PI3K/AKT特异性抑制剂LY294002对胆管癌细胞化疗增敏作用的研究

目的探讨磷脂酰肌醇-3-激酶抑制剂（PI3K）LY294002[22（42吗啉基）282苯基24氢212苯并吡喃242酮]对胆管癌化疗效果的影响。方法将LY294002用于胆管癌细胞系QBC939．MTT法检测单独使用5-Fu、MMC、塞莱西布及联合磷酸化抑制剂LY294002,对体外培养的胆管癌细胞系QBC939的抑制作用;流式细胞技术检测QBC939凋亡抑制率。结果LY294002作用后．5-Fu、MMC及塞莱西布对胆管癌细胞系QBC939抑制作用明显增强．且能提高其凋亡率。结论LY294002能有效提高化疗药物5-Fu、MMC、塞莱西布体外对QBC939细胞抑制作用的敏感性;抑制PI3K介导的信号转导通路,可明显提高胆管癌的化疗效果。     

阻断PI3K/Akt通路增强缺氧环境胰腺癌细胞化疗敏感性的实验研究

目的研究LY294002联合吉西他滨是否能增强缺氧环境下胰腺癌细胞株PANC-1的化疗敏感性。方法缺氧条件下培养胰腺癌细胞株PANC-1,运用MTT检测LY294002联合吉西他滨对胰腺癌细胞株细胞PANC-1生长的影响,运用Western blot检测AKT及磷酸化AKT蛋白表达的水平。结果 MTT检测在缺氧环境下LY294002联合吉西他滨作用的胰腺癌细胞PANC-1与单纯吉西他滨作用组相比细胞生存率显著下降(P=0.003),且具有时间依赖性。Western blot检测显示在缺氧环境下,LY294002联合吉西他滨作用的胰腺癌细胞PANC-1磷酸化AKT蛋白水平与单纯吉西他滨作用组相比显著降低(P=0.002)。结论阻断PI3K/Akt通路能增强缺氧环境下胰腺癌细胞化疗敏感性,为胰腺癌的治疗方法及逆转耐药提供了新的实验依据。     

PI3K／Akt通路和帕金森病

帕金森病是常见的神经系统退行性疾病之一,许多研究表明帕金森病的发病与细胞凋亡有关,而多种信号传导途径参与了细胞凋亡的调控。本义主要对PI3K／Akt通路与帕金森病凋亡的关系予以综述。     

溶血磷脂酸对顺铂诱导卵巢癌细胞凋亡的影响

目的探讨溶血磷脂酸对顺铂诱导的卵巢癌细胞凋亡的影响及机制。方法体外培养卵巢癌细胞株SKOV-3，四甲基偶氯唑蓝（MTT）比色法检测溶血磷脂酸对顺铂诱导的卵巢癌细胞增殖抑制的影响，流式细胞术（FCM）分析溶血磷脂酸对细胞周期变化及细胞凋亡的影响。结果溶血磷脂酸对顺铂诱导的卵巢癌细胞的凋亡有拮抗作用，并且降低G0／G1期的细胞比率。结论溶血磷脂酸可对抗顺铂诱导的卵巢癌细胞株SKOV-3的凋亡。这预示溶血磷脂陵可能是影响卵巢癌化疗耐药的一种重要因素，同时可能与卵巢癌的不良预后有关。     

PI3K/Akt抑制剂联合顺铂治疗肺癌的实验研究

目的 探讨PI3K/Akt抑制剂LY294002[2-(4-吗啉基)-8-苯基-4氢-1-苯并吡喃-4-酮]联合化疗药物顺铂(DDP)治疗肺癌的效应及可能机制,以期为肺癌的临床治疗提供新的理论依据.方法 培养肺腺癌A549细胞,采用MTT方法及流式细胞仪检测LY294002联合DDP对A549细胞增殖及凋亡的影响;通过裸鼠移植瘤实验检测LY294002联合DDP对A549细胞成瘤性的影响.结果 在一定剂量范围内,LY294002与DDP均可抑制A549细胞的生长,其抑制作用呈量-效关系.LY294002与DDP联合作用可增强对A549细胞的抑制作用.LY294002与DDP均可有效的诱导A549细胞凋亡,联用后凋亡率显著增加.裸鼠移植瘤实验显示,LY294002与DDP均可抑制移植瘤的生长,联用后抑瘤率增加.结论 LY294002可增强DDP对A549细胞、裸鼠移植瘤的抗肿瘤作用,该作用可能是通过增强对A549细胞增殖的抑制以及诱导其凋亡来实现的.     

