RNA干扰技术联合抑制胰腺癌细胞系中癌基因K-ras和AKT2表达的实验性研究
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摘要
胰腺癌由于其起病隐匿、易转移、解剖位置复杂、手术困难、对放化疗不敏感等原因,已经成为重要的致死性癌症,寻找有效的基因治疗方法是改善胰腺癌预后的重要措施之一。
本研究观察RNA干扰(RNA interference,RNAi)同时抑制胰腺癌中两个关键性癌基因K-ras和AKT2对于胰腺癌细胞系Panc-1细胞生长和凋亡的影响,探讨其作用机制,为胰腺癌的临床治疗提供新的方法。本研究主要进行了以下几方面的工作:
一、构建并筛选单干扰和双干扰重组质粒载体
1.根据invitrogen公司提供的网上设计工具,设计了两对针对K-ras的干扰序列;连接进入载体后,测序鉴定。
2.设计了四对针对AKT2的干扰序列;连接进入载体后,测序鉴定。
3.瞬时转染人胰腺癌细胞系Panc-1细胞,筛选出有效的抑制K-ras或AKT2的重组质粒载体。
4.采用限制性内切酶方法构建双干扰重组质粒载体。
二、重组质粒载体降低相关癌基因mRNA和蛋白的表达
1.重组质粒anti-kras可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中K-ras mRNA和蛋白的水平。
2.重组质粒anti-akt2可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中AKT2 mRNA和蛋白的水平。
3.重组质粒anti-k+a和anti-a+k可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中K-ras和AKT2 mRNA和蛋白的水平。
三、重组质粒减缓细胞生长,促进细胞凋亡
1.CCK-8方法检测细胞生长曲线。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒的稳定转染细胞生长均减慢,双干扰重组质粒组的生长受抑最显著,与单干扰重组质粒组相比具有统计学差异。
2.克隆形成试验检测细胞的生长增殖能力。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒组的克隆形成能力均下降,双干扰重组质粒组的下降最显著,与单干扰重组质粒组相比具有统计学差异。
3.Heochst方法检测细胞凋亡时细胞核形态的改变。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡小体,双干扰重组质粒组与单干扰质粒组相比,凋亡小体数目增多显著。
4.Annexin V-FITC/PI法检测细胞的早期凋亡。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡,双干扰重组质粒组凋亡细胞比例最高,与单干扰重组质粒组相比具有统计学差异。
5.Caspase-3法检测细胞的凋亡状态。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡蛋白的激活,双干扰重组质粒组凋亡蛋白激活最显著,与单干扰重组质粒组相比具有统计学差异。
四、重组质粒载体降低p-ERK和p-GSK激酶磷酸化水平,减少癌蛋白c-myc含量
1.单干扰重组质粒anti-kras转染后可以有效降低信号通路RAS/MAPK中p-ERK激酶磷酸化水平。
2.单干扰重组质粒anti-akt2转染后可以有效降低信号通路PI3K/AKT中p-GSK激酶磷酸化水平。
3.双干扰重组质粒anti-k+a转染后可以有效降低信号通路RAS/MAPK中p-ERK激酶磷酸化水平和PI3K/AKT中p-GSK激酶磷酸化水平。
4.单干扰重组质粒anti-kras、anti-akt2转染后可以有效降低c-myc蛋白表达;双干扰重组质粒anti-k+a转染后也可以明显降低c-myc蛋白表达,与单干扰重组质粒相比,具有统计学差异。
五、重组质粒载体对动物体内移植瘤生长的影响
1.将各种重组质粒的稳定转染细胞和未处理细胞接种于裸鼠皮下,观察其成瘤能力。结果显示,与未处理组和阴性载体稳定转染细胞系相比,单干扰和双干扰稳定转染细胞系肿瘤生长缓慢,且双干扰稳定转染细胞系肿瘤细胞生长要慢于单干扰组。
2.预先在裸鼠背部皮下种植成瘤,采用PEI转染方法,多点肿瘤内注射两次,观察肿瘤的生长情况,结果显示,与未处理组和单独PEI转染组相比,单干扰处理组肿瘤生长减缓,双干扰处理组肿瘤生长减缓更显著,与单干扰组比较具有统计学差异。
我们的研究表明RNA干扰技术是一种十分有效的基因治疗方法,联合抑制原癌基因K-ras和AKT2可使胰腺癌细胞的生长代谢受到明显的抑制、促进胰腺癌细胞的凋亡;体内实验结果显示联合干扰可以有效抑制肿瘤细胞生长、使其丧失致瘤能力;联合干扰发挥作用的可能机制是同时阻断了两条信号通路RAS/MAPK和PI3K/AKT,诱导促凋亡蛋白BAD发挥促进细胞凋亡作用以及促进c-myc蛋白的降解。本研究的创新点在于采用RNA干扰技术联合抑制两个原癌基因探讨胰腺癌的基因治疗。
Pancreatic ductal adenocarcinoma(PDAC) is exceptionally aggressive with high mortality.Since no effective therapeutic methods have been found up to now,it is urgent to find more powerful method to diagnose the disease earlier and treat it more effectively.
