Clinoptilolite has antiviral, antibacterial, anti-inflammatory, antidiabetic, and anticancer properties due to its biological activities. In various cancer cell culture studies, it has been reported effective against tumour cells and gave positive results in treatment of various tumours in dogs. No study was found on the effects of the nanoparticulate form, nanoclinoptilolite, on cancer cells. The aim of this study was to determine its cytotoxic and apoptotic effects in canine osteosarcoma (OSA) cell culture. Doses at 50% inhibitory concentration were determined by measuring the dose- and duration-dependent cytotoxicity of nanoclinoptilolite on canine D-17 osteosarcoma cells by methylthiazol tetrazolium (MTT) test for 24 h, 48 h, and 72 h. Murine caspase-3 and -7 activity and expression levels of the BAX and BCL2 genes were measured using RT-PCR to investigate the apoptotic effect. Nanoclinoptilolite decreased cell viability and induced caspase-3- and -7-mediated apoptosis in treated canine OSA cells. Furthermore, its application to canine OSA cells downregulated the expression of BCL2 and upregulated the expression of proapoptotic BAX. Clinoptilolite, which was previously demonstrated to have anticancer properties, decreased cell viability effectively and rapidly and increased the apoptotic cell ratio in a novel use in nanoparticle form, exhibiting this effect by increasing the BAX/BCL2 ratio.

OBJECTIVE To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs. ANIMALS 6 healthy Beagles. PROCEDURES Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes. RESULTS Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 10(10) vesicles/mL ± 4.2 × 10(8) vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable. CONCLUSIONS AND CLINICAL RELEVANCE The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.