The Inhibition Of HepG2 Growth By Low-frequency And Low-power Ultrasound With Hydroxyapatite

Objective:To treat human hepatocellular carcinoma HepG2 cells with low-frequency and low-power ultrasound coordinated hydroxyapatite,exploring whether the effect can inhibit the growth of HepG2 cells,and preliminarily to explore the anti-tumor mechanism.Methods:(1)Ultrasound experiment platform was set up,and hydroxyapatite particles were prepared by liquid phase synthesis method.(2)The HepG2 cells were cultured by DMEM high-glucose medium containing hydroxyapatite(200?g/ml)and irradiated by ultrasound with different parameters(f=20 kHz,P=0.1W/cm~2,0.2W/cm~2,0.3W/cm~2,t=30,60,90,120,150,180s,T=0,12,24,48,72h).The growth inhibition rate of HepG2 cells was detected by MTT and trypan blue assay.The biological effects of ultrasound were detected by the uptake of hydroxyapatite particles and calcium ion concentration.And the anti-tumor mechanism was preliminarily explored by cell apoptosis,cell cycle and cell autophagy.The proliferation,migration and invasion ability of treated cells were detected by EdU experiment and Transwell test.(3)Hydroxyapatite particles(200?g/ml)were injected into the tumor of tumor bearing mice,and ultrasound irradiated the hepatoma bearing mice with different parameters(f=20kHz,P=0.1W/cm~2,0.2W/cm~2,0.3W/cm~2,t=1min,5min,10min).The tumor volume was measured,the tumor inhibition rate was calculated.When the tumor inhibition rate was over50%,the ultrasonic radiation was stopped.The tumors were removed and submitted for examination.Cell apoptosis,autophagy and necrosis were investigated in order to explore anti-tumor mechanism.Results:(1)In this section,we established an ultrasonic radiation platform.In the cell experiment,the ultrasonic treatment head was placed in the air-free water,and the cells were radiated by ultrasound.In the animal experiment,a water jacket was placed on the head of the ultrasound treatment which was used to radiate animals with liver cancer.The air-free water was a better couplant to couple with ultrasound,and it can dissipate heat.The synthesized material was hydroxyapatite which was high-purity hydroxyapatite without other impurities.The structure of hydroxyapatite is a short rod,with a small average particle size(a long diameter of 100 nm,a minor diameter of 50 nm),which is in a nano-scale range with a uniform particle size.The distribution of hydroxyapatite is more uniform,with a better dispersion.(2)To treat human hepatocellular carcinoma HepG2 cells with low-frequency and low-power ultrasound coordinated hydroxyapatite.The results of MTT and trypan blue experiments showed that the growth inhibition rate of HepG2 cells increased with the increase of ultrasonic power,the prolongation time of ultrasonic radiation and the prolongation of culture time.The cell growth inhibition rate reached the highest with parameters hydroxyapatite(200?g/ml)and ultrasound(P=0.2W/cm~2,t=90s,T=48h).Meanwhile,using the parameters to treat the cells,the apoptosis rates of ultrasound+hydroxyapatite,ultrasound group,hydroxyapatite group and control group respectively were(34.967±1.973)%,(4.967±1.872)%,(6.867±3.101)%,(3.633±0.569)%.Among them,the apoptotic rate of ultrasound+hydroxyapatite group was significantly higher than that of the other three groups,and the difference was statistically significant(p<0.05).Hydroxyapatite particles uptake amounts of ultrasound+hydroxyapatite group,ultrasound group,hydroxyapatite group and control group were(71943±1847.171)%,(23708±96.348)%,(41532.67±399.830)%and(23473±113.450)%respectively.The amount of ultrasound+hydroxyapatite group was significantly higher than the other three groups,and the difference was statistically significant(p<0.05).The value of calcium ion fluorescence in ultrasound+hydroxyapatite group,ultrasound group,hydroxyapatite group and control group was(3514.33±266.294)%,(2003.33±114.159)%,(2616.33±62.692)%,(1428.33±166.929)%respectively.The cell calcium fluorescence value of ultrasound+hydroxyapatite group was significantly higher than that in the control group,and the difference was statistically significant(p<0.05).The cell proliferation rates of ultrasound+hydroxyapatite group,ultrasound group,hydroxyapatite group and control group were(14.040±1.310)%,(44.092±0.812)%,(47.132±1.455)%and(50.268±1.712)%respectively.The cell proliferation rate of ultrasound+hydroxyapatite group was significantly lower than other three groups,and the difference was statistically significant(P<0.05).The number of cell migration and invasion respectively were(24.83±4.262),(54.33±6.218),(64.00±7.694)and(79.00±8.944),(44.60±4.159),(89.20±6.058),(89.20+6.058),(99.40+7.162)and(123.00+7.382),the number of cell migration and invasion in ultrasound+hydroxyapatite group was significantly lower than other three groups,and the difference was statistically significant(p<0.05).The above results show that low frequency and low power ultrasound synergistic hydroxyapatite can inhibit the growth of HepG2 cells,and ultrasound can promote the hydroxyapatite particles into cells,increasing the intracellular calcium concentration,promoting cell apoptosis,and inhibiting the proliferation,migration and invasion of tumor cells.(3)Ultrasound(f=20kHz,P=0.3W/cm~2,t=10min)coordinated hydroxyapatite(200?g/ml)treated tumor-bearing mice.The inhibitory rate of tumor bearing mice reached 60.35%after 9days treatment.At the same parameters,the apoptosis rate of liver cancer tissue of of ultrasound+hydroxyapatite,ultrasound group,hydroxyapatite group and control group were(31.587±2.363)%,(12.657±3.73)%,(9.703±1.65)%and(4.137±2.54)%.The apoptotic rate of ultrasound+hydroxyapatite group was significantly higher than that of the other three groups,and the difference was statistically significant(p<0.05).The results of HE staining showed that the degree of tumor necrosis in ultrasound+hydroxyapatite group was significantly higher than others three group.No significant histopathological changes in heart,liver,lung,spleen and kidney were observed in organ HE staining.The above results show that low-frequency and low-power ultrasound synergistic hydroxyapatite had the effect of inhibiting the growth of tumors of liver cancer animals,and there is no abnormal histopathological changes in the organs of animals.Conclusion:(1)The treatment of hepatoma HepG2 cells with low-frequency and low-power ultrasound with hydroxyapatite can inhibit the proliferation of HepG2 cells,and can promote cells apoptosis,inhibit migration and invasion.(2)The low-power and low-power ultrasound with hydroxyapatite treated liver cancer tumor bearing mice which can achieve the effect of inhibiting the growth of tumors,and does not cause abnormal organ pathological changes in animals.In summary,low-frequency and low-power ultrasound with hydroxyapatite treated HepG2 cells and hepatoma bearing mice,which can achieve inhibition of HepG2 cell proliferation and inhabit tumor growth in mice.It suggests that the combination of ultrasonic treatment with hydroxyapatite can provide a new safe,effective,and without significant side effects treatment strategy for cancer treatment in the future.