Effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin-treated microenvironment of A549 cell line

Abstract

Objective To investigate the effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin(CBP)-treated A549 cell and its possible mechanisms of inhibiting A549 cell line activity. Methods According to whether SonoVue was used or not, and the different dose of CBP, the groups A-F were arranged as the ultrasound(US) group(group A), the ultrasound combined with microbubbles (USMB) group(group B), the low dose CBP (100 μg/ml)+ US group(group C), the low dose CBP+ USMB group(group D), the high dose CBP (200 μg/ml)+ US group (group E) and the high dose CBP+ USMB group(group F). A549 cells were bathed and washed by a calcium-free buffer, loaded with Ca2+ indicator fluo-4 AM. Real-time images were acquired using laser confocal microscopy. The fluorescence intensity of intracellular calcium ion concentration ([Ca2+ ]i) in individual living cell was observed and the calcium overload was analyzed. Results After ultrasound irradiation, the normalized fluorescence intensity of [Ca2+ ]i increased rapidly, then returned to a new homeostasis (selected cells in groups A, B, E, F) or experienced a second calcium oscillation (some cells in group C and D). All the selected cells in group B and some cells in group C and D exhibited superimposed oscillations. The calcium overloading time in group D was longer than those of any other groups. Four cells in group A experienced delayed calcium oscillations. Compared with group A, the selected cells in other groups exhibited a larger amplitude of calcium oscillation(all P<0.05) and the selected cells in group B and D exhibited calcium oscillation for a longer period of time(all P<0.05). Conclusions In the calcium-free buffer, US, USMB, CBP+ US, CBP+ USMB are direct stimuli of calcium overload in A549 cells. SonoVue, CBP, CBP+ SonoVue are all synergistic stimuli of calcium overload in A549 cells irradiated by ultrasound.US, USMB and CBP may synergistically induce calcium release from intracellular store sites in A549 cells. Calcium overload is a possible mechanism of ultrasound combined with microbubbles in assisting CBP chemotherapy. Key words: Sonication; Microbubbles; Carboplatin; A549 cell; Calcium overload; Cavitation effect