顺铂对人卵巢癌KK细胞周期动力学的影响

顺铂 (c DDP)为广谱抗肿瘤药物 ,但其抗肿瘤作用的分子机制至今仍不甚清楚。已有研究证实 ,在某些细胞系中顺铂的抗肿瘤作用与其诱导的细胞凋亡有关。为此 ,我们以人卵巢癌 KK细胞为研究对象 ,采用流式细胞检测技术 ,探讨顺铂对卵巢癌细胞周期动力学的影响。现报告如下。1　资料与方法1 .1　主要试剂及仪器　顺铂和碘化丙啶 (PI)为 Sig-ma公司产品。RPMI- 1 6 4 0培养基为美国 Promega公司生产。一抗 p5 3单克隆抗体 (DO- 1 )和 p2 1多克隆抗体 (C- 1 9)为美国 Santa Cruz公司产品。流式细胞仪为美国 Beckman Coulter生产的 Epics …     

顺铂腹腔灌注联合泰素静滴对晚期卵巢癌患者腹水癌细胞凋亡及Trail表达的影响

单纯应用顺铂（DDP）腹腔灌注化疗2～3周后腹水难以控制的36例晚期（Ⅲ期）卵巢癌患者,再给予DDP腹腔灌注联合紫杉醇（泰素）静滴。观察其腹水中癌细胞凋亡和Trail的表达及腹水变化情况。结果显示,DDP及泰素联合应用后48、96h及1周时患者腹水中卵巢癌细胞凋亡率和Trail表达均显著升高（P〈0．05）,腹水量也显著减少（P〈0．01）。提示DDP腹腔灌注联合泰素静滴对难以控制腹水增长的晚期卵巢癌有较好的治疗效果。     

DAPT及顺铂对人卵巢癌H08910细胞生物学性能的影响

目的观察γ-分泌酶抑制剂DAPT及顺铂对人卵巢癌H08910细胞增殖、凋亡、侵袭、迁移能力的影响,为卵巢癌治疗提供新思路。方法常规培养H08910细胞至对数生长期,随机分为DAPT组、顺铂组、联合组及对照组,前三组分别以不同质量浓度DAPT、顺铂及DAPT＋顺铂处理,采用四甲基偶氮唑蓝（MIT）比色法检测细胞增殖情况,倒置显微镜观察细胞形态变化,流式细胞仪检测细胞凋亡率,体外迁移及侵袭实验检测细胞迁移及侵袭能力。结果与对照组比较,DAPT组、联合组细胞增殖明显受抑（P均〈0．05）,且呈DAPT作用时间和剂量依赖性,尤以联合组为著（P〈0．05）;倒置显微镜下对照组细胞形态正常,DAPT组及联合组均出现细胞密度减低及凋亡现象;DAPT组、顺铂组、联合组细胞凋亡率均显著高于对照组（P均〈0．05）,尤以联合组为著（P均〈0．05）;与对照组比较,余三组细胞迁移抑制率、细胞侵袭抑制率均显著升高（P均〈0．05）,尤以联合组为著（P均〈0．05）。结论DAPT与顺铂可协同抑制人卵巢癌H08910细胞增殖、促进细胞凋亡、降低细胞侵袭及迁移能力,此为卵巢癌的治疗提供了新思路。     

PI3K/Akt信号转导通路与脑缺血后细胞凋亡

细胞凋亡为脑缺血时细胞死亡的重要形式之一.磷脂酰肌醇-3激酶(phosphoinositide-3 kinase,PI3K)/丝氨酸-苏氨酸蛋白激酶(serine/threonine kinase,Akt)为重要的细胞存活信号通路,c-Jun氨基端激酶(c-jun N-terminal kinase,JNK)为重要的促进细胞凋亡信号通路.这两大通路转导信号的动态平衡维持着生理状态下的细胞生存与凋亡.脑缺血刺激打破了这一生理平衡,导致大量神经细胞凋亡.多种确切的神经保护因素都与增强细胞存活信号的放大或抑制凋亡信号的放大有关,从而维持这2个通路信号的平衡.     

顺铂联合乌苯美司对卵巢癌SKOV3细胞体外生长的作用

目的 探讨不同浓度乌苯美司、顺铂及两药联合对卵巢癌SKOV3细胞体外生长的作用.方法 不同浓度的乌苯美司、顺铂及两药联合作用于体外培养的卵巢癌SKOV-3细胞24 h,MTT法分别检测SKOV-3细胞体外生长的抑制率,联合指数法评价两药对卵巢癌SKOV-3细胞的联合作用.结果 经不同浓度的乌苯美司、顺铂及两药联合作用后,SKOV-3细胞生长受到抑制,且呈剂量依赖性,两者之间具有协同作用.结论 乌苯美司及顺铂单用可以抑制卵巢癌SKOV-3细胞的体外生长,两者联合应用可以增强顺铂对卵巢癌细胞体外生长的抑制作用.     

