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For the first time, this paper aimed to evaluate nine ionophore and synthetic coccidiostat residues in poultry muscle samples, obtained from different production types, by solid-liquid extraction followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The fully validated methodology was successfully applied to a total of 101 chicken and turkey samples obtained from canteens, supermarkets, and home productions in Portugal. Halofuginone, diclazuril, decoquinate, narasin, lasalocid, and salinomycin were detected in 20.8% of the samples. Home raised samples showed a greater frequency, 47.1%. The synthetic coccidiostats halofuginone, diclazuril, and decoquinate were found in averages of 0.7 μg kg-1,2.9 μg kg-1, and 3.7 μg kg-1, respectively, while averages of 1.2 μg kg-1, 1.6 μg kg-1, and 1.3 μg kg-1 were found regarding the ionophores narasin, lasalocid, and salinomycin. As for the risk assessment, values lower than 8.06% of the acceptable daily intake (ADI) were observed, indicating that exposure to coccidiostats through consumption of poultry meat does not represent risk to consumers.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9–24.0 and 4.7–16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2–92.7%(CV

The poor outcomes in retinoblastoma necessitate new treatments. Salinomycin is an attractive candidate, and has demonstrated selective anti-cancer properties in different cancer types. This work addressed the efficacy of salinomycin in retinoblastoma models and probe the associated mechanisms. Cellular functional assays were conducted to determine the effects salinomycin in vitro. Xenograft retinoblastoma mouse model was established to investigate the efficacy of salinomycin in vivo. Biochemical assays were conducted to analyze the mechanism of salinomycin's action focusing on mitochondrial functions, energy reduction-related signaling pathways. Salinomycin has positive effects towards retinoblastoma cells regardless of heterogeneity through suppressing growth and inducing apoptosis. Salinomycin also specifically inhibits cells displaying stemness and highly invasive phenotypes. Using retinoblastoma xenograft mouse model, we show that salinomycin at non-toxic dose effectively inhibits growth and induces apoptosis. Mechanistic studies show that salinomycin inhibits mitochondrial respiration via specifically suppressing complex I and II activities, reduces mitochondrial membrane potential and decreases energy reduction, followed by induction of oxidative stress and damage, AMPK activation and mTOR inhibition. Our study highlights that adding salinomycin to the existing treatment armamentarium for retinoblastoma is beneficial.

The current in vitro study aimed to investigate the effects of a processed sugarcane extract on the viability of avian Eimeria sporozoites. Treatments were applied to hatched sporozoites: 1) without additives (no-treatment control); 2) with ethanol; 3) with salinomycin; 4) with Polygain™. All treatments were incubated in RPMI media containing live sporozoites at 37 °C for 14 h and then the number of viable sporozoites were counted. Compared to the no-treatment control, Polygain™ decreased (P 0.05). In conclusion, Polygain™ could be a potential candidate as an anticoccidial agent., Graphical abstract Image 1

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.

Rationale: Cardiomyopathy is characterized by the deposition of extracellular matrix by activated resident cardiac fibroblasts called myofibroblasts. There are currently no therapeutic approaches to blunt the development of pathological fibrosis and ventricle chamber stiffening that ultimately leads to heart failure. Objective: We undertook a high-throughput screen to identify small molecule inhibitors of myofibroblast activation that might limit the progression of heart failure. We evaluated the therapeutic efficacy of the polyether ionophore salinomycin in patient-derived cardiac fibroblasts and preclinical mouse models of ischemic and nonischemic heart failure. Methods and Results: Here, we demonstrate that salinomycin displays potent anti-fibrotic activity in cardiac fibroblasts obtained from heart failure patients. In preclinical studies, salinomycin prevents cardiac fibrosis and functional decline in mouse models of ischemic and nonischemic heart disease. Remarkably, interventional treatment with salinomycin attenuates preestablished pathological cardiac remodeling secondary to hypertension and limits scar expansion when administered after a severe myocardial infarction. Mechanistically, salinomycin inhibits cardiac fibroblast activation by preventing p38/MAPK (mitogen activated protein kinase) and Rho signaling. Salinomycin also promotes cardiomyocyte survival and improves coronary vessel density, suggesting that cardioprotection conferred by salinomycin occurs via the integration of multiple mechanisms in multiple relevant cardiac cell types. Conclusions: These data establish salinomycin as an antifibrotic agent that targets multiple cardioprotection pathways, thereby holding promise for the treatment of heart failure patients.

Background: It is important to understand the molecular mechanisms involved in cancer drug resistance and to study the activity of new drugs, e.g. salinomycin. Objective: The purpose of the study was to analyze changes in the expression of genes associated with drug resistance in the Ishikawa endometrial cancer cell line when treated with salinomycin. In addition, changes in the level of miRNA potentially regulating these mRNAs were evaluated. Materials and Methods: Endometrial cancer cells were treated with 1 μM of salinomycin for 12, 24 and 48 hours periods. Untreated cells were a control culture. The molecular analysis consists of mRNA and miRNA microarray analysis and the RTqPCR technique. Results: The following was observed about the number of mRNAs differentiating the cell culture exposed to the drug compared to a control culture: H-12 vs. C - 9 mRNAs, H_24 vs. C - 6 mRNAs, and H_48 vs. C - 1 mRNA. It was noted that 4 of the 9 differentiating mRNAs were characteristic for 12 hours of exposure to salinomycin and they correspond to the following genes: TUFT1, ABCB1, MTMR11, and MX2. After 24 hours, 2 mRNAs were characteristic for this time of incubation cells with salinomycin: TUFT1 and MYD88 and after 48 hours, SLC30A5 could also be observed. Discussion: The highest differences in expression were indicated for TUFT1, MTMR11, and SLC30A5. The highest influence probability was determined between TUFT1 and hsa- miR-3188 (FC + 2.48), MTMR11and has-miR-16 (FC -1.74), and between SLC30A5 and hsa-miR-30d (FC -2.01). Conclusions: Salinomycin induces changes in the activity of mRNA and miRNA participating in drug resistance; however, the observed changes in character are the expected result of anti-cancer treatment.

Intra-tumor heterogeneity is widely accepted as one of the key factors, which hinders cancer patients from achieving full recovery. Especially, cancer stem cells (CSCs) may exhibit self-renewal capacity, which makes it harder for complete elimination of tumor. Therefore, simultaneously inhibiting CSCs and non-CSCs in tumors becomes a promising strategy to obtain sustainable anticancer efficacy. Salinomycin (Sal) was reported to be critical to inhibit CSCs. However, the poor bioavailability and catastrophic side effects brought about limitations to clinical practice. To solve this problem, we previously constructed gelatinase-stimuli nanoparticles composed of nontoxic, biocompatible polyethylene glycol-polycaprolactone (PEG-PCL) copolymer with a gelatinase-cleavable peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG) inserted between the two blocks of the copolymer. By applying our “smart” gelatinase-responsive nanoparticles for Sal delivery, we have demonstrated specific accumulation in tumor, anti-CSCs ability and reduced toxicity of Sal-NPs in our previous study. In the present study, we synthesized Sal-Docetaxel-loaded gelatinase-stimuli nanoparticles (Sal-Doc NP) and confirmed single emulsion as the optimal method of producing Sal-Doc NPs (Sal-Doc SE-NP) in comparison with nanoprecipitation. Sal-Doc SE-NPs inhibited both CSCs and non-CSCs in mice transplanted with cervical cancer, and might be associated with enhanced restriction of epithelial-mesenchymal transition (EMT) pathway. Besides, the tumorigenic capacity and growing speed were obviously suppressed in Sal-Doc-SE-NPs-treated group in rechallenge experiment. Our results suggest that Sal-Doc-loaded gelatinase-stimuli nanoparticles could be a promising strategy to enhance antitumor efficacy and reduce side effects by simultaneously suppressing CSCs and non-CSCs.

