奥美拉唑逆转人肺腺癌耐顺铂细胞株A549/DDP耐药性研究
|
摘要
目的:本实验研究V-ATPase非特异性抑制剂—奥美拉唑逆转人肺腺癌耐药株A549/DDP细胞耐药性作用及可能机制。
方法:选用人肺腺癌耐药株A549/DDP为研究对象,采用MTT法测定顺铂(DDP)对A549及A549/DDP细胞的半数抑制浓度(IC50),以此判断A549/DDP细胞的耐药性情况。采用MTT法确定奥美拉唑(Omerprazole,简称OME)非细胞毒性剂量:取低于10%抑制浓度(inhibiting concentration,IC10)作为药物作用浓度:OME4μg/ml。在此浓度逆转剂作用下测定顺铂对A549/DDP细胞的半数抑制浓度(IC50),以此判断OME对耐药细胞药物敏感性的影响。光学显微镜观察细胞形态学改变。运用流式细胞仪(Flow cytometry,FCM)测定加入逆转剂后对A549/DDP细胞株的细胞周期影响。运用流式细胞仪检测有无逆转剂作用下A549/DDP细胞中Bcl-2和PTEN蛋白表达的情况。应用激光扫描共聚焦显微镜(Laser Scanning Confocal Microscopy,LSCM)检测OME处理前后细胞内BCECF-AM(pH探针)荧光强度的变化,间接反映细胞内pH之变化。采用半定量逆转录聚合酶链反应(Reverse Transcription Polymerase Chain Reaction,RT-PCR)检测V-ATPase mRNA在人肺腺癌细胞株A549细胞和耐顺铂细胞株A549/DDP中表达情况。采用半定量逆转录聚合酶链反应检测有无逆转剂作用下A549/DDP细胞中Bcl-2 mRNA和PTEN mRNA表达的情况。
结果:MTT法测定DDP对A549细胞的IC50为1.9μg/ml,DDP对A549/DDP细胞的IC50为43.4μg/ml。OME对A549/DDP细胞的IC10为8.7μg/ml。选取非细胞毒作用浓度为4μg/ml的OME为逆转剂,在经逆转剂预处理24h情况下,DDP对A549/DDP细胞的IC50由43.4μg/ml降至30.1μg/ml,逆转倍数约为1.44,与无逆转剂组相比具有显著性差异(P<0.01)。光镜下显示,空白对照组及阴性对照组细胞形态为梭形或多形性,形态饱满,胞体折光性好,其增殖快速,实验组细胞增大,边缘模糊,胞体折光性减弱。细胞可变圆脱壁,部分细胞胀大破裂。高倍镜下可见细胞明显破碎,培养液中可见到大量细胞碎片。流式细胞仪检测结果表明经OME预处理后细胞周期受到影响,G1期细胞明显增多,S期细胞则相对减少,与无逆转剂组相比具有显著性差异(P<0.01)。其中实验组细胞中OME对细胞周期的影响可能主要以阻滞A549/DDP细胞于G1期为主。流式细胞仪检测加入OME逆转剂组与未加逆转剂组细胞中Bcl-2蛋白表达由1降至0.8,PTEN蛋白表达由1升至1.02。二者各自相比均具有显著性差异(P<0.01)。激光扫描共聚焦显微镜检测OME处理后细胞内BCECF-AM(pH探针)绿色荧光强度明显增强,间接反映OME处理后细胞内pH明显减低。RT-PCR结果显示V-ATPase在A549及A549/DDP中相对表达量分别为0.88和0.99,二者相比具有显著性差异(P<0.01)。RT-PCR结果显示V-ATPase在有无OME逆转剂处理情况下的相对表达量分别为1.05和1.09,二者相比无统计学差异(P>0.05)。RT-PCR结果显示在加入OME逆转剂预处理24h与未OME逆转剂处理情况下两组A549/DDP中Bcl-2的相对表达量由0.9降至0.8,二者相比具有显著性差异(P<0.01);PTEN的相对表达量由0.25升至0.29,二者相比具有显著性差异(P<0.01)。
结论:1、本研究通过体外实验证明了V-ATPase在人肺腺癌耐药细胞株A549/DDP较人肺腺癌细胞株A549细胞中表达增多,为以V-ATPase为作用靶点的而进行逆转肿瘤耐药开辟了一条极具潜力的有效途径。2、本实验表明,通过非细胞毒浓度的OME预处理24小时后,可以使A549/DDP细胞停滞于G1期,并可抑制人肺腺癌耐药株A549/DDP增殖。3、非细胞毒浓度的OME部分逆转人肺腺癌耐药株A549/DDP细胞耐药性的作用机制可能与上调PTEN蛋白和下调Bcl-2蛋白表达有关。4、本实验证实了OME可以作为一种理想的MDR逆转剂进行后续研究。
Objective: To study the effects of omeprazole(OME), Which is a nonspecific inhibitor of V-ATPase, on reversing the cisplatin resistance in human adenocarcinoma of lung cell line A549/DDP, and to study the possible mechanism of reversal of drμg resistance of A549/DDP cell line by OME.
Method: A549 and A549/DDP cells were incubated in cμlture medium in vitro. The DDP or OME effects on survival rates of the cells were determined with MTT assays, which is based on the reduction of a non-toxic water-soluble yellow tetrazolium salt to a purple colored water-insoluble formazan precipitate by the reductive capacity of cytoplasmatic and mitochondrial dehydrogenases present only in living cells. The vital rates(VR) were calcμlated according to the formμla. Based on the VR, 50% inhibitory rate(IC50)was determined by the liner regression of the logarithm of concentration of drμgs and VR for the drμgs. Reversing effect of OME was evaluated by determining cellμlar proliferation with MTT assays. The morphological alterations were confirmed by light microscopy. Distribution of cell cycle were examined by flow cytometry. The intracellμlar Bcl-2 and PTEN fluorescence intensity were measured by flow cytometry (FCM). The intracellμlar pH was detected via Laser Seanning Confocal Microscopy (LSCM). The mRNA expression of V-ATPase, Bcl-2 and PTEN were detected via semiquantitative reverse transcription Polymerase chain reaction (RT-PCR).
