CASPASE-3 expression and activity in PBMCs associate with SARS-CoV-2 infection and clinical features

Cooperation of bisphenol A and leptin in inhibition of caspase-3 expression and activity in OVCAR-3 ovarian cancer cells

e12036 Background: Doxorubicin binds to DNA-associated enzymes, intercalates the base pairs of the DNA and induces apoptosis in cancerous and healthy tissues especially in cardiomyocytes. Caspase mediated apoptosis in cardiomyocytes remains largely unknown. We investigated the role of doxorubicin via caspase system on apoptosis of cardiomyocytes. Methods: H9C2ratcardiomyocytes were incubated with doxorubicin a concentration of 10-6 Mfor 4 or 24 hours to perform gene expression and activity of caspases, respectively. Total RNA isolated and expression analysis were performed by real time PCR assay. Caspase activity was determined by colorimetric caspase assay kit. At least two fold increases in gene expression were accepted statistically significant. Results: In comparison to controls, bothof caspase expression and activity increased in doxorubicin-treated samples. Increase in expression of Caspase 9 and 2 as initiator caspases and caspases 7 and 3 as effector caspases were detected statistically higher than control values. In contrast, expression of caspase 1, 4, 6, 8 and 12 were detected lower than controls. Activity of both caspase 3 and 9 increased significantly. Conclusions: Acute doxorubicin administration caused a significant caspase activation on apoptosis of cardiomyocytes.

Upregulation of Antiviral Factors That Inhibit HIV-1 Infection in Sickle Cell Disease

In vitro and In vivo research have shown that Atractylodes lancea (Thunb) DC. (AL) and its bioactive components -eudesmol and atractylodin could be used to treat cholangiocarcinoma. The study aimed to evaluate the immunomodulatory activity of AL in human subjects. The modulatory effects of AL and b-eudesmol and atractylodin on TNF\(\alpha\) and IL6 expression in PBMCs were measured using real-time PCR. Blood samples were taken from 48 healthy subjects after they were given a single or numerous doses of the standardised AL extract capsule formulation or a placebo. Serum cytokine profiles, lymphocyte subpopulations (B lymphocytes, CD8+ cytotoxic T lymphocytes, CD4+ T-helper lymphocytes, and NK cells), and cytotoxic activity of PBMCs against the cholangiocarcinoma cell line CL-6 were evaluated using cytometric bead array (CBA) with flow cytometry analysis. AL extract at almost all concentrations significantly inhibited both TNF\(\alpha\) and IL6 expression in Con A-mediated inflammation in PBMCs. Only IL6 expression was significantly reduced by \(\beta\)-Eudesmol at all doses. At the lowest dose, atractylodin considerably decreased the expression of both cytokines, whereas at the highest concentration, only IL6 expression was significantly inhibited. The administration of AL at a single oral dose of 1,000 mg appeared to decrease IFNg and IL10 and increase B cells, while significantly increasing NK and CD4+ and CD8+ cells. At 24 hours after dosage, there was a trend of increasing cytotoxic activity of PBMCs (relative to placebo). At 24 hours after dosage, AL at successive doses of 1,000 mg for 21 days tended to reduce the production of all cytokines while significantly suppressing IL17A production. By 24 hours, there was a trend of increased cytotoxic activity in PBMCs, but this trend stopped at 48 hours. The findings support AL's immunomodulatory properties in humans. This activity, together with AL's direct effect in triggering apoptosis in cholangiocarcinoma cells, implies that it could have a role in CCA regulation. Trial Registration: Retrospectively registered on 17 October 2020 [Thai Clinical Trials Registry (TCTR: www.clinical trials.in.th) Number TCTR20201020001]

To study the effect of deltamethrin on the apoptotic rate and the expression of caspase-3 in rat neural cells. Male Wistar rats were randomly divided into 5 groups: control, 5 h, 24 h, 48 h and 5 d exposed groups. Apoptotic rate and the expression of caspase-3 were measured by FACS420 Flow Cytometer; Ac-DEVD-pNa was used as a substrate to detect the activity of caspase-3. Apoptotic rates in 24 h, 48 h and 5 d exposed groups in hippocampus and cerebral cortex [hippocampus: (8.45 +/- 1.02)%, (9.44 +/- 1.14)%, (7.58 +/- 0.75)%; cerebral cortex: (7.90 +/- 0.49)%, (8.01 +/- 0.87)%, (7.97 +/- 0.41)% respectively] were higher than those in the control [hippocampus: (2.97 +/- 0.36)%; cerebral cortex: (3.50 +/- 0.48)%] (P < 0.01); the activity of caspase-3 in 5 h, 24 h and 48 h exposed groups (A(405) nm in hippocampus: 0.389 +/- 0.038, 0.472 +/- 0.041, 0.295 +/- 0.049; A(405) nm in cerebral cortex: 0.321 +/- 0.068, 0.429 +/- 0.077, 0.344 +/- 0.047) and 5 d group of hippocampus (0.246 +/- 0.065) were all higher than those of the control (hippocampus: 0.184 +/- 0.054; cerebral cortex: 0.198 +/- 0.049) (P < 0.05, P < 0.01); the expression of caspase-3 in 5 h, 24 h and 48 h exposed groups increased apparently while 5 d group did not. Exposure to high dose of deltamethrin would affect the apoptosis, the activity and expression of caspase-3 in rat neural cells. The increase in caspase-3 activity and expression occurred before the rising of neuronal apoptotic rate may be the upstream event of apoptosis.