SummaryThis research aimed to examine the effects of thyme, celery and salinomycin on ovarian sex hormones, reproductive traits and antioxidant status during the estrous cycle. Seventy-five mature Barki ewes aged 2–3 years with an average weight of 40 ± 1.5 kg were assigned randomly into five groups (15 head/group). Group 1 was kept as the control; groups 2 and 3 received 20 g/head/day thyme (T) and celery (C) as dried herbs, respectively. Group 4 (T×C) received 10 g thyme + 10 g celery/head/day, and group 5 was treated with salinomycin 1 g/head/day. Blood samples were collected during follicular and luteal phases of the estrous cycle. Thyme and celery and the mixture of T×C increased (P < 0.01) estradiol-17β (E2) during the follicular phase of the estrous cycle, while only the celery group showed a marked (P < 0.001) increase in progesterone (P4) during the luteal phase compared with the control. Salinomycin supplementation decreased (P < 0.05) E2 concentrations during the follicular and luteal phases of the estrous cycle. Supplementation with thyme and celery enhanced (P < 0.001) antioxidant capacity in the luteal phase compared with the follicular stage. The salinomycin group showed increased (P < 0.01) levels of reduced glutathione (GSH) and decreased malondialdehyde (MDA) levels compared with the control group throughout luteal phase. For the interaction between estrous phases and treatments, thyme, celery, and T×C supplementation revealed an increase (P < 0.05) in superoxide dismutase (SOD), GSH, and glutathione disulfide (GSSG) levels compared with the control group during the follicular and luteal phases. Thyme and celery supplementation improved the number of services per conception and fertilization from 1st and 2nd inseminations, respectively. In conclusion, the applied treatment had significant effects on reproductive performance and antioxidant status in ewes throughout the estrous cycle.

Several literature has shown that salinomycin (Sal) is able to kill various types of cancer cells through different signaling pathways. However, its effect on melanoma has seldom been reported. We examined the anti-cancer efficacy of Sal in melanoma cell lines, and found six of eight cell lines were sensitive to Sal. Given the fact that the roles of Sal are diverse in different cancer types, we were eager to figure out the mechanism involved in the current study. We noticed the most sensitive line, SK-Mel-19, showed a typical morphological change after Sal treatment. The autophagy inhibitor, 3-MA, could effectively suppress Sal-induced cell death. It could also facilitate the increase of autophagic markers and reduce the turnover of autophagosomes, which resulted in an aberrant autophagic flux. On the other hand, Sal could stimulate endoplasmic reticulum stress and cause an accumulation of dysfunctional mitochondria. We also discovered a potential correlation between LC3B mRNA level and its sensitivity to Sal in 43 clinical melanoma samples. Overall, our results indicated that Sal could have multiple effect on melanoma cells and induce autophagic cell death in certain kinds of cells, which provided a new insight into the chemotherapy for melanoma.

Studies were conducted to determine the efficacy of synbiotic applications to combat the negative effects of necrotic enteritis (NE). An in vitro study was conducted to test the effect of probiotics species supernatants to decrease Clostridium perfringens (CP) proliferation. Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici culture supernatants decreased the proliferation of CP at 1:1 supernatant-to-pathogen dilution in vitro. Two in vivo studies were conducted to determine the in vivo response of synbiotic supplementation containing the aforementioned probiotic strains on broiler production performance and caecal CP load in broilers induced with NE infection. In experiment 1, 75 broiler chicks were randomly allotted to 3 treatment groups, control (basal diet), ionophore (Salinomycin), and synbiotic (PoultryStar me), from day of hatch, and NE was induced in all birds. There were no significant treatment effects on BW, feed consumption, and feed gain ratio. However, at 35 D, ionophore or synbiotic supplementation increased (P

Salinomycin (SAL) is one of the first discovered inhibitors of human cancer stem cells (CSCs), which acts via blocking the Wnt/β-catenin pathway. However, SAL has not been clinically used to treat human diseases due to its poor aqueous solubility and considerable toxicity. In this study, we developed salinomycin nanocrystals (SAL NCs) to treat colorectal cancer through the inhibitory enhancement of Wnt/β-catenin signaling. The as-prepared SAL NCs exhibited excellent size distribution, stability, and improved water solubility. In vitro cellular uptake and in vivo fluorescence imaging studies showed that SAL NCs increased cellular uptake efficiency compared with free SAL. As a result, SAL NCs exhibited significant higher cytotoxicity, 1.5-3 times better Wnt inhibitory effect, and 10 times better cancer stem cell inhibitory effect than free SAL. Furthermore, compared with free SAL, SAL NCs exhibited 2 times better anti-colon tumor effect in APCmin/+ transgenic mice through oral administration. Our results indicated that SAL NCs with enhanced cellular internalization and tumor tissue accumulation may be a promising agent for colorectal cancer management.

Lead (Pb) exposure induces severe nephrotoxic effects in humans and animals. Herein, we compare the effects of two chelating agents, salinomycin and deferiprone, on Pb-induced renal alterations in mice and in the homeostasis of essential elements. Adult male mice (Institute of Cancer Research (ICR)) were randomized into four groups: control (Ctrl)—untreated mice administered distilled water for 28 days; Pb-exposed group (Pb)—mice administered orally an average daily dose of 80 mg/kg body weight (BW) lead (II) nitrate (Pb(NO3)2) during the first two weeks of the experimental protocol followed by the administration of distilled water for another two weeks; salinomycin-treated (Pb + Sal) group—Pb-exposed mice, administered an average daily dose of 16 mg/kg BW salinomycin for two weeks; deferiprone-treated (Pb + Def) group—Pb-exposed mice, administered an average daily dose of 20 mg/kg BW deferiprone for 14 days. The exposure of mice to Pb induced significant accumulation of the toxic metal in the kidneys and elicited inflammation with leukocyte infiltrations near the glomerulus. Biochemical analysis of the sera revealed that Pb significantly altered the renal function markers. Pb-induced renal toxicity was accompanied by a significant decrease in the endogenous renal concentrations of phosphorous (P), calcium (Ca), copper (Cu) and selenium (Se). In contrast to deferiprone, salinomycin significantly improved renal morphology in Pb-treated mice and decreased the Pb content by 13.62% compared to the Pb-exposed group. There was also a mild decrease in the renal endogenous concentration of magnesium (Mg) and elevation of the renal concentration of iron (Fe) in the salinomycin-treated group compared to controls. Overall, the results demonstrated that salinomycin is a more effective chelating agent for the treatment of Pb-induced alterations in renal morphology compared to deferiprone.

This study was conducted to investigate the effect of insect full-fat meals (Tenebrio molitor and Zophobas morio larvae), added “on top” of a complete diet or calculated into diets, on the growth performance, selected blood, and immune system traits of broiler chickens. 1,000 one-day-old female Ross 308 broiler chicks were used in 2 independent experiments. In the first trial, the birds were randomly assigned to 6 treatments, 10 replicate pens per treatment, and 10 birds per pen, i.e., negative control; positive control with salinomycin addition (60 mg/kg diet), and addition of 0.2% and 0.3% of T. molitor and Z. morio full-fat meals “on top”. In the second experiment, 4 treatments, 10 replicate pens per treatment, and 10 birds per pen were set, i.e., negative control, positive control with salinomycin addition (60 mg/kg diet), and 0.3% of T. molitor and Z. morio full-fat meals calculated in the diets. In both trials the supplementation of insects increased the BWG (Exp. 1: P = 0.024; Exp. 2: P = 0.046) and FI (Exp. 1: P = 0.022; Exp. 2: P = 0.026), and no negative effect on the FCR was recorded in experiment one (P = 0.514), however in second trial insects addition increased FCR values (P = 0.011). In addition, in the first trial, groups fed insects and PC comparing to NC decreased the IgY (P = 0.045) and IgM, (P < 0.001) levels. In the second experiment, IgM levels were also decreased (P < 0.001) in groups fed insects comparing to NC. Moreover, in first trial the IgM levels were negatively correlated to the BWG (r = –0.4845) and FI (r = –0.4986), with statistically significant values (P < 0.001). In conclusion, the current results confirmed that small amount addition (0.2% and 0.3%) of T. molitor and Z. morio full-fat meals to the diet of broiler chickens can improve growth performance and change selected the immune system traits.

