CASP3 Gene Expression Research Articles

An Organic Fraction of Oenothera rosea L'Her Ex. Aiton Prevents Neuroinflammation in a Rat Ischemic Model.

Oenothera rosea L'Her Ex. Aiton, presenting antioxidant and anti-inflammatory activities, is traditionally used to treat bruises and headaches and as a healing agent. This study aimed to investigate whether its organic fraction (EAOr) has neuroprotective properties against neuroinflammation in the context of ischemia/reperfusion. The chemical composition of EAOr was determined using HPLC techniques, and its neuroprotective activities were evaluated in a common carotid-artery ligation model for the induction of ischemia/reperfusion (I/R). The animals were supplemented with EAOR for 15 days. On the last day, the animals were rested for one hour, following which the common carotid-artery ligation procedure was performed to induce I/R. The neurological deficit was evaluated at 24 h after I/R using Bederson's scale, and the relative expression of inflammatory genes and structure of hippocampal neurons were analyzed at 48 h. The chemical analysis revealed five major compounds in EAOr: gallic acid, rutin, ellagic acid, and glucoside and rhamnoside quercetin. EAOr prevented neurological deficit 24 h after I/R; led to the early activation of the AIF and GFAP genes; reduced Nfkb1, IL-1beta, Il-6 and Casp3 gene expression; and protected hippocampal neurons. Our findings demonstrate that EAOr contains polyphenol-type compounds, which could exert a therapeutic effect through the inhibition of neuroinflammation and neuronal death genes, thus maintaining hippocampal neurons.

Apoptosis, Autophagy, and Mitophagy Genes in the CA3 Area in an Ischemic Model of Alzheimer's Disease with 2-Year Survival.

Currently, no evidence exists on the expression of apoptosis (CASP3), autophagy (BECN1), and mitophagy (BNIP3) genes in the CA3 area after ischemia with long-term survival. The goal of the paper was to study changes in above genes expression in CA3 area after ischemia in the period of 6-24 months. In this study, using quantitative RT-PCR, we present the expression of genes associated with neuronal death in a rat ischemic model of Alzheimer's disease. First time, we demonstrated overexpression of the CASP3 gene in CA3 area after ischemia with survival ranging from 0.5 to 2 years. Overexpression of the CASP3 gene was accompanied by a decrease in the activity level of the BECN1 and BNIP3 genes over a period of 0.5 year. Then, during 1-2 years, BNIP3 gene expression increased significantly and coincided with an increase in CASP3 gene expression. However, BECN1 gene expression was variable, increased significantly at 1 and 2 years and was below control values 1.5 years post-ischemia. Our observations suggest that ischemia with long-term survival induces neuronal death in CA3 through activation of caspase 3 in cooperation with the pro-apoptotic gene BNIP3. This study also suggests that the BNIP3 gene regulates caspase-independent pyramidal neuronal death post-ischemia. Thus, caspase-dependent and -independent death of neuronal cells occur post-ischemia in the CA3 area. Our data suggest new role of the BNIP3 gene in the regulation of post-ischemic neuronal death in CA3. This suggests the involvement of the BNIP3 together with the CASP3 in the CA3 in neuronal death post-ischemia.

Chemopreventive Effect of an In Vitro Digested and Fermented Plant Sterol-Enriched Wholemeal Rye Bread in Colon Cancer Cells.

Diet is crucial for the prevention of colorectal cancer. Whole grains are the source of beneficial compounds for this, such as fiber. The enrichment of wholemeal rye bread with plant sterols (PSs) could increase its beneficial effects. This study aimed to assess the potential antiproliferative effect of this enriched food on colon adenocarcinoma cells (Caco-2) compared with a non-enriched one. After a human oral chewing, simulated semi-dynamic gastrointestinal digestion and colonic fermentation in a simgi® system, fermentation liquids (FLs) obtained were used as treatment for cells. Cytotoxicity assay showed that samples diluted 1/5 (v/v) with DMEM are not toxic for non-tumoral cells, whereas they damage tumoral cells. Samples with PS (FLPS) produced a higher chemopreventive effect (vs. blank) in MTT and apoptosis assays, as well as higher gene expression of TP53 and Casp8. Nevertheless, FL0 (without PS) produced a higher chemopreventive effect in a cell cycle and reduced glutathione and calcium assays, besides producing higher gene expression of Casp3 and lower CCND1. The distinct antiproliferative effect of both FLs is attributed to differences in PSs, short chain fatty acids (lower concentration in FLPS vs. FL0) and antioxidant compounds. These results may support wholemeal rye bread consumption as a way of reducing the risk of colorectal cancer development, although further research would be needed.

