inhibition of aberrant p53-PUMA feedback loop activation by intrathecal treatment with si-PUMA and PFT-α prevented IR-induced neuroapoptosis, inflammatory responses and BSCB breakdown by inactivating caspase 3-mediated apoptosis and NF-κB-mediated cytokine release. [ABSTRACT FROM AUTHOR]

Involvement of CD4+ helper T (Th) cells is crucial for CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4+ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2Kb/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy. [ABSTRACT FROM AUTHOR]