Subclinical necrotic enteritis (NE) is primarily caused by the gram-positive bacterium, Clostridium perfringens (Cp). The trend towards removal of in-feed antimicrobials and subsequent increased emergence of infection in poultry has resulted in a wide interest in better understanding of the mechanism behind this disease. The virulence of NE, to a large extent, depends on the virulence of Cp strains. Thus, this study was to assess how 2 different strains of Cp affect performance and gut characteristics of broiler chickens. Ross 308 male broilers (n = 468) were assigned to a 2 × 3 factorial arrangement of treatments with antibiotics (Salinomycin at 72 ppm and zinc bacitracin at 50 ppm -, or +) and challenge (non-challenge, Cp EHE-NE18, or Cp WER-NE36). Oral administration of Eimeria oocysts (day 9) followed by inoculation with 1 mL 108 CFU Cp strains (day 14 and 15) were used to induce NE. Broiler performance was analyzed at day 10, 24, and 35. On day 16, intestinal lesion score and intestinal pH were evaluated and samples of cecal content were analyzed for bacterial counts and short-chain fatty acid concentrations (SCFA). Birds in both challenged groups showed higher feed conversion ratio (FCR), lower weight gain (P < 0.001), increased lesion scores in the jejunum (P < 0.01), and reduced pH in the ileum and cecum (P < 0.01), compared to the non-challenged birds. They also showed decreased numbers of Bacillus spp. (P < 0.001), and Ruminococcus spp. (P < 0.01) in the cecal content. On day 35, the NE36 challenged birds had a lower weight gain (P < 0.001) and higher FCR (P < 0.001) compared to the NE18 challenged birds. Interestingly, cecal Lactobacillus and lactate were increased by the NE challenge (P < 0.001), and to a greater extent in birds challenged with NE36 compared to the NE18 strain (P < 0.001). This study suggests that Cp strains varying in virulence produce different levels of disease in broiler chickens through modulating the gut environment, intestinal microbiota, and SCFA profile to different extents.

Background Two experiments were conducted to compare the growth-promoting (experiment 1), protective, and immunostimulatory effects (experiment 2) of salinomycin, probiotic, a vitamin-selenium complex, and Ferulago angulata hydroalcoholic extract (FAE) against coccidiosis in broilers. In each experiment, 350 1-day-old broiler chickens were equally divided in 7 groups: uninfected negative control (NC); infected positive control (PC); or PC supplemented with salinomycin (Sal); probiotic (Pro); a combination of vitamin E, vitamin C, and selenium (ECSe); 200 mg/kg of FAE (FAE200); or 400 mg/kg of FAE (FAE400). All these groups (except NC) were challenged via oral gavage with oocysts of mixed Eimeria spp. on d 10 (experiment 1) or d 14 (experiment 2). Results In the first trial, all treatments improved growth and feed conversion compared with the PC group, where the best values were noticed in the NC and FAE400 groups throughout the entire experimental period (d 1 to 42). Further, a lower mortality rate (P P Conclusions Considering the results, dietary FAE, especially at high levels of inclusion in broiler diet (400 mg/kg), could beneficially influence growth performance and immune status under coccidiosis challenge, which was comparable to that of probiotic supplement.

Jie Gao,1,2,* Junjie Liu,1,3,* Fangyuan Xie,4,* Ying Lu,3 Chuan Yin,5 Xian Shen1 1Department of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 2Scientific Research Center, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 3Department of Pharmaceutical Sciences, Second Military Medical University, Shanghai, People’s Republic of China; 4Department of Pharmacy, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, People’s Republic of China; 5Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jie Gao; Xian ShenDepartment of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan W Road, Lucheng, Wenzhou, Zhejiang 325000, People’s Republic of ChinaTel +86-0577-88816381; 86-0577-88002709Fax +86-0577-88816381Email gaojiehighclea@163.com; shenxian1963@sina.comPurpose: Conventional chemotherapy is hampered by the presence of breast cancer stem cells (BCSCs). It is crucial to eradicating both the bulky breast cancer cells and BCSCs, using a combination of conventional chemotherapy and anti-CSCs drugs. However, the synergistic ratio of drug combinations cannot be easily maintained in vivo. In our previous studies, we demonstrated that the simultaneous delivery of two drugs via nanoliposomes could maintain the synergistic drug ratio for 12 h in vivo. However, nanoliposomes have the disadvantage of quick drug release, which makes it difficult to maintain the synergistic drug ratio for a long time. Herein, we developed a co-delivery system for docetaxel (DTX)—a first-line chemotherapy drug for breast cancer—and salinomycin (SAL)—an anti-BCSCs drug—in rigid nanoparticles constituted of polylactide-co-glycolide/D-alpha-tocopherol polyethylene glycol 1000 succinate (PLGA/TPGS).Methods: Nanoparticles loaded with SAL and DTX at the optimized ratio (NSD) were prepared by the nanoprecipitation method. The characterization, cellular uptake, and cytotoxicity of nanoparticles were investigated in vitro, and the pharmacokinetics, tissue distribution, antitumor and anti-CSCs activity of nanoparticles were evaluated in vivo.Results: We demonstrated that a SAL/DTX molar ratio of 1:1 was synergistic in MCF-7 cells and MCF-7-MS. Moreover, the enhanced internalization of nanoparticles was observed in MCF-7 cells and MCF-7-MS. Furthermore, the cytotoxicity of NSD against both MCF-7 cells and MCF-7-MS was stronger than the cytotoxicity of any single treatment in vitro. Significantly, NSD could prolong the circulation time and maintain the synergistic ratio of SAL to DTX in vivo for 24 h, thus exhibiting superior tumor targeting and anti-tumor activity compared to other treatments.Conclusion: Co-encapsulation of SAL and DTX in PLGA/TPGS nanoparticles could maintain the synergistic ratio of drugs in vivo in a better manner; thus, providing a promising strategy for synergistic inhibition of breast cancer.Keywords: combined therapy, breast cancer stem cells, docetaxel, salinomycin, nanoparticles

In this study, we present experimental data on the effects of meso-2,3-dimercaptosuccinic acid (DMSA) and tetraethylammonium salt of salinomycinic acid (Sal) on cadmium-induced spleen dysfunction and altered essential metal balance in mice. Sixty-day-old male mice (ICR line) were randomly divided into four groups: untreated control group (Ctrl)-obtained distilled water for 28 days, toxic control group (Cd)-exposed to cadmium acetate dihydrate at average daily dose of 20mg/kg body weight (BW) for 14 days, Cd + DMSA group-obtained cadmium acetate dihydrate as the toxic control group followed by treatment with 20mg/kg BW DMSA for 2 weeks, and Cd + Sal group-mice exposed to cadmium acetate dihydrate at average daily dose of 20mg/kg BW for 2 weeks followed by administration of Sal at an average daily dose of 20mg/kg BW for 2 weeks. The compounds were administered orally via the drinking water of the animals. We found that cadmium exposure caused splenomegaly and reduced the hemoglobin and hematocrit levels and total red blood cell count compared with untreated controls. Cadmium intoxication of mice induced accumulation of the toxic metal ion in the blood and spleen. Alterations in the endogenous levels of calcium (Ca) and iron (Fe) in the spleen of cadmium-exposed mice compared with those in untreated controls were observed. Treatment of cadmium-exposed mice with DMSA or Sal recovered the spleen weight and hematological parameters to normal control values, decreased cadmium concentration in the blood and spleen, and improved splenic architecture. The results prove that Sal is a potential antidote for treatment of Cd-induced spleen dysfunction.

The objective of this study was to assess the effect of dietary yeast products on broiler chickens challenged with salmonella lipopolysaccharide (LPS). The chicks were divided into 8 treatments with 6 replicates and 9 birds per replicate. The treatments consisted of a positive control (PC) [without supplementation and not challenged]; negative control (NC) [without supplementation but challenged]; whole yeast and challenged; yeast cell wall and challenged; yeast glucan and challenged; yeast mannan and challenged; zinc bacitracin and challenged; and Salinomycin and challenged. Whole yeast or Yeast cell wall was included at 2.0 g/kg diet. Yeast glucan or mannan was added at 0.20 g/kg diet. Zinc bacitracin (ZNB) and Salinomycin (SAL) was included at 50 and 60 ppm, respectively. Dietary treatments had no effect (P > 0.05) on feed intake (FI) at day 10. Supplementation with yeast and its derivatives improved (P < 0.05) body weight gain (BWG) and feed conversion ratio (FCR) on day 10. On days 24 and 35, LPS challenge declined FI, BWG, FCR, and flock uniformity (day 28) in the NC group compared to the PC group. Yeast products and antibiotics improved (P < 0.05) FI, BWG, FCR, and flock uniformity in LPS-challenged birds. On day 24, spleen weight increased while bursa weight decreased in the NC group relative to the PC group; this effect was reversed (P < 0.05) by feeding all yeasts and antibiotics. On day 24, application of all the dietary treatments ameliorated the changes observed in white blood cell, lymphocyte and monocyte counts as well as albumin and immunoglobulin G of NC birds. On day 35, all yeasts additives, ZNB and SAL improved (P < 0.05) the meat yield of broilers challenged with LPS. In conclusion, supplementation of diets with yeast and its derivatives can ameliorate the negative effects of salmonella LPS challenge on broiler chicks, thus improving the performance, flock uniformity, and meat yield.

