Subsequent Caspase Activation Research Articles

Mitochondria and cell death.

Mitochondria are cellular factories for energy production, calcium homeostasis and iron metabolism, but they also have an unequivocal and central role in intrinsic apoptosis through the release of cytochrome c. While the subsequent activation of proteolytic caspases ensures that cell death proceeds in the absence of collateral inflammation, other phlogistic cell death pathways have been implicated in using, or engaging, mitochondria. Here we discuss the emerging complexities of intrinsic apoptosis controlled by the BCL-2 family of proteins. We highlight the emerging theory that non-lethal mitochondrial apoptotic signalling has diverse biological roles that impact cancer, innate immunity and ageing. Finally, we delineate the role of mitochondria in other forms of cell death, such as pyroptosis, ferroptosis and necroptosis, and discuss mitochondria as central hubs for the intersection and coordination of cell death signalling pathways, underscoring their potential for therapeutic manipulation.

PKA inhibition kills L-asparaginase-resistant leukemic cells from relapsed acute lymphoblastic leukemia patients.

Despite the success in treating newly diagnosed pediatric acute lymphoblastic leukemia (aLL), the long-term cure rate for the 20% of children who relapse is poor, making relapsed aLL the primary cause of cancer death in children. By unbiased genome-wide retroviral RNAi screening and knockdown studies, we previously discovered opioid receptor mu 1 (OPRM1) as a new aLL cell resistance biomarker for the aLL chemotherapeutic drug, L-asparaginase, i.e., OPRM1 loss triggers L-asparaginase resistance. Indeed, aLL cell OPRM1 level is inversely proportional to L-asparaginase IC50: the lower the OPRM1 level, the higher the L-asparaginase IC50, indicating that aLL cells expressing reduced OPRM1 levels show resistance to L-asparaginase. In the current study, we utilized OPRM1-expressing and -knockdown aLL cells as well as relapsed patient aLL cells to identify candidate targeted therapy for L-asparaginase-resistant aLL. In OPRM1-expressing cells, L-asparaginase induces apoptosis via a cascade of events that include OPRM1-mediated decline in [cAMP]i, downregulation of PKA-mediated BAD S118 phosphorylation that can be reversed by 8-CPT-cAMP, cyt C release from the mitochondria, and subsequent caspase activation and PARP1 cleavage. The critical role of PKA inhibition due to a decrease in [cAMP]i in this apoptotic process is evident in the killing of OPRM1-knockdown and low OPRM1-expressing relapsed patient aLL cells by the PKA inhibitors, H89 and 14-22 amide. These findings demonstrate for the first time that PKA can be targeted to kill aLL cells resistant to L-asparaginase due to OPRM1 loss, and that H89 and 14-22 amide may be utilized to destroy L-asparaginase-resistant patient aLL cells.

Mitochondrial outer membrane protein Samm50 protects against hypoxia-induced cardiac injury by interacting with Shmt2

Cardiac remodeling is a critical process following myocardial infarction (MI), potentially leading to heart failure if untreated. The significance of mitochondrial homeostasis in MI remains insufficiently understood. Samm50 is an essential component of mitochondria. Our study aimed to investigate its role in hypoxia-induced cardiac injury and the underlying mechanisms.First, we observed that Samm50 was dynamically downregulated in mice with MI compared to the control mice. In vitro, Samm50 was also downregulated in oxygen-glucose-deprived neonatal rat cardiomyocytes and fibroblasts. Overexpression and knockdown of Samm50 mitigated and exacerbated cardiac apoptosis and fibrosis, while also improving and worsening mitochondrial homeostasis, respectively.Protein interactions with Samm50 during the protective process were identified via immune-coprecipitation/mass spectroscopy. Mechanistically, serine hydroxymethyltransferase 2 (Shmt2) interacted with Samm50, acting as a crucial element in the protective process by hindering the transfer of Bax from the cytoplasm to the mitochondria and subsequent activation of caspase-3. Inhibition of Shmt2 diminished the protective effect of Samm50 overexpression against cardiac injury. Finally, Samm50 overexpression in vivo mitigated cardiac remodeling and enhanced cardiac function in both acute and chronic MI.In conclusion, Samm50 overexpression mitigated hypoxia-induced cardiac remodeling by inhibiting apoptosis and fibrosis, with Shmt2 acting as a key regulator in this protective process. The Samm50/Shmt2 axis represents a newly discovered mitochondria-related pathway for mitigating hypoxia-induced cardiac injury.

