PTEN对大肠癌细胞增殖与凋亡的影响及其信号机制研究
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摘要
大肠癌(colorectal carcinoma,CRC)是常见的恶性肿瘤,全球每年新增病例近1000000例,492000例死亡。随着经济的进步,生活方式和饮食结构的变化,我国大肠癌每年发病率也快速递增,特别是大城市上升趋势更为显著。大肠癌男女发病率等同,在40岁以后发病危险度随着年龄增加而递增;资料显示我国大肠癌男性发病率约为16.2/100000,女性约为14.5/100000,大肠癌已经成为我国致死性肿瘤中最常见的死亡原因之一。然而,大肠癌的治疗效果并不理想,在所有的病人中约有半数患者治疗失败。大肠癌传统治疗方法,即外科手术治疗、放射治疗和化学药物治疗方法的不断发展和完善,在延长患者生存期和提高患者生存质量方面取得了长足的进步;但是手术和放疗是局部治疗,难以控制肿瘤的复发和转移,化疗药物往往缺乏组织选择性和特异性,对骨髓等正常人体重要器官和组织产生严重的毒副作用,在过去几十年,大肠癌的五年生存率一直徘徊在40～50%左右。因此,探寻大肠上皮细胞癌变,演化和转移的机制,特别是寻找早期肿瘤标志物,以期早期发现和干预大肠癌的生物学进程,对大肠癌的治疗具有重要的意义。
磷脂酰肌醇3—激酶(phosphatidylinositol 3-kinase,PI3K)是一类特异的催化磷脂酰肌醇脂物质的激酶。正常情况下,由其活化而产生的类脂产物3,4-二磷酸磷脂酞肌醇[PI(3,4)P2]和3,4,5-三磷酸磷脂酞肌醇[PI(3,4,5)P3]作为第二信使结合并激活多种细胞内的靶蛋白,形成一个信号级联复合物,最终调节细胞的增殖、分化、存活和迁移等。哺乳动物的PI3K家族主要有三型,在PI3K家族中,IA型PI3K和其下游分子丝氨酸/苏氨酸蛋白激酶Akt(或PKB)所组成的信号通路因其与肿瘤发生发展的相关性,近年来备受瞩目。该通路调节肿瘤细胞的增殖和存活,其活性异常不仅能导致细胞恶性转化,而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关。PTEN是一种定位于10q23.3的抑癌基因,同时具有脂质磷酸酶活性和蛋白磷酸酶活性。作为脂质磷酸酶,PTEN可以使3,4,5-三磷酸磷脂酞肌醇[PI(3,4,5)P3]转化为,4,5-二磷酸磷脂酞肌醇[PI(4,5)P3],从而抑制磷脂酰肌醇3—激酶的活性,减弱PI3K/AKT通路增强蛋白合成、增殖、侵袭和存活的作用。研究表明PTEN突变或表达缺失可以引起PI3K/Akt通路的激活,目前对PTEN的研究,主要集中在肿瘤中PTEN所发生的突变及突变形式,但是对于其在肿瘤中的扩增现象及引起的肿瘤相关生物学特性的变化,则较少报道。
FoxO转录因子是Forkhead转录因子家族成员之一,该家族的共同特征是具有一个长110个氨基酸的保守的DNA结合结构域,称为Fox(Forkhead box)结构域。该家族成员主要受PI3K/Akt通路的磷酸化/去磷酸化修饰,磷酸化/去磷酸化改变FoxO蛋白的亚细胞定位。磷酸化的FoxO蛋白被排出细胞核,其转录活性受到抑制,这是FoxO转录活性受到负性调节的主要机制。肿瘤的发生与细胞周期失控及细胞凋亡机制异常密切相关,其中FoxO蛋白分子可能参与并发挥重要作用:①FoxO活性降低,细胞周期停止减弱,有助于肿瘤的发展;②FoxO活性降低,DNA损伤修复能力受损,可能导致基因组的不稳定;③FoxO蛋白缺乏,细胞凋亡减弱,可能导致肿瘤的扩展;④在人类染色体移位突变的肿瘤中发现有FoxO蛋白分子。目前关于PTEN表达变化引起大肠癌细胞生物学特性的变化与下游通路中FoxO转录因子家族变化的关系,未见报道。
本研究在系统地检测PTEN在大肠黏膜正常组织-大肠息肉-大肠腺瘤-原发性大肠癌组织中的表达差异和分布特点基础上,构建了pc-DNA3.1-PTEN表达质粒,转染大肠癌LoVo细胞和SW480细胞,观察上调大肠癌细胞PTEN表达后对大肠癌细胞增殖、细胞周期及细胞凋亡的影响,同时检测了PI3K通路下游的Akt蛋白、FoxO转录因子家族蛋白及相关细胞周期蛋白的表达变化,探讨PTEN可能参与大肠癌细胞增殖与凋亡的信号通路,旨在为发现大肠癌基因治疗新的靶点提供理论依据。