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

CASP3 gene expression and the role of caspase 3 in the pathogenesis of depressive disorders

BackgroundThe aim of our study was to evaluate the expression of the CASP3 gene at both mRNA and protein levels in patients with depressive disorders and to determine the impact of caspase 3 in the pathogenesis of depression;MethodsA total of 290 subjects, including 190 depressed patients and 100 healthy controls, participated in the study. Socio-demographic and clinical data were collected, and the severity of depressive symptoms was assessed using the Hamilton Depression Rating Scale. Venous blood was collected and gene expression was evaluated using RT-PCR and ELISA at the mRNA and protein levels, respectively;ResultsThe expression of the CASP3 gene was significantly lower in depressed patients compared to healthy controls at both the mRNA and protein levels. Additionally, a positive correlation was observed between CASP3 gene expression and disease duration as well as the number of depressive episodes;ConclusionsFurther studies are needed to investigate the role of caspase 3 in depressive disorders.

Effects of antipsychotics, haloperidol and olanzapine, on the expression of apoptosis-related genes in mouse mHippoE-2 cells and rat hippocampus.

Objective. Modified levels of pro- (caspase3, Bax) and anti-apoptotic (Bcl-2) regulatory proteins have been detected in certain brain areas of schizophrenic patients indicating a possible dysregulation of apoptosis. In the present study, effects of antipsychotics, haloperidol (HAL) and olanzapine (OLA), on the gene expression of caspase3 (casp3), Bax and Bcl-2 were studied in vitro in mouse hippocampal mHippoE-2 cell line and in vivo in the hippocampus of MK-801 animal schizophrenia model with the aim to provide evidence that antipsychotics may affect the activity of apoptosis-related markers. Methods. mHippoE-2 cells were incubated with MK-801 (20 µM), HAL (10 µM), and OLA (10 µM) alone or combined, MK-801+HAL/OLA, for 24, 48, and 72 h. Male Sprague Dawley rats were injected with saline or MK-801 (0.5 mg/kg) for 6 days and since the 7th day, they were treated with vehicle (VEH), HAL (1 mg/kg) or OLA (2 mg/kg) for the next 7 days. The casp3, Bax and Bcl-2 gene expression in mHippoE-2 cells and rat hippocampus was measured by RT-PCR. Results. In mHippoE-2 cells, casp3 gene expression was increased by MK-801 and OLA treatments alone for 48 h, HAL treatment alone for 24 and 72 h, and co-treatment with MK-801+OLA for 24 and 72 h compared to controls. HAL and OLA suppressed the stimulatory effect of MK-801 on casp3 mRNA levels in cells after 48 h of incubation. Bax mRNA levels in mHippoE-2 cells were decreased after HAL treatment for 24 and 48 h, and also after co-treatment with MK-801+HAL for 72 h. In vivo, MK-801 decreased mRNA levels of both pro-apoptotic markers, casp3 and Bax, in hippocampus of VEH-treated rats and Bax mRNA levels in hippocampus of HAL-treated animals. OLA reversed the inhibitory effect of MK-801 on casp3 expression in the VEH-treated animals. Neither MK-801 nor antipsychotics induced changes in the gene expression of anti-apoptotic marker Bcl-2 in mHippoE-2 cells as well as hippocampus of rats. Conclusions. The results of the present study demonstrate that antipsychotics, HAL and OLA, may affect mRNA levels of pro-apoptotic markers in hippocampal cells in vitro, but not in vivo. The obtained data do not clearly support the assumed potentiating role of MK-801 in inducing apoptosis in specific brain areas and a possible protective role of antipsychotics against induction of apoptosis. The obtained data may contribute to a deeper insight into the neurodevelopmental changes connected with schizophrenia.

Network Pharmacology-Based Strategy to Reveal the Mechanism of Cassiae Semen against Cataracts.

