BCL2-associated Agonist Of Cell Death Research Articles

NUP98-BPTF promotes oncogenic transformation through PIM1 upregulation.

Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98-BPTF (NB) fusion in patients with T-cell acute lymphoblastic leukemia (T-ALL) using next-generation sequencing. The FG-repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown. To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline-inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T-ALL cells. NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto-oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB-induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T-ALL cells by inactivating the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion-positive leukemia.

Human Placenta-Derived Mesenchymal Stem Cells Improve Neurological Function in Rats with Intrauterine Hypoxic-Ischaemic Encephalopathy by Reducing Apoptosis and Inflammatory Reactions.

Hypoxic-ischaemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although hypothermia therapy offers some neuroprotection, the recovery of neurological function is limited. Therefore, new synergistic therapies are necessary to improve the prognosis. Mesenchymal stem cell-based therapy is emerging as a promising treatment option for HIE. In this study, we studied the therapeutic efficacy of human placenta-derived mesenchymal stem cells (PD-MSCs) in the HIE rat model and analyzed the underlying therapeutic mechanisms. Rats were divided into 6 groups (n = 9 for each) as follows: control, HIE model, HIE + normal saline, and HIE + PD-MSC transplantation at days 7, 14 and 28 postpartum. Following PD-MSC transplantation, neurological behavior was evaluated using rotarod tests, traction tests, and the Morris water maze test. The degree of brain tissue damage was assessed by histological examination and Nissl staining. Expression levels of apoptosis-related proteins and inflammatory factors were quantified by Western blotting and enzyme-linked immunosorbent assays. Immunofluorescence was used to investigate the ability of PD-MSCs to repair the morphology and function of hippocampal neurons with hypoxic-ischaemic (HI) injury. PD-MSC transplantation enhanced motor coordination and muscle strength in HIE rats. This treatment also improved spatial memory ability by repairing pathological damage and preventing the loss of neurons in the cerebral cortex. The most effective treatment was observed in the HIE + PD-MSC transplantation at day 7 group. Expression levels of microtubule-associated protein-2 (MAP-2), B-cell lymphoma-2 (BCL-2), interleukin (IL)-10, and transforming growth factor (TGF -β1) were significantly higher in the HIE + PD-MSC treatment groups compared to the HIE group, whereas the levels of BCL-2-associated X protein (BAX), BCL-2-associated agonist of cell death (BAD), IL-1β and tumour necrosis factor α (TNF-α) were significantly lower. We demonstrated that intravenous injection of PD-MSC at 7, 14 and 28 days after intrauterine HI damage in a rat model could improve learning, memory, and motor function, possibly by inhibiting apoptosis and inflammatory damage. These findings indicate that autologous PD-MSC therapy could have potential application for the treatment of HIE.

Differences in p38-STAT3-S100A11 signaling after the administration of aristolochic acid I and IVa may account for the disparity in their nephrotoxicity

BackgroundThe safety of herbs containing aristolochic acids (AAs) has become a widespread concern. Previous reports indicate that AAs are highly nephrotoxic and carcinogenic, although there are more than 170 analogues of aristolochic acid. Not all AAs have the same degree of nephrotoxicity or carcinogenicity. Previous studies have found that aristolochic acid IVa (AA-IVa), the principal component of AAs within members of the Aristolochiaceae family, especially Asarum, a commonly used herb in China, has essentially no significant nephrotoxicity. However, several studies, including ours, have shown that aristolochic acid I (AA-I) is clearly nephrotoxic. PurposeThe focus of the study was to elucidate the molecular mechanism responsible for the difference in nephrotoxicity between the AA-I and AA-IVa. Study design/methodMice were administered with AA-I or AA-IVa for 22 weeks through the oral route, followed by a 50-week recovery time. The kidney tissues of mice were extracted at the end of 22 weeks. Pathological examination and proteomic detection (tandem mass tagging (TMT) and phosphorylated proteomics) were performed on the kidney tissue to investigate the key signaling pathways and targets of AAs-induced renal interstitial fibrosis (RIF). The key signaling pathways and targets were verified by Western blot (WB), siRNA transfection, and luciferase assays. ResultsAA-I caused severe nephrotoxicity, high mortality, and extensive RIF. However, the same AA-IVa dosage exhibited almost no nephrotoxicity and does not trigger RIF. The activation of the p38-STAT3-S100A11 signaling pathway and upregulated expression of α smooth muscle actin (α-SMA) and Bcl2-associated agonist of cell death (Bad) proteins could be the molecular mechanism underlying AA-I-induced nephrotoxicity. On the other hand, AA-IVa did not regulate the activation of the p38-STAT3-S100A11 signaling pathway and had relatively little effect on the expression of α-SMA and Bad. Consequently, the difference in the regulation of p38-STAT3-S100A11 pathway, α-SMA, and Bad proteins between AA-I and AA-IVa may be responsible for the divergence in their level of nephrotoxicity. ConclusionThis is the first study to reveal the molecular mechanism underlying the difference in nephrotoxicity between AA-I and AA-IVa. Whether STAT3 is activated or not may be the key factor leading to the difference in nephrotoxicity between AA-I and AA-IVa.