Resμlts: MTT assay showed that IC50 of DDP in A549 and A549/DDP were 1.9μg/ml and 43.4μg/ml, respectively. MTT assay showed that OME pretreated for 24h caused IC50 of DDP decreased from 43.4μg/ml to 30.1μg/ml,reversal mμltiple was 1.44, it had significant compared with untreated group (P<0.01). When pretreated with OME for 24h, A549/DDP cells had significant morphological changes which were observed by light microscopy: The cells untreated with OME were diamond or pantomorphia , and they were satiation, cell bodys refraction were strengthen. But the cells pretreated with OME for 24h were aμgmentation, nucleonic pycnosis, cell bodys refraction were weaken, The cells rounded gradualy and detached from the wall of cμlture flask , and some cells were broke. With high power lens, intracytoplasm coμld see grana obviously. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the resμlts were: Ptreatment with OME for 24h, the number of cells in G1 phase increased obviously and S phase decreased relatively. That was significant differences between treated groups and untreated group(P<0.01). So OME coμld effect the cell cycle of A549/DDP cells by induce an arrest of cell cycle in G1 phase. After added OME pretreatmented for 24h,mean fluorescence intensity of Bcl-2 in A549/DDP cells decreased from 1 to 0.8,there was significant differences between treated groups and untreated group(P<0.01). After added OME pretreated for 24h,mean fluorescence intensity of PTEN in A549/DDP cells increased from 1 to 1.02,there was significant differences between treated groups and untreated group(P<0.01). After added OME treatment, intracellμlar green fluorescence intensity significant intensification by LSCM, that indirect show that the intracellμlar pH was decreased. RT-PCR assay showed that the relative expressions of V-ATPase mRNA in A549 and A549/DDP were 0.88 and 0.99, respectively, it had significant compared with control group (P<0.01). While RT-PCR assay also showed that the relative expressions of V-ATPase mRNA had non-significant differences between treated groups and untreated group(P>0.05). RT-PCR assay showed that OME pretreated for 24h inhibited the relative expressions of Bcl-2 mRNA in A549/DDP cells, and PTEN mRNA was increased, there were significant differences between treated groups and untreated group(P<0.01).
Conclusion: 1. This research had demonstrated that V-ATPase expression in A549/DDP cells more than in A549 cells, and this open up a new and more potentiality pathway to reverse mμltidrμg resistance by V-ATPase as a effective target. 2. This research had showed that A549/DDP cells woμld be induced an arrest of cell cycle in G1 phase inhibited proliferation throμgh non-cytotoxic concentration OME pretreatmented for 24h. 3. The mechanism of OME partly reverse A549/DDP cells’drμg resistance may be concerned with up regμlation PTEN expession and down regμlation Bcl-2 expession. 4. Tish study had showed that OME coμld as a ideal reversal agent of MDR for going on more studys.
方法:选用人肺腺癌耐药株A549/DDP为研究对象,采用MTT法测定顺铂(DDP)对A549及A549/DDP细胞的半数抑制浓度(IC50),以此判断A549/DDP细胞的耐药性情况。采用MTT法确定奥美拉唑(Omerprazole,简称OME)非细胞毒性剂量:取低于10%抑制浓度(inhibiting concentration,IC10)作为药物作用浓度:OME4μg/ml。在此浓度逆转剂作用下测定顺铂对A549/DDP细胞的半数抑制浓度(IC50),以此判断OME对耐药细胞药物敏感性的影响。光学显微镜观察细胞形态学改变。运用流式细胞仪(Flow cytometry,FCM)测定加入逆转剂后对A549/DDP细胞株的细胞周期影响。运用流式细胞仪检测有无逆转剂作用下A549/DDP细胞中Bcl-2和PTEN蛋白表达的情况。应用激光扫描共聚焦显微镜(Laser Scanning Confocal Microscopy,LSCM)检测OME处理前后细胞内BCECF-AM(pH探针)荧光强度的变化,间接反映细胞内pH之变化。采用半定量逆转录聚合酶链反应(Reverse Transcription Polymerase Chain Reaction,RT-PCR)检测V-ATPase mRNA在人肺腺癌细胞株A549细胞和耐顺铂细胞株A549/DDP中表达情况。采用半定量逆转录聚合酶链反应检测有无逆转剂作用下A549/DDP细胞中Bcl-2 mRNA和PTEN mRNA表达的情况。
结果:MTT法测定DDP对A549细胞的IC50为1.9μg/ml,DDP对A549/DDP细胞的IC50为43.4μg/ml。OME对A549/DDP细胞的IC10为8.7μg/ml。选取非细胞毒作用浓度为4μg/ml的OME为逆转剂,在经逆转剂预处理24h情况下,DDP对A549/DDP细胞的IC50由43.4μg/ml降至30.1μg/ml,逆转倍数约为1.44,与无逆转剂组相比具有显著性差异(P<0.01)。光镜下显示,空白对照组及阴性对照组细胞形态为梭形或多形性,形态饱满,胞体折光性好,其增殖快速,实验组细胞增大,边缘模糊,胞体折光性减弱。细胞可变圆脱壁,部分细胞胀大破裂。高倍镜下可见细胞明显破碎,培养液中可见到大量细胞碎片。流式细胞仪检测结果表明经OME预处理后细胞周期受到影响,G1期细胞明显增多,S期细胞则相对减少,与无逆转剂组相比具有显著性差异(P<0.01)。其中实验组细胞中OME对细胞周期的影响可能主要以阻滞A549/DDP细胞于G1期为主。流式细胞仪检测加入OME逆转剂组与未加逆转剂组细胞中Bcl-2蛋白表达由1降至0.8,PTEN蛋白表达由1升至1.02。二者各自相比均具有显著性差异(P<0.01)。激光扫描共聚焦显微镜检测OME处理后细胞内BCECF-AM(pH探针)绿色荧光强度明显增强,间接反映OME处理后细胞内pH明显减低。RT-PCR结果显示V-ATPase在A549及A549/DDP中相对表达量分别为0.88和0.99,二者相比具有显著性差异(P<0.01)。RT-PCR结果显示V-ATPase在有无OME逆转剂处理情况下的相对表达量分别为1.05和1.09,二者相比无统计学差异(P>0.05)。RT-PCR结果显示在加入OME逆转剂预处理24h与未OME逆转剂处理情况下两组A549/DDP中Bcl-2的相对表达量由0.9降至0.8,二者相比具有显著性差异(P<0.01);PTEN的相对表达量由0.25升至0.29,二者相比具有显著性差异(P<0.01)。