材料与方法
一、PTEN在大肠癌癌变过程中的表达及临床意义
应用免疫组织化学法检测PTEN在大肠黏膜正常组织、大肠增生性息肉、大肠腺瘤以及原发性大肠癌组织中的表达情况,并分析PTEN表达与大肠癌临床病理特征的关系。
二、PTEN表达对大肠癌细胞增殖与凋亡的影响
1、转染细胞和建立稳定转染细胞克隆转染实验参照Invitrogen公司的Lipofectamine 2000~(TM)产品说明书进行,转染后24h培养基中加入1000μg/ml G418进行筛选。形成阳性单细胞克隆群落后,用尖吸管吸取单克隆阳性细胞培养,连续培养4周后以含500μg/ml G418的培养基维持培养。
2、pc-DNA3.1-PTEN表达质粒转染后效应检测逆转录聚合酶链式反应(Reverse transcription polymerase chain reaction,RT-PCR)和免疫蛋白质印迹(western blot)检测稳定转染后LoVo细胞和SW480细胞PTEN mRNA和蛋白表达水平的改变。
3、细胞增殖实验稳定细胞株建立后,各组细胞连续培养0h-96h,CCK-8法检测上调PTEN表达对大肠癌细胞LoVo和SW480增殖的影响。
5细胞周期实验流式细胞术检测上调PTEN表达对大肠癌细胞LoVo和SW480细胞周期的影响。
6细胞凋亡实验采用Annexin V-FITC标记的流式细胞术检测上调PTEN表达对5-Fu诱导的大肠癌细胞凋亡的影响。
三、PTEN表达参与大肠癌细胞增殖与凋亡的信号通路研究
Western-Blot检测上调PTEN表达后总蛋白中Akt及p-Akt的表达水平的变化。作为PI3K/Akt信号通路的下游靶点,同时检测FoxO转录因子家族蛋白的表达水平变化,即胞浆蛋白p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,Ser~(253))、p-FoxO4(AFX,Ser~(193))和胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)的表达变化。FoxO转录因子家族参与调节细胞周期与细胞凋亡的变化,Western-Blot进一步检测了细胞周期相关蛋白cyclinD1、cdk4、cdk6、p27/Kipl的表达水平变化。
四、统计学分析
所有结果均经SPSS13.0统计软件统计。免疫组化半定量结果采用等级相关的秩和检验(Kruskal-Wallis test);数据以均数±标准差((?)±s)表示,两组间比较采用t检验,多组间比较采用单因素方差分析(one-way ANOVA),细胞增殖采用重复测量数据的方差分析,组间多重比较LSD方法进行分析,以P＜0.05为差异有统计学意义。
结果
一、PTEN在大肠癌癌变过程中的表达及临床意义
免疫组化结果显示PTEN表达主要定位于大肠病变组织上皮细胞的细胞浆。在大肠正常黏膜、大肠增生性息肉、大肠腺瘤和大肠癌组织中的PTEN蛋白表达有显著性差异(P＜0.05)。统计分析显示在大肠癌中PTEN蛋白与患者的肿瘤分化程度无关(P＞0.05)。
二、上调PTEN表达对大肠癌细胞增殖与凋亡的影响
1、PTEN真核表达质粒pc-DNA3.1-PTEN经酶切鉴定,所插入的片段与预期目的片段大小一致,并经测序证实,BLAST序列对比证实是PTEN基因mRNACDS序列。
2、pc-DNA3.1-PTEN表达质粒和pc-DNA3.1质粒分别转染LoVo细胞和SW480细胞后,经G418抗性筛选得到稳定细胞克隆株,分别命名为:LoVo pc-DNA3.1-PTEN;LoVo pc-DNA3.1;SW480 pc-DNA3.1-PTEN;SW480 pc-DNA3.1。
3、表达质粒稳定转染LoVo细胞和SW480细胞后,经RT-PCR和Western-Blot检测转染后PTEN mRNA和蛋白表达变化情况。与LoVo pc-DNA3.1、SW480pc-DNA3.1相比,LoVo pc-DNA3.1-PTEN和SW480 pc-DNA3.1-PTEN均能够显著增强了PTEN蛋白的表达。