Sulfamethoxazole (SMX) Alters Immune and Apoptotic Endpoints in Developing Zebrafish (Danio rerio)

Sulfamethoxazole (SMX) is a broad-range bacteriostatic antibiotic widely used in animal and fish farming and is also employed in human medicine. These antibiotics can ultimately end up in the aquatic ecosystem and affect non-target organisms such as fish. To discern the effect of SMX on developing zebrafish embryos and larvae, we investigated a broad range of sub-lethal toxicity endpoints. Higher concentrations of SMX affected survivability, caused hatch delay, and induced malformations including edema of the yolk sac, pericardial effusion, bent tail, and curved spine in developing embryos. Lower levels of SMX provoked an inflammatory response in larvae at seven days post fertilization (dpf), as noted by up-regulation of interferon (ifn-γ) and interleukin 1β (il-1β). SMX also increased the expression of genes related to apoptosis, including BCL2-Associated Agonist of Cell Death (bad) and BCL2 Associated X, Apoptosis Regulator (bax) at 50 µg/L and decreased caspase 3 (casp3) expression in a dose-dependent manner. SMX induced hyperactivity in larval fish at 500 and 2500 µg/L based upon the light/dark preference test. Collectively, this study revealed that exposure to SMX can disrupt the immune system by altering host defense mechanisms as well as transcripts related to apoptosis. These data improve understanding of antibiotic chemical toxicity in aquatic organisms and serves as a baseline for in-depth environmental risk assessment of SMX and antibiotics.

Effects of hypoxia and reoxygenation on oxidative stress, histological structure, and apoptosis in a new hypoxia-tolerant variety of blunt snout bream (Megalobrama amblycephala)

A new hypoxia-tolerant variety of blunt snout bream was obtained by successive breeding of the wild population, which markedly improved hypoxia tolerance. In this study, the hypoxia-tolerant variety was exposed to hypoxia (2.0 mg O2·L−1) for 4, 7 days. The contents of blood biochemical indicators including the number of red blood cells (RBC), total cholesterol (T-CHO), total protein (TP), triglyceride (TG), glucose (GLU), and lactic acid (LD) increased significantly (P < 0.05) under hypoxia. The glycogen content in the liver and muscle decreased significantly (P < 0.05) and the LD content in the brain, muscle and liver increased significantly (P < 0.05) under hypoxia. The levels of oxidative stress-related indicators i.e., superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) also changed significantly (P < 0.05) in the heart, liver, and intestine of the new variety under hypoxia. Additionally, hypoxia has caused injuries to the heart, liver, and intestine, but it shows amazing repair ability during reoxygenation. The apoptotic cells and apoptosis rate in the heart, liver, and intestine increased under hypoxia. Under hypoxia, the expression of the B-cell lymphomas 2 (Bcl-2) gene in the heart, liver, and intestine was significantly (P < 0.05) down-regulated, while the expression of the BCL2-associated agonist of cell death (Bad) gene was significantly (P < 0.05) up-regulated. These results are of great significance for enriching the basic data of blunt snout bream new variety in response to hypoxia and promoting the healthy development of its culture industry.

