Nigericin decreases the viability of multidrug-resistant cancer cells and lung tumorspheres and potentiates the effects of cardiac glycosides.

Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the plasticity of cancer cells have been identified as important mechanisms of resistance; therefore, treatments targeting cancer cells independent of stemness phenotype would be much more effective in treating lung cancer. In this article, we have characterized the anticancer effects of the antibiotic Nigericin in cells displaying varying degrees of stemness and resistance to anticancer drugs, arising from (1) routine culture conditions, (2) prolonged periods of serum starvation. These cells are highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea, Colchicine, Obatoclax, Wortmannin, and LY294002, and the multidrug-resistant phenotype of cells growing under prolonged periods of serum starvation is likely the result of extensive rewiring of signaling pathways, and (3) lung tumorspheres that are enriched for cancer stem-like cells. We found that Nigericin potently inhibited the viability of cells growing under routine culture conditions, prolonged periods of serum starvation, and lung tumorspheres. In addition, we found that Nigericin downregulated the expression of key proteins in the Wnt canonical signaling pathway such as LRP6, Wnt5a/b, and ß-catenin, but promotes ß-catenin translocation into the nucleus. The antitumor effects of Nigericin were potentiated by the Wnt activator HLY78 and by therapeutic levels of the US Food and Drug Administration-approved drug Digitoxin and its novel synthetic analog MonoD. We believe that Nigericin may be used in a co-therapy model in combination with other novel chemotherapeutic agents in order to achieve potent inhibition of cancers that display varying degrees of stemness, potentially leading to sustained anticancer effects.

The oxindole Syk inhibitor OXSI-2 blocks nigericin-induced inflammasome signaling and pyroptosis independent of potassium efflux.

The inflammasome is a caspase-1-activating complex that is implicated in a growing number of acute and chronic pathologies. Interest has increased in identifying small molecular inhibitors of inflammasome signaling because of its role in clinically relevant diseases. It was recently reported that the protein tyrosine kinase, Syk, regulates pathogen-induced inflammasome signaling by phosphorylating a molecular switch on the adapter protein ASC. However, several aspects of the role of Syk in inflammasome signaling and the effects of its inhibition remain unclear. The aim of the present study is to explore in detail the effects of the oxindole Syk inhibitor OXSI-2 on various aspects of nigericin-induced inflammasome signaling. Our results indicate that OXSI-2 inhibits inflammasome assembly, caspase-1 activation, IL-1ß processing and release, mitochondrial ROS generation, and pyroptotic cell death. Using a novel live cell potassium sensor we show that Syk inhibition with OXSI-2 has no effect on potassium efflux kinetics and that blockade of potassium efflux with extracellular potassium alters Syk phosphorylation. The effects of OXSI-2 identified in this study provide context for the role of Syk in inflammasome signaling and demonstrate its importance in oxidative signaling upstream of inflammasome activation and downstream of ion flux.

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin.

The K(+),H(+) ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca(2+)-permeable unselective cation channels leading to Ca(2+) entry, increase in cytosolic Ca(2+) activity ([Ca(2+)]i) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3 fluorescence, pHi from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca(2+). The nigericin-induced increase in [Ca(2+)]i and annexin V binding was again significantly blunted but not abolished by the Na(+)/H(+) exchanger inhibitor cariporide (10 µM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca(2+) entry, and involving the cariporide-sensitive Na(+)/H(+) exchanger.

Nigericin selectively targets cancer stem cells in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is prevalent in southern China, northern Africa, and Alaska. The prognosis for NPC patients at early stage is good, while it is poor for patients at late stages. Cancer stem cells (CSCs) have been proposed to be associated with tumor initiation, relapse and metastasis, and the poor prognosis of NPC likely results from residual CSCs after therapy. Study on the therapy targeting CSCs in NPC remains poor, though it received intensive attentions in other cancers. Here, we used NPC cell lines with high and low proportion of CSCs as models to explore the effect of nigericin, an antibiotic, on CSCs. We found that nigericin could selectively target CSCs and sensitize CSCs in NPC to the widely used clinical drug cisplatin both in vitro and in vivo. Moreover, downregulation of the polycomb group protein Bmi-1 may contribute to the inhibitory effect of nigericin on CSCs. Furthermore, by using the in vitro NPC cell models, we found that nigericin could significantly decrease the migration and invasion abilities, which are known to be associated with CSCs. Taken together, our results suggest that nigericin can selectively target CSCs in NPC, which could be a candidate CSCs targeting drug for clinical evaluation.

