Fas Ligand Expression Research Articles

Potential cardioprotective effects of Amentoflavone in doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is an available chemotherapeutic drug for treating various tumors. However, its effectiveness is limited by cardiotoxicity. Amentoflavone (AMF), a natural biflavonoid separated from Cycas thouarsii ethyl acetate fraction, displays promising anticancer, anti-inflammatory, and antioxidant effects. Thus, our research aims to explore whether AMF could boost cardioprotective effects against DOX cardiotoxicity and reveal the potential underlying mechanisms of cardioprotection. Mice were classified into four groups; Normal control, Untreated DOX group, and DOX groups treated with AMF (40 and 80 mg/kg, respectively) intraperitoneal injection daily for four days before doxorubicin administration and for additional three days following DOX administration to assess cardiotoxicity. Echocardiography showed that AMF 80 treated group was protected from DOX cardiotoxicity. Additionally, it alleviated histopathological structural alterations and effectively restored heart weight and body weight ratio. These effects were confirmed biochemically by a substantially reduced serum creatine kinase-MB (CK-MB) and aspartate aminotransferase (AST) levels. AMF effectively restored nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM), and normalized heat shock protein − 27(HSP-27) expression levels compared to the DOX group. Moreover, AMF mitigated oxidative stress conditions and significantly suppressed NADPH oxidase (NOX) expression levels. It also showed significant anti-inflammatory effects via suppressing interleukin-6 (IL-6) expression and decreasing nuclear factor Kabba B (NF-κb) immune-staining. In addition, AMF markedly reduced FAS ligand (FASL) expression and p53 immune staining in cardiac tissue. This study is the first for the in vivo potential beneficial effects of AMF against acute DOX cardiotoxicity, possibly via exerting antioxidant, anti-inflammatory, and anti-apoptotic effects and restoring mitochondrial function.

Fentanyl alleviates intestinal mucosal barrier damage in rats with severe acute pancreatitis by inhibiting the MMP-9/FasL/Fas pathway

Background Fentanyl is an analgesic used against pancreatitis-related pain, while whether it ameliorates severe acute pancreatitis (SAP) has yet to be checked. This study aims to determine fentanyl-delivered effect on SAP and the mechanism underlying this effect. Methods Rat SAP models were established, following fentanyl treatment. The serum activity of amylase (AMY), lipase (LIP), and diamine oxidase (DAO) was detected by enzyme-linked immunosorbent assay (ELISA). Histological examination was performed in the pancreatic and intestinal tissues with hematoxylin-eosin staining. After transfection with matrix metalloproteinase (MMP) 9 overexpression plasmids, Caco-2 monolayers were treated with fentanyl and subsequently exposed to lipopolysaccharide (LPS). The transepithelial electrical resistance (TEER) value was determined in rat intestinal mucosa through an Ussing chamber assisted by Analyze & Acquire, and in Caco-2 cell monolayers through a voltohmmeter. Intestinal mucosa and paracellular permeabilities were determined by fluorescein isothiocyanate (FITC)-labeled dextran assay. The expressions of ZO-1, Occludin, MMP9, Fas and Fas ligand (FasL) in rat intestinal mucosa and/or Caco-2 monolayers were analyzed by qRT-PCR or/and western blot. Results Fentanyl alleviated SAP-related histological alterations in the pancreas and intestines, reduced the elevated levels of SAP-related AMY, LIP, and DAO, but promoted the levels of ZO-1 and Occludin. In SAP rats and Caco-2 monolayers, SAP-related or LPS-induced TEER value decreases, permeability increases, and increases in the expressions of MMP9, Fas, and FasL were reversed partly by fentanyl. Notably, MMP9 overexpression could reverse the above fentanyl-delivered in vitro effects. Conclusions Fentanyl alleviates intestinal mucosal barrier damage in rats with SAP by inhibiting the MMP9/FasL/Fas pathway.

A Combination of Cytokine-Induced Killer Cells With PD-1 Blockade and ALK Inhibitor Showed Substantial Intrinsic Variability Across Non-Small Cell Lung Cancer Cell Lines.

BackgroundCancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve multiple targeted inhibitors. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect of cytokine-induced killer (CIK) cells in combination with ALK and PD-1 inhibitors in vitro on genetically variable NSCLC cell lines.MethodsWe co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) with and without nivolumab (PD-1 inhibitor) and crizotinib (ALK inhibitor). Additionally, we profiled the variability of surface expression multiple immune checkpoints, the concentration of absolute dead cells, intracellular granzyme B on CIK cells using flow cytometry as well as RT-qPCR. ELISA and Western blot were performed to verify the activation of CIK cells.ResultsOur analysis showed that (a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression, (b) this combination strategy showed an effective response on cell viability, IFN-γ production, and intracellular release of granzyme B in CD3+ CD56+ CIK cells, but solely in NCI-H2228, (c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect, and (d) nivolumab induced Foxp3 expression in CD4+CD25+ subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-γ and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis.

Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand

In cell culture environment, some cells adhere firmly to the culture plates and may be vulnerable to cell detachment during passage. Therefore, it is important to harvest cells with a proper detaching method to maintain the viability of cells after detachment. Trypsinization is frequently used for cellular dissociation and detachment. However, most surface proteins and the extracellular matrix are degraded by enzymatic digestion. A mild cell detachment buffer, accutase, is recommended for the replacement of trypsin to dissociate adherent cells and thereby avoid cellular damage. In this study, we demonstrated that use of accutase for cellular detachment may compromise some surface proteins. Compared with ethylenediaminetetraacetic acid (EDTA)-based nonenzymatic cell dissociation buffers, accutase was associated with significant decreases in the surface Fas ligands and Fas receptors. Moreover, we found that accutase may be able to cleave surface Fas ligands into pieces. Our results also illustrated that surface proteins required 20 h to recover after accutase treatment. We demonstrated that using accutase to dissociate adherent cells compromised the expression of Fas ligands and Fas receptors on the cell surface. These findings indicate that it is important to choose suitable cell detachment buffers and allow cells to recover after detachment before experiments.

Induction of ligustrazine apoptosis of A549 cells through activation of death receptor pathway.

BackgroundThe aim of this study was to evaluate the effect of ligustrazine on the apoptosis of A549 cells and clarify the mechanism of ligustrazine-induced apoptosis.MethodsLigustrazine was prepared with medium according to the gradient concentration. Based on a cytotoxicity test, 3 different concentrations of ligustrazine were selected to form low, medium, and high groups, with a 0 mg/mL dose used as the control. The apoptosis degree and Fas (Fas cell surface death receptor) and Fas-L (Fas Ligand) expression were detected by flow cytometry and quantitative polymerase chain reaction (qPCR), respectively; meanwhile, the activity of caspase 8 and caspase 3 was analyzed by enzyme-linked immunosorbent assay (ELISA) and qPCR, respectively.ResultsAfter 24 hours of ligustrazine administration, the survival rate of A549 cells decreased with the increase of drug concentration, while the rate of apoptosis increased with the increase of drug concentration. Meanwhile, Fas and Fas-L expression was found to be significantly increased at both the gene and protein level, which was positively correlated with drug concentration. Furthermore, the expression of caspase 8 and caspase 3 was positively correlated with the concentration of ligustrazine, and there was significant difference compared with the control group.ConclusionsLigustrazine can induce the apoptosis of A549 cells via the upregulation of Fas- and caspase-activating death receptor pathway expression.

Immunoregulatory effects of dental mesenchymal stem cells on T and B lymphocyte responses in primary Sjögren's syndrome.

Background: In this article, theauthorsinvestigatethe modulatory effects of dental mesenchymal stem cells (MSCs) on lymphocyte responses in primary Sjögren's syndrome (pSS), which is an autoimmune disease resultingfromkeratoconjunctivitis sicca and xerostomia. Methods: Mononuclear cells isolated from pSS patients cultured with or without dentalMSCs and analyzed for lymphocyte responses via flow cytometry. Results: Dental-follicle (DF)- and dental-pulp (DP)-MSCs downregulated CD4+ T lymphocyte proliferation by increasing Fas-ligand expression on T lymphocytes and FoxP3 expressing Tregs, and decreasing intracellular IFN-γ and IL-17 secretion in pSS patients. DF-MSCsdecreased the plasma B cell ratio in the favor of naive B cell population in pSS patients' mononuclear cells. Conclusion: DF- and DP-MSCs can be thenew cellular therapeutic candidates for the regulation of immune responses in pSS.

