高强度聚焦超声逆转K562/AO2细胞多药耐药的实验研究
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摘要
背景:肿瘤细胞多药耐药性(Multidrug resistance, MDR)的产生,是目前肿瘤化疗失败的一个主要原因。耐药肿瘤细胞大多有P-gp(P-glycoprotein,P170糖蛋白)蛋白的高表达,作为具有外排泵功能的P-gp蛋白能够能量依赖性地排出包括化疗药物在内的多种底物,对于细胞凋亡等多种细胞生物学行为也有一定的影响,研究MDR的发生机制和有效的逆转MDR已成为肿瘤治疗急需解决的问题。传统的MDR逆转以抑制P-gp的药物为主,但是药物性MDR逆转剂的效率较低,而且毒副作用较大,临床应用难以达到有效的逆转浓度。因此,寻找一种高效、低毒逆转MDR的方法是十分必要的。
目的:研究高强度聚焦超声(High intensity focused ultrasound -HIFU)逆转K562/AO2细胞MDR的效能,并与传统的MDR逆转剂维拉帕米(VRP)比较,初步探讨其逆转MDR作用机制,从而为克服肿瘤细胞MDR提供一种新的、有效的方法和思路。
方法:①超声剂量学研究:固定超声辐照时间或超声声强,应用不同声强或辐照时间的HIFU辐照K562、K562/AO2细胞株后,应用荧光显微镜AnnexinⅤ/PI染色检测细胞的即刻以及48h凋亡情况,用台盼蓝、MTT法检测HIFU对肿瘤细胞的抑制率,了解HIFU剂量-效应关系。②将对数生长期的K562、K562/AO2细胞株分组为:阿霉素(ADM)组、ADM加HIFU辐照(剂量为400W/ cm2×5s)组(HIFU组)、ADM加维拉帕米组(VRP组)。各组分别用MTT法检测K562、K562/AO2细胞各组的抑制率,用流式细胞仪(FCM)检测各组细胞内的ADM浓度, PI染色观察DNA含量的变化,用FCM和荧光显微镜AnnexinⅤ/PI染色检测各组细胞的凋亡情况。③免疫组化检测各组P-gp、PDCD5表达情况,并用Image-Pro Plus5.02软件定量分析。
结果:①当辐照时间一定时(5s)声强越高肿瘤细胞存活率越低,当HIFU声强一定时(400 W/cm2)细胞存活率随HIFU辐照时间延长而下降, HIFU辐照剂量为400W/ cm2×5s时对K562、K562/AO2细胞的存活不产生明显影响,未观察到即刻和延迟的凋亡现象;②在K562细胞株中HIFU、VRP对细胞内ADM浓度没有明显影响,肿瘤细胞增值抑制率和凋亡率HIFU组、VRP组、ADM组各组之间比较P均> 0.05。在K562/AO2细胞株中HIFU、VRP均能提高K562/AO2细胞内ADM浓度,HIFU组、VRP组与ADM组比较P均<0.01, HIFU组与VRP组比较差异无显著性(P > 0.05)。HIFU组、VRP组、ADM组均能抑制K562/AO2细胞的增殖,HIFU组、VRP组与ADM组比较P均<0.01, HIFU组与VRP组比较差异无显著性(P > 0.05)。HIFU组、VRP组、ADM组均能使K562/AO2细胞发生凋亡,细胞周期分析S期细胞比例(SPF)降低,降低幅度HIFU组、VRP组高于ADM组(P均<0.05), HIFU组与VRP组比较差异无显著性(P > 0.05)。③免疫组化的Image-pro plus分析发现: P-gp在K562/AO2表达明显高于K562,HIFU辐照K562/AO2细胞后P-gp表达明显降低。PDCD5在K562/AO2表达明显低于K562,HIFU辐照后K562/AO2细胞PDCD5表达升高。
结论:一定强度剂量的HIFU辐照耐药的肿瘤细胞可以提高化疗药物在耐药肿瘤细胞内的浓度,同时可以增强化疗药物对耐药肿瘤细胞的增值抑制率和凋亡率。HIFU逆转MDR的可能机理是增加了生物膜对于化疗药物的通透性、下调耐药细胞的P-gp表达和上调PDCD5表达,从而改变了耐药肿瘤细胞的凋亡耐受状态,提高了耐药肿瘤细胞对细胞毒药物的敏感性,一定程度上逆转了MDR。虽然在效能比较上HIFU与VRP差异无显著性,但考虑到HIFU的无创、无毒、优良的体外可控性,可以认为HIFU是一种较为理想的MDR逆转方法。
BACKGROUD :High-intensity focused ultrasound is a new technique in tumor therapy,and different dosages show various biological effects. Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Over-expression of P-glycoprotein (ABCB1, MDR1 gene product) on the plasma membrane is frequently observed in drug resistant cancer cells which leading to the failure of chemotherapy. A characteristic feature of P-glycoprotein is its broad substrate specificity. P-glycoprotein transports various hydrophobic compounds out of the cell by utilizing the energy of ATP hydrolysis. Such a diversity of P-glycoprotein substrates poses a significant problem for clinical treatment.P-glycoprotein also modulates drug absorption, bioavailability, tissue disposition , excretion and plays an important role in apoptosis , Multidrug resistance(MDR) of tumor cell results in abortive chemotherapy.Therefore, studying the mechanism of MDR and effectively reversing play a key role in tumor therapy.Traditional MDR reversal agents,the P-gp inhibitor drugs,show relatively lower efficiency but inevitable side effects.accordingly,it’s necessary to develope a better trick that can overcome MDR..
