Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

Antecedent Dietary Glutamine Supplementation Benefits Modulation of Liver Pyroptosis in Mice with Polymicrobial Sepsis.

The liver is the main organ responsible for bacterial and endotoxin clearance. Pyroptosis is a form of proinflammatory programmed cell death activated by caspase-1/11 and gasdermin D (GadD). Pyroptosis protects the host against bacterial infection; however, overactive pyroptosis can lead to organ injury. Glutamine (GLN) is a specific amino acid with anti-inflammatory and immunomodulatory properties. This study investigated the effects of GLN pretreatment on liver pyroptosis in a mouse model of polymicrobial sepsis. Mice were assigned to sham, sepsis control (Sepsis-C), and sepsis GLN (Sepsis-G) groups. The sham and Sepsis-C groups were fed the AIN-93G diet. The Sepsis-G group was provided with identical diet components except that part of the casein was replaced by GLN. After feeding the respective diets for 2 weeks, a cecal ligation and puncture (CLP) procedure was performed in the sepsis groups. An antibiotic was administered after CLP. Mice were sacrificed at either 24 or 72 h after CLP. The results showed that sepsis resulted in upregulated liver caspase-1/11 expression. Compared to the Sepsis-C group, the Sepsis-G group had higher liver caspase-11 and NLRP3 gene expressions at 24 h and lower active caspase-1/11 and cleaved GadD protein levels at 72 h after sepsis. Additionally, liver inflammatory cytokine gene expressions had decreased by 72 h post-CLP. The findings suggest that prophylactic administration of GLN initially upregulated liver pyroptosis to eradicate pathogens, yet the process of pyroptosis was suppressed in the late phase of sepsis. This may have beneficially attenuated liver inflammation and injury in an antibiotic-treated septic condition.

Viburnum pichinchense methanol extract exerts anti-inflammatory effects via targeting the NF-κB and caspase-11 non-canonical inflammasome pathways in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Viburnum pichinchense Benth. Mainly found in Ecuador and Colombia has been ethnopharmacologically utilized as a remedy for various female disorders with kidney inflammation and uterine relaxant. AIM OF THE STUDY: The pharmacological activity of Viburnum pichinchense has never been studied, therefore, this study explored anti-inflammatory activity of Viburnum pichinchense methanol extract (Vp-ME). MATERIALS AND METHODS: Anti-inflammatory activities of Vp-ME were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and HCl/EtOH-induced gastritis mice by MTT assay, nitric oxide (NO) production assay, semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), luciferase reporter assay, Western blotting, and enzyme-linked immunosorbent assays (ELISA). Anti-inflammatory compounds in Vp-ME were identified by high performance liquid chromatography (HPLC). RESULTS: Vp-ME inhibited NO production in RAW264.7 cells stimulated with pam3CSK4, poly I:C or LPS and in LPS-stimulated peritoneal macrophages without cytotoxicity and downregulated mRNA expression of inflammatory enzymes, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6. The anti-inflammatory activity was accomplished by inhibiting nuclear factor-kappa B (NF-κB) transcriptional activation, upstream signaling molecules in the NF-κB pathway, and caspase-11 non-canonical inflammasome in RAW264.7 cells. Moreover, Vp-ME exhibited in vivo anti-inflammatory activity by ameliorating gastritis symptoms, inhibiting iNOS and IL-6 mRNA expression and IκBα activation in mice. HPLC analysis identified resveratrol, quercetin, luteolin, and kaempferol as the anti-inflammatory components in Vp-ME. CONCLUSION: This study demonstrated Vp-ME has the anti-inflammatory activity via targeting NF-κB and caspase-11 non-canonical inflammasome pathways in macrophage-mediated inflammatory responses, suggesting Vp-ME could be developed as anti-inflammatory ethnopharmacological remedies to prevent and treat inflammatory diseases.

Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS.