Differential apoptotic gene expression in the male partners of infertile couples with normal and abnormal sperm parameters.

The purpose of this study was to investigate the cellular and molecular levels of apoptosis induction in three groups of male partners of infertile couples, one featuring subjects with normal sperm parameters and unexplained male infertility (UMI), one including men with abnormal sperm parameters, and one with fertile men as controls. Twenty-five infertile men with abnormal sperm parameters and 25 men with UMI and normal sperm parameters were recruited as experimental group I and experimental group II; 25 fertile men were included as controls. The mRNA levels of Fas, Fas ligand, Caspase 8, Bax, and Bcl2 were measured in the three groups. The cellular rates of early and late apoptosis were assessed using annexin V and propidium iodide staining. The expression of Bax, Bcl2, and the Bax/Bcl2 ratio in experimental group I was significantly higher than that in experimental group II and controls. However, the Bax/Bcl2 ratio was less than 1 among all groups. No significant difference was found among study groups regarding the gene expression of Fas, Fas ligand, and Caspase 8. No significant difference was seen in early apoptotic rates of sperm among study groups. The highest number of necrotic sperm cells was detected in experimental group I. The findings showed that the external pathways of apoptosis were not activated in the absence of external stimuli of sperm apoptosis in ejaculated sperm. Regardless of fertility status, apoptosis gene induction in the internal pathway was associated with abnormalities in sperm motility and/or morphology in men with abnormal parameters.

Effects of 7,12-Dimethylbenz(a)anthracene on Apoptosis of Breast Cancer Cells through Regulating Expressions of Fas Ligand and B-Cell Lymphoma 2

To provide reference basis for the apoptosis of breast cancer cells and the carcinogenesis of breast cancer, the effects of 7,12-dimethylbenz(a)anthracene on apoptosis regulators Fas ligand and B-cell lymphoma-2 were investigated. In this study, 62 female C57BL/6 mice aged from 4 w to 6 w were randomly divided into control group and test group, with 31 mice in each group. The test group was given 7,12-dimethylbenz(a) anthracene solution by gavage at a dose of 50 mg/kg and the control group was given normal saline of equal volume. On the 2nd d after the experiment, all the mice were killed by cervical dislocation. The results showed that after hematoxylin-eosin staining, the tumor cells in the test group were stacked up to form a substantial structure. The expression level of Fas ligand protein in the control group was greatly lower than that in the test group and the positive rate was 20.25 %, which was greatly lower than that of 89.65 % in the test group (p<0.01). The expression level of B-cell lymphoma-2 protein in the mammary gland tissues of mice in the test group was greatly higher than that of the control group and its positive rate was 87.96 %, which was greatly higher than that of 31.48 % in the control group (p<0.01). The expression levels of Fas ligand messenger ribonucleic acid in the mammary gland tissues of mice in the test group and control group were 5.82±4.37 and 1.27±0.12, respectively and there was a statistically obvious difference (p<0.05). The messenger ribonucleic acid expression levels of B-cell lymphoma-2 in the test group and the control group were 18.97±2.65 and 2.02±0.54, respectively, and there was an extremely obvious difference (p<0.01). The apoptosis rate of mammary gland cells in the test group was (19.79±3.53) % and that in the control group was (2.93±0.28) % and there was an extremely obvious difference (p<0.01). It indicated that 7,12-dimethylbenz(a)anthracene inhibited the apoptosis of breast cancer cells by regulating the upregulation of Fas ligand and B-cell lymphoma-2 expression.

