Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

Corilagin attenuates intestinal ischemia/reperfusion injury in mice by inhibiting ferritinophagy-mediated ferroptosis through disrupting NCOA4-ferritin interaction.

AIMS: Intestinal ischemia reperfusion (II/R) is a common clinical emergency. Ferroptosis is reported to play a role in II/R injury. Our previous studies revealed that corilagin significantly attenuates intestinal ischemia/reperfusion injuries. However, the underlying molecular mechanism is unclear and requires further study. MATERIALS AND METHODS: DAO, GSSG/T-GSH, MDA, and Fe2+ were measured by assay kits, 4-HNE was assessed by IHC, and 15-LOX was measured by ELISA. Mitochondrial damage was observed by TEM and cellular oxidation levels were detected by C11-BODIPY 581/591 and DHE probes. LC3, p62, Beclin1, ACSL4, GPX4, NCOA4, and ferritin expression were examined by WB in vivo and in vitro. IF, co-IF, q-PCR, and constructed NCOA4-knock-down IEC-6 cells were used to evaluate the role of NCOA4 in the effect of corilagin against II/R injury. Temporal and nucleoplasmic variations with or without corilagin were observed by WB. Co-IP and molecular docking were used to investigate the NCOA4-ferritin interaction. KEY FINDINGS: Corilagin attenuated II/R-induced ferroptosis both in vitro and in vivo. Further study revealed that the anti-ferroptosis bioactivity of corilagin might be due to the modulation of iron homeostasis via inhibition of ferritinophagy in an NCOA4-dependent manner. SIGNIFICANCE: Corilagin might be a potential therapeutic agent for II/R-induced tissue injury.

Nigericin Boosts Anti-Tumor Immune Response via Inducing Pyroptosis in Triple-Negative Breast Cancer.

Although immune checkpoint inhibitors improved the clinical outcomes of advanced triple negative breast cancer (TBNC) patients, the response rate remains relatively low. Nigericin is an antibiotic derived from Streptomyces hydrophobicus. We found that nigericin caused cell death in TNBC cell lines MDA-MB-231 and 4T1 by inducing concurrent pyroptosis and apoptosis. As nigericin facilitated cellular potassium efflux, we discovered that it caused mitochondrial dysfunction, leading to mitochondrial ROS production, as well as activation of Caspase-1/GSDMD-mediated pyroptosis and Caspase-3-mediated apoptosis in TNBC cells. Notably, nigericin-induced pyroptosis could amplify the anti-tumor immune response by enhancing the infiltration and anti-tumor effect of CD4+ and CD8+ T cells. Moreover, nigericin showed a synergistic therapeutic effect when combined with anti-PD-1 antibody in TNBC treatment. Our study reveals that nigericin may be a promising anti-tumor agent, especially in combination with immune checkpoint inhibitors for advanced TNBC treatment.

Nigericin Induces Apoptosis in Primary Effusion Lymphoma Cells by Mitochondrial Membrane Hyperpolarization and ß-Catenin Destabilization.

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H+ and K+ ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. MATERIALS AND METHODS: We examined the cytotoxic effects of the K+ ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. RESULTS: Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of ß-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. CONCLUSION: Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/ß-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

Sevoflurane preconditioning improves neuroinflammation in cerebral ischemia/reperfusion induced rats through ROS-NLRP3 pathway.

AIM: We aimed to study the influence of sevoflurane on the nucleotide-binding domain and Leucine-rich repeat protein 3 (NLRP3) pathways in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Sixty Sprague-Dawley rats were equally divided into five groups randomly: sham-operated, cerebral I/R, sevoflurane (Sevo), NLRP3 inhibitor-treated (MCC950), and sevoflurane and NLRP3 inducer-treated groups. Rats' neurological functions were assessed using Longa scoring after 24 h of reperfusion, after which they were sacrificed, and cerebral infarction area was determined by triphenyl tetrazolium chloride staining. Pathological changes in damaged portions were assessed using hematoxylin-eosin and Nissl staining, and cell apoptosis was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling staining. Interleukin 1 beta (IL-1ß), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-18), malondialdehyde (MDA), and superoxide dismutase (SOD) levels in brain tissues were determined using enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) levels were analyzed using a ROS assay kit. Protein levels of NLRP3, caspase-1, and IL-1ß were determined by western blot. RESULTS: Neurological function scores, cerebral infarction areas, and neuronal apoptosis index were decreased in the Sevo and MCC950 groups than in the I/R group. IL-1ß, TNF-α, IL-6, IL-18, NLRP3, caspase-1, and IL-1ß levels decreased in the Sevo and MCC950 groups (p < 0.05). ROS and MDA levels increased, but SOD levels increased in the Sevo and MCC950 groups than in the I/R group. NLPR3-inducer nigericin eliminated the protective effects of sevoflurane on cerebral I/R injury in rats. CONCLUSION: Sevoflurane could alleviate cerebral I/R-induced brain damage by inhibiting the ROS-NLRP3 pathway.