结论:1、本研究通过体外实验证明了V-ATPase在人肺腺癌耐药细胞株A549/DDP较人肺腺癌细胞株A549细胞中表达增多,为以V-ATPase为作用靶点的而进行逆转肿瘤耐药开辟了一条极具潜力的有效途径。2、本实验表明,通过非细胞毒浓度的OME预处理24小时后,可以使A549/DDP细胞停滞于G1期,并可抑制人肺腺癌耐药株A549/DDP增殖。3、非细胞毒浓度的OME部分逆转人肺腺癌耐药株A549/DDP细胞耐药性的作用机制可能与上调PTEN蛋白和下调Bcl-2蛋白表达有关。4、本实验证实了OME可以作为一种理想的MDR逆转剂进行后续研究。
Objective: To study the effects of omeprazole(OME), Which is a nonspecific inhibitor of V-ATPase, on reversing the cisplatin resistance in human adenocarcinoma of lung cell line A549/DDP, and to study the possible mechanism of reversal of drμg resistance of A549/DDP cell line by OME.
Method: A549 and A549/DDP cells were incubated in cμlture medium in vitro. The DDP or OME effects on survival rates of the cells were determined with MTT assays, which is based on the reduction of a non-toxic water-soluble yellow tetrazolium salt to a purple colored water-insoluble formazan precipitate by the reductive capacity of cytoplasmatic and mitochondrial dehydrogenases present only in living cells. The vital rates(VR) were calcμlated according to the formμla. Based on the VR, 50% inhibitory rate(IC50)was determined by the liner regression of the logarithm of concentration of drμgs and VR for the drμgs. Reversing effect of OME was evaluated by determining cellμlar proliferation with MTT assays. The morphological alterations were confirmed by light microscopy. Distribution of cell cycle were examined by flow cytometry. The intracellμlar Bcl-2 and PTEN fluorescence intensity were measured by flow cytometry (FCM). The intracellμlar pH was detected via Laser Seanning Confocal Microscopy (LSCM). The mRNA expression of V-ATPase, Bcl-2 and PTEN were detected via semiquantitative reverse transcription Polymerase chain reaction (RT-PCR).
Resμlts: MTT assay showed that IC50 of DDP in A549 and A549/DDP were 1.9μg/ml and 43.4μg/ml, respectively. MTT assay showed that OME pretreated for 24h caused IC50 of DDP decreased from 43.4μg/ml to 30.1μg/ml,reversal mμltiple was 1.44, it had significant compared with untreated group (P<0.01). When pretreated with OME for 24h, A549/DDP cells had significant morphological changes which were observed by light microscopy: The cells untreated with OME were diamond or pantomorphia , and they were satiation, cell bodys refraction were strengthen. But the cells pretreated with OME for 24h were aμgmentation, nucleonic pycnosis, cell bodys refraction were weaken, The cells rounded gradualy and detached from the wall of cμlture flask , and some cells were broke. With high power lens, intracytoplasm coμld see grana obviously. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the resμlts were: Ptreatment with OME for 24h, the number of cells in G1 phase increased obviously and S phase decreased relatively. That was significant differences between treated groups and untreated group(P<0.01). So OME coμld effect the cell cycle of A549/DDP cells by induce an arrest of cell cycle in G1 phase. After added OME pretreatmented for 24h,mean fluorescence intensity of Bcl-2 in A549/DDP cells decreased from 1 to 0.8,there was significant differences between treated groups and untreated group(P<0.01). After added OME pretreated for 24h,mean fluorescence intensity of PTEN in A549/DDP cells increased from 1 to 1.02,there was significant differences between treated groups and untreated group(P<0.01). After added OME treatment, intracellμlar green fluorescence intensity significant intensification by LSCM, that indirect show that the intracellμlar pH was decreased. RT-PCR assay showed that the relative expressions of V-ATPase mRNA in A549 and A549/DDP were 0.88 and 0.99, respectively, it had significant compared with control group (P<0.01). While RT-PCR assay also showed that the relative expressions of V-ATPase mRNA had non-significant differences between treated groups and untreated group(P>0.05). RT-PCR assay showed that OME pretreated for 24h inhibited the relative expressions of Bcl-2 mRNA in A549/DDP cells, and PTEN mRNA was increased, there were significant differences between treated groups and untreated group(P<0.01).
Conclusion: 1. This research had demonstrated that V-ATPase expression in A549/DDP cells more than in A549 cells, and this open up a new and more potentiality pathway to reverse mμltidrμg resistance by V-ATPase as a effective target. 2. This research had showed that A549/DDP cells woμld be induced an arrest of cell cycle in G1 phase inhibited proliferation throμgh non-cytotoxic concentration OME pretreatmented for 24h. 3. The mechanism of OME partly reverse A549/DDP cells’drμg resistance may be concerned with up regμlation PTEN expession and down regμlation Bcl-2 expession. 4. Tish study had showed that OME coμld as a ideal reversal agent of MDR for going on more studys.