Phosphatidylethanolamine-binding protein 4 promotes lung cancer cells proliferation and invasion via PI3K/Akt/mTOR axis

Background

While phosphatidylethanolamine-binding protein 4 (PEBP4) is a key factor in the malignant proliferation and metastasis of tumor cells, the exact regulatory network governing its roles remains unclear. This study was designed to investigate the effect of PEBP4 on PI3K/Akt/mTOR pathway and explore its molecular network that governs the proliferation and metastasis of tumor cells.

Methods

After the recombinant plasmid pcDNA3.1-PEBP4 was constructed, the recombinant plasmid pcDNA3.1-PEBP4 and PEBP4-targeting siRNA were transfected into lung cancer HCC827 cell line. The expressions of PI3K/Akt/mTOR pathway components in HCC827 cells in each group were determined using Western blotting. In the HCC827 cells, the effect of PI3K pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Furthermore, the effect of mTOR inhibitor rapamycin (RAPA) on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of RAPA on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay.

Results

As shown by Western blotting, the protein expressions of p-Akt and phosphorylated mTOR (p-mTOR) were significantly higher in the pcDNA3.1-PEBP4-transfected group than in the normal control group and PEBP4 siRNA group (P<0.05); furthermore, the protein expressions of p-Akt and p-mTOR significantly decreased in the PEBP4 targeting siRNA-transfected group (P<0.05). Treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly inhibited the protein expressions of p-Akt and p-mTOR in HCC827 cells (P<0.05). In contrast, treatment with RAPA only significantly inhibited the protein expression of p-mTOR (P<0.05). As shown by MTT, flow cytometry, and Transwell migration assay, both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and RAPA could significantly lower the viability of HCC827 cells and inhibit their proliferation and invasion (P<0.05); meanwhile, they could reverse the effect of PEBP4 in promoting the proliferation and migration of HCC827 cells (P<0.05).

Conclusions

The overexpression of PEBP4 increases the phosphorylation levels of Akt and mTOR in lung cancer cells. The PI3K/Akt/mTOR signaling axis may be a key molecular pathway via which PEBP4 promotes the proliferation and invasion of non-small cell lung cancer (NSCLC) cells; also, it may serve as a potential therapeutic target.     

PI3K/Akt抑制剂LY294002对胃癌细胞化疗增敏作用的探讨

目的 探讨PI3K/Akt特异性抑制剂LY294002与化疗药物5-Fu及奥沙利铂联合使用对3种胃癌细胞系(MGC803、BGC823和SGC7901)化疗效果的影响.方法 将PI3K/Akt特异性抑制剂LY294002联合化疗药物5-Fu及奥沙利铂作用于3种胃癌细胞系,MTT法检测单独使用5-Fu、奥沙利铂及联合LY294002对体外培养的3种胃癌细胞系的增殖抑制作用,流式细胞术检测细胞凋亡.结果 联合LY294002作用后,5-Fu、奥沙利铂对3种胃癌细胞系的增殖抑制作用明显增强(P<0.05),且凋亡率显著提高(P<0.05).结论结果 LY294002能有效提高化疗药物5-Fu、奥沙利铂体外对胃癌细胞的增殖抑制作用,抑制PI3K/Akt信号转导通路可显著提高胃癌的化疗疗效.     

Loss of PTEN function may account for reduced proliferation pathway sensitivity to LY294002 in human prostate and bladder cancer cells

Purpose  Inhibition of phosphoinositide 3 (PI3)-kinase pathway is attractive for cancer treatment. To examine the role of the phosphatase and tensin homolog (PTEN) in the development of resistance to the treatment. Methods  We cultured human prostate cancer cells (DU145 and PC-3 cells) and bladder cancer cells (EJ-1 and UM-UC-3 cells) with a PI3-kinase inhibitor, LY294002 for more than 6 weeks and cell proliferation was studied. Activation of Akt1 and ERK was examined by immunoblotting. We introduced the wild type PTEN in UM-UC-3 cells and their proliferation along with the signaling pathways was also examined. Results  After 6 weeks, proliferation pathway sensitivity to LY294002 was reduced in cells expressing PTEN, but not in PTEN-null cells. PD98059, a MAPK/ERK kinase inhibitor, significantly inhibited proliferation of PTEN-expressing cells, but not PTEN-null cells. Stable PTEN expression in PTEN-null UM-UC-3 cells increased serum-induced ERK activation and sensitivity to PD98059-treatment, and reduced sensitivity to LY294002 after 6 weeks of exposure. Conclusions  Loss of PTEN function may protect against resistance to PI3-kinase inhibitors through an addiction to the PI3-kinase/Akt pathway.     