OBJECTIVE: This paper studys and evaluates the reversal effect of multidrug resistance by high-intensity focused ultrasound(HIFU) in K562/AO2,and primarily investigated the mechanism. Accordingly,we desiredly develop a promise approach in MDR tumor therapy.
METHODS:①With fixed exposed duration or acoustic intensity,by the means of Trypan blue exclusion and MTT assay,the relationship between tumor cell livability and HIFU dosage that which includes defferent acoustic intensity and duration,was analyzed. ②Logarithmic growth K562 and K562/AO2 cell lines were respectively divided into four groups: contral;adriamycin singlely ; adriamycin combined with verapamil(VRP);adriamycin combined with HIFU administered. Cells proliferation efficiency is evaluated by assay of MTT ;By the way of FCM, intracellular adriamycin concentration,SPF and apoptosis ratio are tested; With the help of AnnexinⅤ/PI,apoptotic cells are observed by fluorescence microscopy.③The semiquantitative analysis of immunohistochemistry results of P-gp and PDCD5 were calculated by Image-pro plus.
RESULT:①With the fixed exposed duratio(n5s), the tumor cell livability cut down when the ultrasound intensity ascends.with the fixed acoustic intensity(400/ cm2), when the duration increased, simultaneously ,the cell survival falls.The HIFU dosage of 400W/ cm2×5s does not influence cell livability;②HIFU and VRP show no significant influence in intracellular adriamycin concentration, cells proliferation and apoptosis ratio in K562 cell strain.but different results were detected in K562/AO2 cell strain,Compare with adriamycin singlely,HIFU and verapamil can significantly enhance intracellular adriamycin concentration,proliferation inhibition ration and apoptosis ratio.but no statistical difference was observed between group HIFU and VRP(P>0.05).③Compared with K562,K562/AO2 expresses higher level P-gp ,but lower level PDCD5. HIFU irradiation results in upregulation of PDCD5 and downregulation of P-gp in K562/AO2.
CONCLSION : HIFU can effectively increase intracellular adriamycin concentration.And consequently,the adriamycin related cytotoxicity that showed in cellular proliferation inhibition and apoptosis ratio in K562/AO2 was sharply ascended..The mechanism of its reversal effect may realize through upregulation of PDCD5 and downregulation of P-gp.Thus,HIFU can reverse the resistance to adriamycin in K562/AO2 cells . Even though no significant difference was observed between HIFU and VRP,but we conclude that HIFU is a relatively ideal reversal agent because of its non-invasion,free toxicity and easy modulation.