[Salinomycin and semduramicin in different concentrations on the broilers eimeriosis control]. / Salinomicina e semduramicina associadas em diferentes concentracoes no controle da eimeriose em frangos de corte.

This study aimed to investigate the association of salinomycin and semduramicin, in different doses, against controlled mixed infection of Eimeria acervulina, E. maxima and E. tenella in broiler chickens. Eight hundred birds were divided into 5 groups (T1: not medicated feed; T2: 30 ppm of salinomycin and 12.5 ppm of semduramicin; T3: 30 ppm of salinomycin and 15 ppm of semduramicin; T4: 40 ppm of salinomycin and 12,5 ppm of semduramicin and T5: 40 ppm of salinomycin and 15 ppm of semduramicin) and inoculated at 15 days of age with sporulated oocysts of E. acervulina, E. maxima and E. tenella in a mixed suspension, through the feed. Performance data and lesion scores were recorded. All treated groups showed statistically better cumulative weight gain at 21 days old. At 35 days old only the T3 group showed significant difference. Cumulative feed conversion showed statistical difference in the groups T4 and T5. The treatment T5 was more effective in the coccidiosis control of E. tenella. T3 and T5 achieved statistical differences in the average lesion scores of the three analyzed species. The association of salinomycin and semduramicin used in lower doses than the usual, showed to be an option in the coccidiosis control in this experiment.

Salinomicina e semduramicina associadas em diferentes concentrações no controle da eimeriose em frangos de corte / Salinomycin and semduramicin in different concentrations on the broilers eimeriosis control

O presente estudo teve por objetivo investigar a associação de salinomicina e semduramicina, em diferentes doses, frente à infecção mista controlada de Eimeria acervulina, E. maxima e E. tenella em frangos de corte. Oitocentas aves foram divididas em 5 grupos (T1: ração não medicada; T2: 30 ppm de salinomicina e 12,5 ppm de semduramicina; T3: 30 ppm de salinomicina e 15 ppm de semduramicina; T4: 40 ppm de salinomicina e 12,5 ppm de semduramicina e T5: 40 ppm de salinomicina e 15 ppm de semduramicina) e inoculadas aos 15 dias de idade com oocistos esporulados de E. acervulina, E. maxima e E. tenella, em inóculo misto, via ração. Parâmetros produtivos e escore de lesões foram registrados. Todos os grupos tratados apresentaram estatisticamente melhores ganhos de peso cumulativo aos 21 dias de vida. Aos 35 dias de vida, somente o grupo T3 apresentou diferença significativa. A conversão alimentar cumulativa apresentou diferença estatística nos grupos T4 e T5. O tratamento T5 foi mais eficaz no controle de E. tenella. T3 e T5 obtiveram diferenças estatísticas no escore médio de lesão das três espécies. O uso de salinomicina, associada a semduramicina, em baixas doses demonstrou uma opção viável no controle da coccidiose neste experimento.

This study aimed to investigate the association of salinomycin and semduramicin, in different doses, against controlled mixed infection of Eimeria acervulina, E. maxima and E. tenella in broiler chickens. Eight hundred birds were divided into 5 groups (T1: not medicated feed; T2: 30 ppm of salinomycin and 12.5 ppm of semduramicin; T3: 30 ppm of salinomycin and 15 ppm of semduramicin; T4: 40 ppm of salinomycin and 12,5 ppm of semduramicin and T5: 40 ppm of salinomycin and 15 ppm of semduramicin) and inoculated at 15 days of age with sporulated oocysts of E. acervulina, E. maxima and E. tenella in a mixed suspension, through the feed. Performance data and lesion scores were recorded. All treated groups showed statistically better cumulative weight gain at 21 days old. At 35 days old only the T3 group showed significant difference. Cumulative feed conversion showed statistical difference in the groups T4 and T5. The treatment T5 was more effective in the coccidiosis control of E. tenella. T3 and T5 achieved statistical differences in the average lesion scores of the three analyzed species. The association of salinomycin and semduramicin used in lower doses than the usual, showed to be an option in the coccidiosis control in this experiment.

Tiamulin and semduramicin: effects of simultaneous administration on performance and health of growing broiler chickens.