Nigericin treatment activates endoplasmic reticulum apoptosis pathway in goldfish kidney leukocytes.

Nigericin has been reported to induce apoptosis and pyroptosis in mammalian models. However, the effects and mechanism underlying the immune responses of teleost HKLs induced by nigericin remain enigmatic. To decipher the mechanism after nigericin treatment, the transcriptomic profile of goldfish HKLs was analyzed. The results demonstrated that a total of 465 differently expressed genes (DEGs) with 275 up-regulated and 190 down-regulated genes were identified between the control and nigericin treated groups. Among them, the top 20 DEG KEGG enrichment pathways were observed including apoptosis pathways. In addition, the expression level of selected genes (ADP4, ADP5, IRE1, MARCC, ALR1, DDX58) by quantitative real-time PCR showed a significant change after treatment with nigericin, which was generally identical to the expression patterns of the transcriptomic data. Furthermore, the treatment could induce cell death of HKLs, which was confirmed by LDH release and annexin V-FITC/PI assays. Taken together, our results support the idea that nigericin treatment might activate the IRE1-JNK apoptosis pathway in goldfish HKLs, which will provide insights into the mechanisms underlying HKLs immunity towards apoptosis or pyroptosis regulation in teleosts.

Nigericin Abrogates Maternal and Embryonic Oxidative Stress in the Streptozotocin-Induced Diabetic Pregnant Rats.

Hyperglycemic exposure in diabetic pregnancy can lead to many developmental changes, such as delayed development, fetal malformations, and fetal/embryo death. These detrimental complications are collectively known as diabetic embryopathy or teratogenesis. The current study focuses to discover the therapeutic properties of the nigericin against the STZ-stimulated diabetic embryopathy via alleviation of maternal and embryonic oxidative stress. The male and female rats at a 1:1 ratio were permitted to mate overnight to establish the course of pregnancy. The pregnant rats were distributed into four groups control, diabetic pregnant (via administering 40 mg/kg of STZ), and diabetic + 10 and 20 mg/kg of nigericin-administered (via oral gavage from days 5 to 12) groups, respectively. The glucose level, urine output, diet intake, and body weight were determined carefully. The embryo and placenta weight and implantation rates were examined, and data were tabulated. The total protein and lipid profiles were assessed using respective kits. The oxidative stress markers and antioxidant enzymes were examined using respective assay kits. The 10 and 20 mg/kg of nigericin treatment decreased the glucose level and urine output and improved the diet intake and body weight gain in diabetic pregnant rats. The nigericin also decreased the total protein, cholesterol, triglycerides, and very-low-density lipoprotein (VLDL) and improved the high-density lipoprotein (HDL) in the serum of pregnant rats. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), and protein carbonyls were decreased by the nigericin in both liver and embryos of the pregnant rats. The levels of glutathione (GSH), total thiols, and activities of catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione S-transferase (GST) were improved by the nigericin in the diabetic pregnant rats. Altogether, these results provide evidence that nigericin treatment remarkably attenuates the diabetes-stimulated embryopathy in rats. The nigericin effectively decreased embryo lethality, reduced glucose and dyslipidemia, and relieves oxidative stress via upregulating the antioxidant enzyme activities. Hence, it can be a talented therapeutic agent to treat diabetic pregnancy-associated complications.