Rotator Cuff Tendinopathy: Pathways of Apoptosis.

Rotator cuff repair is usually successful, but retear is not uncommon. It has been previously identified that there is a higher incidence of apoptosis in the edges of the torn supraspinatus tendon. A prospective cohort study was conducted with 28 patients-14 rotator cuff tear patients, 5 instability patients, and 9 Anterior cruciate ligament reconstruction patients to determine whether there was any increase in several genes implicated in apoptosis, including Fas receptor (FasR), Fas ligand, Aifm-1, Bcl-2, Fadd, Bax, and caspase-3. There was a significant expression of Bax (P=0.2) and FasR (P=0.005) in the edges of torn supraspinatus tendons, and in intact subscapularis tendons, there was a significant expression of caspase-3 (P=0.02) compared with samples from the torn supraspinatus tendon (P=0.04). The cytochrome c pathway, with its subsequent activation of caspase-3, as well as the TRAIL-receptor signaling pathway involving FasR have both been implicated. The elevated expression of Bax supported the model that the Bax to Bcl-2 expression ratio represents a cell death switch. The elevated expression of Bax in the intact subscapularis tissue from rotator cuff tear patients also may confirm that tendinopathy is an ongoing molecular process.

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis

Centriolar satellites are multiprotein aggregates that orbit the centrosome and govern centrosome homeostasis and primary cilia formation. In contrast to the scaffold PCM1, which nucleates centriolar satellites and has been linked to microtubule dynamics, autophagy, and intracellular trafficking, the functions of its interactant CEP131 beyond ciliogenesis remain unclear. Using a knockout strategy in a non-ciliary T-cell line, we report that, although dispensable for centriolar satellite assembly, CEP131 participates in optimal tubulin glycylation and polyglutamylation, and microtubule regrowth. Our unsupervised label-free proteomic analysis by quantitative mass spectrometry further uncovered mitochondrial and apoptotic signatures. CEP131-deficient cells showed an elongated mitochondrial network. Upon cell death inducers targeting mitochondria, knockout cells displayed delayed cytochrome c release from mitochondria, subsequent caspase activation, and apoptosis. This mitochondrial permeabilization defect was intrinsic, and replicable in vitro with isolated organelles. These findings extend CEP131 functions to life-and-death decisions and propose ways to interfere with mitochondrial apoptosis.

Inducible caspase 9-mediated suicide gene therapy using AAV6 vectors in a murine model of breast cancer

Breast carcinoma has one of the highest incidence rates (11.7%), with significant clinical heterogeneity. Although conventional chemotherapy and surgical resection are the current standard of care, the resistance and recurrence, after these interventions, necessitate alternate therapeutic approaches. Cancer gene therapy for breast cancer with the suicide gene is an attractive option due to their directed delivery into the tumor. In this study, we have developed a novel treatment strategy against breast cancer with recombinant adeno-associated virus (AAV) serotype 6 vectors carrying a suicide gene, inducible Caspase 9 (iCasp9). Upon treatment with AAV6-iCasp9 vectors and the chemical inducer of dimerizer, AP20187, the viability of murine breast cancer cells (4T1) was significantly reduced to ∼40%-60% (mock control 100%). Following intratumoral delivery of AAV6-iCasp9 vectors in an orthotopic breast cancer mouse model, we observed a significant increase in iCasp9 transgene expression and a significant reduction in tumor growth rate. At the molecular level, immunohistochemical analysis demonstrated subsequent activation of the effector caspase 3 and cellular death. These data highlight the potential of AAV6-iCasp9-based suicide gene therapy for aggressive breast cancer in patients.

