Heterologous Expression and Auto-Activation of Human Pro-Inflammatory Caspase-1 in Saccharomyces cerevisiae and Comparison to Caspase-8.

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure-function relationships.

miRNA-149 targets PARP-2 in endometrial epithelial and stromal cells to regulate the trophoblast attachment process.

Embryo implantation is a highly complex process involving many regulatory factors, including several micro RNAs (miRNAs/miRs). One miRNA present in the stromal cells of normal endometrium is miR-149, which targets poly (ADP-ribose) polymerase 2 (PARP-2), a gene involved in endometrial receptivity for trophoblast implantation. However, the precise role of miR-149 in the endometrial receptivity during blastocyst implantation is still unknown. We studied miR-149-dependent PARP-2 regulation during trophoblast attachment to endometrial epithelial cells. Using FISH, we found that miR-149 is expressed in mouse endometrial epithelial and stromal cells at implantation and inter-implantation sites. Endometrial receptivity for embryo implantation and attachment is inhibited by the upregulation of miR-149 in the endometrium. Our RT-PCR analysis revealed downregulation of miR-149 in the implantation region of the uterus during the receptive stage (Day 5, 0500 h, p.c.) in the mouse. Under in-vitro conditions, miR-149 overexpression in human endometrial epithelial cells (hEECs) abrogated the human trophoblastic cells spheroid and mouse blastocyst attachment. Subsequently, miR-149 also regulates transformed human endometrial stromal cell (T-hESCs) decidualization by downregulating PARP-2 and upregulating caspase-8 proteins. Overexpression of miR-149 in hEECs and downregulated PARP-2 protein expression, reconfirming that PARP-2 is a downstream target of miR-149 in endometrial cells as well. miR-149 is also able to alter the expression of caspase-8, another PARP-2 regulator. In conclusion, our data indicate that miR-149 is one of the regulators of endometrial receptivity and decidualization for trophoblast implantation, and it exerts the effects by acting on the downstream targets PARP-2 and caspase-8.

Interleukin Enhancer Binding Factor 2 Regulates Cell Viability and Apoptosis of Human Brain Vascular Smooth Muscle Cells.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of intracranial aneurysm (IA) formation and rupture. Interleukin enhancer binding factor 2 (ILF2) is known as the nuclear factor of activated T cells and regulates cell growth. This study was aimed to explore the effects of ILF2 on IA progression. Human brain VSMCs (hBVSMCs) were transfected with pCDNA3.1(+), pCDNA3.1(+)-ILF2, siRNA-negative control, and siRNA-ILF2. The transfection efficiency was then evaluated by determining ILF2 expression. The cell viability and apoptosis were determined using Cell Counting Kit-8 and Annexin V-FITC cell apoptosis assay kit, respectively. Real-time quantification PCR (RT-qPCR) was applied to measure the expression levels of apoptosis-related and inflammation-related genes. Finally, western blot was used to detect the expression level of Fas cell surface death receptor 95 (CD95) and Caspase 8. Overexpression of ILF2 could significantly increase cell viability and decrease cell apoptosis (P < 0.05), while knock-down of ILF2 showed opposite trends for hBVSMCs on cell viability and apoptosis (P < 0.05). RT-qPCR results showed that ILF2 knock-down downregulated the expression levels of BCL2 apoptosis regulator (BCL2), transcriptional regulator Myc-like (c-Myc), and caspase 1 (ICE) whereas upregulated the expression levels of CD95, p21, p53, and interleukin-13 (IL-13). Additionally, the protein expression levels of CD95 and Caspase 8 were significantly decreased after ILF2 overexpression while were significantly increased after ILF2 knock-down (P < 0.05). ILF2 knock-down may inhibit cell viability and promote cell apoptosis of hBVSMCs by regulating the expression levels of apoptosis-related genes and suppressing inflammatory response.

Maternal sciatic nerve administered bupivacaine induces hippocampal cell apoptosis in offspring.