Background and purpose Salinomycin is a well-known inhibitor of human cancer stem cells (CSCs). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models. Experimental approach The inhibitory effect of salinomycin on the Wnt/β-catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real-time PCR. Effects of salinomycin on β-catenin/TCF4E interaction were examined using co-immunoprecipitation and an in vitro GST pull-down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APCmin/+ transgenic mice, and patient-derived colorectal tumour xenografts. Key results Salinomycin blocked β-catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull-down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by β-catenin/LEF1 or β-catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage-independent growth of colorectal cancer cells. In colorectal tumour xenografts and APCmin/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC-related Wnt target genes including LGR5. Conclusions and implications Our study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the β-catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment.

Although salinomycin sodium (SS) has shown in-vitro potential to inhibit cancer stem cell growth and development, its low water solubility makes it a poor candidate as an oral chemotherapeutic agent. To improve the bioavailability of SS, SS was encapsulated here using D-α-tocopherol polyethylene glycol succinate (TPGS)-emulsified poly(lactic-co-glycolic acid) (PLGA) nanoparticles and compared with its parent SS in terms of absorption, pharmacokinetics, and efficacy in suppressing nasopharyngeal carcinomas stem cells. The pharmacokinetics of SS and salinomycin sodium-loaded D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles (SLN) prepared by nanoprecipitation were analyzed in-vivo by timed-interval blood sampling and oral administration of SS and SLN to rats. Sensitive liquid chromatography-mass spectrometry (LC-MS) was developed to quantify plasma drug concentrations. SS and SLN transport in Caco-2 cells was also investigated. The therapeutic efficacy of SS and SLN against cancer stem cells was determined by orally administering the drugs to mice bearing CNE1 and CNE2 nasopharyngeal carcinoma xenografts and then evaluating CD133 cell proportions and tumorsphere formation. The in-vivo trial with rats showed that the Cmax, AUC(0-t), and Tmax for orally administered SLN were all significantly higher than those for SS (P

Cadmium (Cd) is an environmental pollutant shown to induce multi organ dysfunction. In this study we present novel data about the effects of meso-2,3-dimercaptosuccinic acid (DMSA), monensin and salinomycin on the concentration of Cd in skeletal muscles of mice exposed to Cd (II) acetate treatment for 14 days. The impact of Cd and the chelating agents on the endogenous concentrations of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorous (P), selenium (Se) and zinc (Zn) was also investigated. Subacute exposure of mice to Cd (II) acetate resulted in a significant accumulation of the toxic metal ion in the skeletal muscles compared to the untreated controls. Salinomycin most effectively mobilized Cd from the muscles compared to DMSA and monensin. The Cd exposure and the tested chelating agents did not significantly alter the endogenous concentrations of the selected essential elements in mouse muscles. The presented results confirmed that among the tested chelating agents salinomycin is superior as a potential antidote to Cd poisoning.

The anticoccidial influences of various amounts of Cinnamomum verum powder were compared with that of salinomycin as standard synthetic anticoccidial drug on the anticoccidial indicators and production performance in broilers experimentally exposed to coccidiosis. Broiler chicks at 1 d of age (n = 150) were arbitrarily distributed into 6 groups. Birds from groups 1-3 were received the starter and finisher diets plus 2, 4, and 6 g of cinnamon/kg of the diet, respectively. Birds from group 4 were fed the starter and finisher diets plus 66 mg of salinomycin, group 5 constituted the positive control (PC), with the coccidial challenge, and group 6 constituted the negative control (NC), without the coccidial infection, which were both maintained on diets without any cinnamon. The results showed that BW gain, feed conversion ratio, and production efficiency factor declined considerably (P

Rotation with different active ingredients is among the most effective and recommended strategies to preserve the efficacy of anticoccidial drugs and reduce the emergence of resistance. Tools such as anticoccidial sensitivity tests (ASTs) are ideally used to make rational rotation programs and bring benefits to production. The objective of this study was to evaluate the sensitivity of E. acervulina (EA) and E. maxima (EM) from 3 different regions in Brazil, by using four ASTs. Feces samples weighing 6 to 7 kg were collected in the regions of São Paulo, Paraná, and Minas Gerais. Prevalent oocysts from feces were filtered, identified, and quantified to conduct 2 ASTs with EA and 2 with EM. The same experimental design was used in every AST (4 replicates per treatment, with 6 birds each, for a total of 240 birds). Treatment groups were a nonchallenged and nonmedicated control group (T1), a challenged and nonmedicated control group (T2), and the other groups challenged and treated with the following compounds: lasalocid (90 ppm – T3), maduramycin (6 ppm – T4), decoquinate (30 ppm – T5), nicarbazin+semduramicin (66 ppm – T6), monensin (110 ppm – T7), salinomycin (66 ppm – T8), narasin+nicarbazin (100 ppm – T9), and nicarbazin (125 ppm – T10). At the end of each AST (20 d), the percent change (delta value) between the treated group (T3 to T10) and the control group (T2) was calculated for the following variables: body weight gain, feed conversion ratio, lesion score, and an indicator of percentage of optimal anticoccidial activity (POAA) that included T2. Different sensitivity levels of EA and EM isolates could be identified. As a whole, drugs from T5 and T3 groups showed higher delta values when compared to other compounds, whereas the lowest sensitivity levels of these isolates were observed in groups T4 and T7. Despite some limiting factors, ASTs can be a good tool for strategic selection of anticoccidial drugs in order to maintain efficacy and extend the lifespan of these molecules.

In this study, we compare the effects of deferiprone (Def) and tetraethylammonium salt of salinomycinic acid (Sal) on lead (Pb)-induced toxicity in testes of Pb-exposed mice. Mature male ICR mice were allocated into four groups as follows: untreated control mice (ctrl)-received distilled water for 4 weeks; Pb-exposed mice (Pb)-subjected to 14-day Pb (II) nitrate administration at dose 80 mg/kg body weight (b.w.); Pb + Def group-Pb-exposed mice, treated with 20 mg/kg b.w. Def for 2 weeks; and Pb + Sal group-Pb-intoxicated mice, treated with 16 mg/kg b.w. Sal for 14 days. The results demonstrated that Pb exposure significantly increased blood and testicular Pb concentrations, decreased testicular calcium (Ca) content, significantly elevated testicular levels of magnesium (Mg), zinc (Zn), and selenium (Se) but did not significantly affect the endogenous contents of phosphorous (P) and iron (Fe) compared with untreated controls. Pb intoxication induced disorganization of the seminiferous epithelium. Def or Sal administration reduced blood Pb and testicular Pb concentrations in Pb-exposed mice compared with the Pb-intoxicated group. Mg, Zn, and Se concentrations in testes of Pb-exposed mice, treated with Def or Sal, remained higher compared with the untreated controls. Sal significantly increased testicular P concentration compared with untreated controls and significantly elevated the testicular Ca and Fe concentrations compared with the toxic control group. Both chelating agents improved testicular morphology to a great extent. The results demonstrate the potential of both compounds as antidotes for treatment of Pb-induced impairment of male reproductive function.

Pandemic spread of emerging human pathogenic viruses, such as the current SARS-CoV-2, poses both an immediate and future challenge to human health and society. Currently, effective treatment of infection with SARS-CoV-2 is limited and broad spectrum antiviral therapies to meet other emerging pandemics are absent leaving the World population largely unprotected. Here, we have identified distinct members of the family of polyether ionophore antibiotics with potent ability to inhibit SARS-CoV-2 replication and cytopathogenicity in cells. Several compounds from this class displayed more than 100-fold selectivity between viral-induced cytopathogenicity and inhibition of cell viability, however the compound X-206 displayed >500-fold selectivity and was furthermore able to inhibit viral replication even at sub-nM levels. The antiviral mechanism of the polyether ionophores is currently not understood in detail. We demonstrate, e.g. through unbiased bioactivity profiling, that their effects on the host cells differ from those of cationic amphiphiles such as hydroxychloroquine. Collectively, our data suggest that polyether ionophore antibiotics should be subject to further investigations as potential broad-spectrum antiviral agents., Highlights • The polyether ionophore antibiotic X-206 is revealed as a potent inhibitor of the replication of SARS-CoV-2 in vitro. • Other members of the same compound family also display strong antiviral potency although selectivity differs widely. • Morphological profiling allows mechanistic comparison between X-206 and cationic amphiphiles, such as hydroxychloroquine. • Further pre-clinical development of X-206, and other polyether ionophores, as antiviral agents is warranted.