Tumor necrosis factor (TNF)­related apoptosis­inducing ligand (TRAIL) is a member of the TNF superfamily and is an antitumor drug that induces apoptosis in tumor cells with minimal or no effects on normal cells. Here, it is demonstrated that 6­shogaol (6­sho), a bioactive component of ginger, exerted anti­inflammatory and anticancer properties, attenuated tumor cell propagation and induced TRAIL­mediated cell death in liver cancer cells. The current study identified a potential pathway by revealing that TRAIL and 6­sho or chloroquine acted together to trigger reactive oxygen species (ROS) production, to upregulate tumor­suppressor protein 53 (p53) expression and to change the mitochondrial transmembrane potential (MTP). Treatment with N­acetyl­L­cysteine reversed these effects, restoring the MTP and attenuated ROS production and p53 expression. Interestingly, treatment with 6­sho increased p62 and microtubule­associated proteins 1A/1B light chain 3B­II levels, indicating an inhibited autophagy flux. In conclusion, attenuation of 6­sho­induced autophagy flux sensitized cells to TRAIL­induced apoptosis via p53 and ROS, suggesting that the administration of TRAIL in combination with 6­sho may be a suitable therapeutic method for the treatment of TRAIL­resistant Huh7 liver cells.

Rhein triggers apoptosis via induction of endoplasmic reticulum stress, caspase-4 and intracellular calcium in primary human hepatic HL-7702 cells.

Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells.

Influence of Tanshinone IIA on the Apoptosis of Human Esophageal Ec-109 Cells.

The induced-apoptosis effect and mechanism of human esophageal cancer Ec-109 cells via tanshinone IIA was investigated. The Ec-109 cells were cultured in vitro with different concentrations of tanshinone IIA (2 µg/mL, 4 µg/mL, or 8 µg/mL) for 12, 24, 36, and 48 hours. MTT assay was used to evaluate the proliferative inhibition rate of tanshinone IIA on esophageal Ec-109 cells. After 24 hours of culturing in vitro, a control group was assigned. The apoptosis rate was detected by the AO/EB and annexin V-FITC/propidium iodide assay, and the protein levels of Caspase-4 and CHOP were determined by the Western blot technique. MTT data showed that tanshinone IIA could significantly inhibit the proliferation of Ec-109 cells with a dose- and time-dependent mode. Compared with the control group, tanshinone IIA could apparently induce apoptosis of Ec-109 cells, and the level of Caspase-4 and CHOP (p < 0.01) obviously increased. Tanshinone IIA can significantly induce the apoptosis of Ec-109 cells, which may take effect by the stress pathway of the endoplasmic reticulum.

[Effect of Viqi Chutan Recipe on Caspase-4 and DNA-PK of Cell Apoptosis Approach in Transplanted Lung Cancer A549 Cells in Nude Mice].

OBJECTIVE: To study the inhibitory effect of Yiqi Chutan Recipe on the transplanted tumor through endoplasmic reticulum UPR-mediated approach. METHODS: 40 lung cancer A549 cells models transplanted in nude mice were established. On the 7th day of inoculation, mice were randomly divided into model group( saline group) , Cisplatin group (0.002 g/kg), Yiqi Chutan Recipe low dose group (3.0 g/kg), Yiqi Chutan Recipe high dose group(6. 0 g/kg)and Yiqi Chutan Recipe (3.0 g/kg)with Cisplatin group (0.002 g/kg). Each aforementioned group had eight mice. Mice were treated by Yiqi Chutan Recipe to gavage one time a day, for 21 days, and by Cisplatin Injection to intraperitoneal injection one time a day, for 7 days. On the 22th day, all mice were executed to death. Then each tumor's weight and volume were measured, and the expression of Caspase-4 and DNA-PK protein were detected through immunohistochemical method and Western blot method. RESULTS: Compared with model group, the tumors' volume and weight of Yiqi Chutan Recipe high dose group and Yiqi Chutan Recipe with Cisplatin group were decreased, but the expressions of Caspase-4 and DNA-PK protein in tumors were increased (P < 0.01). Yiqi Chutan Recipe with Cisplatin Group had the better effect (P < 0.05). CONCLUSION: Yiqi Chutan Recipe has a certain inhibitory effect on A549 lung cancer in mice and its possible mechanism is relevant to the increase of expression of Caspase-4 and DNA-PK protein.