LY294002联合AD-PTEN对脑胶质瘤细胞增殖和侵袭的影响及机制探讨

目的观察P13K抑制剂LY294002联合含VIEN基因的重组腺病毒（Ad—PTEN）对人脑胶质瘤TJ899细胞增殖和侵袭的影响,并探讨其机制。方法向TJ889分别加入LY294002（LY294002组）、Ad-PTEN＋LY294002（联合组）、Ad—vector病毒（空载组）、DMSO（DMSO组）培养48h。分别采用MTY法、流式细胞术、划痕实验和Tran．swell小室观察TJ899增殖活性和侵袭能力的变化;Western Blot检测TJ899的PTEN／P13K／AKT信号转导通路中相关蛋白。结果与DMSO组、空载组和LY294002组相比,联合组细胞存活速率下降,细胞周期阻滞在G0／G1期,S期减少,细胞凋亡率增加,侵袭和迁移的细胞数减少,PTEN蛋白表达增加,而磷酸化蛋白激酶B、细胞增殖抗原、细胞周期素D1、Bcl-2、基质金属蛋白酶-2、黏着斑激酶蛋白表达显著下降,P均〈0．05。结论LY294002联合Ad-PTEN可明显抑制TJ899的增殖活性和侵袭能力,该作用与抑制P13K／AKT信号通路有关。     

LY294002逆转KB/VCR细胞多药耐药的作用及机制

目的 探讨蛋白激酶抑制剂LY294002对多药耐药细胞KB/VCR的耐药逆转作用及逆转机制.方法 采用MTT法检测LY294002对VCR和ADR的耐药逆转作用,流式细胞仪检测Rh123在KB和KB/VCR细胞内的蓄积,Western blot法检测LY294002对P-gp蛋白表达的影响,DAPI染色检测细胞凋亡.结果 LY294002对化疗药物具有协同增效作用,可以显著逆转KB/VCR的耐药性,能明显减少Rh123的外排,同时降低P-gp的表达.结论 LY294002能够逆转KB/VCR细胞的多药耐药性,不仅能够抑制P-gp功能,而且能够降低P-gp蛋白表达,使肿瘤细胞内化疗药物的浓度增加,提高化疗药物的杀伤作用.     

硼替佐米抑制PI3K/Akt通路增强胃癌细胞对TRAIL诱导凋亡的敏感性

目的:研究硼替佐米对TRAIL诱导胃癌细胞凋亡的影响, 探讨PI3K/Akt通路在TRAIL诱导凋亡中的作用.方法:不同浓度的TRAIL和/或硼替佐米作用于人胃癌细胞系MGC803细胞, MTT法检测细胞存活率, 流式细胞术PI染色检测细胞凋亡.Western blot法检测caspase裂解及p-Akt表达水平的变化.结果:50 nmol/L硼替佐米预处理细胞2 h, 之后予100 μg/L TRAIL继续作用24 h, 细胞存活率明显低于TRAIL单独处理组(35.1%±2.7% vs71.0%±4.3%, P <0.01); 细胞凋亡率明显高于TRAIL单药组(31.3%±2.0% vs 8.2%±0.8%,P <0.01). 20 nmol/L硼替佐米预处理未能增强细胞对TRAIL的敏感性. 进一步研究发现,TRAIL可活化PI3K/Akt通路, 硼替佐米预处理可阻止PI3K/Akt通路的活化, 进而增强细胞对TRAIL诱导凋亡的敏感性.结论:硼替佐米通过抑制TRAIL诱导的PI3K/Ak t通路活化, 增强胃癌MGC803细胞对TRAIL诱导凋亡的敏感性.     

Deguelin, A PI3K/AKT inhibitor, enhances chemosensitivity of leukaemia cells with an active PI3K/AKT pathway

Activation of the phosphoinositide 3 kinase (PI3K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its downregulation, by means of PI3K inhibitors, lowers resistance to various types of therapy in tumour cell lines. Recently, it has been reported that deguelin, a naturally occurring rotenoid, is a powerful inhibitor of PI3K. We investigated whether or not deguelin could enhance the sensitivity to chemotherapeutic drugs of human U937 leukaemia cells and acute myeloid leukaemia (AML) blasts with an activated PI3K/Akt network. Deguelin (10 nmol/l) induced S phase arrest with interference of progression to G2/M, and at 100 nmol/l significantly increased apoptotic cell death of U937. At 10-100 nmol/l concentrations, deguelin downregulated Akt phosphorylation of leukaemia cells and markedly increased sensitivity of U937 cells to etoposide or cytarabine. A 10 nmol/l concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells, whereas it increased sensitivity of AML blasts to cytarabine. Deguelin was less toxic than wortmannin on erythropoietin- and stem cell factor-induced erythropoiesis from CD34+ progenitor cells. Overall, our results indicate that deguelin might be used in the future for increasing sensitivity to therapeutic treatments of leukaemia cells with an active PI3K/Akt signalling network.