Induction of apoptosis and autophagy by calcifying nanoparticles in human bladder cancer cells

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.