ABSTRACT
Calcifying nanoparticles have been linked to various types of human disease, but how they contribute to disease processes is unclear. Here, we examined whether and how calcifying nanoparticles isolated from patients with kidney stones are cytotoxic to human bladder cancer cells. Calcifying nanoparticles were isolated from midstream urine of patients with renal calcium oxalate stones and examined by electron microscopy. Human bladder cancer cells (EJ cells) were cultured in the presence of calcifying nanoparticles or nanohydroxyapatites for 12 and 72 h and examined for toxicity using the Cell Counting Kit-8, for autophagy using transmission electron microscopy and confocal microscopy, and for apoptosis using fluorescence microscopy, transmission electron microscopy, and flow cytometry. Changes in protein expression were analyzed by Western blotting. The results showed that the size and shape of the isolated calcifying nanoparticles were as expected. Calcifying nanoparticles were cytotoxic to EJ cells, more so than nanohydroxyapatites, and this was due, at least in part, to the production of intracellular reactive oxygen species. Transmission electron microscopy showed that calcifying nanoparticles were packaged into vesicles and autolysosomes. Calcifying nanoparticles induced greater autophagy and apoptosis than nanohydroxyapatites. Our findings demonstrate that calcifying nanoparticles can trigger bladder cancer cell injury by boosting reactive oxygen species production and stimulating autophagy and apoptosis.
Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Calcifying Nanoparticles/administration & dosage , Urinary Bladder Neoplasms/drug therapy , Calcifying Nanoparticles/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Flow Cytometry , Humans , Kidney Calculi/chemistry , Kidney Calculi/metabolism , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Reactive Oxygen Species/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
OBJECTIVE: Calcifying nanoparticles (NPs) have been detected recently in calcified human arterial specimens and are involved in the process of calcification. This study was designed to test the hypothesis that human-derived NPs could worsen the response to arterial endothelial injury and induce vascular calcification. METHODS: The right carotid artery of 24 New Zealand rabbits was injured with an angioplasty balloon. Animals were perfused intravenously with saline (100 mL) during the experiment and divided into three groups: group-A, control; group-B, exposed to NPs (2 mL) obtained from calcified aortic valves; and group-C, exposed to NPs (2 mL) and treated postoperatively with atorvastatin (2.5 mg/kg/24 h). At 30 days, both carotid arteries were removed and examined histologically. Blood measurements were monitored during the study. RESULTS: The intimal hyperplasia area was significantly larger in the injured right carotid artery compared with the left unoperated carotid artery in all groups. There was no significant variation in medial area between groups. Morphometrically, the intima/media ratio (IMR) was significantly higher in damaged carotids compared with controls. A significant increase of IMR was found in group-B (1.81 ± 0.41) compared with group-A (0.38 ± 0.59; p = .004) or group-C (0.89 ± 0.79; p = .035). Differences between groups C and A were not significant (p = .064). Calcifications were observed in six animals, all of which had been exposed to NPs (4 in group-B, 2 in group-C, p = .027). Plasma levels of cholesterol and triglycerides remained stable. CONCLUSIONS: This research confirms the ability of systemic inoculation of human-derived NPs to accelerate hyperplasia and stimulate calcification in localized areas of arteries previously submitted to endothelial damage, while it was harmless in healthy arteries. Atorvastatin was demonstrated to slow down this process.
