Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

Silver nanoparticles combat Salmonella Typhimurium: Suppressing intracellular infection and activating dendritic cells.

Salmonella Typhimurium (ST) can hide inside cells, avoid antibiotic therapy and being killed by host's immune system to cause persistent infection in humans and animals. Metal nanoparticles are regarded as an alternative to overcome the above limitations, silver nanoparticles especially have been applied in combating drug-resistant bacteria. However, the therapeutic effects of silver nanoparticles against intracellular infection and their impacts on host immunity remain an area of further investigation. In this work, we synthesized Ganoderma extract-capped silver nanoparticles (Ag@Ge) and explored the therapeutic potential and immune adjuvant effects of Ag@Ge against intracellular ST. Firstly, Ag@Ge had a small particle size of 35.52±7.46 nm, good stability, and biocompatibility. Then, Ag@Ge effectively entered RAW 264.7 cells, suppressed intracellular ST infection. Furthermore, Ag@Ge activated mouse dendritic cells (DCs) in vitro, evidenced by increased phenotypic markers (CD80/CD86/CD40/major compatibility complex II (MHCII)) expression and cytokine and chemokine (interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL-2), and chemokine (C-C motif) receptor-7 (CCR-7)) transcription. More notably, the combination of Ag@Ge with inactivated ST recruited intestinal DCs to mitigate ST infection in mice, evidenced by decreased body weight loss and bacterial loads in the tissues (liver, jejunum, and colon), and improved platelets count. The above findings indicate that Ag@Ge has the potential as an alternative nano-antibiotic against intracellular ST infection.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.

Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis.

Background: Halofuginone (HF)-loaded TPGS polymeric micelles (HTPM) were successfully fabricated using the thin-film hydration technique. HTPM via intravenous injection have been demonstrated to exert an excellent anticancer effect against triple-negative breast cancer (TNBC) cells and subcutaneous xenografts. In the present study, we further explored the potential treatment effect and mechanism of orally administered HTPM alone and in combination with surgical therapy on TNBC in subcutaneous and orthotopic mouse models. Methods: Herein, the stability and in vitro release behavior of HTPM were first evaluated in the simulated gastrointestinal fluids. Caco-2 cell monolayers were then used to investigate the absorption and transport patterns of HF with/without encapsulation in TPGS polymeric micelles. Subsequently, the therapeutic effect of orally administered HTPM was checked on subcutaneous xenografts of TNBC in nude mice. Ultimately, orally administered HTPM, combined with surgical therapy, were utilized to treat orthotopic TNBC in nude mice. Results: Our data confirmed that HTPM exhibited good stability and sustained release in the simulated gastrointestinal fluids. HF was authenticated to be a substrate of P-glycoprotein (P-gp), and its permeability across Caco-2 cell monolayers was markedly enhanced via heightening intracellular absorption and inhibiting P-gp efflux due to encapsulation in TPGS polymeric micelles. Compared with HF alone, HTPM showed stronger tumor-suppressing effects in subcutaneous xenografts of MDA-MB-231 cells when orally administered. Moreover, compared with HTPM or surgical therapy alone, peroral HTPM combined with partial surgical excision synergistically retarded the growth of orthotopic TNBC. Fundamentally, HTPM orally administered at the therapeutic dose did not cause any pathological injury, while HF alone led to weight loss and jejunal bleeding in the investigated mice. Conclusion: Taken together, HTPM could be applied as a potential anticancer agent for TNBC by oral administration.

Deoxynivalenol (Vomitoxin)-Induced Anorexia Is Induced by the Release of Intestinal Hormones in Mice.

Deoxynivalenol (DON), also known as vomitoxin, is a mycotoxin that can cause antifeeding and vomiting in animals. However, the mechanism of DON inducing anorexia is complicated. Studies have shown that intestinal hormones play a significant part in the anorexia caused by DON. We adopted the "modeling of acute antifeeding in mice" as the basic experimental model, and used two methods of gavage and intraperitoneal injection to explore the effect of intestinal hormones on the antifeedant response induced by DON in mice. We found that 1 and 2.5 mg/kg·bw of DON can acutely induce anorexia and increase the plasma intestinal hormones CCK, PYY, GIP, and GLP-1 in mice within 3 h. Direct injection of exogenous intestinal hormones CCK, PYY, GIP, and GLP-1 can trigger anorexia behavior in mice. Furthermore, the PYY receptor antagonist JNJ-31020028, GLP-1 receptor antagonist Exendin(9-39), CCK receptor antagonist Proglumide, GIP receptor antagonist GIP(3-30)NH2 attenuated both intestinal hormone and DON-induced anorectic responses. These results indicate that intestinal hormones play a critical role in the anorexia response induced by DON.

Combined Effect of Deoxynivalenol (DON) and Porcine Circovirus Type 2 (Pcv2) on Inflammatory Cytokine mRNA Expression.

A host's immune system can be invaded by mycotoxin deoxynivalenol (DON) poisoning and porcine circovirus type 2 (PCV2) infections, which affect the host's natural immune function. Pro-inflammatory cytokines, IL-1ß and IL-6, are important regulators in the process of natural immune response, which participate in inflammatory response and enhance immune-mediated tissue damage. Preliminary studies have shown that DON promotes PCV2 infection by activating the MAPK signaling pathway. Here, we explored whether the mRNA expression of IL-1ß and IL-6, induced by the combination of DON and PCV2, would depend on the MAPK signaling pathway. Specific pharmacological antagonists U0126, SP600125 and SB203580, were used to inhibit the activities of ERK, JNK and p38 in the MAPK signaling pathway, respectively. Then, the mRNA expression of IL-1ß and IL-6 in PK-15 cells was detected to explore the effect of the MAPK signaling pathway on IL-1ß and IL-6 mRNA induced by DON and PCV2. The results showed that PK-15 cells treated with DON or PCV2 induced the mRNA expression of IL-1ß and IL-6 in a time- and dose-dependent manner. The combination of DON and PCV2 has an additive effect on inducing the mRNA expression of IL-1ß and IL-6. Additionally, both DON and PCV2 could induce the mRNA expression of IL-1ß and IL-6 via the ERK and the p38 MAPK signal pathways, while PCV2 could induce it via the JNK signal pathway. Taken together, our results suggest that MAPKs play a contributory role in IL-1ß and IL-6 mRNA expression when induced by both DON and PCV2.