BACKGROUND: Bupivacaine, an amid-type local anesthetic, is widely used for clinical patients especially in pregnant women. In addition to neurotoxicity effect of bupivacaine, it can cross the placenta, accumulates in this tissue and retained in fetal tissues. Nevertheless, whether bupivacaine can cause neurotoxicity in fetus remains unclear. Hence, this study was design to investigate the effects of maternal bupivacaine use on fetus hippocampal cell apoptosis and the possible related mechanism. METHODS: On day 15 of pregnancy, sciatic nerve of pregnant wistar rat (180-200 g) were exposed by lateral incision of the right thigh and 0.2 ml of bupivacaine was injected. After their delivery, we randomly selected one male offspring of every mother. On day 30 after of their birth, the rat's hippocampi were isolated for molecular studies. Western blotting was used to examine the expression of cleaved caspase-3, caspase-8 and p-Akt in fetal hippocampus. RESULTS: Our results showed that maternal bupivacaine use caused a significant increment of cleaved caspase-3 and caspase-8 expression in fetal hippocampus compared with the sham group. In addition, maternally administered bupivacaine could significantly decrease hippocampal P.Akt/T.Akt ratio which was concurrent with an increment of cleaved caspase-3 and caspase-8 expression. CONCLUSION: Our data suggest that maternal bupivacaine use increases fetal hippocampal cell apoptosis markers such as caspase 8 and cleaved caspase 3, at least in part, via inhibiting the Akt activation.

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro.

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-ß-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm-oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.

Mutational landscape of penile squamous cell carcinoma in a Chinese population.

Penile squamous cell carcinoma (PSCC) is a malignancy that affects the skin and tissues of the penis, but the knowledge of pathogenesis and carcinogenesis is limited. Here, we characterize the PSCC genomic landscape using whole-exome sequencing. Of the 30 paired blood and tumor samples, we identified recurrent mutations in 11 genes; confirmed previous findings for FAT1 (4/30), HRAS (4/30), NOTCH1 (4/30), TP53 (3/30) and PIK3CA (3/30); and revealed novel candidate driver genes [CASP8 (4/30), SLITRK2 (3/30), FLG (3/30) and TRRAP (3/30)]. Our in vitro experiments suggested CASP8 was involved in mediating TRAIL-induced apoptosis of penile cancer cell lines. We also observed the frequently altered pathways for potential therapeutic implications: alterations in the Notch (30% of sample altered), RTK-RAS (26.7% altered) and Hippo (23.3% altered) pathways accounted for over 50% of tumors. The frequently altered genes (>10%) in these pathways were proved to be expressed in penile tumors by immunohistochemistry assay. These findings provide new insight into the mutational and pathway landscapes of PSCC and suggest potential novel therapeutic opportunities for this malignancy.

Lentivirus-Based Virus-Like Particles Mediate Delivery of Caspase 8 into Breast Cancer Cells and Inhibit Tumor Growth.

OBJECTIVE: Apoptosis plays an important role in both carcinogenesis and cancer treatment. Drugs or treatment strategies that can restore the apoptotic signaling pathways have the potential to eliminate cancer. Caspase 8 (CASP8) plays a vital role in the propagation of an enzymatic cascade that results in cell apoptosis. METHODS AND RESULTS: In this study, the authors investigated the inhibitory effects of a HIV Gag virus-like particles (VLPs) that are incorporated with an active CASP8 (Gag-CASP8-VLPs) on the growth of breast cancer. Their data have shown that Gag-CASP8-VLPs, pseudotyped by the stomatitis virus G protein (VSV-G), can efficiently enter and deliver active CASP8 into breast cancer cells, leading to massive cell apoptosis and death. Interestingly, an injection of Gag-CASP8-VLPs in the tumor tissues of a 4T1 mouse breast cancer model can effectively inhibit tumor growth, and the earlier the Gag-CASP8-VLPs is administered, the more profoundly the tumor growth is inhibited. CONCLUSIONS: Overall, Gag-CASP8-VLPs can deliver CASP8 into breast cancer cells, induce cell apoptosis, and inhibit tumor growth.

Analysis of In Vitro Cyto- and Genotoxicity of Barbatimão Extract on Human Keratinocytes and Fibroblasts.

Barbatimão (Stryphnodendron adstringens, Mart.) is a native Brazilian species used in traditional medicine and some commercial preparations owing to its strong wound-healing activity. However, controversy regarding its use due to safety concerns over the potential genotoxic effect of this plant remains. In order to clarify this issue, the effect of hydroalcoholic extract of barbatimão in vitro on cell viability, DNA damage, and induction of apoptosis in two commercial cell lines of keratinocytes (HaCaT) and fibroblasts (HFF-1) was evaluated. Barbatimão stem bark hydroalcoholic extract (70% ethanol) was obtained and lyophilized for subsequent use in all experiments. The main bioactive molecules quantified by HPLC were gallic acid, caffeic acid, quercetin, catechin, and epigallocatechin gallate (EGCG). Barbatimão (0.024 to 1.99 mg/mL) was found to decrease cellular mortality as compared to the control group. GEMO assay, a noncellular DNA protocol that uses H2O2-exposed calf thymus DNA, revealed not only a genotoxic effect of barbatimão, but also a potential genoprotective action against H2O2-triggered DNA fragmentation. These results indicated that barbatimão at concentrations of 0.49 and 0.99 mg/mL, which are near to the levels found in commercial preparations, exerted an in vitro genoprotective effect on cells by decreasing the levels of DNA oxidation quantified by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and reactive oxygen species (ROS) levels. Gene and protein apoptotic markers, quantified by qRT-PCR (BAX/Bcl-2 genes) and immunoassays (Caspases 3 and 8), respectively, also indicated a decrease in apoptotic events in comparison with control cells. Collectively, the results suggest that barbatimão could exert genoprotective and antiapoptotic effects on human keratinocytes and fibroblasts.

