Up-regulation of PIK3CA promotes metastasis in gastric carcinoma

AIM:To explore expressions of PIK3CA in the progression of gastric cancer from primary to metastasis and its effects on activation of phosphatidylinositol 3-kinase(PI3K)/Akt pathway.METHODS:mRNA and protein levels of PIK3CA were assessed,respectively,by real-time quantitative polymerase chain reaction and immunohistochemistry in specimens of normal gastric mucosa,primary foci and lymph node and distant metastasis of gastric cancer.Akt and phosphorylated Akt protein were also examined by Western blotting in these tissues,in order to analyze the effect of PIK3CA expression level changes on the activation of PI3K/Akt signaling pathway.RESULTS:PIK3CA mRNA in lymph node metastasis were approximately 5 and 2 folds higher,respectively,than that in the corresponding normal gastric mucosa and primary gastric cancer tissues(P<0.05),while no statistical significance was found compared with distant metastasis.Immunohistochemically,PIK3CA protein expression was discovered in 7(35%)specimens of 20 primary foci vs 10(67%)of 15 of lymph node metastasis or 11(61%)of 18 of distant metastasis(35%vs 67%,P=0.015;35%vs 61%,P=0.044).With the increased level of PIK3CA expression,the total Akt protein expression remained almost unchanged,but p-Akt protein was upregulated markedly.CONCLUSION:Increased expression of PIK3CA is expected to be a promising indicator of metastasis in gastric cancer.Up-regulation of PIK3CA may promote the metastasis of gastric cancer through aberrant activation of PI3K/Akt signaling.     

PI3K expression and PIK3CA mutations are related to colorectal cancer metastases

AIM: To assess the significance of phosphatidylinositol 3-kinase (PI3K) in colorectal cancer (CRC) and toxicity of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 in CRC cells with different metastatic abilities.METHODS: Sixty formalin-fixed and paraffin-embedded CRC tumor specimens were investigated. Adjacent normal colonic mucosa specimens from 10 of these cases were selected as controls. PI3K protein was detected by immunohistochemistry and PIK3CA mutations were investigated by gene sequencing analysis. A flow-cytometry-based apoptosis detection kit was used to determine PI3K inhibitor-induced apoptosis in CRC cell lines SW480 and SW620. Expression of phosphorylated protein kinase B in CRC cell lines was detected by Western blotting.RESULTS: There was a significant difference in the proportion of primary lesions (30%, 18/60) vs metastatic lesions (46.7%, 28/60) that were positive for PI3K (P < 0.05). Mutations were detected in exon 9 (13.3%) and exon 20 (8.3%). Out of 60 cases, seven mutations were identified: two hotspot mutations, C.1633G>A resulting in E545A, and C.3140A>G resulting in H1047R; two novel missense mutations C.1624G>A and C.3079G>A; and three synonymous mutations (C.1641G>A, C.1581C>T and C.3027T>A). Exposure of SW480 cells to PI3K inhibitor for 48 h resulted in a significant increase of apoptotic cells in a dose-dependent manner [3.2% apoptotic cells in 0 μmol/L, 4.3% in 5 μmol/L, 6.3% in 10 μmol/L (P < 0.05), and 6.7% in 20 μmol/L (P < 0.05)]. Moreover, PI3K inhibitor induced a similar significant increase of apoptotic cells in the SW620 cell line for 48 h [3.3% apoptotic cells in 0 μmol/L, 13.3% in 5 μmol/L (P < 0.01), 19.2% in 10 μmol/L (P < 0.01), and 21.3% in 20 μmol/L (P < 0.01)].CONCLUSION: High PI3K expression is associated with CRC metastasis. PI3K inhibitor induced apoptosis in CRC cells and displayed strong cytotoxicity for highly metastatic cells. PI3K inhibition may be an effective treatment for CRC.     

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.     

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.     

蛋白酶体抑制剂硼替佐米对胃癌SGC7901细胞的抗肿瘤作用及其机制

目的:研究蛋白酶体抑制剂硼替佐米对胃癌SGC7901细胞凋亡及细胞周期分布的影响,并进一步探讨其作用机制.方法:1-500 nmol/L的硼替佐米作用于人胃癌SGC7901细胞24-48 h,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检测多聚ADP-核糖聚合酶(PARP)和casp...     

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.     