Dyslipidemia initiates keratinocytes proliferation through upregulation of lncRNA NEAT in psoriasis patients

BackgroundPsoriasis is a chronic inflammatory immune-mediated and hyper proliferative skin disorder that has underlying genetic factors. Psoriasis can result from interaction of cytokines between keratinocytes and T-lymphocytes. NEAT is a lncRNA involved in immune modulation and has been previously studied in cancers. This study aims to clarify the unprecedented role of NEAT in psoriasis pathogenesis.MethodsThe study was conducted on 50 healthy control subjects and 50 psoriasis patients. Blood samples from all participants were collected for analysis of their lipid profile. qRT-PCR was done for lncRNA NEAT, TNF-α, VEGF genes expression. The levels of ROS and caspase-3 were estimated by ELISA. ROC analysis was done to detect the diagnostic value of lncRNA NEAT gene expression.ResultsDyslipidemia is more prevalent among psoriasis patients. A significant up regulation in lncRNA NEAT, TNF-α, VEGF genes expression (p value˂0.001) in psoriasis patients in addition to significant increase in ROS and caspase-3 levels (p value˂0.001) in compare to controls. Additionally, a positive significant correlation between TNF-α, ROS, NEAT, caspase-3 and dyslipidemia. NEAT had an area under the curve (AUC) of 0.931 (95% CI 0.844–0.978, p < 0.001).ConclusionDyslipidemia is an initiating signal in psoriasis pathogenesis that creates a state of chronic inflammation and oxidative stress. This state induces keratinocytes proliferation and release of NEAT with subsequent caspase-3 activation to counteract the proliferating cells. NEAT could be considered as a good diagnostic biomarker for psoriasis.Graphical abstract

Endoplasmic Reticulum Stress-Mediated Apoptosis Induced by VR12684 Isolated from Mallotus spodocarpus in Cholangiocarcinoma Cell Line.

Cholangiocarcinoma (CCA) is a poor prognosis of a malignant tumor that has been unresponsive to conventional chemotherapeutic agents. Effective and novel therapeutic agents are urgently needed. VR12684 (isolated from Mallotus spodocarpus) has been reported to exhibit growth inhibitory activities in cancer cell lines. The present study investigated the growth inhibitory mechanisms of this compound in a human CCA cell line (KKU-M156). The effects of VR12684 on anti‑proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by SRB assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) staining and western blot analysis. Treatment with VR12684 decreased cell proliferation in a dose- and time-dependent manner in the KKU-M156 cell line. VR12684 induced cell cycle arrest in the G2 phase in KKU-M156 through down-regulation of cyclin B1 and Cdk1 and up-regulation of p21, p27 and p53 levels. VR12684 induced mitochondria-mediated apoptosis by increasing DNA fragmentation, the Bax/BCL-2 ratio and AIF, and decreasing survivin with subsequent activation of caspase-9 and -3. This compound could induce apoptosis through the endoplasmic reticulum (ER) stress-mediated pathway by up-regulation of GRP78, IRE1α and GADD153 levels leading to down-regulation of Bcl-2 and activation of calpain-1, caspase-7 and -12. These results suggested that VR12684 inhibited KKU-M-156 cell growth by way of cell cycle arrest and induction of apoptosis, at least in part, through the mitochondria- and ER-associated intrinsic pathways. Such compounds warrant evaluation as a candidate for the treatment of human CCA.