Accepted Manuscript version. The Published Journal Article is available on Materials Science and Engineering: C, Volume 122, Article number 111899 (DOI: https://doi.org/10.1016/j.msec.2021.111899). Supplementary Material available free of charge on the article webpage. © 2021. This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT The high rates of aggressiveness, drug resistance and relapse of breast cancer (BC) are mainly attributed to the inability of conventional therapies to equally eradicate bulk differentiated cells and cancer stem cells (CSCs). To improve the effectiveness of BC treatments, we report the in-water synthesis of novel keratin-based nanoformulations, loaded with the CSC-specific drug salinomycin (SAL), the photosensitizer chlorin e6 (Ce6) and vitamin E acetate (SAL/Ce6@kVEs), which combine the capability of releasing SAL with the production of singlet oxygen upon light irradiation. In vitro experiments on BC cell lines and CSC-enriched mammospheres exposed to single or combined therapies showed that SAL/Ce6@kVEs determine synergistic cell killing, limit their self-renewal capacity and decrease the stemness potential by eradication of CSCs. In vivo experiments on zebrafish embryos confirmed the capacity of SAL nanoformulations to interfere with the Wnt/β-catenin signaling pathway, which is dysregulated in BC, thus identifying a target for further translation into pre-clinical models.

Polyether ionophores, with >120 molecules belonging to this group, represent a class of naturally-occurring compounds that exhibit a broad range of pharmacological properties, including promising activity towards a variety of parasites. In this context, salinomycin (SAL) seems to be interesting, as this ionophore has been found to be active against parasites that are responsible for a number of human and animal diseases. On the other hand, less explored is the investigation into the anti-parasitic activity of SAL derivatives. Recently, we identified C1 amides and esters of SAL and its analogue, C20-oxosalinomycin, as promising structures for trypanocidal drug candidates. In search for novel compounds effective against African trypanosomes, the synthetic access to a completely new series of C20-epi-salinomycin (compound 2) analogues is described in this paper. This series includes products obtained via derivatisation of either the C1 carboxyl or the C20 hydroxyl of 2, but also C1/C20 double modified derivatives. The anti-trypanosomal activity as well as the cytotoxic activity of these analogues were evaluated with bloodstream forms of T. brucei and human myeloid HL-60 cells, respectively. It was found that the C20 single modified derivatives 8, 12, and 18 (C20 decanoate, C20 ethyl carbonate, and C20 allophanate of 2, respectively) were the most active compounds in selectively targeting bloodstream-form trypanosomes, with 50% growth inhibition (GI50) values of 0.027–0.043 μM and selectivity indices of 165–353. These results indicate that modification at the C20 position of C20-epi-salinomycin 2 can provide semi-synthetic products with enhanced trypanocidal activity that could be of great value for the development of new drugs to treat African trypanosomiasis.

The study investigated the effect of in-feed administration of dried thyme leaf and celery seed mixture (at 1 : 1 DM basis) compared with salinomycin ionophore on milk production and milk nutritive value of Barki ewes. Thirty ewes (37.5 ± 1.8 kg), divided into 3 treatment groups, were fed: (1) a complete control diet comprising concentrates and fodder maize (Zea mays L.) at 60 : 40 dry matter basis, (2) the control diet plus 20 g of thyme and celery mixture supplementation and (3) the control diet supplemented with 1 g of salinomycin per ewe daily for 90 days. Inclusion of thyme–celery treatment increased (P < 0.05) weight gain, average daily gain, milk yield, milk component yields, and feed efficiency, without affecting milk composition. In addition, the thyme–celery treatment enhanced (P < 0.05) nutrient intake and digestibility, total ruminal volatile fatty acids, branched chain fatty acids, and acetate proportions and decreased ammonia-N concentration. Thyme–celery treatment increased (P < 0.05) serum glucose, thyroxine, and glutamate-pyruvate transaminase concentrations. It is concluded that the thyme and celery mixture (1 : 1 DM basis) at 20 g per lactating ewe daily can replace the salinomycin ionophore. Enhanced feed utilization and lactational performance as well as milk nutritive value for human consumption were observed with the natural additive mixture supplementation.

The aim of the present study was to evaluate the inclusion of narasin, salinomycin, or flavomycin for 140 d on ruminal fermentation parameters, apparent nutrient digestibility, and performance of Nellore cattle offered a forage-based diet. In experiment 1, 32 rumen-cannulated Bos indicus Nellore steers [initial body weight (BW) = 220 ± 12.6 kg] were assigned to individual pens in a randomized complete block design according to their initial shrunk BW. Within block, animals were randomly assigned to 1 of 4 treatments: (1) forage-based diet without feed additives (CON; n = 8), (2) CON diet plus 13 ppm of narasin (NAR; n = 8), (3) CON diet plus 20 ppm of salinomycin (SAL; n = 8), or (4) CON diet plus 3 ppm of flavomycin (FLA; n = 8). The experimental period lasted 140 d and was divided into 5 periods of 28 d each. The inclusion of feed additives did not impact (P ≥ 0.17) dry matter intake (DMI), nutrient intake, and apparent total tract digestibility of nutrients. Nonetheless, steers fed NAR had lower (P < 0.01) molar proportion of acetate compared with CON, SAL, and FLA steers, whereas ruminal acetate tended to be greater (P < 0.09) for SAL vs. CON and FLA, but did not differ (P = 0.68) between CON vs. FLA steers. Ruminal propionate was the highest (P < 0.01) for steers fed NAR and did not differ (P > 0.20) between CON, SAL, and FLA. Consequently, NAR steers had the lowest (P < 0.01) Ac:Pr ratio, whereas Ac:Pr did not differ (P > 0.18) among CON, SAL, and FLA. Total volatile fatty acids were greater (P < 0.04) for NAR and CON vs. SAL and FLA, but did not differ (P > 0.67) among NAR vs. CON and SAL vs. FLA. In experiment 2, 164 Nellore bulls (initial shrunk BW = 299 ± 2.5 kg) were assigned to feedlot pens for 140 d in a randomized complete block design. Within block (n = 10), animals were randomly assigned to the same treatments used in experiment 1. Average daily gain was greater (P < 0.01) in NAR vs. CON, SAL, and FLA bulls, and did not differ (P > 0.12) between CON, SAL, and FLA bulls. Bulls fed NAR had greater (P < 0.02) DMI (as kg/d or % BW) and final shrunk BW compared with CON, SAL, and FLA bulls, whereas DMI and final shrunk BW did not differ (P > 0.26) between CON, SAL, and FLA bulls. Feed efficiency, however, was not impacted (P = 0.51) by any feed additives used herein. Collectively, narasin was the only feed additive that benefited performance and ruminal fermentation of Nellore animals fed a forage-based diet.

Macrophages are sentinels of the immune system, which are often hijacked by tumor cells to assist tumor growth and metastasis. Herein our results showed that low dose salinomycin (SAL) in the range of 10-50 nM could efficiently induce M1 macrophage polarization in a dose- and time- dependent manner in vitro, with 30 nM SAL being optimal to generate M1-type macrophages from RAW246.7 cells. In animal study, intratumorally injected SAL (50 µg/kg) increased proportion of CD86 cells (by 28.9%), and decreased CD206 cells (by 14.2%) in transplant 4T1 tumors, in comparison with PBS group. Thus it resulted in significant regression in tumor growth (20% tumor inhibition) and pulmonary metastasis (reduced the number of metastatic nodes by 58%) in SAL group, whereas lipopolysaccharide (LPS) and paclitaxel (PTX) groups showed comparable number of metastatic lesions and volume of tumor. LPS treatment could as well lead to inflammatory reactions in tumor with SAL group, but resulted in systemic inflammation (elevated levels of IL-1α, IL-1β and TNF-α in serum), and PTX (10 μg/kg) treatment increased both types of macrophages. For the first time, we employed salinomycin below the dose of direct antitumor activity could effectively prime M1 type macrophage stimulation and regress tumor growth and metastasis.