Targeting Multiple Homeostasis-Maintaining Systems by Ionophore Nigericin Is a Novel Approach for Senolysis.

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl-compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases.

Ionophore Antibiotics Inhibit Type II Feline Coronavirus Proliferation In Vitro.

Feline coronaviruses (FCoVs) infect cats worldwide and cause severe systemic diseases, such as feline infectious peritonitis (FIP). FIP has a high mortality rate, and drugs approved by the Food and Drug Administration have been ineffective for the treatment of FIP. Investigating host factors and the functions required for FCoV replication is necessary to develop effective drugs for the treatment of FIP. FCoV utilizes an endosomal trafficking system for cellular entry after binding between the viral spike (S) protein and its receptor. The cellular enzymes that cleave the S protein of FCoV to release the viral genome into the cytosol require an acidic pH optimized in the endosomes by regulating cellular ion concentrations. Ionophore antibiotics are compounds that form complexes with alkali ions to alter the endosomal pH conditions. This study shows that ionophore antibiotics, including valinomycin, salinomycin, and nigericin, inhibit FCoV proliferation in vitro in a dose-dependent manner. These results suggest that ionophore antibiotics should be investigated further as potential broad-spectrum anti-FCoV agents.

Characterization of Pathway-Specific Regulator NigR for High Yield Production of Nigericin in Streptomyces malaysiensis F913.

Nigericin is a polyether antibiotic with potent antibacterial, antifungal, antimalarial and anticancer activity. NigR, the only regulator in the nigericin biosynthetic gene cluster in Streptomyces malaysiensis F913, was identified as a SARP family regulator. Disruption of nigR abolished nigericin biosynthesis, while complementation of nigR restored nigericin production, suggesting that NigR is an essential positive regulator for nigericin biosynthesis. Overexpression of nigR in Streptomyces malaysiensis led to significant increase in nigericin production compared to the wild-type strain. Nigericin production in the overexpression strain was found to reach 0.56 g/L, which may be the highest nigericin titer reported to date. Transcriptional analysis suggested that nigR is required for the transcription of structural genes in the nig gene cluster; quantitative RT-PCR analysis revealed that the expression of structural genes was upregulated in the nigR overexpression strain. Our study suggested that NigR acts in a positive manner to modulate nigericin production by activating transcription of structural genes and provides an effective strategy for scaling up nigericin production.

Ocean acidification impacts sperm swimming performance and pHi in the New Zealand sea urchin Evechinus chloroticus.

In sea urchins, spermatozoa are stored in the gonads in hypercapnic conditions (pH<7.0). During spawning, sperm are diluted in seawater of pH>8.0, and there is an alkalinization of the sperm's internal pH (pHi) through the release of CO2 and H+. Previous research has shown that when pHi is above 7.2-7.3, the dynein ATPase flagellar motors are activated, and the sperm become motile. It has been hypothesized that ocean acidification (OA), which decreases the pH of seawater, may have a narcotic effect on sea urchin sperm by impairing the ability to regulate pHi, resulting in decreased motility and swimming speed. Here, we used data collected from the same individuals to test the relationship between pHi and sperm motility/performance in the New Zealand sea urchin Evechinus chloroticus under near-future (2100) and far-future (2150) atmospheric PCO2 conditions (RCP 8.5: pH 7.77, 7.51). Decreasing seawater pH significantly negatively impacted the proportion of motile sperm, and four of the six computer-assisted sperm analysis (CASA) sperm performance measures. In control conditions, sperm had an activated pHi of 7.52. Evechinus chloroticus sperm could not defend pHi in future OA conditions; there was a stepped decrease in the pHi at pH 7.77, with no significant difference in mean pHi between pH 7.77 and 7.51. Paired measurements in the same males showed a positive relationship between pHi and sperm motility, but with a significant difference in the response between males. Differences in motility and sperm performance in OA conditions may impact fertilization success in a future ocean.

Synergistic Antibacterial Activity of 1-Methyl Ester-Nigericin and Methyl 5-(Hydroxymethyl) Furan-2-Carboxylate Against Proteus spp.