Biological response and cell death signaling pathways modulated by tetrahydroisoquinoline-based aldoximes in human cells

The uncharged 3-hydroxy-2-pyridine aldoximes with protonatable tertiary amines are studied as antidotes in toxic organophosphates (OP) poisoning. Due to some of their specific structural features, we hypothesize that these compounds could exert diverse biological activity beyond their main scope of application. To examine this further, we performed an extensive cell-based assessment to determine their effects on human cells (SH-SY5Y, HEK293, HepG2, HK-2, myoblasts and myotubes) and possible mechanism of action. As our results indicated, aldoxime having a piperidine moiety did not induce significant toxicity up to 300 µM within 24 h, while those with a tetrahydroisoquinoline moiety, in the same concentration range, showed time-dependent effects and stimulated mitochondria-mediated activation of the intrinsic apoptosis pathway through ERK1/2 and p38-MAPK signaling and subsequent activation of initiator caspase 9 and executive caspase 3 accompanied with DNA damage as observed already after 4 h exposure. Mitochondria and fatty acid metabolism were also likely targets of 3-hydroxy-2-pyridine aldoximes with tetrahydroisoquinoline moiety, due to increased phosphorylation of acetyl-CoA carboxylase. In silico analysis predicted kinases as their most probable target class, while pharmacophores modeling additionally predicted the inhibition of a cytochrome P450cam. Overall, if the absence of significant toxicity for piperidine bearing aldoxime highlights the potential of its further studies in medical counter-measures, the observed biological activity of aldoximes with tetrahydroisoquinoline moiety could be indicative for future design of compounds either in a negative context in OP antidotes design, or in a positive one for design of compounds for the treatment of other phenomena like cell proliferating malignancies.

Anti-proliferation and induction of mitochondria-mediated apoptosis by Garcinia hanburyi resin in colorectal cancer cells

IntroductionSeveral parts of Garcinia hanburyi are used in traditional medicine for many purposes. In this study, Garcinia hanburyi resin (GHR) was explored for possible anti-proliferative effects and the underlying mechanism on colorectal cancer (CRC) cells. MethodsGambogic acid (GA) content in GHR was analyzed by HPLC method. The cytotoxicities of GA and GHR were assessed in human CRC cell lines (SW480 and Caco-2) and normal colon cells (CCD841 CoN) using a trypan blue exclusion assay, MTS assay, and cell morphology analysis. Cell cycle and apoptosis at its half maximal inhibitory concentration (IC50) were analyzed using flow cytometry. And, the levels of intrinsic apoptosis-related proteins were measured by Western blot analysis. ResultsGA was the major compound as 71.26% of the GHR. The cell viability of CRC cells was decreased in a time- and dose-dependent manner after exposure to GHR. The selectivity index indicated that GHR had a high degree of selectivity against CRC cells. The same result was obtained for GA treatment. In addition, GHR markedly induced typical apoptotic morphology of CRC cells, but had no obvious effect on normal colon cells. GHR induced apoptosis with the cell cycle arrest at the G2/M phase. An increase in Bax/Bcl-2 ratio and a decrease in procaspase-3 proteins indicated that GHR promoted apoptosis by disrupting the mitochondrial outer membrane permeability and the subsequent activation of caspase-3. ConclusionGHR, which contained GA as an active compound, significantly inhibited CRC cell proliferation via the induction of intrinsic apoptosis, while having low toxicity on normal colon cells. Therefore, GHR could be proposed as a potent candidate for the treatment of CRC.

Abstract 5800: Anti-metastatic potential of pitavastatin in triple-negative breast cancer via targeting breast cancer stem-like properties and STAT3 signaling