Nitidine chloride inhibits proliferation and induces apoptosis in ovarian cancer cells by activating the Fas signalling pathway.

OBJECTIVES: To explore the apoptotic effects and underlying mechanisms of nitidine chloride (NC) in epithelial ovarian cancer. METHODS: The MTT cell proliferation assay was used to detect the inhibitory effects of different concentrations of NC (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 µg/ml) in SKOV3 ovarian carcinoma cells. The number of apoptotic cells was observed by Hoechst staining and measured by flow cytometry. Quantitative PCR was used to measure the expression of Fas, Fas-associated death domain-containing protein (FADD), caspase-8 and caspase-3. RNA interference (RNAi) was used to determine whether caspase-8 played an important role in NC-induced apoptosis. KEY FINDINGS: Nitidine chloride inhibited the proliferation of SKOV3 cells (IC50 = 2.317 ± 0.155 µg/ml) after 24 h of treatment and induced apoptosis (15.9-64.3%). Compared with the control group, a significant increase in Fas, FADD, caspase-8 and caspase-3 gene expression was observed in the NC-treated groups (P < 0.05). After silencing caspase-8 by RNAi, the antiproliferative activity and pro-apoptotic activity of NC in SKOV3 cells decreased (P < 0.05). CONCLUSIONS: Our study showed that NC induced apoptosis in SKOV3 cells by activating the Fas signalling pathway, and caspase-8 played an important role in this process.

Magnetic nanoparticle-mediated gene therapy to induce Fas apoptosis pathway in breast cancer.

CD95 (Fas) is a complex integral protein that can be expressed in many cells. It induces apoptosis when interacted with its ligand CD95L (FasL). However, cancer cells are resistant to CD95-induced apoptosis because of the changes in death domain (DD) of CD95 (procaspase-8 and c-Flip). In this study, magnetic nanoparticles and lipid-based gene transfection methods were performed to provide active Fas expression in breast cancer cells. Plasmid DNA (pDNA), which can express both human Fas and GFP, was transfected to MCF-7 breast cancer cells. Expression of c-FLIP and caspase-8 and effect of monoclonal antibody FasL for apoptosis stimulation were investigated. Also transfection success of methods and effects on surface protein were compared. Western blot results indicated that MCF-7 cells do not express caspase-8 but express large amount of c-FLIPL. Both lipid-based and magnetic nanoparticle-mediated gene transfection methods successfully applied. Caspase-8 apoptosis pathway was activated on transfected cells. Magnetic nanoparticle-mediated gene transfer is a successful non-viral method for transfection, and it does not affect the expression of other cell proteins, such as beta actin and lamin-B1. The raised c-FLIPL concentration in cytosol inhibits apoptosis. However, transfection of CD95-GFP-tagged pDNA significantly increases apoptosis by activating caspase-8 pathway. FasL interaction indicated a slight increase of apoptosis in the transfected cells. The method and pDNA applied in this study have potentials to be used in gene therapy for breast cancer.

Synergistic role of Caspase-8 and Caspase-3 expressions: Prognostic and predictive biomarkers in colorectal cancer.