RNAi is a strong tool to inhibit more than one gene at the same time.Therefore, in this study,we used a plasmid-based RNAi system to evaluate whether simultaneously silencing two oncogenes K-ras and AKT2,which are key players in two distinct signaling pathways-RAS/MAPK and PI3K/AKT,would have a better tumor inhibition effect than silencing a single oncogene.The experimental results both in vitro and in vivo confirmed our hypothesis.The following aspects were studied:
1.Construction of recombinant plasmids,including plasmids that can silence the oncogene K-ras,plasmids that can silence the oncogene AKT2,and plasmids that can silence oncogenes K-ras and AKT2 simultaneously.
2.Effect of the recombinant plasmids on the corresponding mRNA and protein levels in the Panc-1 cells.
3.Effect of the recombinant plasmids on proliferation and colony formation of the Panc-1 cells.
4.Effect of the recombinant plasmids on apoptosis of the Panc-1 cells.
5.Effect of the recombinant plamids on downstream signaling kinases in the Panc-1 cells.
6.In vivo effect of the recombinant plamids on Panc-1 cells.
The results showed that:
1.RNAi inhibited oncogenes K-ras and AKT2 specifically at mRNA and protein levels in Panc-1 cells.
2.Specific inhibition of oncogenes K-ras and/or AKT2 reduced cell proliferating capacity and colony formation of Panc-1 cells.
3.Specific silencing of oncogenes K-ras and/or AKT2 induced apoptosis in Panc-1 cells.
4.Combined inhibition of oncogenes K-ras and/or AKT2 can inhibit the phosphorylation level of p-ERK and p-GSK and reduce the protein level of c-myc protein.
5.Combined inhibition of K-ras and AKT2 inhibited tumor growth in vivo more effectively than inhibition of each of the two genes alone
Conclusion:
Combined silencing of oncogenes K-ras and AKT2 achieved synergistic effects in inhibiting pancreatic cancer cell growth and inducing cell apoptosis in vitro and in vivo,and the possible mechanism may be due to the inhibition of the two signaling pathways of RAS/MAPK and PI3K/AKT simultaneously, inducing the antiapoptic protein BAD and promoting the proteolysis of the oncoprotein c-myc.This result may also offer potential opportunities for clinical cancer therapy.