目的:研究高强度聚焦超声(High intensity focused ultrasound -HIFU)逆转K562/AO2细胞MDR的效能,并与传统的MDR逆转剂维拉帕米(VRP)比较,初步探讨其逆转MDR作用机制,从而为克服肿瘤细胞MDR提供一种新的、有效的方法和思路。
方法:①超声剂量学研究:固定超声辐照时间或超声声强,应用不同声强或辐照时间的HIFU辐照K562、K562/AO2细胞株后,应用荧光显微镜AnnexinⅤ/PI染色检测细胞的即刻以及48h凋亡情况,用台盼蓝、MTT法检测HIFU对肿瘤细胞的抑制率,了解HIFU剂量-效应关系。②将对数生长期的K562、K562/AO2细胞株分组为:阿霉素(ADM)组、ADM加HIFU辐照(剂量为400W/ cm2×5s)组(HIFU组)、ADM加维拉帕米组(VRP组)。各组分别用MTT法检测K562、K562/AO2细胞各组的抑制率,用流式细胞仪(FCM)检测各组细胞内的ADM浓度, PI染色观察DNA含量的变化,用FCM和荧光显微镜AnnexinⅤ/PI染色检测各组细胞的凋亡情况。③免疫组化检测各组P-gp、PDCD5表达情况,并用Image-Pro Plus5.02软件定量分析。
结果:①当辐照时间一定时(5s)声强越高肿瘤细胞存活率越低,当HIFU声强一定时(400 W/cm2)细胞存活率随HIFU辐照时间延长而下降, HIFU辐照剂量为400W/ cm2×5s时对K562、K562/AO2细胞的存活不产生明显影响,未观察到即刻和延迟的凋亡现象;②在K562细胞株中HIFU、VRP对细胞内ADM浓度没有明显影响,肿瘤细胞增值抑制率和凋亡率HIFU组、VRP组、ADM组各组之间比较P均> 0.05。在K562/AO2细胞株中HIFU、VRP均能提高K562/AO2细胞内ADM浓度,HIFU组、VRP组与ADM组比较P均<0.01, HIFU组与VRP组比较差异无显著性(P > 0.05)。HIFU组、VRP组、ADM组均能抑制K562/AO2细胞的增殖,HIFU组、VRP组与ADM组比较P均<0.01, HIFU组与VRP组比较差异无显著性(P > 0.05)。HIFU组、VRP组、ADM组均能使K562/AO2细胞发生凋亡,细胞周期分析S期细胞比例(SPF)降低,降低幅度HIFU组、VRP组高于ADM组(P均<0.05), HIFU组与VRP组比较差异无显著性(P > 0.05)。③免疫组化的Image-pro plus分析发现: P-gp在K562/AO2表达明显高于K562,HIFU辐照K562/AO2细胞后P-gp表达明显降低。PDCD5在K562/AO2表达明显低于K562,HIFU辐照后K562/AO2细胞PDCD5表达升高。
结论:一定强度剂量的HIFU辐照耐药的肿瘤细胞可以提高化疗药物在耐药肿瘤细胞内的浓度,同时可以增强化疗药物对耐药肿瘤细胞的增值抑制率和凋亡率。HIFU逆转MDR的可能机理是增加了生物膜对于化疗药物的通透性、下调耐药细胞的P-gp表达和上调PDCD5表达,从而改变了耐药肿瘤细胞的凋亡耐受状态,提高了耐药肿瘤细胞对细胞毒药物的敏感性,一定程度上逆转了MDR。虽然在效能比较上HIFU与VRP差异无显著性,但考虑到HIFU的无创、无毒、优良的体外可控性,可以认为HIFU是一种较为理想的MDR逆转方法。
BACKGROUD :High-intensity focused ultrasound is a new technique in tumor therapy,and different dosages show various biological effects. Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Over-expression of P-glycoprotein (ABCB1, MDR1 gene product) on the plasma membrane is frequently observed in drug resistant cancer cells which leading to the failure of chemotherapy. A characteristic feature of P-glycoprotein is its broad substrate specificity. P-glycoprotein transports various hydrophobic compounds out of the cell by utilizing the energy of ATP hydrolysis. Such a diversity of P-glycoprotein substrates poses a significant problem for clinical treatment.P-glycoprotein also modulates drug absorption, bioavailability, tissue disposition , excretion and plays an important role in apoptosis , Multidrug resistance(MDR) of tumor cell results in abortive chemotherapy.Therefore, studying the mechanism of MDR and effectively reversing play a key role in tumor therapy.Traditional MDR reversal agents,the P-gp inhibitor drugs,show relatively lower efficiency but inevitable side effects.accordingly,it’s necessary to develope a better trick that can overcome MDR..