The presenilin-dependent gamma-secretase activity, which is responsible for the generation of amyloid beta-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the gamma-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of beta-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the gamma-secretase complex but remains independent of the gamma-secretase catalytic activity.

Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.

This study investigates the simultaneous effect of acrylamide (ACR) and high-fat-intake on the apoptosis in testis cells, and also the expression and activity of caspase-3. Seventy-two male Kunming mice were divided into two blocks and fed with a high-fat diet (crude fat 21.5%) or basic diet (crude fat 4.4%), respectively; and animals in each diet block were exposed to ACR at the dose of 20 mg/kgbw•d or 40 mg/kgbw•d as ACR treated groups or the normal saline as control. Germ cells prepared from testis were stained with Hoechst dye 33258 and paraffin wax sections from testis were suffered to a TUNEL process. Expression of caspase-3 on protein level was investigated using an immunohistochemical analysis assay. The supernatant of unilateral testes were subjected to a Caspase-3 activity kit to determine the activity of Caspase-3 in testis. The concentration of ACR and glycidamide(GA), epoxide of ACR, in plasma and testis were detected by LC-ES/MS/MS analysis. Results based on the morphological changes, percentage of apoptotic cells, and integrated optical density (IOD) of positive amethyst staining which indicates the apoptotic DNA fragmentation, show that apoptosis was induced by acrylamide only; however, acrylamide-induced apoptosis was weakened by high-fat-intake. The protein expression and activity of Caspase-3 were not induced by ACR or high-fat-intake. Moreover, no significant differences of ACR and GA concentration were found between the high-fat and basic diet groups after exposure of ACR. Results indicate that high-fat-intake reverses the effects on apoptosis induced by ACR; and more possibly, apoptosis is induced by a caspase-3-independent mechanism.

A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1)

Breviscapine has been demonstrated to have beneficial effects in ameliorating acute lung injury (ALI), yet its potential therapeutic value and molecular mechanisms in sepsis-induced ALI remain unexplored. We utilized network pharmacology approach to identify the potential targets and mechanisms of breviscapine in treating sepsis-induced ALI. To construct a murine model of sepsis, we performed cecal ligation and puncture (CLP). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) were employed to respectively determine the pathologic changes and levels of inflammatory factors. Neutrophil count and total protein level in bronchoalveolar lavage fluid (BALF) were detected by corresponding kit. Additionally, we utilized flow cytometry, immunofluorescence, Western blotting, and real-time reverse transcription PCR (qRT-PCR) to detect cell apoptosis, protein expression, and gene expression. Finally, we used ELISA kits to detect the activity of myeloperoxidase (MPO) and caspase-8 (CASP8). Breviscapine was revealed to target 81 potential proteins in the treatment of sepsis-induced ALI, while CASP8 was the most important one as demonstrated by network analysis. In vivo experiments demonstrated that breviscapine effectively reduced the severity of sepsis-induced ALI and inflammation, and significantly suppressed neutrophil infiltration in the lung tissues of CLP mice and promoted neutrophil apoptosis in the peripheral blood. In vitro experiments revealed that lipopolysaccharide (LPS)-induced neutrophil apoptosis was inhibited, and the expression and activity of CASP8 were down-regulated. Breviscapine intervention markedly up-regulated the expression and activity of CASP8, consequently activating neutrophil apoptosis and inhibiting inflammatory response by activating the NF-κB signaling pathway. Breviscapine is remarkably effective in improving sepsis-induced ALI, and its mechanism of action may be to induce neutrophil apoptosis, inhibit inflammatory overreaction and reduce its infiltration in pulmonary tissues by up-regulating the expression and activity of CASP8.