Induction of apoptotic cell death in human bladder cancer cells by ethanol extract ofZanthoxylum schinifoliumleaf, through ROS-dependent inactivation of the PI3K/Akt signaling pathway

BACKGROUND/OBJECTIVESZanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells.MATERIALS/METHODSSubsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis.RESULTSEEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADP-ribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability.CONCLUSIONSTaken together, our results indicate that exposure to EEZS exhibits anti-cancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.

Early Response of CD8+ T Cells in COVID-19 Patients.

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

Pivotal Role of Signal-Transducing Adaptor Protein-2 in Pathogenesis of Autoimmune Hepatitis.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein involved in inflammatory and immune responses, such as inflammatory bowel disease and allergic responses. In this study, we investigated the role of STAP-2 in the pathogenesis of autoimmune hepatitis. After intravenous injection of concanavalin A (ConA), STAP-2 knock out (KO) mice showed more severe liver necrosis along with substantial lymphocyte infiltration compared to wild type (WT) mice. Serum alanine aminotransferase levels were significantly higher in ConA-injected STAP-2 KO mice than in WT mice. Levels of interferon-γ (IFN-γ), an important factor for liver necrosis, were also significantly increased in sera of STAP-2 KO mice compared to WT mice after ConA injection. Statistically significant upregulation of Fas ligand (FasL) expression was observed in the livers of ConA-injected STAP-2 KO mice compared to WT mice. In accordance with these results, apoptotic signals were facilitated in STAP-2 KO mice compared to WT mice after ConA injection. Correctively, these results suggest that STAP-2 is involved in the pathogenesis of autoimmune hepatitis by regulating the expression of FasL and the production of IFN-γ.

Expression of proapoptotic receptors Fas/Fas-L on sperm cells and levels of their soluble forms in seminal fluid from patients with varicocele

Varicocele is a condition in which the pampiniform venous plexus in the scrotum dilates abnormally during puberty, affecting testicular growth and semen parameters, and is thought to be a major cause of male infertility. The findings of researches on the presence of the Fas system in sperm are controversial. As the main triggers of apoptosis in the semen of patients with varicocele, in this study, we examined the expression of Fas/Fas-L on sperm cells and also the levels of their soluble forms in seminal plasma.Semen samples were taken from 45 patients with varicocele (study group) and 45 healthy subjects without varicocele (control group) after 3–5 days of ejaculatory abstinence. Flow cytometry was used to examine the expression of Fas and Fas ligand (Fas-L) on Sperm cells. Furthermore, soluble Fas (sFas) and soluble Fas-Ligand (sFas-L) levels in the seminal fluid were determined using an ELISA (enzyme-linked immunosorbent assay).The presence of Fas and Fas-L proteins on the sperm ejaculation surface was not found in the patients with varicocele or the control groups. However, in the case group (3.32 ± 0.31 ng/mL), sFas seminal concentrations were slightly lower than in the control group (5.50 ± 0.36 ng/mL) (p < 0.001). In varicocele patients, there was a strong negative association between seminal sFas and sperm motility.Apoptosis effects through this system on key sperm parameters were not found based on our findings. In varicocele, a reduction in sperm count and motility does not seem to be due to Fas-related mechanisms, but rather to other mechanisms.

Down-regulation of PR/SET domain 10 underlies natural killer cell dysfunction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the world's leading cause of tumor-related mortalities. Natural killer (NK) cells play a critical role at the first immunological defense line against HCC initiation and progression. NK cell dysfunction is therefore an important mechanism for immune evasion of HCC cells. In the present study using a murine HCC model, we revealed the down-regulation of PR/SET Domain 10 (PRDM10) in hepatic NK cells that were phenotypically and functionally exhausted. PRDM10 silencing diminished the expression of natural killer group 2 member D (NKG2D) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), augmented Tcellimmunoglobulin and ITIM domain (TIGIT) expression, and decreased the expression of interferon (IFN)-γ, perforin and granzyme B in normal hepatic NK cells in vitro. Consistently, PRDM10-deficient NK cells exhibited impaired cytotoxicity on target cells. In contrast, PRDM10 over-expression promoted NKG2D and Fas ligand(FasL) expression, reduced CD96 expression and enhanced transcripts of IFN-γ, perforin and granzyme B in NK cells in vivo. Moreover, PRDM10 silencing and PRDM10 over-expression down-regulated and up-regulated Eomesodermin (Eomes) expression, respectively. In summary, this study reveals PRDM10 down-regulation as a novel mechanism underlying NK cell dysfunction and identifies PRDM10 as a supporting factor of NK cell function.