BACKGROUND: Expressions of Caspase-8 and Caspase-3 have been identified as important markers in many malignant tumors, but their roles in colorectal cancer (CRC) have not been confirmed. The purpose of this study was to investigate the role of Caspase-8 and Caspase-3 in CRC. METHODS: We enrolled 470 CRC patients in this study. Archival paraffin-embedded CRC tissue samples were used to construct tissue microarray (TMA), expressions of Caspase-8 and Caspase-3 that were stained by immunohistochemistry. Prognostic and predictive role of Caspase-8 and Caspase-3 expressions, alone or united, were evaluated by univariate and multivariate analysis respectively. RESULTS: In comparison with adjacent normal tissues, Caspase-8 and Caspase-3 protein levels were upregulated in CRC tissues significantly, furthermore, high expressions of Caspase-8 and Caspase-3 were correlated with decreased overall survival (OS) (p< 0.05), and also with unfavorable clinicopathologic characteristics. Cox regression analysis showed that high Caspase-8 and Caspase-3 expressions were independent negative markers of OS. CONCLUSION: Caspase-8 and Caspase-3 expressions in tumor tissues are novel candidate prognostic markers for CRC patients. It was the first time to be identified that Caspase-8 and Caspase-3 expressions had synergistic role as efficient prognostic indicators for CRC patients.

The 3-methyl-4-nitrophenol (PNMC) compromises airway epithelial barrier function.

BACKGROUND AND AIMS: It is recognized that the air pollution is associated with the pathogenesis of airway diseases. This study aims to elucidate the role of the 3-methyl-4-nitrophenol (PNMC), one of the components of diesel-exhaust particles, in compromising the airway epithelial barrier integrity. METHODS: A549 cells, an airway epithelial cell line, were cultured to monolayers to be used as an in vitro epithelial barrier model. BALB/c mice were treated with nasal drops containing PNMC to test the effects of PNMC on alternating the airway epithelial barrier functions. RESULTS: Exposure of mice to PNMC induced nasal epithelial cell apoptosis and increased the permeability of the nasal epithelial barrier. PNMC increased casp8 and casp3 activities in nasal epithelial cells. Exposure to PNMC up regulated Fas and FasL expression in airway epithelial cells. Inhibition of caspase abolished the PNMC-induced airway epithelial barrier dysfunction. CONCLUSION: Exposure of airway mucosa to PNMC induces epithelial cell apoptosis and compromises the epithelial barrier function, which can be prevented by the inhibition of caspases.

Induction of Apoptosis in HepaRG Cell Line by Aloe-Emodin through Generation of Reactive Oxygen Species and the Mitochondrial Pathway.

BACKGROUND/AIMS: Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone), an anthraquinone active compounds, is isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. The aim of this study was to determine potential cytotoxic effects of aloe-emodin on HepaRG cells and to define the underlying mechanism. METHODS: MTT was used to evaluate cell viability. Apoptotic cell death was analyzed via Annexin V-FITC/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by flow cytometry, while the expression of apoptosis-related proteins was determined by Western blot analysis. RESULTS: Treatment with aloe-emodin significantly reduced cell viability and induced apoptosis in HepaRG cells in a dose- and time-dependent manner. It provoked ROS generation and depolarization of MMP in HepaRG cells when compared with controls. Aloe-emodin dose-dependently increased release of mitochondrial cytochrome c, and levels of Fas, p53, p21, Bax/Bcl-2 ratio, as well as activation of caspase-3, caspase-8, caspase-9, and subsequent cleavage of poly(ADP-ribose)polymerase (PARP). It also induced S-phase cell cycle arrest by increasing the expression of p21 and cyclin E proteins while significantly decreasing the expression of cyclin A and CDK2. CONCLUSION: These results suggest that aloe-emodin inhibits cell proliferation and induces apoptosis in HepaRG cells, most probably through a mechanism involving both Fas death pathway and the mitochondrial pathway by generation of ROS. These findings underscore the need for risk assessment of human exposure to aloe-emodin.

SLPI promotes the gastric cancer growth and metastasis by regulating the expression of P53, Bcl-2 and Caspase-8.

OBJECTIVE: The incidence of gastric cancer is very high all over the world, but the mechanism of the occurrence and development of gastric cancer is unclear. Secretory leukocyte protease inhibitor (SLPI) is overexpressed in gastric, lung and ovarian cancers, which accelerates the metastasis of cancer cells. In this research, we mainly explored the expression level and possible mechanism of SLPI in gastric cancer. PATIENTS AND METHODS: The expression and clinical significance of SLPI in 68 cases of gastric cancer tissues and adjacent tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) assay was used to detect the proliferation ability of gastric cancer cell lines. In addition, we used Western blot to clarify the relationship between SLPI and metastasis. RESULTS: Compared with the adjacent tissues, we found that SLPI was highly expressed in gastric cancer tissues. We also found that the expression of SLPI was in significant correlation with the survival time, clinical classification and size of the tumor. What's more, SLPI could promote the proliferation and metastasis of gastric cancer by regulating P53, Bcl-2 and Caspase-8 expression through apoptosis signaling pathway. CONCLUSIONS: We concluded that SLPI was closely related with to invasion and metastasis of gastric cancer. Perhaps we can hopefully find new targets for the treatment of gastric cancer.