本研究观察RNA干扰(RNA interference,RNAi)同时抑制胰腺癌中两个关键性癌基因K-ras和AKT2对于胰腺癌细胞系Panc-1细胞生长和凋亡的影响,探讨其作用机制,为胰腺癌的临床治疗提供新的方法。本研究主要进行了以下几方面的工作:
一、构建并筛选单干扰和双干扰重组质粒载体
1.根据invitrogen公司提供的网上设计工具,设计了两对针对K-ras的干扰序列;连接进入载体后,测序鉴定。
2.设计了四对针对AKT2的干扰序列;连接进入载体后,测序鉴定。
3.瞬时转染人胰腺癌细胞系Panc-1细胞,筛选出有效的抑制K-ras或AKT2的重组质粒载体。
4.采用限制性内切酶方法构建双干扰重组质粒载体。
二、重组质粒载体降低相关癌基因mRNA和蛋白的表达
1.重组质粒anti-kras可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中K-ras mRNA和蛋白的水平。
2.重组质粒anti-akt2可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中AKT2 mRNA和蛋白的水平。
3.重组质粒anti-k+a和anti-a+k可以有效和特异性地减少胰腺癌细胞系Panc-1细胞中K-ras和AKT2 mRNA和蛋白的水平。
三、重组质粒减缓细胞生长,促进细胞凋亡
1.CCK-8方法检测细胞生长曲线。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒的稳定转染细胞生长均减慢,双干扰重组质粒组的生长受抑最显著,与单干扰重组质粒组相比具有统计学差异。
2.克隆形成试验检测细胞的生长增殖能力。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒组的克隆形成能力均下降,双干扰重组质粒组的下降最显著,与单干扰重组质粒组相比具有统计学差异。
3.Heochst方法检测细胞凋亡时细胞核形态的改变。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡小体,双干扰重组质粒组与单干扰质粒组相比,凋亡小体数目增多显著。
4.Annexin V-FITC/PI法检测细胞的早期凋亡。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡,双干扰重组质粒组凋亡细胞比例最高,与单干扰重组质粒组相比具有统计学差异。
5.Caspase-3法检测细胞的凋亡状态。结果显示,相对于未处理组和阴性载体对照组,所有重组质粒转染的细胞均出现凋亡蛋白的激活,双干扰重组质粒组凋亡蛋白激活最显著,与单干扰重组质粒组相比具有统计学差异。
四、重组质粒载体降低p-ERK和p-GSK激酶磷酸化水平,减少癌蛋白c-myc含量
1.单干扰重组质粒anti-kras转染后可以有效降低信号通路RAS/MAPK中p-ERK激酶磷酸化水平。
2.单干扰重组质粒anti-akt2转染后可以有效降低信号通路PI3K/AKT中p-GSK激酶磷酸化水平。
3.双干扰重组质粒anti-k+a转染后可以有效降低信号通路RAS/MAPK中p-ERK激酶磷酸化水平和PI3K/AKT中p-GSK激酶磷酸化水平。
4.单干扰重组质粒anti-kras、anti-akt2转染后可以有效降低c-myc蛋白表达;双干扰重组质粒anti-k+a转染后也可以明显降低c-myc蛋白表达,与单干扰重组质粒相比,具有统计学差异。
五、重组质粒载体对动物体内移植瘤生长的影响
1.将各种重组质粒的稳定转染细胞和未处理细胞接种于裸鼠皮下,观察其成瘤能力。结果显示,与未处理组和阴性载体稳定转染细胞系相比,单干扰和双干扰稳定转染细胞系肿瘤生长缓慢,且双干扰稳定转染细胞系肿瘤细胞生长要慢于单干扰组。
2.预先在裸鼠背部皮下种植成瘤,采用PEI转染方法,多点肿瘤内注射两次,观察肿瘤的生长情况,结果显示,与未处理组和单独PEI转染组相比,单干扰处理组肿瘤生长减缓,双干扰处理组肿瘤生长减缓更显著,与单干扰组比较具有统计学差异。
我们的研究表明RNA干扰技术是一种十分有效的基因治疗方法,联合抑制原癌基因K-ras和AKT2可使胰腺癌细胞的生长代谢受到明显的抑制、促进胰腺癌细胞的凋亡;体内实验结果显示联合干扰可以有效抑制肿瘤细胞生长、使其丧失致瘤能力;联合干扰发挥作用的可能机制是同时阻断了两条信号通路RAS/MAPK和PI3K/AKT,诱导促凋亡蛋白BAD发挥促进细胞凋亡作用以及促进c-myc蛋白的降解。本研究的创新点在于采用RNA干扰技术联合抑制两个原癌基因探讨胰腺癌的基因治疗。
Pancreatic ductal adenocarcinoma(PDAC) is exceptionally aggressive with high mortality.Since no effective therapeutic methods have been found up to now,it is urgent to find more powerful method to diagnose the disease earlier and treat it more effectively.