辛伐他汀对冠心病患者内皮祖细胞增殖的影响及其机制初步探讨

目的:观察辛伐他汀对冠心病患者外周血内皮祖细胞(EPCs)增殖的影响并初步探讨其机制。方法:冠心病患者28例,随机分为辛伐他汀组和对照组,前者给予辛伐他汀40mg/d口服,治疗前及治疗后2、4周抽取外周血,采用密度梯度离心法获取单个核细胞,培养7d后对贴壁的细胞进行分析。以激光共聚焦显微镜鉴定为FITC标记的荆豆凝集素Ⅰ和Dil(一种亲脂性碳化青荧光染料)标记的乙酰化低密度脂蛋白双染色阳性细胞为EPCs。计数法比较二组EPCs数目,并对病人血浆低密度脂蛋白-胆固醇(LDL-C)水平和EPCs数进行相关分析。另取10例冠心病患者血培养EPCs,加入辛伐他汀、PI3K/Akt信号转导通路抑制剂Wortmannin后观察对其增殖的影响。结果:辛伐他汀组病人治疗后2周、4周外周血中EPCs明显上升(P<0.01),其变化与LDL-C变化无相关性(P>0.05),体外实验辛伐他汀 Wortmannin组的血EPC数目较辛伐他汀组明显下降(P<0.01)。结论:辛伐他汀能刺激EPCs的增殖,这种作用能被Wortmannin阻断。故辛伐他汀作用可能与激活PI3K/Akt信号转导通路有关。     

PI3K／Akt信号途径参与高糖诱导的肾小管上皮细胞转分化及对分化抑制因子2表达的影响

目的观察PI3K／Akt在高糖诱导的肾小管上皮细胞转分化中的作用及对分化抑制因子2（1d2）表达的影响。方法大鼠肾小管上皮细胞随机分成3组：对照（Con）组,高糖（H—Glu）组和PI3K抑制剂Wortmannin（Wort）组。Con组加正常培养液,限GIu组在培养液中加30mmol／L葡萄糖,Wort组用Wortmannin预处理1h后,加30mmol／L葡萄糖。24h后用免疫细胞化学、Western blot法和RT-PCR法检测α平滑肌肌动蛋白（α-SMA）、p-Akt和Id2的表达。结果Con组α-SMA、p-Akt阳性表达细胞很少,绝大部分细胞出现Id2阳性表达。与COn组比较,H-Glu组出现α-SMA和p-Akt表达升高,Id2表达降低（P〈0．05）。与H-Glu组比较,Wort组α-SMA和p-Akt表达下降,Id2表达升高（P〈0．05）。结论PI3K／Akt可能通过抑制Id2基因表达,参与高糖诱导的大鼠肾小管上皮细胞转分化。     

Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?

Frequent activation of phosphatidylinositol-3 kinases (PI3K)/Akt/mTOR signaling pathway in gastric cancer (GC) is gaining immense popularity with identification of mutations and/or amplifications of PIK3CA gene or loss of function of PTEN, a tumor suppressor protein, to name a few; both playing a crucial role in regulating this pathway. These aberrations result in dysregulation of this pathway eventually leading to gastric oncogenesis, hence, there is a need for targeted therapy for more effective anticancer treatment. Several inhibitors are currently in either preclinical or clinical stages for treatment of solid tumors like GC. With so many inhibitors under development, further studies on predictive biomarkers are needed to measure the specificity of any therapeutic intervention. Herein, we review the common dysregulation of PI3K/Akt/mTOR pathway in GC and the various types of single or dual pathway inhibitors under development that might have a superior role in GC treatment. We also summarize the recent developments in identification of predictive biomarkers and propose use of predictive biomarkers to facilitate more personalized cancer therapy with effective PI3K/Akt/mTOR pathway inhibition.     

粒细胞集落刺激因子通过PI3K-Akt通路抑制缺血再灌注损伤

目的:研究经冠状动脉(冠脉)内注入粒细胞集落刺激因子(G-CSF)对大鼠心肌缺血再灌注损伤的预防作用,及其与PI3K-Akt信号转导途径的关系。方法:制备大鼠心肌缺血再灌注模型。再灌注同时经冠脉内给予G-CSF或G-CSF+PI3K激酶抑制剂(LY294002),灌注120min后应用TUNEL法检测细胞凋亡,免疫组织化学法观察凋亡相关蛋白Bax及Bcl-2及caspase-3表达情况,免疫印迹检测总-Akt(t-Akt)及磷酸化Akt(p-Akt)表达。结果:缺血再灌注同时给予冠脉内G-CSF治疗能够显著减轻缺血区心肌细胞凋亡,降低Bax、caspase-3的表达,增高心肌细胞内Bcl-2表达,同时p-Akt的蛋白水平显著增高(P<0.01)。LY294002阻断Akt活化后,抑制了G-CSF诱导的抗心肌细胞凋亡作用。结论:缺血再灌注同时冠脉内给予G-CSF可保护梗死区残存的心肌细胞,减少缺血再灌注所诱导的心肌细胞凋亡,其保护机制与PI3K-Akt信号通路有关。     

Knockin of mutant PIK3CA activates multiple oncogenic pathways

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3β is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.     