The pleuromutilin antibiotic tiamulin (TIA) is known to produce a variety of negative interactive effects when it is administered in combination with several anticoccidial ionophores. A 35-d growth study was performed in cages to evaluate the compatibility of TIA when it was administered concurrently with the poly-ether ionophore anticoccidial semduramicin (SEM). Tiamulin and SEM, both alone and in combination, were administered to 10 replicates of female broilers arranged in a completely randomized block design. Tiamulin was administered in drinking water (250 mg of TIA/kg of water) from d 15 through 19 of the study, whereas SEM was incorporated in feed (25 mg/kg) from placement to the conclusion of the test. Water consumption was determined during the period of concurrent administration of the drugs and weekly measurements of feed intake and bird performance were recorded. In addition, hematocrit, blood cell counts, serum protein, albumin, glucose, uric acid, electrolytes, and activities of several enzymes were determined from blood samples taken at d 35. Results indicated that simultaneous administration of TIA and SEM during the third week of the trial reduced water and feed intake resulting in a temporary growth depression. Feed efficiency was transiently affected during the period of coadministration. However, during the fourth week of the test, negative effects in body weight were not observed for any treatment and feed conversion improved for birds concurrently receiving TIA + SEM. By the termination of the experiment, no adverse effects were observed in final performance for any treatment. Histopathological and hematological parameters were unaffected by treatment at d 35 of the test. These results demonstrated that simultaneous administration of TIA and SEM produced only temporary impairments of water and feed consumption that transiently influenced performance. Neither mortality nor long-term effects on performance variables occurred in broilers.

Phenotypic and genotypic changes in Salmonella enterica subsp. enterica serotype typhimurium during passage in intestines of broiler chickens fed on diets that included ionophore anticoccidial supplements.

The effect of continuous in-feed administration of anticoccidial agents on antimicrobial sensitivity and the level of bacterial shedding in poultry experimentally infected with Salmonella enterica subsp. enterica serotype Typhimurium definitive type 104 (DT104) were investigated. On day 0, 1,200 1-day-old Salmonella-free broiler chicks were placed into 50 pens, and the pens were randomly allocated to one of five treatments: nonsupplemented (negative control; T1), monensin at 120 mg/kg of diet (T2), salinomycin at 60 mg/kg of diet (T3), semduramicin at 20 mg/kg of diet (T4), or semduramicin at 25 mg/kg of diet (T5). Each bird was inoculated with a well-characterized strain of serotype Typhimurium DT104 on day 10. On day 49, the birds were euthanatized humanely. Bulk fecal samples were collected on days 13, 43, and 48 and were examined for organisms which had acquired resistance. The genetic basis of acquired resistance was determined from representative samples of isolates. Of 784 Salmonella-selective plates supplemented with antimicrobial agents, only 33 showed growth. These isolates came from all treatment regimens, including the nonsupplemented control. A number of phenotypic changes were observed; these included changes in motility, phage type, and agglutination properties. Supplementation of the diet with an anticoccidial drug does not appear to affect antimicrobial resistance or the level of excretion of salmonellae. Most of the changes observed do not seem to be related to the presence of a supplement in feed. Salmonellae appear to be capable of acquiring antimicrobial resistance and phenotypic changes independently of specific antimicrobial selection pressures.

The influence of withdrawal time on the performance of broiler chickens fed semduramicin.

Three 49-d experiments, with a total of 6,528 male broiler chicks in floor pens, were conducted to test the hypothesis that Semduramicin feeding-time affects the body weight gain, feed consumption, and feed conversion ratio of broilers. Semduramicin ionophore was added to corn and soybean meal-based diets at the recommended level, 25 mg/kg for 0,34,39, or 42 d. Thus, three withdrawal times were employed (7, 10, and 15 d) during each experiment. Significant differences among experiments were observed for body weight gain, feed intake, and feed conversion ratio, but no significant differences due to Semduramicin were noted in body weight or feed intake. There was only one treatment by experiment interaction found for 0-to-34-d feed intake (P = 0.028), but it was not evident for 0 to 39 d (P = 0.818) or any other times. Feeding Semduramicin with a 10- or 15-d withdrawal period resulted in an improvement in feed conversion of about 0.04 units.

Studies on semduramicin and nutritional responses: 2. methionine levels.