RNAi is a strong tool to inhibit more than one gene at the same time.Therefore, in this study,we used a plasmid-based RNAi system to evaluate whether simultaneously silencing two oncogenes K-ras and AKT2,which are key players in two distinct signaling pathways-RAS/MAPK and PI3K/AKT,would have a better tumor inhibition effect than silencing a single oncogene.The experimental results both in vitro and in vivo confirmed our hypothesis.The following aspects were studied:
1.Construction of recombinant plasmids,including plasmids that can silence the oncogene K-ras,plasmids that can silence the oncogene AKT2,and plasmids that can silence oncogenes K-ras and AKT2 simultaneously.
2.Effect of the recombinant plasmids on the corresponding mRNA and protein levels in the Panc-1 cells.
3.Effect of the recombinant plasmids on proliferation and colony formation of the Panc-1 cells.
4.Effect of the recombinant plasmids on apoptosis of the Panc-1 cells.
5.Effect of the recombinant plamids on downstream signaling kinases in the Panc-1 cells.
6.In vivo effect of the recombinant plamids on Panc-1 cells.
The results showed that:
1.RNAi inhibited oncogenes K-ras and AKT2 specifically at mRNA and protein levels in Panc-1 cells.
2.Specific inhibition of oncogenes K-ras and/or AKT2 reduced cell proliferating capacity and colony formation of Panc-1 cells.
3.Specific silencing of oncogenes K-ras and/or AKT2 induced apoptosis in Panc-1 cells.
4.Combined inhibition of oncogenes K-ras and/or AKT2 can inhibit the phosphorylation level of p-ERK and p-GSK and reduce the protein level of c-myc protein.
5.Combined inhibition of K-ras and AKT2 inhibited tumor growth in vivo more effectively than inhibition of each of the two genes alone
Conclusion:
Combined silencing of oncogenes K-ras and AKT2 achieved synergistic effects in inhibiting pancreatic cancer cell growth and inducing cell apoptosis in vitro and in vivo,and the possible mechanism may be due to the inhibition of the two signaling pathways of RAS/MAPK and PI3K/AKT simultaneously, inducing the antiapoptic protein BAD and promoting the proteolysis of the oncoprotein c-myc.This result may also offer potential opportunities for clinical cancer therapy.
引文
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38.WANG Xiao-juan,DAI Guo-yi.Antisense RNA of survivin sensitive doxorubicin to pancreatic cancer cell line Panc-1.Acta Acad Med NeiMongol,2005,27:3180-183.
39.Guan Hai-tao,Xue Xing-huan,Wang Xi-jing.Effect of enhancing the radiosensitivity of pancreatic cancer cell line PC-2 by siRNA against Survivin.Mordern Oncology,2006,114(14):436-439.
40.Lammering G,Lin PS,Gontessa JN,et al.Adenovirus mediated-overexpression of dominant negtive epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensensitized human carcinoma and human maligant glioma cells.Int J Radiat Oncol Biol Phys,2001,51 (3):775-784.
41.Pulukuri Sai MuraliKrishnal,Christopher S.Gondi1,Sajani S.et al.RNA Interference-Directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion,Survival and Tumorigenicity In vivo.J Biol Chem,2005,280(43):36529-36540.
42.Fisher G H,Wellen S L,Klimstra D,et al.Induction and apoptopic regression of lung adenocarcinomas by regulation of a K-RAS transgene in the presence and absence of tumor suppressor genes .Genes Dev,2001,15:3249-3262.
43.Jackson E L,Willis N,Merk K,et al.Analysis of lung tumor inflation and progression using conditional expression of oncogenic K-ras.Genes Dev,2001,15:3243-3248.
44.Agami R,Bernards R.Distinct inhibition and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage.Cell,2000,102:55-66.