IGF-1对Rh1肉瘤细胞PI3K/Akt/mTOR信号通路的影响

目的 探讨胰岛素样生长因子(IGF)1对Rh1肉瘤细胞生长活性和PI3 K/Akt/mTOR信号通路的背景变化.方法 常规细胞培养,用无血清培养基消除内源性因子影响27h,再用IGF-1(终浓度为10 ng/ml)刺激72 h,流式细胞仪检测细胞生长活性;另外Western印迹方法观察IGF-1刺激细胞5、10、20、30和60min后Akt(s473)、S6的动态变化.结果 与对照组相比,IGF-1可促进Rh1细胞存活.IGF-1刺激不同时间后S6磷酸化则随着时间的延长逐渐增强;IGF-1亦导致Akt(s473)位点的磷酸化,随时间的延长,磷酸化Akt在5min时达高峰,此后逐渐减弱.结论 Akt、S6等是PI3K/Akt/mTOR信号通路中的重要信号分子,对Rhl细胞而言,在IGF-1刺激下S6有逐渐增强的变化,Akt (s473)位点磷酸化则有减弱的动态变化.     

PI3K/Akt信号通路对肝癌细胞系HepG2中SP细胞的影响

[目的]探讨PI3K/Akt信号通路对肝癌细胞系HepG2中肿瘤干细胞比例及干细胞特性的影响.[方法]使用PI3K/Akt通路抑制剂处理HepG2细胞后,使用流式技术分析HepG2细胞系中的侧群（SP）细胞的变化.软琼脂克隆形成实验检测PI3K/Akt抑制剂对HepG2细胞中SP细胞和非SP细胞成克隆能力的影响.[结果]HepG2细胞中存在SP细胞,经过LY294002处理后,SP细胞比例下降.LY294002可以显著降低SP细胞的软琼脂成克隆能力,对非SP细胞的软琼脂成克隆能力影响不明显.[结论]HepG2细胞中的SP细胞具有干细胞特性,PI3K/Akt信号通路对HepG2细胞中SP细胞的维持起重要作用,抑制PI3K/Akt信号通路后HepG2细胞中的SP细胞比例明显减低,并能显著抑制SP细胞的增殖速度、软琼脂成克隆能力,增加SP细胞对化疗药物的敏感性,为更加深入地了解肝癌干细胞的特性以及探索针对肿瘤干细胞的治疗提供理论依据.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

血管紧张素(1-7)抑制异丙肾上腺素诱导的H9c2心肌细胞凋亡

目的观察血管紧张素(1-7)[Ang(1-7)]对心肌细胞凋亡的影响,探讨其可能的作用机制。方法应用异丙肾上腺素(ISO)处理H9c2心肌细胞12 h建立心肌细胞凋亡模型,Ang(1-7)或PI3K抑制剂LY294002与H9c2心肌细胞共处理12 h观察对ISO诱导的心肌细胞凋亡的影响。显微镜下观察H9c2心肌细胞生长情况,采用MTS法检测各组细胞的相对细胞活性,TUNEL法检测各组细胞凋亡率。JC-1荧光探针检测线粒体膜电位,Western blot检测cleaved Caspase-3、p-Akt及Akt蛋白的表达量。结果 ISO呈浓度依赖性抑制H9c2心肌细胞的相对细胞活性,Ang(1-7)呈浓度依赖性逆转ISO诱导的H9c2心肌细胞相对活性的降低;与对照组比较,ISO组细胞凋亡率及cleaved Caspase-3蛋白表达量显著增加,线粒体膜电位和p-Akt蛋白表达量显著降低;Ang(1-7)可抑制ISO诱导的H9c2心肌细胞凋亡率的增加,减少cleaved Caspase-3蛋白的表达,增加线粒体膜电位和p-Akt蛋白表达量。LY294002预处理后Ang(1-7)对ISO诱导的H9c2心肌细胞的保护作用明显减弱,表现为心肌细胞凋亡率及cleaved Caspase-3蛋白的表达量明显增加,p-Akt蛋白表达量减少。结论 ISO能诱导H9c2心肌细胞线粒体途径的细胞凋亡,而Ang(1-7)能抑制ISO诱导的线粒体途径的细胞凋亡。PI3K/Akt信号通路可能在Ang(1-7)抑制ISO诱导的H9c2心肌细胞凋亡中起到关键作用。     

Obesity, the PI3K/Akt signal pathway and colon cancer

Obesity is currently reaching epidemic levels worldwide and is a major predisposing factor for a variety of life-threatening diseases including diabetes, hypertension and cardiovascular diseases. Recently, it has also been suggested to be linked with cancer.
Epidemiological studies have shown that obesity increases the risk of colon cancer by 1.5–2 fold with obesity-associated colon cancer accounting for 14–35% of total incidence. Several factors, altered in obesity, may be important in cancer development including increased levels of blood insulin, insulin-like growth factor I, leptin, TNF-α, IL-6 as well as decreased adiponectin. A unifying characteristic of all these factors is that they increase the activity of the PI3K/Akt signal pathway. The PI3K/Akt signal pathway in turn activates signals for cell survival, cell growth and cell cycle leading to carcinogenesis. Here we review the evidence that PI3K/Akt and its downstream targets are important in obesity-associated colon cancer and thus, that targeted inhibition of this pathway could be employed for the prevention of obesity-associated colon cancer and incorporated into the therapy regime for those with irremovable colon cancers.