Abstract Triple-negative breast cancer (TNBC) is the most deadly and aggressive phenotype, with a higher rate of metastatic recurrence. TNBC does yet have a suitable treatment option other than cytotoxic anticancer drugs. Although pitavastatin has been shown to exert anti-proliferative effects and cytotoxicity in various types of cancer cells, the precise mechanisms underlying pitavastatin’s anti-cancer effects in TNBC have not been fully elucidated. We sought to investigate the mechanism of pitavastatin-induced apoptosis and its effects on cancer stem cell (CSC)-like characteristics in TNBC. Exposure to pitavastatin induced mitochondria-mediated apoptotic cell death in BT549 and 4T1 cells. Mitochondrial dysfunction was accompanied with a robust production of reactive oxygen species (ROS) and collapse of mitochondrial membrane potential (MMP), resulting in subsequent activation of caspase-3/-7 and PARP cleavage. Pitavastatin effectively suppressed CSC-like properties in TNBC via targeting CD44+/CD24- and CD49f+/CD24- phenotypes, as well as impediment of mammosphere formation in vitro. This phenomenon was accompanied with dysregulation of STAT3 survival pathway, concomitant with significant downregulation of cyclin D1, survivin and vimentin. Pitavastatin effectively targets both the proliferating TNBC tumor cells and CSCs via the dysregulation of STAT3 and suppression of CSC-like properties, markedly reducing angiogenesis and tumor growth, coinciding with decreased Ki-67 expression. It is noteworthy that pitavastatin considerably suppressed metastasis, coinciding with significant reduction of MMP-2, MMP-9 and VEGF in the circulating blood of mice. Our findings highlight that pitavastatin may be potentially effective for the treatment of metastatic TNBC. Citation Format: Dongmi Ko, Juyeon Seo, Seongjae Kim, Soeun Park, Minsu Park, Kee Dal Nam, Yong koo Kang, Sora Seock, Eunsun Jung, Yoon-Jae Kim, Jaeyoun Park, Ji Young Kim, Jae Hong Seo. Anti-metastatic potential of pitavastatin in triple-negative breast cancer via targeting breast cancer stem-like properties and STAT3 signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5800.

SAR-guided development of indole-matrine hybrids as potential anticancer agents via mitochondrial stress/cytochrome c/caspase 3 signaling pathway

Matrine is a clinically used adjuvant anticancer drug, yet its mild potency limited its application. To improve the anticancer activity of matrine, a total of 31 indole-matrine hybrids were constructed in four rounds of SAR-guided iterative structural optimization process. All of the synthesized compounds were evaluated for their antiproliferative activities against a panel of four human cancer cell lines (Hela, MCF-7, SGC-7901, HepG2) and two normal cell lines (GES-1, LO2). The most active hybrid 8g exhibited the anticancer IC50 values of 0.9 to 1.2 μM, which was 3-magnitude of orders more potent than matrine. 8g also showed better selectivity towards cancer cells with the selectivity index value raised from 1.5 to 6.2. Mechanistic studies demonstrated a mitochondrial distribution for 8g by intracellular click chemistry approaches, which led to the discovery that 8g strongly induced mitochondrial stress, as evidenced by impaired energy metabolism, depolarized mitochondrial membrane potential, overload of mitochondrial calcium and escalated ROS production. 8g-induced mitochondrial stress further led to the release of cytochrome c and subsequent activation of caspase 3, which significantly promoted cellular death and inhibited colony formation.

Schisanhenol Attenuates OxLDL-Induced Endothelial Dysfunction via an AMPK-Dependent Mechanism.

Atherosclerotic cardiovascular diseases, commonly known as the formation of fibrofatty lesions in the artery wall, are the leading causes of death globally. Oxidized low-density lipoprotein (oxLDL) is one of the major components of atherosclerotic plaques. It is evident that dietary supplementation containing sources of antioxidants can prevent atherogenic diseases. Schisanhenol (SAL), a dibenzocyclooctene lignin, has been shown to attenuate oxLDL-induced apoptosis and the generation of reactive oxygen species (ROS) in endothelial cells. However, the underlying molecular mechanisms are still largely unknown. In this study, human umbilical vein endothelial cells (HUVECs) were pre-treated with SAL and oxLDL. Our results showed that adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was enhanced in cells pre-treated with SAL in time-dependent and dose-dependent manners. Subsequently, oxLDL-induced AMPK dephosphorylation and protein kinase C (PKC) phosphorylation were significantly reversed in the presence of SAL. In addition, SAL treatment led to an inhibiting effect on the oxLDL-induced membrane assembly of NADPH oxidase subunits, and a similar effect was observed in ROS generation. This effect was further confirmed using knockdown AMPK with small interfering RNA (siRNA) and pharmaceutical reagents, such as the AMPK activator (AICAR), PKC inhibitor (Gö 6983), and ROS inhibitor (DPI). Furthermore, the oxLDL-induced intracellular calcium rise and the potential collapse of the mitochondrial membrane reduced the Bcl-2/Bax ratio, and released cytochrome c from the mitochondria, leading to the subsequent activation of caspase-3 in HUVECs, which were also markedly suppressed by SAL pretreatment. The results mentioned above may provide additional insights into the possible molecular mechanisms underlying the cardiovascular protective effects of SAL.