Cassiae semen (CS) is one of the most well-known herbs used in the treatment of cataracts in China. However, the potential mechanisms of its anticataract effects have not been fully explored. In this study, network pharmacology was used to investigate the potential mechanism underlying the actions of CS against cataracts, and molecular docking was performed to analyze the binding activity of proteins and compounds. qPCR was performed to detect the mRNA level of genes, and the cell apoptotic rate was measured using flow cytometry. We identified 13 active compounds from CS and 105 targets, as well as 238 cataract-related targets. PPI networks were constructed, and fifty key targets were obtained. These key targets were enriched in the regulation of transcription, apoptotic process, and signal transduction pathways. Molecular docking demonstrated that the compounds of CS exhibited good affinity to some critical targets. Furthermore, CS prevented the apoptosis of human lens epithelial cells induced by UVB lights by decreasing the gene expression of CASP3, ESR1, and TP53 and increasing the CRYAB gene expression. The present study attempted to explain the mechanisms for the effects of CS in the prevention and treatment of cataracts and provided an effective strategy to investigate active ingredients from natural medicines. Further studies are required to verify these findings via in vivo and in vitro experiments.

Investigation of the Effect of Lactobacillus Brevis Bacteria on the Expression of Rel A, IKB, and Casp3 Genes in HT29 Colon Cancer Cells

Aims Studies have shown that probiotic bacteria inhibit the onset and progression of carcinogenesis through different pathways. Our objective in this study was to determine the effect of probiotic bacteria on the expression of growth-related genes Rel A, IKB, and Casp3 in HT29 colon cancer cells Methods & Materials In this study, the Lactobacillus brevis probiotic bacteria were first cultured, and after the supply of media conditioning, they were treated on HT29 cancer cells. The bacterium’s cytotoxic effect (bacterial T cells) was investigated using a microculture tetrazolium test (MTT). DNA was extracted from the treated cells, and a DNA Ladder assay was performed. Also, the 4′,6-diamidino-2-phenylindole (DAPI) test was performed to show cell apoptosis. After ribonucleic acid (RNA) extraction and complementary DNA (cDNA) preparation to determine the mechanism of the effect of this bacterium on cellular signaling, the expression of growth-related genes Rel A, IKB, and Cas3 was measured using a real-time polymerase chain reaction (PCR) method. Findings The microculture tetrazolium (MTT) test showed that L. b bacteria inhibit HT29 cells’ proliferation, induce apoptosis in these cells, and inhibit Rel A gene proliferation by increasing IKB gene expression. Also, 4′,6-diamidino-2-phenylindole (DAPI), and DNA ladder assay following the treatment of HT29 cells regarding the mentioned bacteria showed qualitative changes in cell apoptosis. In addition, real-time polymerase chain reaction (PCR) results showed that L. b increased Casp3 gene expression in HT29 colon cancer cells (P=0.038). Conclusion Our findings indicate that L. b stimulates the apoptotic cell signaling pathway in HT29 colon cancer cells. It can be used as a new treatment strategy or therapy for colon cancer treatment.

Involvement of Caspase-3 Pathway in Anti-cancer Properties of Genistein in AGS Gastric Cancer Cell Line is Still Enigmatic

Background: Genistein is an isoflavone that has been reported to have various anti-cancer properties. Objectives: This study aimed to reveal whether or not the anti-cancer properties of genistein in AGS gastric cancer cell line were mediated through caspase-3 enzyme. Methods: AGS gastric cancer cells were treated with different concentrations of genistein for 12, 24, and 48 hours and, then, the viability of the cells and IC50 were determined. To determine the effect of genistein on AGS cell migration potency, the wound healing assay was performed. The genistein-induced apoptosis in AGS gastric cells was evaluated by flow cytometry. Caspase-3 gene (CASP3) expression level and its enzyme activity level were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and colorimetric techniques, respectively. Results: The IC50 value was calculated as 70 µM concentration for 24 hours of incubation with genistein. Genistein significantly reduced AGS cell migration compared to the untreated control cells (P<0.001). Genistein increased the early and late apoptosis of the cells (P<0.001) and upregulated the caspase-3 gene expression (P<0.001), but did not significantly enhance the caspase-3 enzyme activity in treated cells. Conclusion: Genistein exhibited anti-cancer effects on AGS cells to some extent by reducing cellular migration, increasing apoptosis, and upregulating CASP3 gene expression; however, it did not alter the caspse-3 activity. Therefore, it was recommended that more studies should be carried out to delineate the role of caspase-3 in health benefits attributed to the genistein.

