OBJECTIVE The aim of the study was to examine the reversal effects of ultrasound （US） on the MDR in HepG2/ADM, a HepG2 cell line resistant to Adriamycin （ADM）, and to study the mechanism of US action.METHODS Using...OBJECTIVE The aim of the study was to examine the reversal effects of ultrasound （US） on the MDR in HepG2/ADM, a HepG2 cell line resistant to Adriamycin （ADM）, and to study the mechanism of US action.METHODS Using the MTT assay, the effects of US on MDR in HepG2/ADR cells were studied. Before and after the treatment with 0.5 W/cm^2 low intensity ultrasound （LIUS）, the expression of the MDR-related genes, mdrl, mrp and lrp was assayed with the reverse transcriptase polymerase chain reaction （RT-PCR） and the levels of their respective protein expression determined by flow cytometry. By using confocal laser scanning microscopy （CLSM）, we examined the intracellular daunorubicin （DNR） distribution, and the effects on the cells of treatment with US or DNR.RESULTS LIUS significantly reversed MDR in HepG2/ADR cells. After treatment with LIUS at 0.5 W/cm^2, chemosensitivity to ADM and DNR increased 3.35-fold and 2.81-fold, respectively. The reversal activity by LIUS plus verapamil （VER） was stronger than with either US or VER alone. After treatment with 0.5 W/cm^2, the expression of both the MDR1 and the MRP mRNA genes began to decline （P 〈 0.01 and P 〈 0.05, respectively）; the expression of LRP showed no significant changes. Changes in the expression of the P-glycoprotein （P-gp） and MRP were similar to those of their mRNA expressions. Results of the CLSM showed that administration of US （0. 5 W/cm^2） or VER （15.7 uM） with DNR to HepGa/ADM cells showed a significant change in the distribution of DNR in the cells.CONCLUSION Our results show that LIUS can reverse MDR. The reversal effects are stronger than those of either US or VER alone, when combined with VER administration. As LIUS is noninvasive causing no toxicity, it might have potential for clinical application. The reversal mechanism needs further study.展开更多
目的人参皂苷Rh2能抑制多种恶性肿瘤细胞增殖,但缺少Rh2对多药耐药的肝癌细胞的敏感性研究。文中主要探讨人参皂苷Rh2对人肝癌细胞株HepG2/ADM多药耐药性[盐酸多柔比星(ADM)、5-氟尿嘧啶(5-Fu)、顺铂(DDP)、长春新碱(VCR)]的逆转作用及...目的人参皂苷Rh2能抑制多种恶性肿瘤细胞增殖,但缺少Rh2对多药耐药的肝癌细胞的敏感性研究。文中主要探讨人参皂苷Rh2对人肝癌细胞株HepG2/ADM多药耐药性[盐酸多柔比星(ADM)、5-氟尿嘧啶(5-Fu)、顺铂(DDP)、长春新碱(VCR)]的逆转作用及机制。方法 MTT法检测(0~250μg/m L)Rh2对HepG2/ADM细胞活力的影响;MTT法筛选最佳逆转耐药的Rh2浓度。细胞分为空白对照组、ADM组和ADM+40μg/m LRh2组。空白对照组:不加药物处理;ADM组:给予ADM处理48 h;ADM+40μg/m LRh2组:给予40μg/m L的Rh2预处理30 min后,给予ADM处理48 h。流式细胞术检测Rh2对细胞内Rh-123荧光强度的影响;RT-PCR法检测MDR1基因的表达;Western blot检测P-gp、Bax、Bcl-2、cleved caspase-3蛋白水平。结果与HepG2细胞相比,HepG2/ADM对ADM、DDP、5-FU、VCR 4种化疗药物的耐药指数分别为32.95、4.63、4.20、4.81。经过40μg/m L的Rh2作用HepG2/ADM细胞48 h后,耐药细胞对4种化疗药的敏感性增强且IC50明显下降,其耐药性的逆转倍数分别为3.70、3.53、2.64、2.55倍。耐药细胞内储留的Rh-123的荧光强度通过流式细胞仪检测结果显示,与ADM组比较,加入40μg/m L Rh2后,细胞内Rh-123的荧光强度明显增高(65.83±1.78 vs 78.21±1.26,P<0.01)。RT-PCR结果显示,ADM+40μg/m L Rh2组MDR1表达较ADM组显著降低(0.48±0.02 vs 0.86±0.05,P<0.05)。Western blot结果显示,ADM+40μg/m L Rh2组P-gp蛋白水平亦较ADM组明显降低(0.97±0.04 vs 1.91±0.03,P<0.01);ADM+40μg/m L Rh2组Bax和cleaved caspase-3表达较ADM组明显增加(1.76±0.04 vs 1.25±0.02,38.26±5.45 vs 0.42±0.04,P<0.05),同时发现Bcl-2表达明显减少(1.25±0.05 vs 1.86±0.03,P<0.05)。结论人参皂苷Rh2能有效逆转HepG2/ADM细胞的多药耐药性,其作用机制可能与降低MDR1、P-gp表达、增加细胞内药物积累以及介导Bax/Bcl-2信号通路有关。展开更多