A series of studies was carried out to determine the anticoccidial effects of a product derived from plant material sourced from Quillaja saponaria and Yucca schidigera. These plants are known to contain high concentrations of triterpenoid and steroidal saponins, substances that are known to display an array of biological effects. Battery tests involving individual Eimeria acervulina, Eimeria maxima, and Eimeria tenella infections and graded levels of a quillaja/yucca combination (QY) (0, 200, 250, and 300 ppm) were conducted. Body weight gain, coccidial lesion scores, and total oocysts per gram of feces (OPG) were used to evaluate anticoccidial effects. In addition, three floor pen trials evaluated the effects of 250 ppm QY in the control coccidial infections. The first pen trial measured the effects of 250 ppm QY, both alone and in combination with 66 ppm salinomycin (Sal), in a 2 3 2 factorial treatment arrangement. Two additional 42-day pen studies assessed the effects 250 ppm QY in birds vaccinated for coccidiosis. Data from the three battery trials indicated that at doses of 250 ppm QY or more, weight gain was improved, E. acervulina and E. tenella lesion scores were reduced, and OPG was lowered. In general, OPG was reduced by about 50% across all species by 250 and 300 ppm QY. Results of the pen study indicated that 250 ppm QY and Sal, when fed individually, reduced OPG and lesion scores and improved final performance. However, when QY and Sal were administered concurrently, further significant reductions in OPG occurred. The final performance of broilers vaccinated for coccidiosis was also improved at 250 ppm QY, as was OPG at both 21 and 28 days. Thus, at QY doses of 250 ppm or more, anticoccidial activity was evident but lacked the potency exhibited by many standard anticoccidials. When combined with either Sal or a live coccidiosis vaccine, QY improved the anticoccidial effects and performance of these anticoccidial methods.

Salinomycin is a polyether antibiotic showing anticancer activity. There are many reports of its toxicity to animals but little is known about the potential adverse effects in humans. The action of the drug may be connected to its metabolism. That is why we investigated the cytotoxicity of salinomycin and pathways of its biotransformation using human primary hepatocytes, human hepatoma cells (HepG2), and the mouse fibroblast cell line (Balb/c 3T3). The cytotoxicity of salinomycin was time-dependent, concentration-dependent, and cell-dependent with primary hepatocytes being the most resistant. Among the studied models, primary hepatocytes were the only ones to efficiently metabolize salinomycin but even they were saturated at higher concentrations. The main route of biotransformation was monooxygenation leading to the formation of monohydroxysalinomycin, dihydroxysalinomycin, and trihydroxysalinomycin. Tiamulin, which is a known inhibitor of CYP450 izoenzymes, synergistically induced cytotoxicity of salinomycin in all cell types, including non-metabolising fibroblasts. Therefore, the pharmacokinetic interaction cannot fully explain tiamulin impact on salinomycin toxicity.

The study aims to combine the delivery of two anticancer drugs to target both proliferating cancer cells and dormant cancer stem cells (CSCs) present in colorectal cancer. Two drugs were selected and encapsulated in lipid nanocapsules: SN38, the active form of irinotecan, which is unstable in the plasma but active against replicating cells, and salinomycin, a highly toxic ionophore active against cancer stem cells that is not suitable for clinical use. Using an engineered medium that enhanced the ratio of CSCs in HCT116 cell cultures, we demonstrated by clonogenicity tests and in sphere assays that Salinomycin acts mainly on CSCs, while SN38 acts mainly on proliferating cancer cells. In a preclinical murine CRC model, encapsulation of both drugs in lipid nanocapsules reduced their toxicity, including hemolysis, and led to a higher survival than what was observed following treatment with single drugs or non-encapsulated drugs. Nanoparticles loaded with an anticancer drug and salinomycin were effective against the therapy-resistant dormant CSCs and cancer cells.

Qin Wang,1,* Fangcen Liu,2,* Lifeng Wang,1 Chen Xie,3 Puyuan Wu,1 Shiyao Du,1 Shujuan Zhou,1 Zhichen Sun,1 Qin Liu,1 Lixia Yu,1 Baorui Liu,1 Rutian Li1 1The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing 210008, People’s Republic of China; 2The Comprehensive Cancer Centre, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing, People’s Republic of China; 3Key Laboratory for Organic Electronics and Information Displays, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing 210023, People’s Republic of China*These authors contributed equally to this workCorrespondence: Rutian Li; Baorui LiuThe Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, 321 Zhongshan Road, Nanjing 210008, People’s Republic of ChinaTel +86-25-83107081Fax +86-25-83317016Email rutianli@nju.edu.cn; baoruiliu@nju.edu.cnBackground: Cervical cancer stem cells (CCSCs) represent a subpopulation of tumor cells that possess self-renewal capacity and numerous intrinsic mechanisms of resistance to conventional chemotherapy and radiotherapy. These cells play a crucial role in relapse and metastasis of cervical cancer. Therefore, eradication of CCSCs is the primary objective in cervical cancer therapy. Salinomycin (Sal) is an agent used for the elimination of cancer stem cells (CSCs); however, the occurrence of several side effects hinders its application. Nanoscale drug-delivery systems offer great promise for the diagnosis and treatment of tumors. These systems can be used to reduce the side effects of Sal and improve clinical benefit.Methods: Sal-loaded polyethylene glycol-peptide-polycaprolactone nanoparticles (Sal NPs) were fabricated under mild and non-toxic conditions. The real-time biodistribution of Sal NPs was investigated through non-invasive near-infrared fluorescent imaging. The efficacy of tumor growth inhibition by Sal NPs was evaluated using tumor xenografts in nude mice. Flow cytometry, immunohistochemistry, and Western blotting were used to detect the apoptosis of CSCs after treatment with Sal NPs. Immunohistochemistry and Western blotting were used to examine epithelial–mesenchymal transition (epithelial interstitial transformation) signal-related molecules.Results: Sal NPs exhibited antitumor efficacy against cervical cancers by inducing apoptosis of CCSCs and inhibiting the epithelial–mesenchymal transition pathway. Besides, tumor pieces resected from Sal NP-treated mice showed decreased reseeding ability and growth speed, further demonstrating the significant inhibitory ability of Sal NPs against CSCs. Moreover, owing to targeted delivery based on the gelatinase-responsive strategy, Sal NPs was more effective and tolerable than free Sal.Conclusion: To the best of our knowledge, this is the first study to show that CCSC-targeted Sal NPs provide a potential approach to selectively target and efficiently eradicate CCSCs. This renders them a promising strategy to improve the therapeutic effect against cervical cancer.Keywords: nanoparticles, salinomycin, tumor-targeted delivery, cancer stem cells, epithelial interstitial transformation

Drug combination is considered to be the cornerstone of cancer treatment. Simultaneous administration of two or more drugs but at lower doses not only increases cytotoxic effects on tumor cells, but also reduces side effects and possibly overcomes drug resistance. Salinomycin is a well-known cancer stem cell killer, and dichloroacetate is a pyruvate dehydrogenase kinase inhibitor that exclusively targets cells with altered mitochondrial activity, a characteristic being common to most of the cancer cells. In our recent study, we have demonstrated that salinomycin exerted a cytotoxic effect on colorectal carcinoma cells in the 2D and 3D cultures and provided evidence that the mechanism of their synergy was mediated by dichloroacetate-dependent inhibition of the activity of multidrug resistance proteins. In the current work, we confirmed the synergistic cytotoxic properties of salinomycin and dichloroacetate in the 2D and 3D cultures of Lewis lung carcinoma (LLC1) cells. To verify if a synergistic effect of these compounds persisted in vivo, we performed series of experiments using a syngeneic LLC1-C57BL/6 mouse model and demonstrated that combination therapy with salinomycin and DCA increased the survival rate of allografted mice, inhibited metastatic site formation and reduced the populations of cancer stem cells as well as cells that underwent the epithelial-to-mesenchymal transition. Our results demonstrate that a synergistic effect of salinomycin and dichloroacetate exists not only in vitro but also in vivo and suggest their benefits in the treatment of metastatic cancers.