Hyperglycaemia-associated Caspase-3 predicts diabetes and coronary artery disease events.

BackgroundApoptosis is central in both diabetes and atherosclerosis, linked to pancreatic beta cell death and plaque progression. Circulating Caspase-3 has also been associated with diabetes and coronary calcium score. Here, we explored if soluble Caspase-3 (sCaspase-3) is associated with cardio-metabolic risk factors and predicts incidence of diabetes and coronary artery disease (CAD).MethodsClinical data and plasma from 4637 individuals from the Malmö Diet and Cancer cohort were studied. Plasma sCaspase-3 was measured by a Proximity Extension Assay. National registers were used to identify diabetes and CAD events during follow-up. Type 2 diabetes risk variants and expression quantitative trait loci (eQTL) for sCaspase-3 were retrieved from the DIAGRAM consortium and the Genotype-Tissue Expression project.ResultsHbA1c was the factor with the strongest association with sCaspase-3 (r = 0.18, P = 1.3x10−36). During follow-up 666 individuals developed diabetes and 648 individuals suffered from CAD. Increasing sCaspase-3 was associated with a higher risk of developing diabetes (hazard ratio (HR) 1.18 per 1unit; P = 7 × 10−5) and CAD (HR 1.2 per 1 unit, P = 1 × 10−4) during follow-up. A genetic variant rs60780116, located upstream of CASP3, showed strong association with type 2 diabetes (OR 1.06, 95%CI 1.04–1.07, P = 8.4 × 10−11). An eQTL was identified between this variant and gene expression of CASP3, where the allele positively correlated with type 2 diabetes was associated with increased CASP3 expression in blood.ConclusionsThe present study provides evidence for plasma sCaspase-3 as a marker of cardio-metabolic risk factors and as a predictor of future diabetes and CAD in a cohort without cardiovascular disease or diabetes at baseline.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via β-adrenergic signaling. However, the potential of β-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the β-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

Abstract 2156: Genomics and molecular analysis of Aurora kinase B (AURKB) in response to antiretroviral (ARV) treatment in lung cancer

Abstract Aurora kinase B (AURKB) is essential for normal physiological processes. However, aberrant AURKB expression has been reported in various cancers, including lung cancer. Lung cancer is a leading non-AIDS defining cancer (NADCs) and the relationship between the use of antiretrovirals (ARVs) and lung cancer is poorly understood. The anti-proliferative effects of Efavirenz (EFV) and Lopinavir/ritonavir (LPV/r) have also been documented. This study aimed at determining the genomic and molecular roles of AURKB in lung cancer in response to ARV treatment. For this purpose, cellular (xCELLigence RTCA), molecular (RT-qPCR) and bioinformatics tools were used in normal fibroblasts (MRC-5) and adenocarcinoma (A549) lung cells. Real-Time Cell Analysis (RTCA) data confirmed the anti-proliferative effects of ARVs in a time-dependent manner. AURKB gene expression was elevated in A549 compared to MRC-5 in non-ARV treated. Interestingly, p53 and CASP3 gene expression was downregulated. Contrarily, ARV treatment resulted in the formation of a negative signaling loop due to AURKB being downregulated, while CASP3 and p53 were upregulated. KEGG analysis demonstrated AURKB, CASP3 and p53 as cell cycle related genes. STRING database searches showed a strong protein-protein interaction (PPI) between AURKB and p53, and CAP3 and p53, but a weak PPI between AURKB and CASP3. Gene Ontology (GO) analysis revealed a decline in serine/threonine kinase (AURKB) activity in cancer cells in response to ARV treatment. Although not yet confirmed, AURKB displays oncogene features, while its overexpression has been shown to deregulate p53. The association between the use of ARVs and lung carcinogenesis remains to be fully comprehended and targeting AURKB signaling may help shed light on this relationship. Citation Format: Rahaba Makgotso Marima, Rodney Hull, Zodwa Dlamini, Clement Penny. Genomics and molecular analysis of Aurora kinase B (AURKB) in response to antiretroviral (ARV) treatment in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2156.

Secoisolariciresinol diglucoside induces caspase-3-mediated apoptosis in monolayer and spheroid cultures of human colon carcinoma cells.

