| Objective: Optimize ultrasound irradiation mode, intensity, irradiation time, and theconcentration of microbubbles to determine the best parameters for induced apoptosisof DU145prostate cancer cells using ultrasound combined with SonoVue; Observethe effects on growth of DU145cells and explore the mechanism; Establish a modelfor DU145human prostate cancer xenografts in nude mice, and observe the effects onxenografts growth and caveolin-1expression.Methods:(1) Irradiate DUl45cell suspension using pulsed or continuous wave mode oflow-frequency ultrasound irradiation, and observe changes membrane permeabilitythrough detection of FD500staining rate by fluorescence microscopy, cellproliferation by MTT assay, and apoptosis rate by flow cytometry.(2) Using orthogonal design method, three factors (ultrasound intensity;irradiation time; microbubble density) were explored with three values for each factor(ultrasound intensity:0.15,0.30, and0.45W/cm2, irradiation time:10,20and30s,and microbubble density:10,20and50%). Each sample was irradiated for24h usingone of the combination of parameter and analyzed by flow cytometry to determineapoptosis rate, from which optimal irradiation parameters for treatment were derived.(3) Ultrasound irradiation of DU145cell suspension was carried out using theoptimized parameters. Samples were divided into six groups: control, SonoVue,ultrasound group, ultrasound combined with SonoVue, methyl-β-cyclodextrin, andcholesterol group. For methyl-β-cyclodextrin group, methyl-β-cyclodextrin was addedto cells24h before the ultrasound irradiation. For the cholesterol group, cells werefirst treated with methyl-β-cyclodextrin for24h, washed three times using cold PBS, treated with RPMI-1640medium and cholesterol, and cultured for another24h. Cellgrowth curve and apoptosis rate were obtained by MTT assay and flow cytometryrespectively. Western-blots were used to detect the expression levels of caveolin-1,phospho-Akt, and the vascular endothelial growth factor.(4) To establish a xenograft model in nude mice for DU145human prostatecancer cells, the32tumor-bearing mice were randomly divided into4groups: control,SonoVue, ultrasound group, and ultrasound combined with SonoVue group. The micewere irradiated with ultrasound (frequency:80kHz and intensity:0.5W/cm2), thegrowth of tumor was monitored for3consecutive weeks to obtain growth curve. Theexpression of caveolin-1in tumor tissue was detected by Western-blot andimmunohistochemical methods, while the expression of caveolin-1expression in theserum was analyzed by ELISA.Results:(1) Compared with the control group, the percentages of FD500fluorescence-stained cells were increased in two patterns of ultrasound exposuregroups. The rate of FD500staining positive was more higher in pulse wave patternthan that of continuous wave, the difference was statistically significant (P<0.01). Thecell proliferation was slightly inhibited by pulsed ultrasound, and the inhibitory effectwas significantly enhanced by continuous ultrasound (P<0.05). The rate of DU145cell apoptosis increased in two patterns of ultrasound treatment groups compared withthe control group; the continuous ultrasound promoted cell apoptosis significantlythan that of pulsed ultrasound, the difference had significant statistical significance(P<0.01).(2) The effect on the cell apoptosis of the factors, ultrasound intensity>irradiation time> microbubble density. The level of impact of various factors:ultrasound intensity:0.45W/cm2>0.30W/cm2>0.15W/cm2; irradiation time:30s>20s>10s; microbubble density:20%>50%>10%.(3) Cell growth curve showed cells in the control group and SonoVue groupgrew rapidly, while cells in the ultrasound group and ultrasound combined with SonoVue cells grew relatively slowly. After incubation for48h, ultrasound orespecially, ultrasound combined with SonoVue significantly inhibited cell growth(P<0.05). Compared with ultrasound combined with SonoVue group,methyl-β-cyclodextrin inhibited the growth of DU145cells. Cholesterol can partiallyreverse the effect of methyl-β-cyclodextrin. Flow cytometry showed that differencesin apoptosis rate observed for the ultrasound group and ultrasound combined withSonoVue group, when compared with the control group, are statistically significant(P<0.05). Moreover, apoptosis rate difference between the ultrasound group andultrasound SonoVue group is also statistically significant (P<0.05). Compared withthe ultrasound combined with SonoVue group, methyl-β-cyclodextrin significantlypromoted the apoptosis of DU145cells, and the difference between the two groups isstatistically significant (P<0.05). However, cholesterol partially reversed the effects ofmethyl-β-cyclodextrin in promoting apoptosis. Western-blots showed that theexpression levels of caveolin-1, phospho-Akt and the vascular endothelial growthfactor decreased significantly in the ultrasound combined with SonoVue group, butslightly in the ultrasound group (P<0.05). Compared with ultrasound combined withSonoVue group, the expression levels of caveolin-1, phospho-Akt and the vascularendothelial growth factor decreased significantly in the methyl-β-cyclodextrin group(P<0.05). Compared to the methyl-β-cyclodextrin group, expression levels of theseproteins slightly increased in the cholesterol group.(4) Treatment with ultrasound alone produced tumor volumes and weightsreduction compared with control and SonoVue group. Combined ultrasound andSonoVue treatment significantly inhibited the growth of xenografts and producedgreater tumor volumes and weights regression than either treatment alone (p<0.05).Results were shown by Western-blot and immunehistochemistry that theultrasound-treated mice had significant reduction in expression levels of caveolin-1protein, compared with the control (p<0.05). Levels of caveolin-1were furtherreduced when combined with ultrasound and SonoVue as compared to the control(p<0.01). Ultrasound alone treatment only slightly reduced the caveolin-1concentrations in comparison with the control, and the difference was not statistically significant (p>0.05). Serum caveolin-1concentrations were lower in the combinationof ultrasound and SonoVue group than they were in control group (p<0.05).Conclusions:(1) Low-frequency ultrasound using different radiation patterns can affect thebiological behaviour of prostate cancer DU145cells. For gene transfection or drugtransport, pulsed ultrasound better increase the cell membrane permeability; Appliedin the treatment of purpose, continuous ultrasound can inhibit tumor growth.(2) Low-frequency ultrasound combined with SonoVue could induce apoptosisof prostate cancer DU145cells. The optimal combination was ultrasound intensity0.45W/cm2, irradiation time30s, microbubble density20%.(3) Low-frequency ultrasound combined with SonoVue could inhibit theproliferation of prostate cancer DU145cells and its mechanism might be associatedwith disturbing the pathway of caveolin-1, phospho-Akt, vascular endothelial growthfactor signaling axis.(4) Low-frequency ultrasound combined with SonoVue could inhibit the growthof prostate cancer DU145xenografts in nude mice and the inhibitory effect mighthave certain relation to down-regulating the expression of caveolin-1. |
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