Effect of four fluoroquinolones on the viability of bladder cancer cells in 2D and 3D cultures.

Introduction: The anticancer properties of fluoroquinolones and the high concentrations they achieve in urine may help in bladder cancer therapy. This study aimed to analyze the properties of 4 fluoroquinolones as potential candidates for supportive treatment of bladder cancer. Methods: Comparative analyses were performed on the cytotoxic effects of norfloxacin, enrofloxacin, moxifloxacin, and ofloxacin on normal and cancer urothelial cell lines. In 2D culture, the cytotoxic properties of fluoroquinolones were evaluated using MTT assay, real-time cell growth analysis, fluorescence and light microscopy, flow cytometry, and molecular analysis. In 3D culture, the properties of fluoroquinolones were tested using luminescence assays and confocal microscopy. Results and Discussion: All tested fluoroquinolones in 2D culture decreased the viability of both tested cell lines in a dose- and timedependent manner. Lower concentrations did not influence cell morphology and cytoskeletal organization. In higher concentrations, destruction of the actin cytoskeleton and shrinkage of the nucleus was visible. Flow cytometry analysis showed cell cycle inhibition of bladder cancer cell lines in the G2/M phase. This influence was minimal in the case of normal urothelium cells. In both tested cell lines, increases in the number of late apoptotic cells were observed. Molecular analysis showed variable expression of studied genes depending on the drug and concentration. In 3D culture, tested drugs were effective only in the highest tested concentrations which was accompanied by caspase 3/7 activation and cytoskeleton degradation. This effect was hardly visible in non-cancer cell lines. According to the data, norfloxacin and enrofloxacin had the most promising properties. These two fluoroquinolones exhibited the highest cytotoxic properties against both tested cell lines. In the case of norfloxacin, almost all calculated LC values for bladder cancer cell lines were achievable in the urine. Enrofloxacin and norfloxacin can be used to support chemotherapy in bladder cancer patients.

Avian Cell Culture Models to Study Immunomodulatory Properties of Bioactive Products.

Antimicrobial resistance is becoming a greater danger to both human and animal health, reducing the capacity to treat bacterial infections and increasing the risk of morbidity and mortality from resistant bacteria. Antimicrobial efficacy in the treatment of bacterial infections is still a major concern in both veterinary and human medicine. Antimicrobials can be replaced with bioactive products. Only a small number of plant species have been studied in respect to their bioactive compounds. More research is needed to characterize and evaluate the therapeutic properties of the plant extracts. Due to the more and more common phenomenon of antimicrobial resistance, poultry farming requires the use of natural alternatives to veterinary antibiotics that have an immunomodulatory effect. These include a variety of bioactive products, such as plant extracts, essential oils, probiotics, prebiotics, and synbiotics. This article presents several studies on bioactive products and their immunomodulatory effects tested in vitro and ex vivo using various avian cell culture models. Primary cell cultures that have been established to study the immune response in chickens include peripheral blood mononuclear cells (PBMCs), intestinal epithelial cells (IEC), and bone marrow-derived dendritic cells (BMDCs). Chicken lymphatic lines that can be used to study immune responses are mainly: chicken B cells infected with avian leukemia RAV-1 virus (DT40), macrophage-like cell line (HD11), and a spleen-derived macrophage cell line (MQ-NCSU). Ex vivo organ cultures combine in vitro and in vivo studies, as this model is based on fragments of organs or tissues grown in vitro. As such, it mimics the natural reactions of organisms, but under controlled conditions. Most ex vivo organ cultures of chickens are derived from the ileum and are used to model the interaction between the gastrointestinal tract and the microbiota. In conclusion, the use of in vitro and ex vivo models allows for numerous experimental replications in a short period, with little or no ethical constraints and limited confounding factors.

In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin.

The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo's feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.