Salubrinal protects against cigarette smoke extract-induced HBEpC apoptosis likely via regulating the activity of PERK-eIF2α signaling pathway.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress plays an important role in cigarette smoke extract (CSE)-induced apoptotic cell death, which is an important pathogenic factor of chronic obstructive pulmonary disease (COPD). The aim of this study was to explore the role of the PERK-eIF2 pathway in CSE-induced human bronchial epithelial (HBE) cell apoptosis and to evaluate the protective effects and possible mechanism of salubrinal (Sal) on CSE-induced HBE cell apoptosis. METHODS: Normal human bronchial epithelial cells (HBEpC) were cultured and then treated with CSE alone or together with Sal or preincubated with or without PERK siRNA. Expressions of p-PERK/PERK, p-eIF2α/eIF2α, and caspase 3 and 4 were detected with PCR, Western blot, and immunofluorescence. Apoptosis was detected using AnnexinV-PI flow cytometry. RESULTS: CSE induced apoptotic cell death and caused a dynamic change in PERK-eIF2α pathway activity following the course of CSE exposure. The knockdown of PERK suppressed the expression of both PERK and p-eIF2a and caused a great increase in cell apoptosis. Sal could eliminate the effects of PERK knockdown, protecting the cells against the CSE insult, and this protection was accomplished through maintaining the homeostasis of PERK- eIF2α pathway. CONCLUSIONS: PERK-eIF2α pathway mediates the CSE-induced HBE cell apoptosis. The intactness of PERK-eIF2α pathway is crucial for HBE cell survival under CSE insult. Sal can protect against CSE-induced HBE cell apoptosis, and this effect is likely achieved through maintaining the homeostasis of PERK- eIF2α pathway.

Altholactone induces apoptotic cell death in human colorectal cancer cells.

Resistance of colorectal cancer (CRC) to the available chemotherapy reveals the demand for identification of new anticancer agents. We evaluated the antitumour potential of altholactone, a naturally occurring bioactive compound isolated from Goniothalamus spp. (Annonaceae) hooks, against CRC cells. Antitumour activity of altholactone was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the propidium iodide method. Apoptosis mediators involved were assessed using biochemical inhibitors and Western blotting analysis. Results revealed that altholactone induced varying degrees of apoptosis in CRC cells but not in normal fibroblasts. Dissection of the altholactone-induced apoptotic signalling pathway revealed that altholactone activated caspase-dependent and -independent apoptotic pathways. Activation of caspase-4 appeared to be the initiating event in the caspase-dependent apoptotic pathway. Pre-treatment of CRC cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited activation of caspase-4 and altholactone-induced apoptosis. These results indicate that altholactone induces selective cytotoxicity against colon carcinoma cells and warrants further clinical evaluation.

In vitro and in vivo effects of water extract of white cocoa tea (Camellia ptilophylla) against human prostate cancer.

PURPOSE: We evaluated the chemotherapeutic effect of water extract of white cocoa tea (WCTE) against human prostate cancer (PCa) in vitro and in vivo. METHODS: Cell viability and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Western blotting was performed to determine changes in levels of various proteins. Effect of WCTE was determined in athymic nude mice implanted with PC-3 cells. RESULTS: Treatment with WCTE (100-150 microg/ml) inhibited cell proliferation, which correlated with G2/M phase arrest in PC-3 cells. WCTE treatment to PC-3 cells resulted in (1) induction of WAF1/p21 and KIP1/p27, (2) decrease in cyclins D1, D2 and E, (3) decrease in cyclin-dependent kinase (cdk) 2, 4 and 6, (4) induction of Bax and down-regulation of Bcl-2, (5) decrease in procaspase-3, -8, (6) inhibition of nuclear translocation and phosphorylation of NF-kappaB and activation of IKKalpha, and (7) inhibition of phosphorylation and degradation of IkappaBalpha. Oral administration of WCTE (0.1 and 0.2%, wt/vol) to athymic nude mice resulted in greater than 50% inhibition of tumor growth. There was a decrease in expressions of cyclin D1, Bcl-2 and p-NF-kappaB and an increase in WAF1/p21 and Bax in tumor tissues of mice. CONCLUSION: WCTE can be a useful chemotherapeutic agent against human PCa.

Endoplasmic reticulum stress associated with caspases-4 and -2 mediates korbazol-induced B-chronic lymphocytic leukemia cell apoptosis.