Differences in the mechanisms of action of BDE-47 and its metabolites on OVCAR-3 and MCF-7 cell apoptosis.

Data concerning possible carcinogenic action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent tissues are limited. Our earlier studies showed that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) stimulated OVCAR-3 and MCF-7 cell proliferation, while its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) increased estrogen receptors protein expression and extracellular signal-regulated kinase 1/2 and protein kinase Cα phosphorylation in these cell lines. In addition to cell proliferative disorder, a failure in the regulation of apoptosis can also lead to the formation and development of tumors. Therefore, in the present study, we investigated the effect of BDE-47 and its metabolites (2.5-50 ng ml-1 ) on the expression of apoptosis regulatory genes and proteins, caspase-8 and -9 activity and DNA fragmentation induced by extracellular signal-regulated kinase inhibitor (PD098059) and protein kinase Cα inhibitor (GÓ§ 6976) in ovarian (OVCAR-3) and breast (MCF-7) cancer cells. In OVCAR-3 cells, BDE-47 upregulated expression of most of the investigated genes and increased protein expression of tumor necrosis factor (TNF)-α, TNF receptor 1, caspase-6, Bcl-xl and caspase-8 activity. Whereas in MCF-7 cells, BDE-47 resulted in the downregulation of most of the investigated genes, and decreased caspase-8 and -9 activity. In both OVCAR-3 and MCF-7 cells, the expression of most of the investigated genes were downregulated by metabolites. Exposure of OVCAR-3 cells to 5-OH-BDE-47 corresponded with a decrease in the protein expression of caspase-6, caspase-9 and Bcl-xl and treatment with 6-OH-BDE-47 decreased Bcl-xl and TNF receptor 1 expression in OVCAR-3 cells and caspase-9 expression in MCF-7 cells. Hydroxylated metabolites of BDE-47 have strong inhibitory effects on apoptosis in ovarian and breast tumor cells and thus should be considered potential carcinogens in hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Raloxifene on the Proliferation and Apoptosis of Human Aortic Valve Interstitial Cells.

We aimed to explore the effects of raloxifene (RAL) on the proliferation and apoptosis of human aortic valve interstitial cells (AVICs). Different concentrations of RAL were used to act on AVICs. MTS kit is used to test the effects of different concentrations of RAL on the proliferation of AVICs. Cell cycle and apoptosis test used flow cytometry after seven-day treatment. The relative expression levels of caspase-3 and caspase-8 are tested with RT-qPCR and Western blot. The results of MTS testing revealed that the absorbance value (OD value) of the cells in the concentration groups of 10 and 100 nmol/L RAL at a wavelength of 490 nm at five, seven, and nine days significantly decreased compared with that in the control group. Meanwhile, the results of flow cytometry of the cells collected after seven days showed that the ratio of the S stage and the cell apoptosis rate of AVICs can be significantly reduced by RAL in the concentration groups of 10 and 100 nmol/L. The mRNA and protein expressions of caspase-3 and caspase-8 were significantly decreased compared with those in the control group. This study laid the foundation for further treatment of aortic valve disease by using RAL.

Myenteric plexuses atrophy in the vicinity of colorectal cancer tissue is not caused by apoptosis or necrosis.