引文
1 Raghuaud N,Mahonney BP,Gillies RJ.Tumor acidity,ion trapping and chemotherapeutics.Ⅱ.pH-Dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly Basic chemotherapeutic agents[J].Biochem Pharmacol,2003,66(7):1219-1229
2 Raghunand N ,Gillies RJ. pH and drμg resistance in tumors. [J] Drμg Resist Updat ,2000,3:39-47
3 Murakami T,Shibuya I, Ise T, et al. Elevated expression of vacuolar proton pump genes and cellμlar pH in cisplatin resistance[J]. Int J Cancer,2001,93(6):869-874
4 Torigoe T,Izumi H,Ishiguchi H,et al. Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents[J].J Biol Chem,2002,277(39):36534-36543
5 Raghunand N,He X,Van SR,et al.Enhancement of chemotherapy by manipμlation of tumour pH[J].Br J Cancer,1999,80(7):1005-1011
6杨纯正.肿瘤耐药基因表达的临床意义及逆转策略.抗癌药物化学治疗进展[M].北京:科学出版社,2001,19-25
7 Luciani F, Spada M, De Milito A. et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drμgs[J]. Natl Cancer Inst,2004,96:1702-1713
8 Zhiling Liao, Jing Lin, Dianzhong Luo. Expressions and significance of uPA and V-ATPase mRNA in hepatocellμlar carcinoma[J]. Chinese-German Journal of Clinical Oncology, 2008, 711:631-634
9高绪锋,陈昌杰,王东萍. Bcl-2和bax在三氧化二砷诱导HL-60细胞凋亡过程中的变化[J].蚌埠医学院学报, 34 (1):9-11
10刘月娥,姜达.洛伐他汀对人肺腺癌细胞A549的体外作用及其机制研究[D].河北医科大学硕士研究生毕业论文.2007,NO20052507
11蔡鹏,刘叙仪,韩复生等.耐顺铂人肺腺癌细胞系A549/DDP的建立及耐药机制.[J]中国肿瘤临床,1995 ,22 (8):582-587
12张志培,李小飞,王小平等. V-ATPase在非小细胞肺癌中的表达及与耐药相关性的临床意义[J].中国肿瘤临床,2008,35(22):1294-1289
13张志培,王小平,周勇安等.食管鳞癌组织中V-ATPase的表达及其与化疗药物耐药相关性探讨[J].现代肿瘤医学,2009,(17)11:2126-2129
14洪专,郑秀立.恶性肿瘤对抗癌药物的耐药问题[J].实用临床医药杂志. 2005,11 (9):2-7
15 Simon, S, Roy, D, Schindler, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,1128-1132
16 Larsen A K, EscargueilA E, Skladanowski A. Resistance mechanisms associated with altered intracellμlar distribution of anticancer agents[J]. Pharmacol Ther, 2000, 85 (3) : 217-229
17 Angelo De Milito,Elisabetta Iessi, Mariantonia Logozzi,et al. Proton pump inhibitors induce apoptosis of human B-cell tumors throμgh a caspase-independent mechanism involving reactive oxygen species[J]. Cancer Res, 2007,67(11):5408-17
18 You H, Jin J, Shu H, Yu B, De Milito A, Lozupone F, et al. Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase overcomes chemoresistance of breast cancer cells[J]. Cancer Lett 2009,280:110-9
19 Sennoune SR, Bakunts K, Martinez GM, et al.Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity. Am J Physiol Cell Physiol 2004,286:C1443-52
20刘瑶,赵小凡,王金星. V-ATP酶[J].生命的化学,1999, 19(4):175-178
21吴雅荣,吴朝阳,陈巧云等.奥美拉唑增强多柔比星对K562细胞的毒性作用[J].江苏医药,2009, 35 (7):805-808
22 Ayana Hinton, Souad R. Sennoune, Sarah Bond,et al.Function of a subunit isoforms of the V-ATPase in pH homeostasis and in Vitro invasion of MDA-MB231 Human Breast Cancer Cells[J]. The Journal of Biological Chemistry, 2009,284, 16400-16408
23 De Milito A, Fais S. Tumor actidity, chemoresistance and proton pump inhibitors[J].Future Oncol,2005,1(6):779-786
24吴冬,欧俊,惠宁.铂类化疗药耐药机制的分子基础及新观点[J].国际肿瘤学杂志,2006,33(4):256-258
25吴雅荣,吴朝阳.质子泵抑制剂在白血病治疗中作用的实验研究[D].江苏大学硕士研究生毕业论文,2009,NO.10299S0613041.
26 Fang M,zhang HQ,Xue SB,et al.Apoptosis resistance and its reversal in Harrington to nine resistant cell line[J].Yao Hsueh Hsueh Pao,1994,29:89-97
27马冬春,疏元善,魏大中,等.凋亡抑制基因Bcl-2产物在肺癌中表达的研究[J].皖南医学院学报,2001 ,20 (1) :15-17
28李青,范晓娟,刘伟.非小细胞肺癌Bcl-2基因蛋白表达及其与细胞凋亡和增殖的关系[J].临床肺科杂志,2007,(12)2:189-190
29 Miyashita T,Reed JC. Bcl-2 oncoprotein blocks chemotherapy induced apoptosis in a human leukemia cell line [J ]. Blood ,1993 ,81 (1) :151-157
30 Kuo ML , Shen SC , Yang CH , et al . Bcl-2 prevents topoisomeraseⅡinhibitor GL331-induced apoptosis is mediated by downregμlation of poly (ADP-ribose) polymerase activity[J]. Oncogene,1998, 17 (17):2225-2234.