Apoptotic effects of secoisolariciresinol diglucoside (SDG) in 2D and 3D cultures of SW480 cells were investigated. 40-200μM SDG was used and IC50 values were determined for three different time intervals as 24, 48, or 72hr for further experiments. BrdU, TUNEL, AIF, and caspase-3 stainings were used. SDG inhibited cell proliferation almost half and half for all time intervals in 2D and 3D cultures and also, induced apoptosis. Apoptotic cell percentages in the control group for 24, 48, and 72hr were 27.00%, 29.00%, and 28.00%, respectively, while in the SDG treatment group were 59.00%, 61.00%, and 62.00%, respectively. In the spheroid cell culture, apoptotic cell percentages in the control group for 24, 48, and 72hr were 6.90%, 7.20%, and 7.10%, respectively, while in the SDG treatment group were 19.50%, 19.50%, and 20.70%, respectively. Caspase-3 and AIF antibodies were used to indicate caspase-dependent and -independent apoptotic pathways. Significant increases were seen in both AIF and caspase-3 stainings when compared to the control group but caspase-3 staining results were significantly greater when compared to the AIF staining at all time intervals (p<.05). To prove this, CASP3 gene expression was evaluated by RT-qPCR. Unlike staining results, there was no statistically significant change at 24hr in 2D and 3D cultures. But, significant upregulation at 48 (2.32-fold in 2D and 2.46-fold in 3D) and 72hr (5.04-fold in 2D and 6.45-fold in 3D) were seen. PRACTICAL APPLICATIONS: Colon cancer is one of the most prevalent cancer in the developed countries and its etiology is complex. Although the underlying mechanisms are mostly unknown, the link between diet and colon cancer is known and dietary habits can promote cancer or protect against it. In recent years, flaxseed is accepted as a significant functional food ingredient and feeding with it could help in to prevent cancer. Secoisolariciresinol diglucoside is a flaxseed lignan and is metabolized to mammalian lignans by the gut. In the present study, SDG was evaluated for its apoptotic effects in colon carcinoma cell line via monolayer and spheroid cultures using immunohistochemical and gene expression techniques. Findings of this study suggest that SDG may protect against cancers and in particularly against colon cancer and further investigations has to be carried out for detailed underlying mechanisms.

Adenosine A1 Receptor Antagonist Up-regulates Casp3 and Stimulates Apoptosis Rate in Breast Cancer Cell Line T47D

Background: Adenosine receptor family, especially A1 type is-overexpressed in breast-derived tumor cells and the P53 gene is mutant in some of these cells while the casps gene is of wild type as well. The aim of this study was to evaluate the effect of the A1 receptor function on cell programmed death or proliferation, as well as the relationship between this receptor stimulation/inhibition and caspase 3 (casp3) expression in T47D cell line that has a mutant and non-functional P53 gene. Materials and Methods: The expression of casps3 was measured by real-time polymerase chain reaction and then flow cytometery and MTT assay were used to assess the apoptotic and proliferation cell rate after the treatment of T47D cells with specific agonist N6-cyclopentyladenosine (CPA) and antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) of this receptor 24, 48, and 72 hours after treatment. Result: Our results indicated that DPCPX significantly induces apoptosis in T47D cells and the rate of survival cell after the reduction of this treatment, especially 72 hours after treatment. Finally, the expression of casp3 was up-regulated by DPCPX treatment, especially in 72 hours while CPA treatment had opposite results (P&gt;0.05). Conclusion: In general, DPCPX could up-regulate casp3 gene expression and subsequently increase the apoptosis rate in T47D cells with casp3 expression without the P53 gene interference. Therefore, adenosine A1 receptor antagonists may be introduced as anti-cancer agents.