45.Zamore P D,TuschI T,Sharp P A,et al.RNAi:Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell,2000,102:25-33.
46.McCormick F.Ras GTPase activating protein:signal transmitter and signal terminater.Cell,1989,56:5-8.
47.McCormick F.GTP binding and growth control.Curr Opin Cell Biol,1990,2:181-184.
48.Chin L,Tarn A,Pomerantz J,et al.Essential role for oncogenic Ras in tumor maintenance.Nature,1999,400:468-472.
49.Murthy SS,Tosolini A,Taguchi T,et al.Mapping of AKT3,encoding a member of the AKT protein kinase B family,to human and rodent chromo somes by fluorescence in situ hybridization.Cytogenet Cell Genet,2000,88 (122):382401.
50.Testa JR,Bellaco saal.AKT plays a central role in tumorigenesis.Proc Natl A cad Sci USA,2001,98 (20):109832-109851.
51.Xu X,Sakon M,N agano H,et al.AKT2 expression correlates with progno sis of human hepatocellular carcinoma.Oncol Rep,2004,11 (1):252321.
52.V ivanco I,Sawyers CL.The phosphatidylinositol kinase AKT pathway in human cancer.Nat Rev Cancer,2002,2 (7):4892-5011.
53.Yamamo to S,Tomita Y,Hoshida Y,et al.Prognostic significance of activated AKT expression in pancreatic ductal adenocarcinoma.Clin Cancer Res,2004,10 (8):28462-28501.
54.Pearson G,Robinson F,Beers Gibson T,et al.Mitogen activated protein (MAP) kinase pathways:regulation and physiological functions.Endocr Rev,2001,22 (2):1532183.
55.Friess H,Kleeff J,Korc M,et al.Molecular aspects of pancreatic cancer and future perspectives.Dig Surg,1999,16:281-90.
56.Reddy SA.Signaling pathways in pancreatic cancer.Cancer J,2001,7:274-86.
57.Takayuki Asano,Yixin Yao,Jijiang Zhu,et al.The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kB and c-Myc in pancreatic cancer cells.Oncogene,2004,23:8571-8580.
58.Maira SM,Galetic I,Brazil DP,et al.Carboxyl-terminal modulator protein (CTMP),a negative regulator of PKB/Akt and v-Akt at the plasma membrane.Science,2001,294:374-80.
59.Elicia Peneul,Steven Martin.Tranformation by V-src:Ras-MAPK and PI3K-mTOR mediated parallel pathways.Moleculer biology of the cell,1999,10:1693-1703.
60.Lincoln A,Edwards,Maite Verreault,et al.Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells.Mol Cancer Ther,2006,5:645-54.
61.Kian-Huat Lim,Christopher M.Counter.Reduction in the requirement of oncogenic Ras signalling to activation of PI3K/AKT pathway during tumor maintenance.Cancer cell,2005,8:381-392.
62.Yang E,Zha J,Jockel J,et al.Bad,a heterodimeric partner for Bcl-XL and Bcl-2,displaces Bax and promotes cell death.Cell,1995,80:285-291.
63.Bonni A,Brunet A,West A.E,et al.Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms.Science,1999,286:1358-1362.
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66.She Q,Solit O.The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells.Cancer cell,2005,10:287-297.
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69.Schleger C,Verbeke C,Hildenbrand R,et al.c-MYC activation in primary and metastatic ductal adenocarcinoma of the pancreas:incidence,mechanisms,and clinical significance.Mod Pathol,2002, 15:462-469.
70.Henriksson M,Bakardjiev A,Klein G,et al.Phosphorylation sites mapping in the N-terminal domain of c-myc modulate its transforming potential.Oncogene,1993,8:3199-209.
71.Gregory MA,Qi Y,Hann SR.Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization.J Biol Chem,2003,278:51606-12.
72.Sears RG.The life cycle of C-myc:frpm synthesis to degradation.Cell Cycle,2004,3:1133-7.
73.Sears R,Nuckolls F,Haura E,et al.Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability.Genes Dev,2000,14:2501-2514.