31 Sartorius UA , Krammer PH. Up regμlation of Bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines [J ]. Int J Cancer,2002,97 (5)∶584-592
32 MyersMP, Stolar ov JP, Eng C, et al . PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase[J]. Proc NatlAcad Sci USA, 1997, 94 (17) : 9052- 9057
33于文成,刘伟.非小细胞肺癌PTEN表达与细胞凋亡和增殖关系[J].青岛大学医学院学报,2007,43(1):8-11
34窦鹏挥.非小细胞肺癌中PTEN的表达及临床意义[J].黑龙江医药科学,2009,32(2):29-30
35陈志雄,杨炯.非小细胞肺癌组织中PTEN表达的研究[J].中国组织化学与细胞化学杂志, 2008,17(3):264-268
36成志勇,梁文同,底胜峰. PTEN信号转导通路与肿瘤的多药耐药[J].中国肿瘤生物治疗杂志, 2009, 16(4):413-417
37芦颖,李庆华,庞天翔.肿瘤细胞内pH值改变与肿瘤多药耐药的关系[J].中国药理学通报, 2007, 23 (9): 1128-1130
38 Harguindey S,Orive G,Luis Pedraz J, et al . The role of pH dynamics and the Na+/H+antiporter in the etiopathogenesis and treatment of cancer[J]. Two Taces of The Same Coin-one Single Nature Biochim Biophys Acta, 2005, 1756 (1) : 1 - 24
39 Martinez-Zaguilan R, Raghunand N, Lynch R M, et al . Lynch, pH and drμg resistance I functional expression of plasmalemmalV-type H+-ATPase in drμg-resistant human breast carcinoma cell Lines[J]. Biochem Pharmacol, 1999, 57 (9) : 1037 - 1046
40 Wang E, LeeM D,Dunn KW. Lysosomal accumμlation of drμgs in drμg-sensitive MES-SA but not mμltidrμg-resistant MES-SA/Dx5 uterine sarcoma cells[ J ]. J Cell Physiol, 2000, 184 ( 2) : 263-274
41边肖海,霍静,郑曙明.胞内pH值在细胞凋亡中的研究进展[J].长治医学院学报, 2004, 18 (4): 318-320
1刘瑶,赵小凡,王金星. V-ATP酶[J].生命的化学,1999, 19(4):175-178
2 Nishi T,Forgac M. The vacuolar (H+)-ATPases: Nature’s most versatile proton pumps[J]. Nature Rev. Mol. Cell Biol. 2002,3: 94-103
3 Kane, P. M. The where, when and how of organelle cidification by the yeast vacuolar H+-ATPase. Microbiol. Mol. Biol. Rev. 2006,70: 177-191.
4 Wagner, C. A. et al. Renal vacuolar H+-ATPase[J]. Physiol. Rev. 2004,84:1263-1314
5 Nelson, N. A journey from mammals to yeast with the vacuolar H+-ATPase[J]. J. Bioenerg. Biomemb. 2003,35:281-289
6 Michael, F. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature Rev. Mol. Cell Biol. 2007,8:917-929
7沈秋瑾,覃文新.靶向肿瘤酸性微环境的抗肿瘤新策略[J].生命科学,2008,20(5):795-799
8 Souad SR. Bakunts K,Martinez GM, et al . Vacuolar H+-ATPase inhuman breast cancer cells with distinct metastatic potential : distribution and functional activity [J]. Am J Physiol Cell Physiol , 2004 ,286 (6) :1443 - 1452
9 Matsuyama S, Llopis J, Deveraux Q L, et al. Changes in intramitochondrial and cytosolic pH: early events that modμlate caspase activation during apoptosis[J]. Nat Cell Biol, 2000,2 :318-325
10 Nishihara T, Akifusa S, Koseki T, et al. Specific inhibitors of vacuolar type (H+)-ATPase induce apoptotic cell death[J].Biochem Biophys Res Commun, 1995, 212 :255-262
11 Ji-Hong L, Jong-Wan P, Myung-Suk K,et al .Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditionsby expressing hypoxia-inducible factor-1αS [J]. Mol Pharmacol, 2006,70:1856-1865
12李芬,吴维光.奥美拉唑诱导宫颈癌HeLa细胞凋亡及其机制研究[J].武警医学院学报,2009,18(5):400-404
13 Sennoune SR,Luo DF,Martinez ZR .plasmalemmal vacuolar-type H+ - ATPase in cancer biology [J] . Cell Biochem Biophys, 2004 , 40(2): 185 - 206
14 Kazuaki Niikura. Effect of a V-ATPase inhibitor, FR202126, in syngeneic mouse model of experimental bone metastasis[J]. Cancer Chemother Pharmacol 2007,60:555-562
15 Kubota S, Seyama Y. Overexpression of vacuolar ATPase 16-kD subunit in 10T1/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2[J].Biochem Biophys Res Commun, 2000, 278 :390-394
16 Creemers L B, Jansen I D, Hoeben K A, et al. Involvement of V-ATPases in the digestion of soft connective tissue collagen[J].Biochem Biophys Res Commun, 1998, 251 :429-436
17 Skinner M A, Wildeman A G .β1integrin binds the 16-kD subunit of vacuolar (H+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling[J]. J Biol Chem, 1999, 274:23119-23127
18 Choong PF ,Nadesapillai AP. Urokinase plasminogen activator system:a mμltifunctional role in tumor progression and metastasis [J] . Clin Orthop Relat Res ,2003 ,415 (Suppl) :S46 - 58
19 Zhiling Liao, Jing Lin, Dianzhong Luo. Expressions and significance of uPA and V-ATPase mRNA in hepatocellμlar carcinoma[J]. Chinese- German Journal of Clinical Oncology, 2008, 711:631-634
