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.

Methyl halide transferase-based gas reporters for quantification of filamentous bacteria in microdroplet emulsions.

The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 106 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.

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.

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.

Antitumor effects of anlotinib in thyroid cancer.

There is no effective treatment for patients with poorly differentiated papillary thyroid cancer or anaplastic thyroid cancer (ATC). Anlotinib, a multi-kinase inhibitor, has already shown antitumor effects in various types of carcinoma in a phase I clinical trial. In this study, we aimed to better understand the effect and efficacy of anlotinib against thyroid carcinoma cells in vitro and in vivo. We found that anlotinib inhibits the cell viability of papillary thyroid cancer and ATC cell lines, likely due to abnormal spindle assembly, G2/M arrest, and activation of TP53 upon anlotinib treatment. Moreover, anlotinib suppresses the migration of thyroid cancer cells in vitro and the growth of xenograft thyroid tumors in mice. Our data demonstrate that anlotinib has significant anticancer activity in thyroid cancer, and potentially offers an effective therapeutic strategy for patients of advanced thyroid cancer type.

[Transurethral seminal vesiculoscopy for the treatment of vesiculitis with hemospermia:A report of 64 cases].

Objective: To explore the treatment of vesiculitis with hemospermia by transurethral seminal vesiculoscopy. Methods: We treated 64 cases of vesiculitis with hemospermia by transurethral seminal vesiculoscopy. During the operation,we removed the stones and inflammatory substances and collected seminal vesicle fluid to be cultured for bacteria,ureaplasma urealyticum(UU),chlamydia trachomatis(CT),and mycoplasma hominis(MH),followed by infusion of levofloxacin at 0. 3 g/100 ml into the seminal vesicle. Regular follow-up was conducted post-operatively. Results: All the operations were successfully accomplished, the operation time averaging(40 ± 15) min(25- 50 min). The ejaculatory duct opening was observed on the verumontanum surface in the posterior urethra in 2 cases, abnormal passages found in the prostatic utricle in 8 cases, and seminal vesicle fenestration from the prostatic utricle conducted in the other 54 cases(32 by seminal vesiculoscopy and 22 with holmium laser). Stones were seen in the prostatic utricle in 5 cases, in the seminal vesicle in 6 cases, and in both the prostatic utricle and seminal vesicle in 2 cases. Culture of the seminal vesicle fluid showed the acinetobacter to be positive in 1 case and UU, CT, and MH to be negative. At 3 months after surgery, hemospermia was cured in 52 cases, relieved in 8,and unimproved in 4. Conclusion: Seminal vesicle fenestration drainage by transurethral seminal vesiculoscopy for the treatment of vesiculitis with hemospermia has the advantages of short operation time, high effectiveness and no obvious complications and can also be employed for the examination of the seminal vesicle as well as removal of stones and inflammatory substances.