Enhanced Apoptosis in Choroidal Tissues in Lens-Induced Myopia Guinea Pigs by Activating the RASA1 Signaling Pathway.

PurposeThis study aimed to explore the role of the RAS p21 protein activator 1 (RASA1) signaling pathway in apoptosis in choroid tissues from guinea pigs with negative lens-induced myopia (LIM).MethodsBiometric measurements were performed to examine refractive status, ocular parameters, and choroidal thickness (ChT) after myopia induction. The choroidal morphology was observed by hematoxylin and eosin (H&E) staining and TUNEL assay. The expression of the RASA1 signaling pathway at the mRNA and protein levels in choroidal tissues was measured by real-time quantitative PCR (qPCR) and western blot assays.ResultsCompared with the normal control (NC) group, the ocular length of the guinea pigs in LIM increased remarkably, as did the myopic refraction. ChT decreased after myopia induction. H&E staining showed that the thickness and laxity of the choroidal tissues in LIM were strikingly reduced. The number of apoptotic cells in the LIM eyes was increased. Moreover, qPCR and western blot assays showed that the expression levels of both RASA1 and BCL-2-associated agonist of cell death (BAD) were higher in the LIM group than in the NC group, whereas the expression level of B-cell lymphoma 2 (BCL-2) was decreased after 2 weeks of experimental myopia. However, the trend of RASA1, BAD, and BCL-2 expression was reversed after 4 weeks of experimental myopia compared with levels after 2 weeks of experimental myopia.ConclusionsResults showed that the RASA1 signaling pathway is activated in choroid tissues in myopic guinea pigs. Activated RASA1 signaling induces high BAD expression and low BCL-2 expression, which in turn promotes apoptosis and ultimately causes ChT thinning in myopic guinea pigs.

Administration of 4‑hexylresorcinol increases p53‑mediated transcriptional activity in oral cancer cells with the p53 mutation.

The p53 mutation is inherent in over 50% of human cancers. In head and neck squamous cell carcinoma, the p53 mutation is associated with a poor prognosis. 4-Hexylresorcinol (4HR) is a pharmacologic chaperone. The present study aimed to investigate the effect of 4HR on p53 transcriptional activity in oral carcinoma cells with p53 mutations. To identify conformational changes induced by 4HR administration, peptides including the DNA-binding domain from mutant and wild-type p53 were synthesized, and Fourier transform infrared spectroscopy was performed. To determine the effect of 4HR on p53 mutant carcinoma cells, western blot analysis, p53 transcriptional activity analysis, MTT assay and apoptosis immunocytochemistry were performed. The YD-15 cell line has a mutation in the DNA binding domain of p53 (Glu258Ala). When p53 Ala-258 was coupled by 4HR, the p53 Ala-258 structure lost its original conformation and approached a conformation similar to that of p53 Glu-258. In the cell experiments, 4HR administration to p53 mutant cells increased p53 transcriptional activity and the expression levels of apoptosis-associated proteins such as B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX) and BCL2-associated agonist of cell death (BAD). Accordingly, 4HR administration on YD-15 cells decreased cell viability and increased apoptosis. In conclusion, 4HR is a potential substance for use in the recovery of loss-of-function in mutant p53 as a pharmacologic chaperone.

Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension.

Pulmonary hypertension (PH) is a vascular disease characterized by elevated pulmonary arterial pressure, leading to right ventricular failure and death. Pathogenic features of PH include endothelial apoptosis and vascular inflammation, which drive vascular remodeling and increased pulmonary arterial pressure. Re-analysis of the whole transcriptome sequencing comparing human pulmonary arterial endothelial cells (PAECs) isolated from PH and control patients identified AREG, which encodes Amphiregulin, as a key endothelial survival factor. PAECs from PH patients and mice exhibited down-regulation of AREG and its receptor epidermal growth factor receptor (EGFR). Moreover, the deficiency of AREG and EGFR in ECs in vivo and in vitro heightened inflammatory leukocyte recruitment, cytokine production, and endothelial apoptosis, as well as diminished angiogenesis. Correspondingly, hypoxic mice lacking Egfr in ECs (cdh5 cre/+ Egfr fl/fl) displayed elevated RVSP and pulmonary remodeling. Computational analysis identified NCOA6, PHB2, and RRP1B as putative genes regulating AREG in endothelial cells. The master transcription factor of hypoxia HIF-1⍺ binds to the promoter regions of these genes and up-regulates their expression in hypoxia. Silencing of these genes in cultured PAECs decreased inflammation and apoptosis, and increased angiogenesis in hypoxic conditions. Our pathway analysis and gene silencing experiments revealed that BCL2-associated agonist of cell death (BAD) is a downstream mediator of AREG BAD silencing in ECs lacking AREG mitigated inflammation and apoptosis, and suppressed tube formation. In conclusion, loss of Amphiregulin and its receptor EGFR in PH is a crucial step in the pathogenesis of PH, promoting pulmonary endothelial cell death, influx of inflammatory myeloid cells, and vascular remodeling.

Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K+ channel complex-mediated cellular apoptosis.

Complexes formed with α5-integrins and the voltage-gated potassium (K+ ) channel KCNB1 (Kv2.1), known as IKCs, transduce the electrical activity at the plasma membrane into biochemical events that impinge on cytoskeletal remodeling, cell differentiation, and migration. However, when cells are subject to stress of oxidative nature IKCs turn toxic and cause inflammation and death. Here, biochemical, pharmacological, and cell viability evidence demonstrates that in response to oxidative insults, IKCs activate an apoptotic Mitogen-activated protein kinase/extracellular signal-regulated kinase (Ras-MAPK) signaling pathway. Simultaneously, wild-type (WT) KCNB1 channels sequester protein kinase B (Akt) causing dephosphorylation of BCL2-associated agonist of cell death (BAD), a major sentinel of apoptosis progression. In contrast, IKCs formed with C73A KCNB1 variant that does not induce apoptosis (IKCC73A ), do not sequester Akt and thus are able to engage cell survival mechanisms. Taken together, these data suggest that apoptotic and survival forces co-exist in IKCs. Integrins send death signals through Ras-MAPK and KCNB1 channels simultaneously sabotage survival mechanisms. Thus, the combined action of integrins and KCNB1 channels advances life or death.

Abstract 9770: Endothelial Loss of Amphiregulin Drives Inflammation and Endothelial Apoptosis in Pulmonary Hypertension

Pulmonary hypertension (PH) is a vascular disease characterized by elevated pulmonary arterial pressure (PAP), leading to right ventricular failure and death. Pathogenic features of PH include endothelial apoptosis and vascular inflammation, which drive vascular remodeling and increased PAP. Re-analysis of the whole transcriptome sequencing comparing human pulmonary arterial endothelial cells (PAECs) isolated from PH and control patients identified AREG , which encodes amphiregulin, as a key endothelial survival factor. PAECs from PH patients and mice exhibited downregulation of AREG and its receptor epidermal growth factor receptor (EGFR). Moreover, the deficiency of AREG and EGFR in ECs in vivo and in vitro heightened inflammatory leukocyte recruitment, cytokine production and endothelial apoptosis, and diminished angiogenesis. Correspondingly, hypoxic mice lacking Egfr in ECs ( cdh5 cre/+ Egfr fl/fl ) displayed elevated RVSP, Fulton index, and pulmonary remodeling. Computational analysis identified NCOA6, PHB2, and RRP1B as putative genes regulating AREG in endothelial cells. The master transcription factor of hypoxia HIF-1αbinds to the promoter regions of these genes and upregulate their expression in hypoxia. Silencing of these genes in cultured PAECs decreased inflammation and apoptosis, and increased angiogenesis in hypoxic conditions. Our pathway analysis and gene silencing experiments revealed that BCL2-associated agonist of cell death (BAD) is a downstream mediator of AREG . BAD silencing in ECs lacking AREG , mitigated inflammation and apoptosis and suppressed tube formation. In conclusion, loss of amphiregulin and its receptor EGFR in PH is a crucial step in the pathogenesis of PH, promoting pulmonary endothelial cell death, vascular recruitment of inflammatory myeloid cells, and vascular remodeling.

