Experimental Study On Effects Of Ultrasound Combined With Microbubbles On Intracellular Ca²⁺ Homeostasis In Carboplatin-treated Microenvironment Of A549 Cell Line

BackgroundAlthough carboplatin is a first-line chemotherapy drug for lung cancer,the side effects and drug resistance limit its clinical application.Ultrasound has been widely used to enhance drug delivery and assist chemotherapy.Due to the sensitivity of different tumor cell line to ultrasound is various,the biological effects of ultrasound and microbubbles are random to some degree,and the energy parameters of the ultrasonic instruments are not exactly same in different studies,at present,no unified standard has been obtained.The results cannot be compared to each other accurately.Intracellular calcium ion concentration([Ca²⁺]_i)is a double-edged sword that regulates the kinetics of sonoporation and the fate of sonoporated cell.It is not only an initiator for membrane resealing by endocytosis or exocytosis,but also a trigger for apoptosis.There is a close relationship between Ca²⁺and the efficacy and safety of ultrasound cavitation mediated drug delivery.ObjectivesTo investigate the appropriate dose of carboplatin and ultrasonic energy parameters for ultrasound assisting carboplatin therapy on A549 cell line in vitro.To investigate the effects of ultrasound combined with microbubbles on intracellular Ca²⁺homeostasis in carboplatin（CBP）-treated A549 cell and the possible mechanisms of inhibiting A549 cell line activity.Methods1.A549 cells were planted in 96-well plates and treated with carboplatin in a concentration gradient of 0-300μg/ml for 24 h.Then 10μl CCK-8 was added to each well and cells were incubated for 1.5 h.The absorbance at 450 nm was measured to calculate the cell inhibition rate.The IC50 of carboplatin for 24 h was calculated using the Graphpad Prism 5software with drug dose-effect model.IC25 was chosen as the appropriate dose.2.6 groups were set as follows:the groups of Control,US,USMB,CBP,CBP+US,CBP+USMB.After 24 h,cell viability was measured by CCK-8 assay.Differences of survival rates were analyzed using SPSS 24.0,and appropriate ultrasound intensity was determined.3.Optical inverted microscope was used to observe the morphological changes of cells in each group.4.The groups A-F were the ultrasound（US）group（group A）,the ultrasound combined with microbubbles（USMB）group（group B）,the low dose CBP（100μg/ml）+US group（group C）,the low dose CBP+USMB group（group D）,the high dose CBP（200μg/ml）+US group（group E）and the high dose CBP+USMB group（group F）.A549 cells were bathed and washed in 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 in individual living cells was observed and the calcium overload was analyzed.Results1.The IC50 and IC25 of carboplatin for 24 h was 65.72μg/ml,50μg/ml respectively,the latter of which was used as the suitable dose in the following experiement.2.At the intensity of 0.6W/cm²,there were no significant difference among the US,USMB and Control groups（P=0.142）.However,at the same intensity,the cell viabilities of CBP+US,CBP+USMB and the CBP groups were significantly different from each other（all P<0.05）.No significant difference of the cell viabilities was found among the five CBP+USMB（0.2-1.0W/cm²）groups（P=0.691）.The inhibitory effect was ranked as CBP+USMB group>CBP+US group>CBP group>USMB group>US group.3.Under the microscopy,cells were irregular in shape and adherence was poor in the treatment groups,of which the CBP+USMB group was the most significant one.4.After ultrasound irradiation,the normalized fluorescence intensity of intracellular Ca²⁺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 groups C and D）.All the selected cells in group B and some cells in groups C and D exhibited superimposed oscillations.Four cells in group A experienced delayed calcium oscillations.The calcium overloading time of group D was longer than that of any other groups.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 groups B and D exhibited calcium oscillation for a longer period of time（all P<0.05）.Conclusions1.50μg/ml carboplatin under ultrasound intensity of 0.6W/cm² with microbubbles is the optimal combination for CBP+USMB therapy for A549 cell line in vitro.2.Ultrasound combined with microbubbles can assist carboplatin and inhibit the viability of A549 cells.This finding will become a promising strategy for cancer therapy.3.In the calcium-free buffer,US,USMB,CBP+US,CBP+USMB are direct stimuli of calcium overload.SonoVue,CBP,CBP+SonoVue in combination with ultrasound irradiation,are synergistic stimuli of calcium overload.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 carboplatin chemotherapy.