INTRODUCTION: The previously performed studies showed that the presence of colorectal cancer (CRC) tumor is associated with the atrophy of myenteric plexuses in the vicinity of cancer invasion; however, the possible mechanisms of this phenomenon are not known. The aim of the present study was to determine whether the atrophic changes of the enteric nervous system (ENS) within an intestine wall of the CRC patients were caused by apoptosis or necrosis and whether they were associated with changes in the number of galanin-immunore-active (GAL-Ir) neurons. MATERIAL AND METHODS: Samples of the large intestine wall located close to the CRC invasion and control, distally-located part of the colon, were collected from 9 CRC patients. The size of ENS plexuses and the number of neurons were compared. Triple immunofluorescent staining was used to visualize the co-expression of caspase 3 (CASP3) or caspase 8 (CASP8) with GAL and protein gene-product 9.5 (PGP 9.5, panneuronal marker) in the submucosal and myenteric ENS plexuses. The cells expressing myeloperoxidase (MPO, marker of neutrophils) and CD68 (marker of macrophages) were detected by immunohistochemistry around/in myenteric plexuses (MPs) and in the muscularis externa of the colon wall in the vicinity of tumor invasion. RESULTS: Myenteric plexuses in the vicinity of the CRC tissue were significantly smaller and had lower number of neurons per plexus than distantly located plexuses. The number of CASP8- and CASP3-Ir neurons in the ENS plexuses was similar in the colon wall both close to and distally from tumor invasion. The number of CASP8-Ir neurons within MPs located close to CRC invasion was higher than of CASP3-Ir neurons. The percentage of neurons co-expressing CASP8 and GAL in myenteric plexuses close and distantly from tumor was three-fold lower than of those co-expressing CASP3 and GAL. The mean number of neutrophils and macrophages inside and around myenteric plexuses located close to tumor invasion was higher or similar, respectively, as compared with adjacent muscularis externa. CONCLUSIONS: The atrophy of myenteric plexuses in the vicinity of CRC invasion is not caused by apoptosis or necrosis. The differences in the proportions of neurons expressing galanin and the studied caspases suggest as yet unknown role of this neuropeptide in the mechanisms of neuron's atrophy in MPs located close to CRC tumor.

Oxidative stress and cell death in the cerebral cortex as a long-term consequence of neonatal hypoglycemia.

Neonatal hypoglycemia limits glucose supply to cells leading to long-term consequences in brain function. The present study evaluated antioxidant and cell death factors' alterations in cerebral cortex of 1-month-old rats exposed to neonatal hypoglycemia. Gene expression studies by real-time PCR were carried out using gene-specific TaqMan probes. Fluorescent dyes were used for immunohistochemistry and nuclear staining and imaged by confocal microscope. Total antioxidant level and expression of antioxidant enzymes - superoxide dismutase (SOD) and gluthathione peroxide (GPx) - mRNA was significantly reduced along with high peroxide level in the cerebral cortex of 1-month-old rats exposed to neonatal hypoglycemia. Real-time PCR analysis showed an upregulation of Bax, caspase 3, and caspase 8 gene expression. Confocal imaging with TOPRO-3 staining and immunohistochemistry with caspase 3 antibody indicated cell death activation. The reduced free radical scavenging capability coupled with the expression of key factors involved in cell death pathway points to the possibility of oxidative stress in the cortex of 1-month-old rats exposed to neonatal hypoglycemia. The observed results indicate the effects of neonatal hypoglycemia in determining the antioxidant capability of cerebral cortex in a later stage of life.

Alterations in blood pressure, antioxidant status and caspase 8 expression in cobalt chloride-induced cardio-renal dysfunction are reversed by Ocimum gratissimum and gallic acid in Wistar rats.

The protective abilities of the chloroform extract of Ocimum gratissimum (COG) and gallic acid against cobalt chloride (CoCl2) - induced cardiac and renal toxicity were evaluated. Rats were exposed to CoCl2 (350ppm) for 7 days, either alone, or in combination with COG (100 and 200mg/kg) or gallic acid (120mg/kg). CoCl2 given alone, caused significant increases (p<0.05) in oxidative stress parameters (hydrogen peroxide, H2O2 and malondialdehyde, MDA) and increased expression of the apoptotic initiator caspase 8 in the heart and kidneys. There was significant reduction (p<0.05) in reduced glutathione (GSH) in cardiac and renal tissues; reduction in superoxide dismutase (SOD) activity in the kidneys and adaptive increases in Glutathione S-transferase (GST) and catalase (CAT). CoCl2 also produced significant reduction (p<0.05) in systolic (SBP), diastolic (DBP) and mean arterial (MAP) blood pressures. Oral COG and gallic acid treatment significantly reduced (p<0.05) the levels of H2O2 and MDA; with reduced expression of caspase 8 and restoration of GSH levels, GPx, SOD and CAT activities, howbeit, to varying degrees in the heart and kidneys. COG (200mg/kg) was most effective in restoring the blood pressures in the rats to near control levels. CoCl2-induced histopathological lesions including myocardial infarction and inflammation and renal tubular necrosis and inflammation were effectively ameliorated by the treatments administered. This study provides evidence for the protective roles of O. gratissimum and gallic acid by modulation of CoCl2-induced alterations in blood pressure, antioxidant status and pro-apoptotic caspase 8 in Wistar rats.

The pyrrolo-1,5-benzoxazepine, PBOX-15, enhances TRAIL­induced apoptosis by upregulation of DR5 and downregulation of core cell survival proteins in acute lymphoblastic leukaemia cells.

Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.