Phosphoproteomic Analysis of Primary Myeloma Patient Samples Identifies Distinct Phosphorylation Signatures Correlating with Chemo-Sensitivity Profiles in an Ex Vivo Drug Sensitivity Testing Platform

Introduction: Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow resulting in end-organ damage. Despite an extensive increase in the five-year survival rate in recent years, MM is still considered an incurable disease as patients will repeatedly relapse and develop resistance to current chemotherapies. A key focus for the personalization of myeloma therapy is understanding the biological mechanisms of drug resistance and identifying clinically useful biomarkers of therapeutic response. Highly efficient techniques for the enrichment of phosphorylated peptides followed by high resolution mass spectrometry facilitates the quantitation of thousands of site-specific phosphorylation events. Here, we have performed a phosphoproteomic analysis on MM cell lysates stratified based on their ex vivo drug response profiles to advance our understanding of drug resistance mechanisms.Materials and Methods: CD138 + plasma cells were isolated from 20 adult MM patient bone marrow aspirates at diagnosis (n=7) or relapse (n=13). Samples were grouped based on ex vivo drug sensitivity and resistance testing (DSRT) as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III), highly resistant (Group IV) [1]. For the phosphoproteomic analysis, peptides were generated and purified using the filter aided sample preparation (FASP) protocol. Peptide tandem mass tag (TMT) labelling, Fe 3+ immobilized metal ion affinity chromatography (IMAC), synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) was performed. Nonenriched peptides were used for proteomic analysis. Resulting data was analysed using MaxQuant, followed by normalization of phosphosite intensities using the internal reference scale (IRS) method, and statistical analysis using Perseus. Functional enrichment and kinase enrichment analyses were performed on significant phosphoproteins using g:profiler and KEA2, respectively.Results: Our quantitative MS-based phosphoproteomic analysis identified 2,945 phosphorylation sites on 2,232 phosphopeptides from 690 phosphoproteins. Of these phosphorylation sites, 176 were significantly changed between all four DSRT groups and 267 were significantly changed between Group I and Group IV (False Discovery Rate (FDR) < 0.05). Hierarchical clustering was performed to highlight the distinct phosphoproteomic profiles associated with each DSRT group, of which the very sensitive (Group I) and very resistant (Group IV) subgroups demonstrated a well-defined separation (Fig. 1A, 1B). KEGG pathway analysis and gene ontology (GO) analysis of significantly increased phosphorylated proteins in Group IV compared to Group I MM patients demonstrated an increased phosphorylation of proteins associated with tight junctions, the Rap1 signalling pathway and the phosphatidylinositol signalling system; indicating an upregulation of cell adhesion associated processes in drug resistant MM. Phosphoproteins increased in abundance in Group I compared to Group IV MM patients revealed an increased phosphorylation of proteins involved in translation and RNA processing including the spliceosome, RNA transport and RNA binding pathways (Fig. 1C). We identified filamin A serine 2152, RAS guanyl-releasing protein 2 serine 576 and proto-oncogene tyrosine-protein kinase Src serine 17 as increased in Group IV MM, and nuclease-sensitive element-binding protein 1 (YBX1) serine 165, CD44 serine 697 and Bcl2-associated agonist of cell death (BAD) serine 99 as increased in Group I MM. KEA of the upregulated phosphoproteome in Group IV revealed an enrichment of cyclin dependent kinase 1 (CDK1) and ribosomal s6 kinases (RPS6K) whereas casein kinase 2 (CK2) and the glycolysis-associated kinases were enriched in Group I (Fig. 1D).Conclusion: Our study has generated a phosphoproteomic dataset demonstrating distinct phosphorylation signatures associated with drug sensitivity in clinical MM plasma cells. The identification of phosphorylation events associated with drug resistance provides a basis for further exploration of these events and associated signalling pathways to further understand drug resistance mechanisms in MM and identify potential biomarkers of therapeutic response and targets for drug re-sensitization in MM.