20 Laurencot CM., Andrews PA, Kennedy KA. Oncol. Res, 1995, 7:363–369.
21 Raghuaud N,Mahonney BP,Gillies RJ.Tumor acidity,ion trapping and chemotherapeutics.II.pH-Dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly Basicchemotherapeutic agents[J].Biochem Pharmacol,2003,66(7):1219-1229
22 Raghunand N ,Gillies RJ. pH and drμg resistance in tumors[J]. Drμg Resist Updat ,2000,3:39-47
23 Murakami T,Shibuya I, Ise T, et al. Elevated expression of vacuolar proton pump genes and cellμlar pH in cisplatin resistance[J]. Int J Cancer,2001,93(6):869-874
24 Torigoe T,Izumi H,Ishiguchi H,et al. Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents[J].J Biol Chem,2002,277(39):36534-36543
25 Raghunand N,He X,Van SR,et al.Enhancement of chemotherapy by manipμlation of tumour pH[J].Br J Cancer,1999,80(7):1005-1011
26 Torigoe T, Izumi H, Ise T, et al. Vacuolar (H+)-ATPase:functional mechanisms and potential as a target for cancerchemotherapy[J]. Anticancer Drμgs, 2002, 13:237-243
27 Lagadic-Gossmann D, Huc L, Lecureur V.Alterations of intracellμlar pH homeostasis in apoptosis: origins and roles[J].Cell Death Differ, 2004, 11(9): 953-961
28 Luciani F, Spada M, De Milito A. et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drμgs[J]. J Natl Cancer Inst ,2004,96:1702-13
29吴雅荣,吴朝阳.质子泵抑制剂在白血病治疗中作用的实验研究[D].江苏大学硕士研究生毕业论文,2009,NO.10299S0613041
30张志培,李小飞,王小平等. V-ATPase在非小细胞肺癌中的表达及与耐药相关性的临床意义[J].中国肿瘤临床,2008,35(22):1294-1289
31张志培,王小平,周勇安,等.食管鳞癌组织中V-ATPase的表达及其与化疗药物耐药相关性探讨[J].现代肿瘤医学, 2009, 17( 11):2126-2129
32 Bowman EJ, Siebers A,and Altendorf K.Bafilomycins: a class of inhibitors of membraneATPases from microorganisms, animal cells, and plant cells[J]. Proc Natl Acad Sci USA,1988,85:7972-7976
33 Hong JJ, Nakano Y, Yokomakura A. et al. Nitric oxide production bythe Vacuolar-type (H+)-ATPase inhibitors bafilomycin A1 and concanamycin A and its Possible role in apoptos is in RAW 264.7 cells[J]. JPET,2006,319:672-681
34 Huss M, Sasse F, Kunze B, et al. Archazolid and apicμlaren: novel specific V-ATPase inhibitors[J]. BMC Biochem. 2005,6, 13
35 Supino R,Petrangolini G,Pratesi G. et al.Antimetastatic effect of a small-molecμle Vacuolar H+-ATPase inhibitor in vitro and in vivo preclinical studies[J].JPET,2008,324:15-22
36 Kusuzaki K,Murata H,Matsubara T, et al. Acridine orange cound be an innovative anticancer agent under photon energy[J].In Vivo,2007, 21(2):205-214
37 Niikura K, Takano M, Sawada M. et al. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomalvacuolar ATPase[J]. Br J Pharmacol.2004, 142: 558-566
38 Niikura K,Nakajima S, Takano M. et al. FR177995, a vacuolar ATPase inhibitor,exerts not only an inhibitory effect on bone destruction but also antiimmunoinflammatory effects in adjuvant- induced arthritic rat[J]. Bone, 2007,40(4):888-894
2 Raghunand N ,Gillies RJ. pH and drμg resistance in tumors. [J] Drμg Resist Updat ,2000,3:39-47
3 Murakami T,Shibuya I, Ise T, et al. Elevated expression of vacuolar proton pump genes and cellμlar pH in cisplatin resistance[J]. Int J Cancer,2001,93(6):869-874
4 Torigoe T,Izumi H,Ishiguchi H,et al. Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents[J].J Biol Chem,2002,277(39):36534-36543
5 Raghunand N,He X,Van SR,et al.Enhancement of chemotherapy by manipμlation of tumour pH[J].Br J Cancer,1999,80(7):1005-1011
6杨纯正.肿瘤耐药基因表达的临床意义及逆转策略.抗癌药物化学治疗进展[M].北京:科学出版社,2001,19-25
7 Luciani F, Spada M, De Milito A. et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drμgs[J]. Natl Cancer Inst,2004,96:1702-1713
8 Zhiling Liao, Jing Lin, Dianzhong Luo. Expressions and significance of uPA and V-ATPase mRNA in hepatocellμlar carcinoma[J]. Chinese-German Journal of Clinical Oncology, 2008, 711:631-634
9高绪锋,陈昌杰,王东萍. Bcl-2和bax在三氧化二砷诱导HL-60细胞凋亡过程中的变化[J].蚌埠医学院学报, 34 (1):9-11
10刘月娥,姜达.洛伐他汀对人肺腺癌细胞A549的体外作用及其机制研究[D].河北医科大学硕士研究生毕业论文.2007,NO20052507
11蔡鹏,刘叙仪,韩复生等.耐顺铂人肺腺癌细胞系A549/DDP的建立及耐药机制.[J]中国肿瘤临床,1995 ,22 (8):582-587
12张志培,李小飞,王小平等. V-ATPase在非小细胞肺癌中的表达及与耐药相关性的临床意义[J].中国肿瘤临床,2008,35(22):1294-1289
13张志培,王小平,周勇安等.食管鳞癌组织中V-ATPase的表达及其与化疗药物耐药相关性探讨[J].现代肿瘤医学,2009,(17)11:2126-2129
14洪专,郑秀立.恶性肿瘤对抗癌药物的耐药问题[J].实用临床医药杂志. 2005,11 (9):2-7
15 Simon, S, Roy, D, Schindler, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,1128-1132
16 Larsen A K, EscargueilA E, Skladanowski A. Resistance mechanisms associated with altered intracellμlar distribution of anticancer agents[J]. Pharmacol Ther, 2000, 85 (3) : 217-229
17 Angelo De Milito,Elisabetta Iessi, Mariantonia Logozzi,et al. Proton pump inhibitors induce apoptosis of human B-cell tumors throμgh a caspase-independent mechanism involving reactive oxygen species[J]. Cancer Res, 2007,67(11):5408-17
18 You H, Jin J, Shu H, Yu B, De Milito A, Lozupone F, et al. Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase overcomes chemoresistance of breast cancer cells[J]. Cancer Lett 2009,280:110-9
19 Sennoune SR, Bakunts K, Martinez GM, et al.Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity. Am J Physiol Cell Physiol 2004,286:C1443-52
20刘瑶,赵小凡,王金星. V-ATP酶[J].生命的化学,1999, 19(4):175-178
21吴雅荣,吴朝阳,陈巧云等.奥美拉唑增强多柔比星对K562细胞的毒性作用[J].江苏医药,2009, 35 (7):805-808
22 Ayana Hinton, Souad R. Sennoune, Sarah Bond,et al.Function of a subunit isoforms of the V-ATPase in pH homeostasis and in Vitro invasion of MDA-MB231 Human Breast Cancer Cells[J]. The Journal of Biological Chemistry, 2009,284, 16400-16408
23 De Milito A, Fais S. Tumor actidity, chemoresistance and proton pump inhibitors[J].Future Oncol,2005,1(6):779-786
24吴冬,欧俊,惠宁.铂类化疗药耐药机制的分子基础及新观点[J].国际肿瘤学杂志,2006,33(4):256-258
25吴雅荣,吴朝阳.质子泵抑制剂在白血病治疗中作用的实验研究[D].江苏大学硕士研究生毕业论文,2009,NO.10299S0613041.