PURPOSE: B-cell chronic lymphocytic leukemia (B-CLL) is an incurable disease that rapidly develops drug resistance. Therefore there is a need for identifying new agents that will improve the therapeutic outcome. Korbazol is a natural product known to exert cytotoxic effect on the in vitro survival of leukemic cells. The aim of this study was to investigate the mechanism of korbazol-induced apoptosis in B-CLL leukemic cells. METHODS: peripheral blood mononuclear cells from 10 B-CLL patients were used for assessing the effect of caspase inhibitors and chelator of intracellular Ca(2)+. RESULTS: cell death rate induced by the tested compound was decreased with the caspase-3 inhibitor Ac-DEVD-CHO, and the inhibitors of caspase-2 (Z-VDVAD-FMK) and -4 (ZYVAD- FMK), but not with the caspase-9 inhibitor z-LEHD-FMK and caspase-8 inhibitor z-IETD-FMK. No significant release of cytochrome C (cyt C) from mitochondria to the cytosol of B-CLL cells treated with korbazol was observed. Moreover, chelating of intracellular Ca(2)+ with BAPTA-AM almost completely abolished the cytotoxic effect of korbazol. CONCLUSION: engagement of caspases-2 and -4 and mobilization of intracellular Ca(2)+ indicate involvement of endoplasmic reticulum (ER) stress in apoptosis induced by korbazol.

Altered redox state of luminal pyridine nucleotides facilitates the sensitivity towards oxidative injury and leads to endoplasmic reticulum stress dependent autophagy in HepG2 cells.

Maintenance of the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum - an important pro-survival factor in the cell - is ensured by the concerted action of glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase. The mechanism by which the redox imbalance leads to cell death was investigated in HepG2 cells. The chemical inhibition of the glucose-6-phosphate transporter, the silencing of hexose-6-phosphate dehydrogenase and/or the glucose-6-phosphate transporter, or the oxidation of luminal NADPH by themselves did not cause a significant loss of cell viability. However, these treatments caused ER calcium store depletion. If these treatments were supplemented with the administration of a subliminal dose of the oxidizing agent menadione, endoplasmic reticulum vacuolization and a loss of viability were observed. Combined treatments resulted in the activation of ATF6 and procaspase-4, and in the induction of Grp78 and CHOP. In spite of the presence of UPR markers and proapoptotic signaling the effector caspases - caspase-3 and caspase-7 - were not active. On the other hand, an elevation of the autophagy marker LC3B was observed. Immunohistochemistry revealed a punctuated distribution of LC3B II, coinciding with the vacuolization of the endoplasmic reticulum. The results suggest that altered redox state of endoplasmic reticulum luminal pyridine nucleotides sensitizes the cell to autophagy.

Oxidative and endoplasmic reticulum stress signaling are involved in dehydrocostuslactone-mediated apoptosis in human non-small cell lung cancer cells.

This study investigates the anticancer effect of dehydrocostuslactone (DHE), a medicinal plant-derived sesquiterpene lactone, on human non-small cell lung cancer cell lines, A549, NCI-H460 and NCI-H520. Our results show that DHE inhibits the proliferation of A549, NCI-H460 and NCI-H520 cells. DHE-induced apoptosis in both A549 and NCI-H460 cells. DHE triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol-calcium levels, PKR-like ER kinase (PERK) phosphorylation, inositol requiring protein 1 (IRE1) and CHOP/GADD153 upregulation, X-box transcription factor-1 (XBP-1) mRNA splicing, and caspase-4 activation. The release of calcium triggered the production of ROS, which further enhances calcium overloading and subsequently activates p38, JNK and ERK1/2. Both IRE1 miRNA transfection and BAPTA-AM pretreatment inhibit DHE-mediated apoptosis, supporting the hypothesis that DHE induces cell death through ER stress. Importantly, a novel anticancer agent for the treatment of non-small cell lung cancer, and is supported by animal studies which have shown a dramatic 50% reduction in tumor size after 28 days of treatment. This study demonstrates that DHE may be a novel anticancer agent for the treatment of non-small cell lung cancer.

Calcium and survivin are involved in the induction of apoptosis by dihydroartemisinin in human lung cancer SPC-A-1 cells.

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial drug. Recent studies suggest that it also has anticancer effects. The present study investigated the apoptosis activity of DHA in cultured human lung cancer cells by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay and flow cytometry. Intracellular free calcium concentrations in the lung cancer cells were evaluated by laser scanning confocal microscopy using Fura-3/AM as probe. The observations also indicated that DHA downregulated the mRNA and protein expression level of survivin in the lung cancer cell line SPC-A-1 cells, whereas it did not affect those of caspase-4. These results demonstrated that DHA can induce apoptosis of lung cancer cell line SPC-A-1 cells and that calcium and survivin participated in the apoptotic signalling pathways.