Expression and functional analysis of the BCL2-Associated agonist of cell death (BAD) gene in grass carp (Ctenopharyngodon idella) during bacterial infection

The BCL2-associated agonist of cell death protein is a key participant in apoptosis dependent on mitochondria and in disease progression that involves the regulation of cell death, such as tumorigenesis, diabetes, sepsis shock, and epilepsy. Nevertheless, the mechanisms underlying the immune responses to teleost BAD bacterial infection and mitochondrial-dependent apoptosis remains unclear. In order to elucidate the mechanisms involved, in this study, a Ctenopharyngodon idella (grass carp) BAD gene named GcBAD1 was firstly cloned and characterized. The results indicated that the ORF (open reading frame) of GcBAD1 was 438 bp in length, encoding a 145-amino acid putative protein of 16.66 kDa. This deduced amino acid sequence has a better identity than another teleost species according to a phylogenetic analysis, and contains a Bcl2-BAD-1 domain. In healthy grass carp fish, the mRNA transcripts of GcBAD1 were widely present in the studied tissues, which could be ranked as follows; spleen > brain > middle-kidney > head-kidney > liver > gills > intestines > heart and muscle. In addition, during infection by Aeromonas hydrophila and Staphylococcus aureus, the mRNA transcription and protein levels expression of GcBAD1 in the head-kidney, spleen, and liver tissues of the fish were significantly up-regulated. Moreover, when the C. idellus kidney cell line (CIK) cells were incubated with Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), the GcBAD1 expression transcripts were also significantly up-regulated. Additionally, overexpression of GcBAD1 in CIK cells was able to activate apoptosis-related genes, including those encoding p53, Cytochrome C (CytoC), caspase-3, and caspase-9. Besides, in the TUNEL assays, when pEGFP-BAD1 was over-expressed, the number of red signals associated with apoptosis were significantly increased in the CIK cells infected with LPS or LTA at 12 h. This study demonstrates that GcBAD1 has a significant role in the mitochondrial apoptosis pathway of grass carp's innate immunity. Our findings provide new insight into the potential mechanisms of teleost antibacterial immunity.

Differentiation-promoting and Protective Effects of the Fractions of Various Ginseng Species in C2C12 Cells

Background: The effects of Panax ginseng C. A. Meyer in protecting and promoting the differentiation of muscle cells have been reported. However, the influences of various ginseng species and various fractions were not investigated.<BR>Methods and Results: In this study, these effects on muscle cells were confirmed using butanol and water fractions of various ginseng species. The fractions of P. ginseng were confirmed to have a cytoprotective effect on C2C12 cells. The myogenin expression level was significantly higher by 1.5 times or more, in the P. ginseng fraction groups than in the other groups, confirming that the P. ginseng fractions promoted cell differentiation. Both fractions of P. ginseng significantly reduced the rate of production of reactive oxygen species when compared with that of the control group. In the mechanism study, P. ginseng fractions tended to decrease muscle RING-finger protein-1 (MuRF-1), AMP-activated protein kinase (AMPK), forkhead box O3α (Foxo3α), and the expression of the BCL2-associated agonist of cell death (BAD). In particular, AMPK was significantly reduced in the P. ginseng water fraction group when compared to that in the butanol fraction and control groups.<BR>Conclusions: The results of this study confirmed that P. ginseng has superior cell differentiation-promoting and protective effects on muscle cells when compared with the effects of Panax quinquefolium and Panax notoginseng.

Candidone Inhibits Migration and Invasion, and Induces Apoptosis in HepG2 Cells.

The aim of the study was to investigate the inhibitory activity of candidone, the active constituent of Derris (D.) indica, on the proliferation, migration, and invasiveness of human hepatoblastoma (HepG2) cells. Cancer cell death was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis-associated morphological changes were observed by phase contrast microscopy. Additionally, Western blotting was used to study protein expression following treatment with candidone, and transwell migration and invasion assays were used for observing cancer cell migration and invasiveness, respectively. The results suggest that candidone possesses potent inhibitory activity against HepG2 cells (concentration, 100 µM; 24 h treatment). Cancer cells treated with candidone exhibited apoptosis-associated changes, including detachment, cell shrinkage and death. Furthermore, candidone was shown to promote cell death by activating caspase-3 and -9, and decreasing the expression of antiapoptotic proteins, including p65, induced myeloid leukemia cell differentiation protein Mcl-1, B-cell lymphoma 2 (Bcl2), Bcl2-associated agonist of cell death and survivin. Moreover, candidone inhibited the migration and invasion abilities of HepG2 cells and decreased the levels of proteins associated with these processes, including phospho-p38 and active matrix metallopeptidase 9. Collectively, the results of the present study indicate that candidone is able to inhibit the proliferation, migration and invasive potential of HepG2 cells.