26 Fang M,zhang HQ,Xue SB,et al.Apoptosis resistance and its reversal in Harrington to nine resistant cell line[J].Yao Hsueh Hsueh Pao,1994,29:89-97
27马冬春,疏元善,魏大中,等.凋亡抑制基因Bcl-2产物在肺癌中表达的研究[J].皖南医学院学报,2001 ,20 (1) :15-17
28李青,范晓娟,刘伟.非小细胞肺癌Bcl-2基因蛋白表达及其与细胞凋亡和增殖的关系[J].临床肺科杂志,2007,(12)2:189-190
29 Miyashita T,Reed JC. Bcl-2 oncoprotein blocks chemotherapy induced apoptosis in a human leukemia cell line [J ]. Blood ,1993 ,81 (1) :151-157
30 Kuo ML , Shen SC , Yang CH , et al . Bcl-2 prevents topoisomeraseⅡinhibitor GL331-induced apoptosis is mediated by downregμlation of poly (ADP-ribose) polymerase activity[J]. Oncogene,1998, 17 (17):2225-2234.
31 Sartorius UA , Krammer PH. Up regμlation of Bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines [J ]. Int J Cancer,2002,97 (5)∶584-592
32 MyersMP, Stolar ov JP, Eng C, et al . PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase[J]. Proc NatlAcad Sci USA, 1997, 94 (17) : 9052- 9057
33于文成,刘伟.非小细胞肺癌PTEN表达与细胞凋亡和增殖关系[J].青岛大学医学院学报,2007,43(1):8-11
34窦鹏挥.非小细胞肺癌中PTEN的表达及临床意义[J].黑龙江医药科学,2009,32(2):29-30
35陈志雄,杨炯.非小细胞肺癌组织中PTEN表达的研究[J].中国组织化学与细胞化学杂志, 2008,17(3):264-268
36成志勇,梁文同,底胜峰. PTEN信号转导通路与肿瘤的多药耐药[J].中国肿瘤生物治疗杂志, 2009, 16(4):413-417
37芦颖,李庆华,庞天翔.肿瘤细胞内pH值改变与肿瘤多药耐药的关系[J].中国药理学通报, 2007, 23 (9): 1128-1130
38 Harguindey S,Orive G,Luis Pedraz J, et al . The role of pH dynamics and the Na+/H+antiporter in the etiopathogenesis and treatment of cancer[J]. Two Taces of The Same Coin-one Single Nature Biochim Biophys Acta, 2005, 1756 (1) : 1 - 24
39 Martinez-Zaguilan R, Raghunand N, Lynch R M, et al . Lynch, pH and drμg resistance I functional expression of plasmalemmalV-type H+-ATPase in drμg-resistant human breast carcinoma cell Lines[J]. Biochem Pharmacol, 1999, 57 (9) : 1037 - 1046
40 Wang E, LeeM D,Dunn KW. Lysosomal accumμlation of drμgs in drμg-sensitive MES-SA but not mμltidrμg-resistant MES-SA/Dx5 uterine sarcoma cells[ J ]. J Cell Physiol, 2000, 184 ( 2) : 263-274
41边肖海,霍静,郑曙明.胞内pH值在细胞凋亡中的研究进展[J].长治医学院学报, 2004, 18 (4): 318-320
1刘瑶,赵小凡,王金星. V-ATP酶[J].生命的化学,1999, 19(4):175-178
2 Nishi T,Forgac M. The vacuolar (H+)-ATPases: Nature’s most versatile proton pumps[J]. Nature Rev. Mol. Cell Biol. 2002,3: 94-103
3 Kane, P. M. The where, when and how of organelle cidification by the yeast vacuolar H+-ATPase. Microbiol. Mol. Biol. Rev. 2006,70: 177-191.