OBJECTIVE: This paper studys and evaluates the reversal effect of multidrug resistance by high-intensity focused ultrasound(HIFU) in K562/AO2,and primarily investigated the mechanism. Accordingly,we desiredly develop a promise approach in MDR tumor therapy.
METHODS:①With fixed exposed duration or acoustic intensity,by the means of Trypan blue exclusion and MTT assay,the relationship between tumor cell livability and HIFU dosage that which includes defferent acoustic intensity and duration,was analyzed. ②Logarithmic growth K562 and K562/AO2 cell lines were respectively divided into four groups: contral;adriamycin singlely ; adriamycin combined with verapamil(VRP);adriamycin combined with HIFU administered. Cells proliferation efficiency is evaluated by assay of MTT ;By the way of FCM, intracellular adriamycin concentration,SPF and apoptosis ratio are tested; With the help of AnnexinⅤ/PI,apoptotic cells are observed by fluorescence microscopy.③The semiquantitative analysis of immunohistochemistry results of P-gp and PDCD5 were calculated by Image-pro plus.
RESULT:①With the fixed exposed duratio(n5s), the tumor cell livability cut down when the ultrasound intensity ascends.with the fixed acoustic intensity(400/ cm2), when the duration increased, simultaneously ,the cell survival falls.The HIFU dosage of 400W/ cm2×5s does not influence cell livability;②HIFU and VRP show no significant influence in intracellular adriamycin concentration, cells proliferation and apoptosis ratio in K562 cell strain.but different results were detected in K562/AO2 cell strain,Compare with adriamycin singlely,HIFU and verapamil can significantly enhance intracellular adriamycin concentration,proliferation inhibition ration and apoptosis ratio.but no statistical difference was observed between group HIFU and VRP(P>0.05).③Compared with K562,K562/AO2 expresses higher level P-gp ,but lower level PDCD5. HIFU irradiation results in upregulation of PDCD5 and downregulation of P-gp in K562/AO2.
CONCLSION : HIFU can effectively increase intracellular adriamycin concentration.And consequently,the adriamycin related cytotoxicity that showed in cellular proliferation inhibition and apoptosis ratio in K562/AO2 was sharply ascended..The mechanism of its reversal effect may realize through upregulation of PDCD5 and downregulation of P-gp.Thus,HIFU can reverse the resistance to adriamycin in K562/AO2 cells . Even though no significant difference was observed between HIFU and VRP,but we conclude that HIFU is a relatively ideal reversal agent because of its non-invasion,free toxicity and easy modulation.
引文
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[1]王树滨,彭晖,齐静.肿瘤耐药及逆转耐药的新策略. [J]中国肿瘤.2001;9(6):269-270.
[2]戴春岭,符立梧.肿瘤多药耐药逆转剂的研究进展. [J]中国药理学通报, 2005,May; 21(5) : 513～8
[3]缪泽鸿,丁健. 抗肿瘤多药耐药的研究进展[J].癌症,2003,22(8):882-892
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[7]Kennedy JE. High-intensity focused ultrasound in the treatment of solid tumours .[J]Nat Rev Cancer. 2005 Apr;5(4):321-7.
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[11]叶欣,费兴波. 高强度聚焦超声治疗肝癌后免疫功能的变化[J].中国肿瘤临床与康复,2005,12(1):40-43。
[12]邹玉红,叶 欣,杨宪勇。高强度聚焦超声热疗法对原发性肝癌患者 T 淋巴细胞亚群和 NK 细胞的影响. [J]中国临床康复 第 9 卷第 2 期,2005-1-16
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[20]Carol Lovegrove. High-intensity focused ultrasound shows promise for noninvasive tumor ablation[J]. Nature Clinical Practice Oncology, 2006 January,Vol 3 No1.