<b>Background and Objective:</b> Synergistic combinations of antimicrobial agents with different mechanisms of action are successful approaches for combating bacterial infections. This study aimed to evaluate the synergistic effect of 1-methyl ester-nigericin <b>(1)</b> and methyl 5-(hydroxymethyl) furan-2-carboxylate <b>(2)</b> against <i>Proteus</i> spp., isolates. <b>Materials and Methods:</b> The synergistic antimicrobial activity of the compounds was tested by the checkerboard method and time-kill curves. To estimate the interaction between the compounds, the Fractional Inhibitory Concentration Index (FICI) of the combination was calculated. The cytotoxic activity of the compounds in combination was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on LLC-MK2 cell lines. The reduction percentage of biofilms was obtained using the colourimetric method. <b>Results:</b> The MIC values for compounds <b>1</b> and <b>2</b> against test bacteria ranged from 39.06-78.12 µg mL¹ and from 78.12-156.25 µg mL¹, respectively. The MIC was reduced to 1-8th as a result of the combination of compounds <b>1</b> and <b>2</b>. After 4-24 hrs of treatment with ½ MIC of compounds <b>1</b> and <b>2</b>, the killing rate (in CFU mL¹) increased to a greater degree than observed with either test compound alone. The combination of compounds <b>1</b> and <b>2</b> showed a synergistic effect with FICI of 0.50 and 0.28. The synergistic combination of compounds <b>1</b> and <b>2</b> was effective on the biofilm reduction of <i>Proteus</i> <i>vulgaris</i> NP16 (85.72%) and NP47 (89.14%). <b>Conclusion:</b> This study recommends compounds <b>1</b> and <b>2</b> in combination as a potential alternative treatment agent for <i>Proteus</i> spp. infections.

Senkyunolide A inhibits the progression of osteoarthritis by inhibiting the NLRP3 signalling pathway.

CONTEXT: Osteoarthritis (OA) is a degenerative disease. Senkyunolide A (SenA) is an important phthalide from Ligusticum chuanxiong Hort (Umbelliferae) with anti-spasmodic and neuroprotective effects. OBJECTIVE: We explored the effect of SenA on IL-1ß-stimulated chondrocytes and OA mice. MATERIALS AND METHODS: Chondrocytes were stimulated by IL-1ß (10 ng/mL) to establish an OA model in vitro. Cells were treated with SenA (20, 40, 80 and 160 µg/mL) for 48 h. The in vivo OA model was established by cutting off the medial meniscus tibial ligament (MMTL) at right knee incision of male C57BL/6 mice. One week after surgery, mice were injected with SenA (intraperitoneally one week) and divided into four groups (n = 6 per group): Sham, OA, OA + SenA 20 mg/kg and OA + SenA 40 mg/kg. The OA progression was examined by haematoxylin and eosin (H&E) staining. RESULTS: SenA treatment increased cell viability (33%), proliferation (71%), inhibited apoptosis (21%), decreased levels of catabolic marker proteins (MMP13, 23%; ADAMTS4, 31%; ADAMTS5, 19%), increased levels of anabolic marker proteins (IGF-1, 57%; aggrecan, 75%; Col2a1, 48%), reduced levels of inflammation cytokines (TNF-α, 31%; IL-6, 19%; IL-18, 20%) and decreased levels of NLRP3 (21%), ASC (20%) and caspase-1 (29%) of chondrocytes. However, NLRP3 agonist nigericin increased levels of MMP13 (55%), ADAMTS4 (70%), ADAMTS5 (53%), decreased levels of IGF-1 (36%), aggrecan (26%), Col2a1 (25%), inhibited proliferation (61%) and promoted apoptosis (76%). DISCUSSION AND CONCLUSIONS: SenA alleviates OA progression by inhibiting NLRP3 signalling pathways. These findings provide an experimental basis for the clinical application of drugs in the treatment of OA.

The inhibiting effect of Aspirin Triggered-Resolvin D1 in non-canonical pyroptosis in rats with acute keratitis.