OBJECTIVE To evaluate the potential efficacy of low-intensity ultrasound （US） in combination with anticancer drugs to reverse multidrug resistance （MDR）in nude mice. METHODS A total of 40 male and female athymic n...OBJECTIVE To evaluate the potential efficacy of low-intensity ultrasound （US） in combination with anticancer drugs to reverse multidrug resistance （MDR）in nude mice. METHODS A total of 40 male and female athymic nude mice were inoculated subcutaneously with 5×10^6 HepG2/ADM and HepG2 cells. Ultrasound with pulsed irradiation at an average intensity of 0.5 W/cm^2 was given to the tumor area 10 rain after administration of adriamycin （ADM）. The tumor 3 dimensional diameters were measured by calipers before and after treatment, and the tumor growth indexes （TGI） calculated. RT-PCR was used to detect the gene levels of the HepG2/ADM cells. Immunohistochemical analyses for MDR proteins were conducted on the tumor tissues. RESULTS The ultrasonic treatment resulted in an average reduction in the tumor volume of 62% one month later. The relative mRNA levels of MDR1 and MRP were significantly different among the following 4 groups： untreated group as control, ADM treated; US treated; and ADM plus US treated. The mRNA levels of mdrl and mrp were down-regulated in the US groups compared to those of the non-ultrasound groups by multiple comparisons. The relative mRNA levels of Irp expression were not significantly changed. The results of immunohistochemistry indicated that tumor tissue from animals treated with US had remarkably low mdrl and mrp expression. CONCLUSION The results showed that low-intensity US can effectively reduce the size of adriamycin-resistant human hepotacarcinoma in a nude mouse model, and support the efficacy of US to overcome multiple mechanisms of drug resistance.展开更多
基金a grant from National Natural Science Foundation of China (No.30200060)
文摘OBJECTIVE The aim of the study was to examine the reversal effects of ultrasound （US） on the MDR in HepG2/ADM, a HepG2 cell line resistant to Adriamycin （ADM）, and to study the mechanism of US action.METHODS Using the MTT assay, the effects of US on MDR in HepG2/ADR cells were studied. Before and after the treatment with 0.5 W/cm^2 low intensity ultrasound （LIUS）, the expression of the MDR-related genes, mdrl, mrp and lrp was assayed with the reverse transcriptase polymerase chain reaction （RT-PCR） and the levels of their respective protein expression determined by flow cytometry. By using confocal laser scanning microscopy （CLSM）, we examined the intracellular daunorubicin （DNR） distribution, and the effects on the cells of treatment with US or DNR.RESULTS LIUS significantly reversed MDR in HepG2/ADR cells. After treatment with LIUS at 0.5 W/cm^2, chemosensitivity to ADM and DNR increased 3.35-fold and 2.81-fold, respectively. The reversal activity by LIUS plus verapamil （VER） was stronger than with either US or VER alone. After treatment with 0.5 W/cm^2, the expression of both the MDR1 and the MRP mRNA genes began to decline （P 〈 0.01 and P 〈 0.05, respectively）; the expression of LRP showed no significant changes. Changes in the expression of the P-glycoprotein （P-gp） and MRP were similar to those of their mRNA expressions. Results of the CLSM showed that administration of US （0. 5 W/cm^2） or VER （15.7 uM） with DNR to HepGa/ADM cells showed a significant change in the distribution of DNR in the cells.CONCLUSION Our results show that LIUS can reverse MDR. The reversal effects are stronger than those of either US or VER alone, when combined with VER administration. As LIUS is noninvasive causing no toxicity, it might have potential for clinical application. The reversal mechanism needs further study.