74.Fabbrini M.S,Carpani D,Bello-Rivero I,et al.The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells:internalization is toxin mediated.FASEB J,1997,11:1169-1176.
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36.Duxbury MS,Ito H,Benoit E,et al.RNA interference demonstrates a novel role for *integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.Clin Cancer Res,2005,11(9):3433-8.
37.Hanahan D,Weinberg RA.The hallmarks of cancer.Cell,2000,100:57-70.
38.WANG Xiao-juan,DAI Guo-yi.Antisense RNA of survivin sensitive doxorubicin to pancreatic cancer cell line Panc-1.Acta Acad Med NeiMongol,2005,27:3180-183.
39.Guan Hai-tao,Xue Xing-huan,Wang Xi-jing.Effect of enhancing the radiosensitivity of pancreatic cancer cell line PC-2 by siRNA against Survivin.Mordern Oncology,2006,114(14):436-439.
40.Lammering G,Lin PS,Gontessa JN,et al.Adenovirus mediated-overexpression of dominant negtive epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensensitized human carcinoma and human maligant glioma cells.Int J Radiat Oncol Biol Phys,2001,51 (3):775-784.
41.Pulukuri Sai MuraliKrishnal,Christopher S.Gondi1,Sajani S.et al.RNA Interference-Directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion,Survival and Tumorigenicity In vivo.J Biol Chem,2005,280(43):36529-36540.
42.Fisher G H,Wellen S L,Klimstra D,et al.Induction and apoptopic regression of lung adenocarcinomas by regulation of a K-RAS transgene in the presence and absence of tumor suppressor genes .Genes Dev,2001,15:3249-3262.
43.Jackson E L,Willis N,Merk K,et al.Analysis of lung tumor inflation and progression using conditional expression of oncogenic K-ras.Genes Dev,2001,15:3243-3248.
44.Agami R,Bernards R.Distinct inhibition and maintenance mechanisms cooperate to induce G1 cell cycle arrest in response to DNA damage.Cell,2000,102:55-66.
45.Zamore P D,TuschI T,Sharp P A,et al.RNAi:Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell,2000,102:25-33.
46.McCormick F.Ras GTPase activating protein:signal transmitter and signal terminater.Cell,1989,56:5-8.
47.McCormick F.GTP binding and growth control.Curr Opin Cell Biol,1990,2:181-184.
48.Chin L,Tarn A,Pomerantz J,et al.Essential role for oncogenic Ras in tumor maintenance.Nature,1999,400:468-472.
49.Murthy SS,Tosolini A,Taguchi T,et al.Mapping of AKT3,encoding a member of the AKT protein kinase B family,to human and rodent chromo somes by fluorescence in situ hybridization.Cytogenet Cell Genet,2000,88 (122):382401.
50.Testa JR,Bellaco saal.AKT plays a central role in tumorigenesis.Proc Natl A cad Sci USA,2001,98 (20):109832-109851.
51.Xu X,Sakon M,N agano H,et al.AKT2 expression correlates with progno sis of human hepatocellular carcinoma.Oncol Rep,2004,11 (1):252321.
52.V ivanco I,Sawyers CL.The phosphatidylinositol kinase AKT pathway in human cancer.Nat Rev Cancer,2002,2 (7):4892-5011.
53.Yamamo to S,Tomita Y,Hoshida Y,et al.Prognostic significance of activated AKT expression in pancreatic ductal adenocarcinoma.Clin Cancer Res,2004,10 (8):28462-28501.
54.Pearson G,Robinson F,Beers Gibson T,et al.Mitogen activated protein (MAP) kinase pathways:regulation and physiological functions.Endocr Rev,2001,22 (2):1532183.
55.Friess H,Kleeff J,Korc M,et al.Molecular aspects of pancreatic cancer and future perspectives.Dig Surg,1999,16:281-90.
56.Reddy SA.Signaling pathways in pancreatic cancer.Cancer J,2001,7:274-86.
57.Takayuki Asano,Yixin Yao,Jijiang Zhu,et al.The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kB and c-Myc in pancreatic cancer cells.Oncogene,2004,23:8571-8580.