Prebiotics and phytogenics functional additives in low fish meal and fish oil based diets for European sea bass (Dicentrarchus labrax): Effects on stress and immune responses

The use of terrestrial raw materials to replace fish meal (FM) and fish oil (FO) in marine fish diets may affect fish growth performance and health. In the last years functional additives have been profiled as good candidates to reduce the effects on health and disease resistance derived from this replacement, via reinforcement of the fish immune system. In the present study, three isoenergetic and isonitrogenous diets with low FM and FO (10% and 6% respectively) were tested based on supplementation either with 0.5% galactomannanoligosaccharides (GMOS diet) or 0.02% of a mixture of essential oils (PHYTO diet), a non-supplemented diet was defined as a control diet. Fish were fed the experimental diets in triplicate for 9 weeks and then they were subjected to a stress by confinement as a single challenge (C treatment) or combined with an experimental intestinal infection with Vibrio anguillarum (CI treatment). Along the challenge test, selected stress and immunological parameters were evaluated at 2, 24 and 168h after C or CI challenges. As stress indicators, circulating plasma cortisol and glucose concentrations were analyzed as well as the relative gene expression of cyp11b hydroxylase, hypoxia inducible factor, steroidogenic acute regulatory protein, heat shock protein 70 and heat shock protein 90 (cyp11b, hif-1α, StAR, hsp70 and hsp90). As immune markers, serum and skin mucus lysozyme, bactericidal and peroxidase activities were measured, as well as gene expression of Caspase-3 (casp-3) and interleukin 1β (il-1ß).The use of functional additives induced a significant (p < 0.05) reduction of circulating plasma cortisol concentration when confinement was the unique challenge test applied. Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1β gene expression 2h after stress test, whereas StAR expression was significantly (p < 0.05) up-regulated. However, when combination of confinement stress and infection was applied (CI treatment), the use of PHYTO significantly (p < 0.05) down-regulated StAR and casp-3 gene expression 2h after challenge test, denoting that PHYTO diet reinforced fish capacity of stress response via protection of head kidney leucocytes from stress-related apoptotic processes, with lower caspase-3 gene expression and a higher il-1β gene expression when an infection occurs. Additionally, dietary supplementation with GMOS and PHYTO compounds increased fish serum lysozyme after infection. Both functional additives entailed a better capability of the animals to cope with infection in European sea bass when fed low FM and FO diets.

Analysis of the expression of BAX, BCL2, BIRC6, CASP3, CASP9 apoptosis genes during the first episode of schizophrenia.

The aim of the study was the analysis of (1) the level of BAX,BCL2,BIRC6,CASP3, CASP9 apoptosis genes expression in schizophrenic patients and in the control group, and (2) the relationships between the genes expression level and the morphological and biochemical parameters, as well as the severity of psychopathological symptoms. The study included 21 patients diagnosed with schizophrenia according to ICD-10 and 26 healthy subjects. The following parameters were assessed: genes expression in peripheral blood lymphocytes, laboratory parameters and severity of psychopathological symptoms (using the PANSS). The expression of the BCL2 gene and the CASP3 gene was significantly higher in schizophrenic patients compared to the controls. A significant positive correlation was found between the BAX gene expression level and neutrophil, lymphocyte and monocyte counts as well as the severity of negative symptoms (PANSS-N), between BCL2 and CASP9 genes expression and the monocyte count, and between the CASP3 gene expression and the lymphocyte count. (1) Significantly higher expression of BCL2 and CASP3 genes in schizophrenic patients compared to the controls proves the intensification of the apoptosis process, fitting in the theory of increased apoptosis in the pathophysiology of schizophrenia. (2) Significant correlations between the BAX gene expression and differential blood count parameters (leucocyte, neutrophil, lymphocyte, monocyte count) in the group of schizophrenic patients suggest a relationship with immune dysregulation and confirm the presence of apoptosis in peripheral blood lymphocytes. (3) The BAX gene expression may be indicative of the severity of negative symptoms in schizophrenia. (4) The analysis of the intercorrelation of studied genes expression indicates that BAX and CASP3 genes were the most active in the apoptosis process in the study group.

Pyroptotic and apoptotic cell death in iNOS and nNOS overexpressing K562 cells: A mechanistic insight

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1β release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.