PURPOSE: To investigate the effect of Aspirin Triggered-Resolvin D1 (AT-RvD1) as an anti-pyroptosis and anti-inflammatory agent on lipopolysaccharide (LPS) induced acute keratitis in Wistar rats. METHODS: Acute keratitis in rats were induced by LPS stromal injection. Inflammatory reaction was measured by clinical score and histological observations. The non-canonical pyroptosis, the role of AT-RvD1 and Docosahexaenoic Acid (DHA) on non-canonical pyroptosis, were verified by quantification real-time PCR (qRT-PCR) and Western-blot. Besides, Human corneal epithelial cells (HCECs) primed with LPS, were stimulated with Nigericin, AT-RvD1 and necrosulfonamide (NSA), a Gasdermin-D (GSDMD) inhibitor separately. CCK-8 tests and flow cytometry were conducted to evaluate the cell viability and death ratio. And the marker of non-canonical pyroptosis were verified by Western blot. RESULTS: AT-RvD1 and DHA both alleviated the inflammation of rat cornea through inhibiting the expression of Caspase-11 and p30 which was triggered by LPS. Meanwhile, the activation of Caspase-4 and p30 were also significantly suppressed by AT-RvD1 in vitro, which is consistent with the results in rats. CONCLUSIONS: The non-canonical pyroptosis signaling pathways played an important role in rats with acute keratitis. In addition, AT-RvD1 can exert as an anti-inflammatory activity by inhibiting the non-canonical pyroptosis. Hence, it may be a promising and safe agent in treating acute keratitis.

Nigericin exerts anticancer effects through inhibition of the SRC/STAT3/BCL-2 in osteosarcoma.

The treatment of osteosarcoma has reached a bottleneck period in recent 30 years, there is an urgent need to find new drugs and treatment methods. Nigericin, an antibiotic derived from Streptomyces hygroscopicus, has exerted promising antitumoral effect in various tumors. The anticancer effect of Nigericin in human osteosarcoma has never been reported. In the present study, we explored the anticancer effects of Nigericin in osteosarcoma in vitro and in vivo. Our results showed that nigericin treatment significantly reduced tumor cell proliferation in dose-dependent and time-dependent in human osteosarcoma cells. Nigericin can inhibit cell growth of osteosarcoma cells, in addition to S-phase cycle arrest, the nigericin induces apoptosis. Furthermore, bioinformatics predicted that Nigericin exerts anticancer effects through inhibiting SRC/STAT3 signaling pathway in osteosarcoma. The direct binding between SRC and activator of transcription 3 (STAT3) was confirmed by Western blot. Nigericin can down regulate STAT3 and Bcl-2. In order to further elucidate the inhibitory effect of nigericin on SRC/STAT3/Bcl-2 signal transduction mechanism, we established human osteosarcoma cancer cells stably expressing STAT3. Western blot confirmed that nigericin exerts anticancer effects on human osteosarcoma cancer cells by directly targeting STAT3. In addition, Nigericin can significantly inhibit tumor migration and invasion. Finally, Nigericin inhibits tumor growth in a mouse osteosarcoma model. The nigericin targeting the SRC/STAT3/BCL-2 signaling pathway may provide new insights into the molecular mechanism of nigericin on cancer cells and suggest its possible clinical application in osteosarcoma.

The ASC Speck and NLRP3 Inflammasome Function Are Spatially and Temporally Distinct.

Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein, we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation but facilitates and maximizes IL-1ß processing. Furthermore, the NLRP3 agonists H2O2 and MSU elicited IL-1ß maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size, while speck numbers and IL-1ß processing are negatively correlated, cyclical and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.

Combination of nigericin with cisplatin enhances the inhibitory effect of cisplatin on epithelial ovarian cancer metastasis by inhibiting slug expression via the Wnt/ß-catenin signalling pathway.