58.Maira SM,Galetic I,Brazil DP,et al.Carboxyl-terminal modulator protein (CTMP),a negative regulator of PKB/Akt and v-Akt at the plasma membrane.Science,2001,294:374-80.
59.Elicia Peneul,Steven Martin.Tranformation by V-src:Ras-MAPK and PI3K-mTOR mediated parallel pathways.Moleculer biology of the cell,1999,10:1693-1703.
60.Lincoln A,Edwards,Maite Verreault,et al.Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells.Mol Cancer Ther,2006,5:645-54.
61.Kian-Huat Lim,Christopher M.Counter.Reduction in the requirement of oncogenic Ras signalling to activation of PI3K/AKT pathway during tumor maintenance.Cancer cell,2005,8:381-392.
62.Yang E,Zha J,Jockel J,et al.Bad,a heterodimeric partner for Bcl-XL and Bcl-2,displaces Bax and promotes cell death.Cell,1995,80:285-291.
63.Bonni A,Brunet A,West A.E,et al.Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms.Science,1999,286:1358-1362.
64.Zha J,Harada H,Yang E,et al.Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L).Cell,1996,87:619-628.
65.Datta S.R,Dudek H,Tao X,et al.Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.Cell,1997,91:231-241.
66.She Q,Solit O.The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells.Cancer cell,2005,10:287-297.
67.Steiner P,Rudolph B,Muller D,et al.The functions of Myc in cell cycle progression and apoptosis.Prog Cell Cycle Res,1996,2:73-82.
68.Nagy A,Kozma L,Kiss I,et al.Copy number of cancer genes predict tumor grade and survival of pancreatic cancer patients.Anticancer Res.2001.21:1321-1325.
69.Schleger C,Verbeke C,Hildenbrand R,et al.c-MYC activation in primary and metastatic ductal adenocarcinoma of the pancreas:incidence,mechanisms,and clinical significance.Mod Pathol,2002, 15:462-469.
70.Henriksson M,Bakardjiev A,Klein G,et al.Phosphorylation sites mapping in the N-terminal domain of c-myc modulate its transforming potential.Oncogene,1993,8:3199-209.
71.Gregory MA,Qi Y,Hann SR.Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization.J Biol Chem,2003,278:51606-12.
72.Sears RG.The life cycle of C-myc:frpm synthesis to degradation.Cell Cycle,2004,3:1133-7.
73.Sears R,Nuckolls F,Haura E,et al.Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability.Genes Dev,2000,14:2501-2514.
74.Fabbrini M.S,Carpani D,Bello-Rivero I,et al.The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells:internalization is toxin mediated.FASEB J,1997,11:1169-1176.
1.Cantley LC.The phosphoinositide 3-kinase pathway.Science.2002.296:1655-1657.
2.Vivanco,Sawyers CL.The phosphotidylinositol 3-Kinase-AKT pathway in human cancer.Nat Rev Cancer.2002.2:489-501.
3.Czech MP.PIP2 and PIP3:complex roles at the cell surface.Cell.2000.17;100(6):603-6.
4.Dummler B,Hemmings BA.Physiological roles of PKB/Akt isoforms in development of disease.Biochem Soc Trans.2007.35(Pt2):231-5.
5.Song G,Ouyang G,Bao S.The activation of Akt/PKB signaling pathway and cell survival.J Cell Mol Med.2005 Jan-Mar;9(1):59-71.
6.Stauffer F,Holzer P,Garc(?)a-Echeverr(?)a C,et al.Blocking the PI3K/PKB pathway in tumor cells.Curr Med Chem Anticancer Agents.2005.5(5):449-62.
7.Osaki M,Oshimura M,Ito H,et al.PI3K-Akt pathway:its functions and alterations in human cancer.Apoptosis.2004.9(6):667-76.
8.Fresno Vara JA,Casado E,et al.PI3K/Akt signalling pathway and cancer.Cancer Treat Rev.2004.30(2):193-204.
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