Methods to Activate the NLRP3 Inflammasome.

The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors.

Quercetin 3-O-rutinoside prevents radiation induced oxidative damage and inflammation by coordinated regulation of Nrf2/ NF-κB/ NLRP3- inflammasome signaling in gastrointestine

BackgroundIrradiation causes extensive alteration in the cellular homeostasis leading to severe organ damage and dysfunction. The lack of understanding of the mechanism conferring radioprotection is often considered a major limitation in the development of agents that suppress the damaging effects of radiation. PurposeIn this study, we demonstrate that Quercetin 3-O-rutinoside (Q-3-R), a flavonoid, can significantly protect the gastrointestine against gamma radiation induced damage. MethodsQ-3-R (10 mg/kg body weight) treated C57BL/6 mice were investigated for anti-oxidant status, regulations of critical targets of Nrf2, NF-κB signaling and the inflammasome complex in the irradiated intestine. ResultsProphylactic administration of Q-3-R stabilized radiation induced reactive oxygen species, maintained total antioxidant and glutathione levels in the intestine. Q-3-R pretreatment activated Nrf2, supported its nuclear translocation and upregulated cytoprotective proteins, HO-1, thioredoxin1, GST and GCLC. The key inflammatory mediator, NF-kB p65 was found down-regulated in Q-3-R treated jejunum. NF-kB p65 downregulation caused suppression of genes of the inflammasome complex, pro-inflammatory cytokine (IL-6 and TNF-α) expression and myeloperoxidase activity. ML385, a specific Nrf2 inhibitor that, when used at a concentration of 30 mg/kg prevented Q-3-R mediated activation of Nrf2 and upregulated NF-kB, suggesting that Q-3-R mediates protection against radiation primarily via Nrf2 activation. Q-3-R also prevented intestinal apoptosis by inhibiting mitochondrial translocation of cytochrome c into the cytosol and subsequent activation of caspase-3. Molecular docking studies confirmed the interactions of Q-3-R with key antioxidant and anti-inflammatory proteins. ConclusionThe findings suggest that Q-3-R effectively coordinated the regulation of cellular anti-oxidant and inflammatory response, thereby minimizing apoptotic event in the irradiated intestine.

Total extract of Abelmoschus manihot L. alleviates uric acid-induced renal tubular epithelial injury via inhibition of caspase-8/caspase-3/NLRP3/GSDME signaling.