Personalized cancer vaccine strategy elicits polyfunctional T cells and demonstrates clinical benefits in ovarian cancer

T cells are important for controlling ovarian cancer (OC). We previously demonstrated that combinatorial use of a personalized whole-tumor lysate-pulsed dendritic cell vaccine (OCDC), bevacizumab (Bev), and cyclophosphamide (Cy) elicited neoantigen-specific T cells and prolonged OC survival. Here, we hypothesize that adding acetylsalicylic acid (ASA) and low-dose interleukin (IL)-2 would increase the vaccine efficacy in a recurrent advanced OC phase I trial (NCT01132014). By adding ASA and low-dose IL-2 to the OCDC-Bev-Cy combinatorial regimen, we elicited vaccine-specific T-cell responses that positively correlated with patients’ prolonged time-to-progression and overall survival. In the ID8 ovarian model, animals receiving the same regimen showed prolonged survival, increased tumor-infiltrating perforin-producing T cells, increased neoantigen-specific CD8+ T cells, and reduced endothelial Fas ligand expression and tumor-infiltrating T-regulatory cells. This combinatorial strategy was efficacious and also highlighted the predictive value of the ID8 model for future ovarian trial development.

Interleukin-18 in Inflammatory Kidney Disease.

Interleukin (IL)-18, a member of the IL-1 superfamily, is a pro-inflammatory cytokine that is structurally similar to IL-1β. IL-18 promotes the production of interferon gamma (IFN-γ) and strongly induces a Th1 response. IL-18 drives the same myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway as IL-1β. In physiological conditions, IL-18 is regulated by the endogenous inhibitor IL-18 binding protein (IL-18BP), and the activity of IL-18 is balanced. It is reported that in several inflammatory diseases, the IL-18 activity is unbalanced, and IL-18 neutralization by IL-18BP is insufficient. IL-18 acts synergistically with IL-12 to induce the production of IFN-γ as a Th1 cytokine, and IL-18 acts alone to induce the production of Th2 cytokines such as IL-4 and IL-13. In addition, IL-18 alone enhances natural killer (NK) cell activity and FAS ligand expression. The biological and pathological roles of IL-18 have been studied in many diseases. Here we review the knowledge regarding IL-18 signaling and the role of IL-18 in inflammatory kidney diseases. Findings on renal injury in coronavirus disease 2019 (COVID-19) and its association with IL-18 will also be presented.

Histological changes in placental rat apoptosis via FasL and cytochrome c by the nano-herbal Zanthoxylum acanthopodium.

Zanthoxylum acanthopodium has several biological activities, such as antimicrobial, anti-inflammatory, and antioxidant properties. This strong antioxidant herb can be used as a drug for hypertension. FasL and cytochrome c expression play roles in the apoptotic pathway in the placenta. This study focused on the histological change in apoptosis via cytochrome c and Fas ligand expression by investigating whether Zanthoxylum acanthopodium (ZA) fruits affect apoptosis. The present study consisted of five treatments: Normal pregnant rats (C), Hypertension rats (C+), hypertension rats+extra virgin olive oil (EVOO) (T1), Hypertension rats+ZA (T2), and hypertension rats+EVOO+ZA (T3). Hypertension was induced in rats by injecting 3 mlml of 6% NaCl. Nanoherbal of ZA (100mg/kg BW) and EVOO (1ml) were given on the 13th-19th days of pregnancy. Pregnant rats were dissected on the 20th day of pregnancy by cervical dislocation. ELISA assays were performed for the analysis of HSP-70 expression. Immunohistochemistry and TUNEL assays were used to analyse the histological changes in placental tissue. The results showed that cytochrome c and FasL protein exposure levels in the labyrinth, basal, and yolk sac zones were increased during hypertensive pregnancy (P<0.0001) in rats. The administration of nanoherbal of ZA decreased the expression of cytochrome c and FasL. A significant difference was found in the combination of nanoherbal of ZA and EVOO.