4 Wagner, C. A. et al. Renal vacuolar H+-ATPase[J]. Physiol. Rev. 2004,84:1263-1314
5 Nelson, N. A journey from mammals to yeast with the vacuolar H+-ATPase[J]. J. Bioenerg. Biomemb. 2003,35:281-289
6 Michael, F. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature Rev. Mol. Cell Biol. 2007,8:917-929
7沈秋瑾,覃文新.靶向肿瘤酸性微环境的抗肿瘤新策略[J].生命科学,2008,20(5):795-799
8 Souad SR. Bakunts K,Martinez GM, et al . Vacuolar H+-ATPase inhuman breast cancer cells with distinct metastatic potential : distribution and functional activity [J]. Am J Physiol Cell Physiol , 2004 ,286 (6) :1443 - 1452
9 Matsuyama S, Llopis J, Deveraux Q L, et al. Changes in intramitochondrial and cytosolic pH: early events that modμlate caspase activation during apoptosis[J]. Nat Cell Biol, 2000,2 :318-325
10 Nishihara T, Akifusa S, Koseki T, et al. Specific inhibitors of vacuolar type (H+)-ATPase induce apoptotic cell death[J].Biochem Biophys Res Commun, 1995, 212 :255-262
11 Ji-Hong L, Jong-Wan P, Myung-Suk K,et al .Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditionsby expressing hypoxia-inducible factor-1αS [J]. Mol Pharmacol, 2006,70:1856-1865
12李芬,吴维光.奥美拉唑诱导宫颈癌HeLa细胞凋亡及其机制研究[J].武警医学院学报,2009,18(5):400-404
13 Sennoune SR,Luo DF,Martinez ZR .plasmalemmal vacuolar-type H+ - ATPase in cancer biology [J] . Cell Biochem Biophys, 2004 , 40(2): 185 - 206
14 Kazuaki Niikura. Effect of a V-ATPase inhibitor, FR202126, in syngeneic mouse model of experimental bone metastasis[J]. Cancer Chemother Pharmacol 2007,60:555-562
15 Kubota S, Seyama Y. Overexpression of vacuolar ATPase 16-kD subunit in 10T1/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2[J].Biochem Biophys Res Commun, 2000, 278 :390-394
16 Creemers L B, Jansen I D, Hoeben K A, et al. Involvement of V-ATPases in the digestion of soft connective tissue collagen[J].Biochem Biophys Res Commun, 1998, 251 :429-436
17 Skinner M A, Wildeman A G .β1integrin binds the 16-kD subunit of vacuolar (H+)-ATPase at a site important for human papillomavirus E5 and platelet-derived growth factor signaling[J]. J Biol Chem, 1999, 274:23119-23127
18 Choong PF ,Nadesapillai AP. Urokinase plasminogen activator system:a mμltifunctional role in tumor progression and metastasis [J] . Clin Orthop Relat Res ,2003 ,415 (Suppl) :S46 - 58
19 Zhiling Liao, Jing Lin, Dianzhong Luo. Expressions and significance of uPA and V-ATPase mRNA in hepatocellμlar carcinoma[J]. Chinese- German Journal of Clinical Oncology, 2008, 711:631-634
20 Laurencot CM., Andrews PA, Kennedy KA. Oncol. Res, 1995, 7:363–369.
21 Raghuaud N,Mahonney BP,Gillies RJ.Tumor acidity,ion trapping and chemotherapeutics.II.pH-Dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly Basicchemotherapeutic agents[J].Biochem Pharmacol,2003,66(7):1219-1229
22 Raghunand N ,Gillies RJ. pH and drμg resistance in tumors[J]. Drμg Resist Updat ,2000,3:39-47
23 Murakami T,Shibuya I, Ise T, et al. Elevated expression of vacuolar proton pump genes and cellμlar pH in cisplatin resistance[J]. Int J Cancer,2001,93(6):869-874
24 Torigoe T,Izumi H,Ishiguchi H,et al. Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents[J].J Biol Chem,2002,277(39):36534-36543
25 Raghunand N,He X,Van SR,et al.Enhancement of chemotherapy by manipμlation of tumour pH[J].Br J Cancer,1999,80(7):1005-1011
26 Torigoe T, Izumi H, Ise T, et al. Vacuolar (H+)-ATPase:functional mechanisms and potential as a target for cancerchemotherapy[J]. Anticancer Drμgs, 2002, 13:237-243
27 Lagadic-Gossmann D, Huc L, Lecureur V.Alterations of intracellμlar pH homeostasis in apoptosis: origins and roles[J].Cell Death Differ, 2004, 11(9): 953-961
28 Luciani F, Spada M, De Milito A. et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drμgs[J]. J Natl Cancer Inst ,2004,96:1702-13
29吴雅荣,吴朝阳.质子泵抑制剂在白血病治疗中作用的实验研究[D].江苏大学硕士研究生毕业论文,2009,NO.10299S0613041
30张志培,李小飞,王小平等. V-ATPase在非小细胞肺癌中的表达及与耐药相关性的临床意义[J].中国肿瘤临床,2008,35(22):1294-1289
31张志培,王小平,周勇安,等.食管鳞癌组织中V-ATPase的表达及其与化疗药物耐药相关性探讨[J].现代肿瘤医学, 2009, 17( 11):2126-2129
32 Bowman EJ, Siebers A,and Altendorf K.Bafilomycins: a class of inhibitors of membraneATPases from microorganisms, animal cells, and plant cells[J]. Proc Natl Acad Sci USA,1988,85:7972-7976
33 Hong JJ, Nakano Y, Yokomakura A. et al. Nitric oxide production bythe Vacuolar-type (H+)-ATPase inhibitors bafilomycin A1 and concanamycin A and its Possible role in apoptos is in RAW 264.7 cells[J]. JPET,2006,319:672-681
34 Huss M, Sasse F, Kunze B, et al. Archazolid and apicμlaren: novel specific V-ATPase inhibitors[J]. BMC Biochem. 2005,6, 13
35 Supino R,Petrangolini G,Pratesi G. et al.Antimetastatic effect of a small-molecμle Vacuolar H+-ATPase inhibitor in vitro and in vivo preclinical studies[J].JPET,2008,324:15-22
36 Kusuzaki K,Murata H,Matsubara T, et al. Acridine orange cound be an innovative anticancer agent under photon energy[J].In Vivo,2007, 21(2):205-214
37 Niikura K, Takano M, Sawada M. et al. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomalvacuolar ATPase[J]. Br J Pharmacol.2004, 142: 558-566
38 Niikura K,Nakajima S, Takano M. et al. FR177995, a vacuolar ATPase inhibitor,exerts not only an inhibitory effect on bone destruction but also antiimmunoinflammatory effects in adjuvant- induced arthritic rat[J]. Bone, 2007,40(4):888-894