Purpose: The incidence of uric acid (UA)-induced kidney injury is increasing owing to the high incidence of hyperuricemia in recent years. The flower of Abelmoschus manihot (Linneus) Medik is a traditional Chinese medicinal herb widely used in the treatment of some kidney diseases. In our previous study, we reported that the total extract of A. manihot L. flower (TEA) attenuated adriamycin-induced renal tubular cell injury. In this study, we aimed to evaluate the role of TEA in UA-induced tubular cell injury. Methods: Normal rat proximal epithelial NRK-52E cells were incubated with UA to mimic hyperuricemia conditions. The role of TEA in the renal tubular cells was also assessed. The cellular morphology was observed using phase-contrast microscopy, and cell viability was analyzed using the Cell Counting kit-8. Living and dead cells were stained using a Calcein-AM/PI double stain kit. The release of lactate dehydrogenase (LDH) was analyzed by LDH cytotoxicity Assay Kit. The expression of target proteins was analyzed using western blot analysis. Results: UA triggered NRK-52E cell injury, as evidenced by morphological changes, detachment of cells from the bottom, cell swelling, large bubbles blowing from cell membrane and loss of cell viability. UA increased release of LDH. UA induced the expression of p-ERK1/2 and the subsequent activation of caspase-8, caspase-3, and NLRP3 inflammasomes. Pyroptosis was elicited by UA after gasdermin E N-terminal (GSDME-NT) was cleaved from gasdermin E (GSDME). Z-DEVD-FMK, a caspase-3 inhibitor, suppressed the expression of both NLRP3 and GSDME-NT, but not that of caspase-8. INF39, an NLRP3 inhibitor, altered the expression of GSDME-NT expression, but not that caspase-3 and caspase-8. TEA alleviated UA-induced cell injury by suppressing ERK1/2/caspase-8/caspase-3/NLRP3/GSDME signaling. Conclusion: GSDME-mediated pyroptosis was involved in UA-induced renal tubular cell injury. This is the first study to report that TEA protects renal tubular epithelial cells against UA by inhibiting the ERK/1/2/caspase-8/caspase-3/NLRP3/GSDME pathway.

ER stress-induced cell death proceeds independently of the TRAIL-R2 signaling axis in pancreatic \u03b2 cells

Prolonged ER stress and the associated unfolded protein response (UPR) can trigger programmed cell death. Studies in cancer cell lines demonstrated that the intracellular accumulation of TRAIL receptor-2 (TRAIL-R2) and the subsequent activation of caspase-8 contribute significantly to apoptosis induction upon ER stress. While this might motivate therapeutic strategies that promote cancer cell death through ER stress-induced caspase-8 activation, it could also support the unwanted demise of non-cancer cells. Here, we therefore investigated if TRAIL-R2 dependent signaling towards apoptosis can be induced in pancreatic β cells, whose loss by prolonged ER stress is associated with the onset of diabetes. Interestingly, we found that elevated ER stress in these cells does not result in TRAIL-R2 transcriptional induction or elevated protein levels, and that the barely detectable expression of TRAIL-R2 is insufficient to allow TRAIL-induced apoptosis to proceed. Overall, this indicates that apoptotic cell death upon ER stress most likely proceeds independent of TRAIL-R2 in pancreatic β cells. Our findings therefore point to differences in ER stress response and death decision-making between cancer cells and pancreatic β cells and also have implications for future targeted treatment strategies that need to differentiate between ER stress susceptibility of cancer cells and pancreatic β cells.

Interaction between the inflammasome and commensal microorganisms in gastrointestinal health and disease.

The inflammasome is a cytosolic multiprotein complex that plays a crucial role in inflammation and cell death. The sensor proteins in the inflammasome complex detect various microbial and endogenous stimuli, leading to subsequent caspase activation. The activation of caspases results in the maturation of pro‐inflammatory cytokines IL‐1β and IL‐18 or pyroptosis. Inflammasome dysfunction is associated with the pathogenesis of various diseases, including autoimmune disease and cancer. It appears that the interactions between the gut microbiota and the inflammasome play crucial roles in the gastrointestinal tract. The gut microbiota induces the expression and activation of inflammasome proteins, which contribute to both homeostasis and disease in the gut. Likewise, although controversial, mounting evidence suggests that inflammasome activation can modulate the composition of the gut microbiota, which, in turn, affects disease progression. In this review, we summarize the current concepts and recent insights linking the inflammasome and gut commensal microorganisms. We describe how the reciprocal interaction between the inflammasome and the commensal microbiota relates to physiological and pathophysiological consequences in the host.

Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction

Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.