EGFR signaling promotes resistance to CHK1 inhibitor prexasertib in triple negative breast cancer.

Aim: Innate resistance to the CHK1 inhibitor prexasertib has been described, but resistance mechanisms are not understood. We aimed to determine the role epidermal growth factor receptor (EGFR) plays in innate resistance to prexasertib in triple negative breast cancer (TNBC).Methods: Using a panel of pre-clinical TNBC cell lines, we measured the sensitivity to prexasertib. We examined the effect activation of EGFR had on prexasertib sensitivity. We measured the synergy of dual blockade of EGFR with erlotinib and CHK1 with prexasertib in TNBC cell lines and xenografts.Results: EGFR overexpression and activation increased resistance to CHK1 inhibition by prexasertib. EGFR promoted the phosphorylation of BCL2-associated agonist of cell death (BAD), inactivating its pro-apoptotic functions. Inhibition of EGFR reversed BAD phosphorylation, increasing sensitivity to prexasertib.Conclusion: The use of prexasertib as a monotherapy in TNBC has been limited due to modest clinical responses. We demonstrated that EGFR activation contributes to innate resistance to prexasertib in TNBC and potentially other cancers. EGFR expression status should be considered in clinical trials examining prexasertib’s use as a monotherapy or combination therapy.

I2 imidazoline receptor modulation protects aged SAMP8 mice against cognitive decline by suppressing the calcineurin pathway.

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I2-IRs) are increased in the brain in Alzheimer's disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I2-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-D aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3β, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1), was increased in B06-treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I2 imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP8 mice.

Animal Models of Metabolic Epilepsy and Epilepsy Associated Metabolic Dysfunction: A Systematic Review.

Epilepsy is a serious neurological disorder affecting around 70 million people globally and is characterized by spontaneous recurrent seizures. Recent evidence indicates that dysfunction in metabolic processes can lead to the alteration of neuronal and network excitability, thereby contributing to epileptogenesis. Developing a suitable animal model that can recapitulate all the clinical phenotypes of human metabolic epilepsy (ME) is crucial yet challenging. The specific environment of many symptoms as well as the primary state of the applicable neurobiology, genetics, and lack of valid biomarkers/diagnostic tests are the key factors that hinder the process of developing a suitable animal model. The present systematic review summarizes the current state of available animal models of metabolic dysfunction associated with epileptic disorders. A systematic search was performed by using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) model. A range of electronic databases, including google scholar, Springer, PubMed, ScienceDirect, and Scopus, were scanned between January 2000 and April 2020. Based on the selection criteria, 23 eligible articles were chosen and are discussed in the current review. Critical analysis of the selected literature delineated several available approaches that have been modeled into metabolic epilepsy and pointed out several drawbacks associated with the currently available models. The result describes available models of metabolic dysfunction associated with epileptic disorder, such as mitochondrial respiration deficits, Lafora disease (LD) model-altered glycogen metabolism, causing epilepsy, glucose transporter 1 (GLUT1) deficiency, adiponectin responsive seizures, phospholipid dysfunction, glutaric aciduria, mitochondrial disorders, pyruvate dehydrogenase (PDH) α-subunit gene (PDHA1), pyridoxine dependent epilepsy (PDE), BCL2-associated agonist of cell death (BAD), Kcna1 knock out (KO), and long noncoding RNAs (lncRNA) cancer susceptibility candidate 2 (lncRNA CASC2). Finally, the review highlights certain focus areas that may increase the possibilities of developing more suitable animal models and underscores the importance of the rationalization of animal models and evaluation methods for studying ME. The review also suggests the pressing need of developing precise robust animal models and evaluation methods for investigating ME.

Leishmania aethiopica cell-to-cell spreading involves caspase-3, AkT, and NF-κB but not PKC-δ activation and involves uptake of LAMP-1-positive bodies containing parasites.