Imperatorin improves in vitro porcine embryo development by reducing oxidative stress and autophagy

Imperatorin (IMP), a furanocoumarin derivative with many biological properties and pharmacological activities, is widely used as an antibacterial, anti-inflammatory, antiviral, anticancer, cardiovascular and neuroprotective agent. The purpose of this study was to explore the effects of IMP on early embryo development in pigs as well as the potential mechanisms. Our results showed that IMP can enhance the developmental competence of porcine early embryos. Supplementation of in vitro culture medium with 40 μM IMP significantly increased the blastocyst rate and total cell number. At the same time, apoptosis of blastocysts was also significantly decreased in the supplemented group compared with the control group, in accordance with the subsequent results of FAS and CASP3 gene expression analysis. Furthermore, IMP attenuated intracellular reactive oxygen species (ROS) generation, increased fluorescein diacetate (FDA) and glutathione (GSH) levels. Importantly, IMP not only improved the activity of mitochondria but also inhibited the occurrence of autophagy. In addition, pluripotency-related genes (OCT4, NANOG, and SOX2) and a growth and metabolism regulatory gene (mTOR) were upregulated after IMP supplementation on Day 7. These results demonstrate that IMP exerts a beneficial effect on preimplantation embryo development by reducing oxidative stress and autophagy.

Doublecotin-Like Kinase 1 Increases Chemoresistance of Colorectal Cancer Cells through the Anti-Apoptosis Pathway.

Colorectal Cancer (CRC) is the third most common cancer diagnosed and the second leading cause of cancer-related deaths in the United States. Cancer Stem Cells (CSCs) are believed to be the primary reason for the recurrence of CRC. Specific stem cell marker, doublecortin-like kinase 1 (DCLK1) plays critical roles in the tumorigenesis and progression of CRC. Up-regulation of DCLK1 is correlated with poor prognosis. Whether DCLK1 is correlated with enhanced chemoresistance of CRC cells is unclear. We aim to reveal the association of DCLK1 with chemoresistance of CRC cells and the underlying molecular mechanisms. Stable DCLK1 over-expression cells (DCLK1+) were established using the HCT116 cells (WT). DCLK1+ and WT cells were treated with 5-Fluorouracil (5-Fu) at different doses for 24 or 48 hours. MTT assay was used to evaluate cell viability and IC50 of 5-Fu was determined. Quantitative real-time PCR was applied to determine the gene expression of caspase-3 (casp-3), casp-4, and casp-10. Cleaved casp-3 expression was investigated using Western blot and immunofluorescence. Our results demonstrated that IC50 of 5-Fu for the DCLK1+ cells was significantly higher than that of the WT cells for both 24 and 48-hour treatment (p=0.002 and 0.048 respectively), indicating increased chemoresistance of the DCLK1+ cells. Gene expression of casp-3, casp-4, and casp-10 were significantly inhibited in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (p=7.616e-08, 1.575e-05 and 5.307e-08, respectively). Cleaved casp-3 amount and casp-3 positive cells were significantly decreased in the DCLK1+ cells after 5-Fu treatment compared to the WT cells (p=0.015). In conclusion, our results demonstrated that DCLK1 overexpression enhanced the chemoresistance of CRC cells to 5-Fu treatment by suppressing gene expression of key caspases in the apoptosis pathway and activation of the apoptosis pathway. DCLK1 can be an intriguing therapeutic target for the effective treatment of CRC patients.

SkQ1 Controls CASP3 Gene Expression and Caspase-3-Like Activity in the Brain of Rats under Oxidative Stress.

Here, we studied the effect of the mitochondria-targeted antioxidant SkQ1 (plastoquinone cationic derivative) on the CASP3 gene expression and caspase-3 activity in rat cerebral cortex and brain mitochondria under normal conditions and in oxidative stress induced by hyperbaric oxygenation (HBO). Under physiological conditions, SkQ1 administration (50 nmol/kg, 5 days) did not affect the CASP3 gene expression and caspase-3-like activity in the cortical cells, as well as caspase-3-like activity in brain mitochondria, but caused a moderate decrease in the content of primary products of lipid peroxidation (LPO) and an increase in the reduced glutathione (GSH) level. HBO-induced oxidative stress (0.5 MPa, 90 min) was accompanied by significant upregulation of CASP3 mRNA and caspase-3-like activity in the cerebral cortex, activation of the mitochondrial enzyme with simultaneous decrease in the GSH content, increase in the glutathione reductase activity, and stimulation of LPO. Administration of SkQ1 before the HBO session maintained the basal levels of the CASP3 gene expression and enzyme activity in the cerebral cortex cells and led to the normalization of caspase-3-like activity and redox parameters in brain mitochondria. We hypothesize that SkQ1 protects brain cells from the HBO-induced oxidative stress due to its antioxidant activity and stimulation of antiapoptotic mechanisms.