文摘目的人参皂苷Rh2能抑制多种恶性肿瘤细胞增殖,但缺少Rh2对多药耐药的肝癌细胞的敏感性研究。文中主要探讨人参皂苷Rh2对人肝癌细胞株HepG2/ADM多药耐药性[盐酸多柔比星(ADM)、5-氟尿嘧啶(5-Fu)、顺铂(DDP)、长春新碱(VCR)]的逆转作用及机制。方法 MTT法检测(0~250μg/m L)Rh2对HepG2/ADM细胞活力的影响;MTT法筛选最佳逆转耐药的Rh2浓度。细胞分为空白对照组、ADM组和ADM+40μg/m LRh2组。空白对照组:不加药物处理;ADM组:给予ADM处理48 h;ADM+40μg/m LRh2组:给予40μg/m L的Rh2预处理30 min后,给予ADM处理48 h。流式细胞术检测Rh2对细胞内Rh-123荧光强度的影响;RT-PCR法检测MDR1基因的表达;Western blot检测P-gp、Bax、Bcl-2、cleved caspase-3蛋白水平。结果与HepG2细胞相比,HepG2/ADM对ADM、DDP、5-FU、VCR 4种化疗药物的耐药指数分别为32.95、4.63、4.20、4.81。经过40μg/m L的Rh2作用HepG2/ADM细胞48 h后,耐药细胞对4种化疗药的敏感性增强且IC50明显下降,其耐药性的逆转倍数分别为3.70、3.53、2.64、2.55倍。耐药细胞内储留的Rh-123的荧光强度通过流式细胞仪检测结果显示,与ADM组比较,加入40μg/m L Rh2后,细胞内Rh-123的荧光强度明显增高(65.83±1.78 vs 78.21±1.26,P<0.01)。RT-PCR结果显示,ADM+40μg/m L Rh2组MDR1表达较ADM组显著降低(0.48±0.02 vs 0.86±0.05,P<0.05)。Western blot结果显示,ADM+40μg/m L Rh2组P-gp蛋白水平亦较ADM组明显降低(0.97±0.04 vs 1.91±0.03,P<0.01);ADM+40μg/m L Rh2组Bax和cleaved caspase-3表达较ADM组明显增加(1.76±0.04 vs 1.25±0.02,38.26±5.45 vs 0.42±0.04,P<0.05),同时发现Bcl-2表达明显减少(1.25±0.05 vs 1.86±0.03,P<0.05)。结论人参皂苷Rh2能有效逆转HepG2/ADM细胞的多药耐药性,其作用机制可能与降低MDR1、P-gp表达、增加细胞内药物积累以及介导Bax/Bcl-2信号通路有关。
基金This work was supported by a grant from theNational Natural Science Foundation of China(No.30200060).
文摘OBJECTIVE To evaluate the potential efficacy of low-intensity ultrasound （US） in combination with anticancer drugs to reverse multidrug resistance （MDR）in nude mice. METHODS A total of 40 male and female athymic nude mice were inoculated subcutaneously with 5×10^6 HepG2/ADM and HepG2 cells. Ultrasound with pulsed irradiation at an average intensity of 0.5 W/cm^2 was given to the tumor area 10 rain after administration of adriamycin （ADM）. The tumor 3 dimensional diameters were measured by calipers before and after treatment, and the tumor growth indexes （TGI） calculated. RT-PCR was used to detect the gene levels of the HepG2/ADM cells. Immunohistochemical analyses for MDR proteins were conducted on the tumor tissues. RESULTS The ultrasonic treatment resulted in an average reduction in the tumor volume of 62% one month later. The relative mRNA levels of MDR1 and MRP were significantly different among the following 4 groups： untreated group as control, ADM treated; US treated; and ADM plus US treated. The mRNA levels of mdrl and mrp were down-regulated in the US groups compared to those of the non-ultrasound groups by multiple comparisons. The relative mRNA levels of Irp expression were not significantly changed. The results of immunohistochemistry indicated that tumor tissue from animals treated with US had remarkably low mdrl and mrp expression. CONCLUSION The results showed that low-intensity US can effectively reduce the size of adriamycin-resistant human hepotacarcinoma in a nude mouse model, and support the efficacy of US to overcome multiple mechanisms of drug resistance.