Development of human leishmaniasis is dependent on the ability of intracellular Leishmania parasites to spread and enter macrophages. The mechanism through which free promastigotes and amastigotes bind and enter host macrophages has been previously investigated; however, little is known about intracellular trafficking and cell-to-cell spreading. In this study, the mechanism involved in the spreading of Leishmaniaaethiopica and Leishmaniamexicana was investigated. A significant increase in phosphatidylserine (PS) exhibition, cytochrome C release, and active caspase-3 expression was detected (P<0.05) during L.aethiopica, but not L.mexicana spreading. A decrease (P<0.05) of protein kinase B (Akt) protein and BCL2-associated agonist of cell death (BAD) phosphorylation was also observed. The nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB) signaling pathway and pro-apoptotic protein protein kinase C delta (PKC-δ) were downregulated while inhibition of caspase-3 activation prevented L.aethiopica spreading. Overall suggesting that L.aethiopica induces host cell's apoptosis during spreading in a caspase-3-dependent manner. The trafficking of amastigotes within macrophages following cell-to-cell spreading differed from that of axenic parasites and involved co-localization with lysosomal-associated membrane protein 1 (LAMP-1) within 10min postinfection. Interestingly, following infection with axenic amastigotes and promastigotes, co-localization of parasites with LAMP-1-positive structures took place at 1 and 4h, respectively, suggesting that the membrane coat and LAMP-1 protein were derived from the donor cell. Collectively, these findings indicate that host cell apoptosis, demonstrated by PS exhibition, caspase-3 activation, cytochrome C release, downregulation of Akt, BAD phosphorylation, NF-kB activation, and independent of PKC-δ expression, is involved in L.aethiopica spreading. Moreover, L.aethiopica parasites associate with LAMP-rich structures when taken up by neighboring macrophages.

Expression of the BAD pathway is a marker of triple-negative status and poor outcome

Triple-negative breast cancer (TNBC) has few therapeutic targets, making nonspecific chemotherapy the main treatment. Therapies enhancing cancer cell sensitivity to cytotoxic agents could significantly improve patient outcomes. A BCL2-associated agonist of cell death (BAD) pathway gene expression signature (BPGES) was derived using principal component analysis (PCA) and evaluated for associations with the TNBC phenotype and clinical outcomes. Immunohistochemistry was used to determine the relative expression levels of phospho-BAD isoforms in tumour samples. Cell survival assays evaluated the effects of BAD pathway inhibition on chemo-sensitivity. BPGES score was associated with TNBC status and overall survival (OS) in breast cancer samples of the Moffitt Total Cancer Care dataset and The Cancer Genome Atlas (TCGA). TNBC tumours were enriched for the expression of phospho-BAD isoforms. Further, the BPGES was associated with TNBC status in breast cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE). Targeted inhibition of kinases known to phosphorylate BAD protein resulted in increased sensitivity to platinum agents in TNBC cell lines compared to non-TNBC cell lines. The BAD pathway is associated with triple-negative status and OS. TNBC tumours were enriched for the expression of phosphorylated BAD protein compared to non-TNBC tumours. These findings suggest that the BAD pathway it is an important determinant of TNBC clinical outcomes.

AXL Downstream Targeting Unravels Synergistic Drug Combinations in Ovarian Carcinoma Cells.

Platinum-based therapy represents the main pharmacological treatment for ovarian carcinoma. Since molecular targeting of receptor tyrosine kinases (RTK) affects factors that may modulate drug response, the aim of this study was to examine whether downstream targets of AXL RTK could be exploited to improve cell response to cisplatin. Inhibitors of p38 (SB203580) and of signal transducer and activator of transcription 3 (stattic) were employed in combination with cisplatin in ovarian carcinoma cell lines. Apoptosis assay and western blot analysis were performed to evaluate cell response after treatment. SB203580 produced a synergistic effect in combination with cisplatin in cisplatin-resistant IGROV-1/Pt1 cells. In addition, a favorable drug interaction was observed in A2780 cells when pre-incubated with cisplatin prior to stattic. The analysis of cell response after combined treatment showed down-regulation of the pro-apoptotic protein BCL2-associated agonist of cell death (BAD). Our results support the notion that downstream targets of AXL in ovarian carcinoma cells can be exploited to increase cisplatin activity in ovarian carcinoma models.