The present study was undertaken to investigate the antiparasitic activity of extracellular products of Streptomyces albus. Bioactivity-guided isolation of chloroform extracts affording a compound showing potent activity. The structure of the compound was elucidated as salinomycin (SAL) by EI-MS, 1H NMR and 13C NMR. In vitro test showed that SAL has potent anti-parasitic efficacy against theronts of Ichthyophthirius multifiliis with 10 min, 1, 2, 3 and 4 h (effective concentration) EC50 (95% confidence intervals) of 2.12 (2.22–2.02), 1.93 (1.98–1.88), 1.42 (1.47–1.37), 1.35 (1.41–1.31) and 1.11 (1.21–1.01) mg L−1. In vitro antiparasitic assays revealed that SAL could be 100% effective against I. multifiliis encysted tomonts at a concentration of 8.0 mg L−1. In vivo test demonstrated that the number of I. multifiliis trophonts on Erythroculter ilishaeformis treated with SAL was markedly lower than that of control group at 10 days after exposed to theronts (P < 0.05). In the control group, 80% mortality was observed owing to heavy I. multifiliis infection at 10 days. On the other hand, only 30.0% mortality was recorded in the group treated with 8.0 mg L−1 SAL. The median lethal dose (LD50) of SAL for E. ilishaeformis was 32.9 mg L−1.

Salinomycin (SAL) is a polyether antibiotic, which is commonly used as a coccidiostat and has recently shown to exhibit anticancer activity. The toxic action of the drug may be connected with the extent and routes of its biotransformation. The cytotoxic potential of SAL and its combination with tiamulin and prednisolone was investigated using three cell models from rat: primary hepatocytes, hepatoma cells (FaO) and myoblasts (L6). The four biochemical endpoints were assessed: mitochondrial and lysosomal activity, total cell protein content and membrane integrity. The metabolites of SAL in the medium from cell cultures were determined using LC-MS/MS. The cytotoxicity of SAL was time-, concentration- and cells dependent. The most sensitive endpoint was the inhibition of lysosomal activity. Tiamulin increased SAL cytotoxicity, whereas the opposite results were observed for prednisolone. Primary hepatocytes were the most efficient in SAL biotransformation both in terms of its intensity and number of produced metabolites. The range of the cytotoxicity and mode of salinomycin interaction with tiamulin and prednisolone cannot be explained by the biotransformation alone.

Two experiments were conducted to investigate the effects of tannic acid extract (TAE) formulations on the performance and intestinal health of male Cobb × Cobb 500 broilers exposed to coccidiosis. In the first experiment, 320 broiler chicks were randomly assigned to 5 treatments with 8 replicates. Treatments included non-medicated, uninfected (NC); non-medicated, infected (PC); salinomycin (SAL, 66 mg/kg); tannic acid (TA, 0.5 g/kg) and TAE (TAE, 0.5 g/kg). On d 14, all groups (except NC) were orally inoculated with Eimeria acervulina, E. maxima and E. tenella oocysts. Intestinal lesion scores, fecal oocyst counts (OPG) and performance were evaluated on d 20. The PC had greater lesions and higher FCR than infected, supplemented groups. Only TAE reduced OPG compared to PC (P0.05). In the second experiment, 3,000 broiler chicks were vaccinated on day of hatch with live coccidial oocysts, then randomly assigned to 5 treatments with 15 replicates. Treatments included non-medicated (CNT); salinomycin (SAL, 66 mg/kg); robenidine (ROB, 33 mg/kg); TAE (0.5 g/kg) and TAE with Bacillus coagulans (TAE+BC, 0.5 g/kg). On d 29, a subset of pens (n = 20) were challenged with a mixed Eimeria spp. oral inoculum; performance, lesions and OPG were evaluated on d 35. An immune challenge was created in half the pens by issuing broilers feed without supplementation materials during the challenge. For the non-challenged pens (n = 55), performance was measured up to d 49. Performance of non-challenged, vaccinated-CNT birds was improved with all treatments at d 21 and d 49. Among the challenged birds, withdrawal of SAL or ROB resulted in FCR similar to the challenged CNT group (P0.05), whereas withdrawal of TAE or TAE+BC maintained improved FCR compared to challenged-CNT birds (P0.05). These findings indicate supplementation of TAE and TAE+BC with coccidiosis vaccination can be considered as a potential alternative strategy to address coccidiosis in broiler chickens.

Aims: Recently, salinomycin (SAL) has been reported to inhibit proliferation and induce apoptosis in various tumors. The aim of this study was to deliver SAL to orthotopic model of pancreatic cancer by the aid of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Methods: The NPs were physico-chemically characterized and evaluated for cytotoxicity on luciferase-transduced AsPC-1 cells in vitro as well as implanted orthotopically into the pancreas of nude mice. Results: SAL (3.5 mg/kg every other day) blocked tumor growth by 52% compared to the control group after 3 weeks of therapy. Western blotting of tumor protein extracts indicated that SAL treatment leads to up-regulation of E-cadherin, β-catenin, and transforming growth factor beta receptor (TGFβR) expressions in AsPC-1 orthotopic tumor. Noteworthy, immunofluorescence staining of adjacent tumor sections showed that treatment with SAL NPs cause significant apoptosis in the tumor cells rather than the stroma. Further investigations also revealed that TGFβR2 over-expression was induced in stroma cells after treatment with SAL NPs. Conclusion: These results highlight SAL-loaded PLGA NPs as a promising system for pancreatic cancer treatment, while the mechanistic questions need to be subsequently tested.

To improve the druggability of salinomycin, a 20-epi-amino-20-deoxysalinomycin derivatives library was synthesized with high efficacy from which a few salinomycin derivatives with high potency and selectivity were identified through comprehensive cytotoxicity assay, including a fluorine-19 magnetic resonance sensitive tool molecule. Using a K-ras cellular model, salinomycin and its derivatives showed different molecular mode of action from literature reports. These results would be valuable for developing salinomycin-based cancer therapy.

Since Salinomycin (Sal) emerged its ability to target breast cancer stem cells in 2009, numerous experiments have been carried out to test Sal’s anticancer effects. What deserve to be mentioned is that Sal can efficiently induce proliferation inhibition, cell death and metastasis suppression against human cancers from different origins both in vivo and in vitro without causing serious side effects as the conventional chemotherapeutical drugs on the body. There may be novel cell death pathways involving the anticancer effects of Sal except the conventional pathways, such as autophagic pathway. This review is focused on how autophagy involves the effects of Sal, trying to describe clearly and systematically why autophagy plays a vital role in predominant anticancer effects of Sal, including its distinctive characteristic. Based on recent advances, we present evidence that a dual role of Sal involving in autophagy may account for its unique anticancer effects - the preference for cancer cells. Further researches are required to confirm the authenticity of this suppose in order to develop an ideal anticancer drug.

Salinomycin, as a polyether ionophore antibiotic, is extensively used as a feed additive against coccidiosis in poultry and as a growth promoter of ruminants worldwide. Owing to its narrow therapeutic index, numerous intoxication have been reported in target/non-target animals by overdosage, misuse or drug interactions as well as human who consumed salinomycin accidently. Salinomycin-induced cardiotoxicity in chicken and non-target animals is considered as a major contributor to animal death. In the current study, we aim to elucidate the underlying mechanism of its myocardial toxicity using primary chicken myocardial cell as an in vitro model. The results showed that salinomycin altered cellular morphology and induced cell death in a concentration-dependent manner. Salinomycin treatment elevated the permeability of the cell membrane and leaded to the efflux of enzymes, including creatine kinase (CK) and lactate dehydrogenase (LDH). Flow cytometry analysis indicated the number of apoptotic cells increased significantly by salinomycin exposure. Furthermore, caspase-3 and caspase-9 were activated at gene and protein level rather than caspase-8, along with the up-regulation of apoptosis genes Bax, Cytochrome C, Apoptotic peptidase activating factor 1 (Apaf-1) and the down-regulation of Bcl-2. Salinomycin-induced mitochondrial dysfunction was accompanied by the significant decrease of mitochondrial membrane potential (MMP) and the severe ultrastructure damage. In conclusion, these findings suggest that the toxic dose of salinomycin induces severe cardiomyocytes death through mitochondria mediated apoptosis pathway.