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[1]王树滨,彭晖,齐静.肿瘤耐药及逆转耐药的新策略. [J]中国肿瘤.2001;9(6):269-270.
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[3]缪泽鸿,丁健. 抗肿瘤多药耐药的研究进展[J].癌症,2003,22(8):882-892
[4]Hiroshi Omote,Marwan K. Interaction of Transported Drugs with the Lipid Bilayer and P-Glycoprotein through a Solvation Exchange Mechanism[J]. Biophys. J. 2006 90: 4046-4059
[5]Tsuruo T, Tomida A Multidrug resistance.Anticancer Drugs[J]. 1995 Apr;6(2):213-8
[6] 叶欣,费兴波,何申戌.高强度聚焦超声治疗肿瘤.[J].国外医学肿瘤学分册,2004。31(1):38-40。
[7]Kennedy JE. High-intensity focused ultrasound in the treatment of solid tumours .[J]Nat Rev Cancer. 2005 Apr;5(4):321-7.
[8]Zhenlin Hu a, Xiao Yi Yang b, Yunbo Liu a, etal.Release of endogenous danger signals from HIFU-treated tumor cells and their stimulatory effects on APCs . [J]Biochemical and Biophysical Research Communications 335 (2005) 124–131
[9]Sandra L Poliachik, Wayne L. etal.The relation between cavitation and platelet aggregation during exposure to high-intensity focused ultrasound. [J]Ultrasound in Med. & Biol., Vol. 30, No. 2, pp. 261–269, 2004.
[10] Vesna Zderic a,Amid Keshavarzi a,etal. Hemorrhage control in arteries using high-intensity focused ultrasound: A survival study[J] .Ultras.2005.08.002
[11]叶欣,费兴波. 高强度聚焦超声治疗肝癌后免疫功能的变化[J].中国肿瘤临床与康复,2005,12(1):40-43。
[12]邹玉红,叶 欣,杨宪勇。高强度聚焦超声热疗法对原发性肝癌患者 T 淋巴细胞亚群和 NK 细胞的影响. [J]中国临床康复 第 9 卷第 2 期,2005-1-16
[13]Feng Wu,Zhi-Biao Wang,Pei Lu,etal.Activated anti-tumor immunity in cancer patients after high-intensity focused ultrasound ablation. [J] Ultrasound in Med. & Biol., Vol. 30, No. 9, pp. 1217–1222, 2004.
[14]武睿毅,王国民,曾昭冲.高强度聚集超声治疗小鼠前列腺癌皮下移植瘤的疗效. [J].复旦学报(医学版)2004 年 1 月,31(1)
[15]Zeng Jun-Qun , Wang Guo-Min, Yao Bo, Wang Gong-Xian and He Shen-Xu.Short-term results of 89 cases of rectal carcinoma treated with high-intensity focused ultrasound and low-dose radiotherapy[J]. Ultrasound in Medicine & Biology Volume 30, Issue 1 , January 2004, Pages 57-60
[16]Philippe apparel,Laura curiel. Synergistic inhibitory effect of high-intensity focused ultrasound combined with chemotherapy on Dunning adenocarcinoma[J]. 2005 BJU International(95,881-885)
[17]翟宝进,邵泽勇,伍峰.HIFU 逆转人肝癌细胞 HepG2/ADM 多药耐药的实验研究[J].癌症.2003 22(12):1284-1288
[18]Tinghe Yu, Kai Hu, Jin Bai,et al. Reversal of adriamycin resistance in ovarian carcinoma cell line by combination of verapamil and low-level ultrasound[J].Ultrasonics Sonochemistry 2003 10 :37–40
[19]Yang Liu, Kevin Lillehei,Wesley N. Overcoming MDR by Ultrasound-Induced Hyperthermia[J]. Biochem Biophys Res Commun. 2001,Nov 23,289(1):62-8
[20]Carol Lovegrove. High-intensity focused ultrasound shows promise for noninvasive tumor ablation[J]. Nature Clinical Practice Oncology, 2006 January,Vol 3 No1.