Three experiments were conducted to determine whether feeding semduramicin at recommended levels would affect the broiler chicken's response to dietary methionine. In Experiment 1, three levels of methionine (0, 0.15, 0.30%) were fed to chicks in battery brooders. In Experiment 2, two levels of protein (18 and 22%) and three levels of methionine (0, 0.15, 0.30%) were fed to chicks in floor pens from 18 to 44 d. In Experiment 3, two methionine supplements were fed during each of the starter (0 and 0.21%, 0 to 18 d) and grower (0 and 0.10%, 18 to 35 d) periods to chicks in floor pens. In all experiments, male commercial broiler chicks were used, and all diets were fed with and without 25 mg/kg diet of semduramicin. The basal diets were based on corn, soybean meal, and poultry oil. In Experiment 1, there was a growth and feed conversion ratio response to methionine supplements, but there was no effect of semduramicin on growth nor any semduramicin by methionine supplement interaction. In Experiment 2, at 44 d, protein and methionine levels both influenced feed conversion ratios, but semduramicin did not. Feeding 22 vs 18% protein increased carcass and breast muscle yields and decreased abdominal fat pad weights. The results of Experiments 2 and 3 were very similar. Overall, the effects of semduramicin on growth and processing parameters were small and not significant, but considerable benefits in performance and carcass parameters could be realized by feeding the higher levels of protein or methionine.

Studies on semduramicin and nutritional responses. 1. Level and source of protein.

An experiment was conducted to determine whether feeding semduramicin at recommended levels (25 mg/kg) would affect the broiler chicken's response to dietary protein; and to determine whether protein source (all vegetable, corn and soybean meal, vs 12% high animal protein ingredients) would affect the response to semduramicin. Semduramicin was fed to half of 1,584 male Ross x Ross broilers in floor pens during the growing phase (18 to 35 d). Three protein fortification levels were also fed with protein and amino acid minimum restrictions at 80, 100, and 120% of NRC (1994) recommendations. At 35 d, semduramicin was found to cause a slight growth depression in body weight gain only when low protein levels were fed (P = 0.051). After a 7-d withdrawal period, compensatory gains occurred (P = 0.006) such that there was no interaction effect of semduramicin by protein level for 42-d body weight (P = 0.75). Birds fed the diets containing high animal protein were slightly heavier than those fed control diets containing all-vegetable protein (2.40+/-0.02 vs 2.36+/-0.01 kg/bird; P = 0.059). Semduramicin feeding did not affect feed consumption (2.43+/-0.03 vs 2.40+/-0.02 kg per bird during the growing period when it was fed) so that overall feed conversion ratios were identical to two decimal places (1.90+/-0.02 vs 1.90+0.02). Neither did semduramicin affect feathering score or weight of the No. 8 primary feather, except that feather weight tended to be improved by semduramicin feeding with the diets containing ingredients high in animal protein (P = 0.067).

Effect of semduramicin, salinomycin, and monensin on performance, shank pigmentation, and coccidial lesions in broiler chickens in floor pens.

The effects of 25 ppm semduramicin, 66 ppm salinomycin, 110 ppm monensin, and unmedicated treatments on performance, shank pigmentation, and coccidial lesion scores in broiler chickens were evaluated in two floorpen trials in the United States. On day 24 of each test, birds in each treatment were inoculated via the feed with a mixture of recent field isolates of Eimeria spp. at a dose rate calculated to provide 2 x 10(5) E. acervulina, 3 x 10(4) E. maxima, and 2 x 10(4) E. tenella sporulated oocysts per bird. Weight gain and feed conversion were significantly (P < or = 0.05) improved in the semduramicin-treated broilers in comparison with the monensin-treated and unmedicated broilers. These performance variables for the salinomycin-treated birds were intermediate between the semduramicin- and monensin-treated birds. Shank pigmentation scores were significantly (P < or = 0.05) improved in the three anticoccidial treatments compared with unmedicated birds, with the highest scores (P < or = 0.05) occurring in the semduramicin-treated broilers. Semduramicin was more efficacious (P < or = 0.05) than salinomycin in controlling upper intestinal lesions and more efficacious than monensin in controlling mid-intestinal lesions. All three drugs were comparable in controlling lesions in the ceca.

Anticoccidial efficacy of semduramicin. 1. Evaluation against field isolates by dose titration in battery tests.

Semduramicin (AVIAX), a novel polyether ionophore, was titrated in a series of five battery tests at 20, 25, and 30 ppm in feed to determine the optimum level for use. Twelve-day-old broiler chicks were medicated for 48 h prior to inoculation in each 9-day test. The inocula included monospecific field isolates of Eimeria tenella, Eimeria brunetti, Eimeria necatrix, and Eimeria maxima, and a mixture of these species with Eimeria acervulina and Eimeria mitis. The numbers of oocysts inoculated were selected after titration of each species and the mixture of species. All three concentrations of semduramicin significantly (P < .05) reduced coccidiosis mortality and lesion scores and achieved lower feed:gain ratios and greater weight gains than the infected, unmediated treatments. A concentration of 25 ppm semduramicin was determined to be optimal based on improved lesion control compared with 20 ppm and improved weight gain compared with 30 ppm.