Epithelial ovarian cancer (EOC) is the most lethal cancer among female genital tumours. Standard therapies, including postoperative chemotherapy, exhibit high proportions of recurrence and resistance. Novel therapeutic strategies are combined with chemotherapy. Emerging studies have demonstrated that nigericin, an H+, K+ and Pb2+ ionophore, exhibits promising anticancer activity in various types of malignancy, such as colorectal and epithelial ovarian cancer. Our previous study suggested that nigericin could regulate EOC cell proliferation, migration and invasion, and may be a novel chemotherapy candidate for EOC. However, to the best of our knowledge, the effects of combined therapy with cisplatin, and the associated underlying mechanisms, are not yet fully understood. The present study aimed to clarify the effects of combined chemical therapy with nigericin and cisplatin on EOC cells and to reveal its mechanism. Wound healing, Transwell, cell viability and colony formation assays were used to measure the migration, invasion and proliferation of EOC cells. Western blotting was used to detect protein expression. A slug overexpression lentivirus was used to create a slug overexpression model in SK-OV-3 cells. Small interfering RNA was used to knock down slug expression. Nigericin combined with cisplatin enhanced the inhibitory effects of cisplatin on the migration and colony formation of EOC cells. Nigericin also enhanced the inhibitory effects of cisplatin on the expression levels of MMP7, as well as the inhibitory effects of cisplatin on the expression levels of ß-catenin and GSK-3ß, indicating that nigericin and cisplatin regulated in the Wnt/ß-catenin signalling pathway. When slug was knocked down, the effect of nigericin was weakened. Overexpression of slug could repress the inhibitory effect of nigericin on the Wnt/ß-catenin signalling pathway. Furthermore, nigericin inhibited slug expression by enhancing its modification through small ubiquitin-like modifiers (SUMOs; referred to as SUMOylation). Overall, the present results demonstrated that nigericin combined with cisplatin might serve as a novel therapeutic strategy in patients with metastatic EOC because the combined therapy had higher effectiveness than single drug use. The underlying mechanism of combined therapy maybe the enhanced inhibitory effect of slug through its nigericin-induced SUMOylation.

NLRP3 Triggers Attenuate Lipocalin-2 Expression Independent with Inflammasome Activation.

Lipocalin-2 (LCN2), a small secretory glycoprotein, is upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 inhibits bacterial growth by iron sequestration and regulates the innate immune system. Inflammasome activates the inflammatory caspases leading to pyroptosis and cytokine maturation. This study examined the effects of inflammasome activation on LCN2 secretion in response to TLR signaling. The triggers of NLRP3 inflammasome activation attenuated LCN2 secretion while it induced interleukin-1ß in mouse macrophages. In mice, NLRP3 inflammasome activation inhibited TLR-mediated LCN2 secretion. The inhibition of NLRP3 triggers on LCN2 secretion was caused by the inhibited transcription and translation of LCN2. At the same time, no changes in the other cytokines and IκBζ, a well-known transcriptional factor of Lcn2 transcription, were observed. Overall, NLRP3 triggers are a regulator of LCN2 expression suggesting a new linkage of inflammasome activation and LCN2 secretion in the innate immunity.

Evidence of nigericin as a potential therapeutic candidate for cancers: A review.

Emerging studies have shown that nigericin, an H+, K+ and Pb2+ ionophore, has exhibited a promising anti-cancer activity in various cancers. However, its anti-cancer mechanisms have not been fully elucidated. In this review, the recent progresses on the use of nigericin in human cancers have been summarized. By exchanging H+ and K+ across cell membranes, nigericin shows promising anti-cancer activities in in vitro and in vivo as a single agent or in combination with other anti-cancer drugs through decreasing intracellular pH (pHi). The underlying mechanisms of nigericin also include the inactivation of Wnt/ß-catenin signals, blockade of Androgen Receptor (AR) signaling, and activation of Stress-Activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) signaling pathways. In many cancers, nigericin is proved to specifically target putative Cancer Stem Cells (CSCs), and its synergistic effects on photodynamic therapy are also reported. Other mechanisms of nigericin including influencing the mitochondrial membrane potentials, inducing an increase in drug accumulation and autophagy, controlling insulin accumulation in nuclei, and increasing the cytotoxic activity of liposome-entrapped drugs, are also discussed. Notably, the potential adverse effects such as teratogenic effects, insulin resistance and eryptosis shall not be ignored. Taken together, these reports suggest that treatment of cancer cells with nigericin may offer a novel therapeutic strategy and future potential of translation to clinics.

Role of Annexin A1 in NLRP3 Inflammasome Activation in Murine Neutrophils.

This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1ß production by WT neutrophils after nigericin and ATP stimulation. However, IL-1ß release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1ß release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1ß production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.