The objective of this study was to characterize differences in the cecal microbiota of chickens vaccinated for coccidiosis or receiving salinomycin in the diet. In this study, 140 male 1-day-old broiler chickens were divided in 2 groups: vaccine group (live vaccine) vaccinated at the first day and salinomycin group (125 ppm/kg since the first day until 35 d of age). Each treatment was composed for 7 replicates of 10 birds per pen. At 28 d, the cecal content of one bird per replicate was collected for microbiota analysis. The genetic sequencing was conducted by the Miseq Illumina platform. Vaccine group showed lower body weight, weight gain, and poorer feed conversion in the total period (P < 0.05). Bacterial 16S rRNA genes were classified as 3 major phyla (Bacteroidetes, Firmicutes, and Proteobacteria), accounting for more than 98% of the total bacterial community. The microbiota complexity in the cecal was estimated based on the α-diversity indices. The vaccine did not reduce species richness and diversity (P > 0.05). The richness distribution in the salinomycin group was larger and more uniform than the vaccinated birds. Salinomycin group was related to the enrichment of Bacteroidetes, whereas Firmicutes and Proteobacteria phyla were in greater proportions in the vaccine group. The last phylum includes a wide variety of pathogenic bacteria. The vaccine did not decrease the species richness but decreased the percentage of Bacteroidetes, a phylum composed by genera that produce short-chain fatty acids improving intestinal health. Vaccine group also had higher Proteobacteria phylum, which may help explain its poorer performance.

Cancer stem cells (CSCs) in gastric cancer (GC) have been established recently as key therapeutic targets for the successful treatment of GC. Emerging evidence suggests that both CSCs and cancer cells should be eradicated to achieve optimal therapeutic efficacy. In the present study, salinomycin, which has been reported to kill CSCs, was used in combination with docetaxel, a chemotherapeutic drug that is used as first-line therapy in GC, to eradicate both GC stem cells (SCs) and cancer cells. Salinomycin and docetaxel were loaded separately into poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles of ∼140 nm with a narrow size distribution, high drug loading, and sustained drug release. GC SCs were isolated by magnetic-activated cell sorting on the basis of CD44 expression as the CSC phenotype. CD44 GC SCs showed the characteristics of CSCs, including increased SC gene expression, tumorsphere formation capacity, and tumorigenicity in nude mice. We found that both salinomycin and salinomycin-loaded nanoparticles (salinomycin-NPs) could selectively eradicate GC SCs, as reflected by reduced tumorsphere formation capacity and the frequency of CD44 GC cells, whereas docetaxel and docetaxel-loaded nanoparticles (docetaxel-NPs) could significantly eradicate GC cells. In nude mice bearing GC xenografts, salinomycin-NPs and salinomycin significantly decreased the intratumor population of GC SCs. Notably, salinomycin-NPs combined with docetaxel-NPs suppressed tumor growth more effectively than did salinomycin combined with docetaxel, single salinomycin-NPs, or docetaxel-NPs. Therefore, salinomycin-NPs combined with docetaxel-NPs represent a promising strategy for the treatment of GC by eradicating both GC SCs and cancer cells.

Chemotherapeutic treatment of human and animal trypanosomiasis is unsatisfactory because only a few drugs are available. As these drugs have poor efficacy and cause adverse reactions, more effective and tolerable medications are needed. As the polyether ionophore antibiotic lasalocid acid is used as medicated feed additive in cattle, the compound was tested for its trypanocidal and cytotoxic activity against bloodstream forms of Trypanosoma brucei and human myeloid HL-60 cells. The concentrations required of lasalocid acid to reduce the growth rate of trypanosomes by 50% and to kill the parasites were 1.75 and 10 μM, respectively. The ionophore displayed also cytotoxic activity against HL-60 cells but the human cells were about 10 to 14 times less sensitive indicating moderate selectivity. As the trypanocidal mechanism of action of polyether ionophore antibiotics is due to a sodium influx-induced cell swelling, the effect of lasalocid acid on cell volume change in bloodstream-form trypanosomes was investigated. Interestingly, lasalocid acid induced a much faster cell swelling in trypanosomes than the more trypanocidal related ionophore salinomycin. These results support further investigations of lasalocid acid and derivatives thereof as potential agents against African trypanosomiasis. Electronic supplementary material The online version of this article (10.1007/s00436-017-5624-6) contains supplementary material, which is available to authorized users.

The aim of this study was to investigate the effect of dietary supplementation with nisin alone or in combination with salinomycin or monensin on broiler chickens in terms of growth performance, selected blood parameters, digestive enzyme activity, apparent nutrient digestibility, and tibiotarsus mineralization, as well as selected gastrointestinal tract (GIT) organ weights, intestinal length, and central immune organ weights. Two independent experiments, each including 400 one-day-old female Ross 308 chicks differing in ionophore coccidiostats, i.e., salinomycin and monensin supplementation, were conducted. The following treatments were applied: experiment 1: NA—no additives, SAL—salinomycin (60 mg/kg diet), NIS—nisin (2,700 IU/kg diet), SAL+NIS—salinomycin (60 mg/kg diet) and nisin (2,700 IU/kg diet); experiment 2: NA—no additives, MON—monensin (100 mg/kg diet), NIS—nisin (2,700 IU/kg diet) and MON+NIS—monensin (100 mg/kg diet) and nisin (2,700 IU/kg diet). The addition of nisin with or without ionophores to the birds’ diet improved broiler growth performance in terms of BWG and FCR (days 1 to 14) and BWG and FI (15 to 35 d; 1 to 35 d). Salinomycin showed effects similar to those of nisin influence on growth performance (1 to 35 d), while monensin supplementation resulted in lower BWG. Moreover, no additive effect between nisin and ionophores was observed. Nisin and salinomycin had no influence on the serum concentration of selected hormones and other blood biochemical parameters except glucose, which was reduced by nisin. A decrease in lipase activity was observed during nisin and salinomycin supplementation, while the apparent ileal digestibility of fat was not affected. However, the digestibility of crude protein increased with nisin administration. Additionally, the effects of nisin on decreasing the weight and length of GIT segments were observed. Supplementation with nisin and monensin was not associated with a negative impact on tibiotarsus mineralization and the immune organ index. This study suggests that nisin may be used in broiler nutrition as a growth promotor, with no negative influence on the bird's metabolism or immune status.

The presence of mycotoxins in broiler feed can have deleterious effects on the wellbeing of the animals and their performance. Mycotoxin binders are feed additives that aim to adsorb mycotoxins in the intestinal tract and thereby prevent the oral absorption of the mycotoxin. The simultaneous administration of coccidiostats and/or antimicrobials with mycotoxin binders might lead to a reduced oral bioavailability of these veterinary medicinal products. This paper describes the influence of 3 mycotoxin binders (i.e., clay 1 containing montmorillonite, mica, and feldspars; clay 2 containing montmorillonite and quartz; and yeast 1 being a modified glucomannan fraction of inactivated yeast cells) and activated carbon on the oral bioavailability and pharmacokinetic parameters of the antimicrobials doxycycline and tylosin, and the coccidiostats diclazuril and salinomycin. A feeding study with 40 15 day-old broilers was performed evaluating the effects of long-term feeding 2 g mycotoxin binder/kg of feed. The birds were randomly divided into 5 groups of 8 birds each, i.e., a control group receiving no binder and 4 test groups receiving either clay 1, clay 2, yeast 1, or activated carbon mixed in the feed. After 15 d of feeding, both the control and each test group were administered doxycycline, tylosin, diclazuril, and salinomycin, consecutively, respecting a wash-out period of 2 to 3 d between each administration. The 4 medicinal products were dosed using a single bolus administration directly in the crop. After each bolus administration, blood was collected for plasma analysis and calculation of the main pharmacokinetic parameters and relative oral bioavailability (F = area under the plasma concentration-time curve (AUC0-8 h) in the test groups/AUC0-8 h in the control group)*100). No effects were observed of any of the mycotoxin binders on the relative oral bioavailability of the coccidiostats (i.e., F between 82 and 101% and 79 and 93% for diclazuril and salinomycin, respectively). Also, no significant effects could be noticed of any of the mycotoxin binders on the relative oral bioavailability of the antimicrobials doxycycline and tylosin (i.e., F between 67 and 83% and between 43 and 104%, respectively).