Encapsulating Halofuginone Hydrobromide in TPGS Polymeric Micelles Enhances Efficacy Against Triple-Negative Breast Cancer Cells.

BACKGROUND: Halofuginone hydrobromide (HF) is a synthetic analogue of the naturally occurring quinazolinone alkaloid febrifugine, which has potential therapeutic effects against breast cancer, however, its poor water solubility greatly limits its pharmaceutical application. D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of vitamin E, which can self-assemble to form polymeric micelles (PMs) for encapsulating insoluble anti-tumor drugs, thereby effectively enhancing their anti-cancer effects. METHODS: HF-loaded TPGS PMs (HTPMs) were manufactured using a thin-film hydration technique, followed by a series of characterizations, including the hydrodynamic diameter (HD), zeta potential (ZP), stability, drug loading (DL), encapsulation efficiency (EE), and in vitro drug release. The anti-cancer effects and potential mechanism of HTPMs were investigated in the breast cell lines MDA-MB-231 and MCF-7, and normal breast epithelial cell line Eph-ev. The breast cancer-bearing BALB/c nude mouse model was successfully established by subcutaneous injection of MDA-MB-231 cells and used to evaluate the in vivo therapeutic effect and safety of the HTPMs. RESULTS: The optimized HTPMs had an HD of 17.8±0.5 nm and ZP of 14.40±0.1 mV. These PMs exhibited DL of 12.94 ± 0.46% and EE of 90.6 ± 0.85%, along with excellent storage stability, dilution tolerance and sustained drug release in pH-dependent manner within 24 h compared to free HF. Additionally, the HTPMs had stronger inhibitory effects than free HF and paclitaxel against MDA-MB-231 triple-negative breast cancer cells, and little toxicity in normal breast epithelial Eph-ev cells. The HTPMs induced cell cycle arrest and apoptosis of MDA-MB-231 by disrupting the mitochondrial membrane potential and enhancing reactive oxygen species formation. Evaluation of in vivo anti-tumor efficacy demonstrated that HTPMs exerted a stronger tumor inhibition rate (68.17%) than free HF, and exhibited excellent biocompatibility. CONCLUSION: The findings from this study indicate that HTPMs holds great clinical potential for treating triple-negative breast cancer.

Maduramicin triggers methuosis-like cell death in primary chicken myocardial cells.

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.

Maduramicin induces apoptosis in chicken myocardial cells via intrinsic and extrinsic pathways.

Maduramicin is one of the most extensively used anticoccidial drugs for the treatment of Eimeria spp. infections. However, overdosage, misuse and drug interactions have resulted in the development of ionophore toxic syndrome. Heart and skeletal muscles have been identified as the main target organs of toxicity. In the present study, primary chicken myocardial cells were isolated to investigate the toxicity and underlying mechanisms of maduramicin. Our results showed that maduramicin causes morphological changes and a decrease in the viability of chicken myocardial cells. Annexin V-FITC/PI and 4',6-diamidino-2-phenylindole (DAPI) staining showed a significant increase in the number of apoptotic cells. Furthermore, caspases-3/8/9 were activated at the gene and protein levels and this was accompanied by the upregulation of apoptosis-related genes, including bcl-2, bax, and cytochrome C. Treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (z-VAD-fmk) ameliorated the apoptosis and cytotoxicity. Furthermore, intracellular Ca2+ and reactive oxygen species (ROS) were elevated, whereas mitochondrial membrane potential (MMP) and intracellular glutathione (GSH) decreased with exposure to maduramicin. The antioxidant N-acetyl-cysteine (NAC) had no significant effect on maduramicin-induced cytotoxicity and apoptosis. Taken together, our findings demonstrate that maduramicin is cytotoxic to primary chicken myocardial cells via caspase dependent and independent apoptotic pathways.

Transcriptome profile analysis reveals cardiotoxicity of maduramicin in primary chicken myocardial cells.

Maduramicin, an excellent ionophore antibiotic, is extensively used to control coccidiosis in poultry. Numerous maduramicin intoxications have been reported in farm animal and human due to its relatively narrow safety range, with necrosis or degeneration of cardiac and skeletal muscles as hallmark. To date, the mechanisms of maduramicin-induced cardiotoxicity remain unclear in chicken and other animals. Maduramicin (5 µg/mL)-treated primary chicken myocardial cells were used for RNA sequencing (RNA-Seq) and bioinformatics analysis in this study. A total of 1442 differential expressed genes were identified. 810 genes were up-regulated and the rest 632 genes were down-regulated. Transcriptome analysis revealed that the cytokine-cytokine receptor interaction, apoptosis, calcium signal and cytoplasmic vacuolization pathways were significantly affected. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that gene expression patterns were consistent with RNA-Seq analysis. Pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8), apoptosis ratios, cleaved caspase-3, intracellular calcium level and Ca2+-ATPase activity were elevated after maduramicin (0.05, 0.5 and 5 µg/mL) treatment. Massive vacuole formation was found in the cytoplasm by morphology and transmission electron microscopy observation. Taken together, the results suggested that maduramicin exerted its cardiotoxicity by multiple molecular mechanisms in primary chicken myocardial cells.