Protective immune response against P32 oncogenic peptide-pulsed PBMCs in mouse models of breast cancer

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

Epigenetic Mechanisms Underlying HIV-Infection Induced Susceptibility of CD4+ T Cells to Enhanced Activation-Induced FasL Expression and Cell Death.

Chronic immune activation and CD4 T cell depletion are significant pathogenic features of HIV infection. Expression of Fas ligand (FasL), a key mediator of activation-induced cell death in T cells, is elevated in people living with HIV-1 infection (PLWH). However, the epigenetic mechanisms underlying the enhanced induction of FasL expression in CD4 T lymphocytes in PLWH are not completely elucidated. Hence, the current work examined the effect of HIV infection on FasL promoter-associated histone modifications and transcriptional regulation in CD4 T lymphocytes in PLWH. Flow cytometric analysis was performed to examine the Fas-FasL expression on total CD4 T cells and naïve/memory CD4 T cell subsets. Epigenetic FasL promoter histone modifications were investigated by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis using freshly isolated total CD4 T lymphocytes from HIV-1 infected and noninfected individuals. All naïve/memory CD4 T cell subsets from PLWH showed markedly greater frequency of FasL expression. Notably, examination of functional outcome of FasL/Fas co-expression demonstrated the preferential susceptibility of Tcm and Tem subsets to activation-induced apoptosis. Importantly, these CD4 T cells collectively demonstrated a distinct FasL promoter histone profile involving a coordinated cross-talk between histone H3 modifications leading to enhanced FasL gene expression. Specifically, levels of transcriptionally permissive histone H3K4-trimethylation (H3K4Me3) and histone H3K9-acetylation (H3K9Ac) were increased, with a concomitant decrease in the repressive H3K9-trimethylation (H3K9Me3). The present work demonstrates that epigenetic mechanisms involving promoter-histone modifications regulate transcriptional competence and FasL expression in CD4 T cells from PLWH and render them susceptible to activation-induced cell death.

Induction of ROS-dependent apoptotic cell death by platycodin D in human prostate cancer PC3 cells

Background and objective: Platycodin D (PD), a triterpenoid saponin isolated from an edible and medicinal plant Platycodon grandiflorum, possesses multiple pharmacological properties. The purpose of this study is to investigate the effect of PD on the growth of PC3 human prostate cancer cells and the underlying molecular mechanisms.Materials and methods: Cell viability, apoptosis and mitochondrial membrane potential (MMP) were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, nuclear staining and flow cytometry analysis. To investigate the mechanism of anti-cancer activity of PD, expression of apoptosis regulatory protein, caspase activity, and generation of intracellular reactive oxygen species (ROS) were determined.Results: PD treatment reduced PC3 cell proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL) and pro-apoptotic Bax, and decreased expression of anti-apoptotic Bcl-2 and truncation of Bid. PD also inhibited expression of c-FLIP and members of inhibitor of apoptosis protein family, and activated caspases, resulting in an increase in poly (ADP-ribose) polymerase cleavage. However, in the presence of a pan-caspase inhibitor, PD-mediated growth inhibition and apoptosis were significantly protected. PD also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by PD treatment, which was significantly attenuated by the ROS scavenger N-acetyl-L-cysteine (NAC). Furthermore, NAC fully suppressed PD-induced apoptotic events and cytotoxicity.Conclusions: The results of this study show that PD had chemopreventive potential through the induction of ROS-dependent apoptosis in PC3 cells, and that this compound could be useful for developing an effective and selective natural source to inhibit cancer cell proliferation.

Lumican silencing alleviates tumor necrosis factor-α-induced nucleus pulposus cell inflammation and senescence by inhibiting apoptosis signal regulating kinase 1/p38 signaling pathway via inactivating Fas ligand expression

ABSTRACT A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and β-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.