Increased cAMP-PKA signaling pathway activation is involved in up-regulation of CTLA-4 expression in CD4+ T cells in acute SIVmac239-infected Chinese rhesus macaques

Objective To investigate the expression of apoptosis regulatory factor Survivin and caspase-3 in epithelial ovarian tumor and its clinical significance.Methods The expression of proteins Survivin and caspase-3 in 40 cases of epithelial ovarian tumor,10 cases of borderline epithelial ovarian tumora and 10 cases of benign ethelial tumor were detected by immunohistochemistry; then the clinical and pathological features were analyzed.Results There was a negative correlation of Survivin and caspase-3 expression in epithelial ovarian tumor; the expression rate of Survivin in epithelial ovarian cancer was 72.5％ (29/40),and it was higher than that in epithelial ovarian tumor and borderline epithelial ovarian tumor(P ＜ 0.05).The expression rate of caspase-3 in epithelial ovarian cancer was 0.5％(2/40),and it was more lower than that in epithelial ovarian tumor and borderline epithelial ovarian tumor(P ＜0.01).Conclusions The expression of caspase-3 is inhibited by Survivin,which results in the ovarian cancer cells multiplication,apoptosis obstruction and tumor generation; The expression of Survivin gene happens in the early stage of ovarian cancer malignant transformation; The expression of Survivin is closely correlated with tumor cell differentiation and proliferation,and its high expression can be the identification marker of unfavourable prognosis; The expression level of caspase-3 in ovarian cancer is quite low,which makes it a new index to identify the benign and malignant ovarian tumor; The qualitative and quantitative analysis of Survivin,caspase-3 and PCNA may become the index of curative effect of drugs. Key words: Survivin; Caspase-3; Ovarian cancer

Aquaporins (AQPs) have been found to be associated with a number of diseases. However, the role of AQP-1 in the pathogenesis of osteoarthritis remains unclear. We previously found that AQP-1 expression was upregulated in osteoarthritic cartilage and strongly correlated with caspase-3 expression and activity. The aim of this study was to further investigate the association of AQP-1 expression with chondrocyte apoptosis in a rat model of osteoarthritis, using RNA interference to knock down AQP-1. For this purspose, 72 male Sprague-Dawley rats were randomly assigned to 3 groups as follows: the control group not treated surgically (n=24), the sham-operated group (n=24), and the osteoarthritis group (n=24). Osteoarthritis was induced by amputating the anterior cruciate ligament and medial collateral ligament and partially excising the medial meniscus. Chondrocytes from the rats with osteoarthritis were isolated and cultured. shRNAs were used to knock down AQP-1 expression in the cultured chondrocytes. The expression of AQP-1 and caspase-3 was determined by reverse transcription-quantitative polymerase chain reaction. Caspase-3 activity was measured using a caspase-3 colorimetric assay. The rats in our model of osteoarthritis exhibited severe cartilage damage. The knockdown of AQP-1 decreased caspase-3 expression and activity in the cultured chondrocytes. In addition, the expression of AQP-1 positively correlated with caspase-3 expression and activity. Thus, the findings of our study, suggest that AQP-1 promotes caspase-3 activation and thereby contributes to chondrocyte apoptosis and to the development of osteoarthritis.

To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro. SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential (MMP) levels, and analyzing reactive oxygen species (ROS) concentrations were analyzed by flow cytometry. Cytochrome C (Cyt C), apoptosis-inducing factor (AIF), endonuclease G (Endo G), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point (Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits. Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/mL for 1, 2, 4, 6, or 8 h and showed a time- and concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/mL melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h (n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99% (n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42 (n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control (5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor (Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group (1067.0 ± 132.5 U/g vs 8695.7 ± 449.1 U/g). The expression of the Cyt C, Endo G, and AIF proteins in SGC-7901 cells was significantly higher than those in the control (P < 0.05), while the expression of the Smac/Diablo protein was significantly lower than the control group after melittin exposure (P < 0.01). Ac-DEVD-CHO did not, however, have any effect on the expression of caspase-8 and FAS in the SGC-7901 cells. Melittin can induce apoptosis of human gastric cancer (GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC.

The gene expression of mTOR, autophagy-related ULK1, caspase 3, CDK-inhibitor p21, and TNF α was measured in the peripheral blood of osteoarthritic (OA) patients at different stages of the disease aiming to establish a gene expression profile that might indicate the activity of the disease and joint destruction. Whole blood of 65 OA outpatients, 27 end-stage OA patients, 27 healthy volunteers, and knee articular cartilages of 28 end-stage OA patients and 26 healthy subjects were examined. OA outpatients were subjected to clinical testing, ultrasonography, and radiographic and WOMAC scoring. Protein levels of p70-S6K, p21, and caspase 3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. Upregulation of mTOR gene expression was observed in PBMCs of 42 OA outpatients (“High mTOR expression subset”) and in PBMCs and articular cartilages of all end-stage OA patients. A positive correlation between mTOR gene expression in PBMCs and cartilage was observed in the end-stage OA patients. 23 OA outpatients in the “Low mTOR expression subset” exhibited significantly lower mTOR gene expression in PBMCs compared to healthy controls. These “Low mTOR” subset subjects experienced significantly more pain upon walking, and standing and increased total joint stiffness versus “High mTOR” subset, while the latter more often exhibited synovitis. The protein concentrations of p70-S6K, p21, and caspase 3 in PBMCs were significantly lower in the “Low” subset versus “High” subset and end-stage subjects. Increases in the expression of mTOR in PBMCs of OA patients are related to disease activity, being associated with synovitis more than with pain.