4、应用CCK-8法连续检测各组细胞0h-96h的增值情况,结果显示,与阴性对照组细胞相比,24h、48h、72h、96h时转染pc-DNA3.1-PTEN表达质粒的LoVo、SW480细胞增殖速度显著减慢(P＜0.05),说明上调PTEN蛋白表达可显著减慢LoVo细胞和SW480细胞的恶性增值。
5、稳定转染后行流式细胞术检测细胞周期,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞出现显著的G1期细胞阻滞(P＜0.05),同时伴有显著的S期细胞数量减少(P＜0.05)。说明上调PTEN蛋白表达可显著抑制大肠癌细胞周期进展,减慢大肠癌细胞的恶性增殖速度。
6、细胞凋亡实验显示,未用5-Fu处理的各组细胞培养48h均未检测到明显的Annexin V阳性凋亡细胞,LoVo细胞和SW480细胞分别用0.01μmol/ml 5-Fu和0.03μmol/ml 5-Fu处理48h后,阴性对照组细胞均未出现明显的细胞凋亡,而转染pc-DNA3.1-PTEN的LoVo细胞和SW480细胞组均出现显著的Annexin V阳性凋亡细胞(P＜0.05),说明上调PTEN蛋白表达可以显著增加大肠癌细胞对化疗药物5-Fu的敏感性。
三、PTEN表达参与大肠癌细胞增殖与凋亡的信号通路研究
通过Western-Blot检测PI3K下游参与细胞增殖与凋亡的信号通路蛋白的变化。结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的Akt和p-Akt的表达量明显降低(P＜0.05),说明上调PTEN蛋白表达可以显著降低有活性的Akt蛋白的表达。作为Akt的下游靶蛋白,FoxO转录因子家族参与细胞增殖与凋亡的调节,Western-Blot结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)表达量显著增加(P＜0.05),而相应的胞浆磷酸化蛋白p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,Ser~(253))、p-FoxO4(AFX,Ser~(193))表达量显著降低(P＜0.05),说明上调PTEN蛋白表达可以促进FoxO转录因子家族的胞浆胞核移位,使其胞核表达增加,从而促进下游参与细胞增殖与细胞凋亡的靶基因的转录。Western-Blot进一步检测了FoxO转录因子家族蛋白表达变化对细胞周期相关蛋白的影响,结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的细胞周期相关蛋白cyclinD1、cdk4、cdk6表达显著降低(P＜0.05),而细胞周期素依赖性激酶抑制剂p27/Kipl表达水平显著增加(P＜0.05)。这些结果提示上调PTEN蛋白表达可以通过激活下游的FoxO转录因子家族成员,并引起细胞周期相关蛋白的改变,从而参与调节大肠癌细胞周期阻滞和对5-Fu诱导的细胞凋亡敏感性增加等生物学行为的改变。
结论
1、免疫组织化学检测结果显示PTEN表达从大肠正常黏膜-大肠增生性息肉-大肠腺瘤-大肠癌依次降低,提示PTEN在大肠癌的发生发展中起重要作用。
2、上调PTEN蛋白表达可以抑制大肠癌LoVo细胞和SW480细胞的增殖。
3、上调PTEN蛋白表达可以引起大肠癌细胞G1期阻滞,S期细胞数量减少,抑制恶性增殖。
4、上调PTEN蛋白表达可以增强大肠癌LoVo细胞和SW480细胞对5-Fu的敏感性。
5、上调PTEN蛋白表达可以降低Akt的活性,进而增强了FoxO转录因子家族的活性。FoxO转录因子家族的活性的增加抑制了促细胞周期进展相关蛋白的表达。提示上调PTEN蛋白表达引起的大肠癌细胞周期阻滞和对5-Fu诱导凋亡敏感性的增加是与下游FoxO转录因子家族的激活密切相关的。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world.The prognosis of CRCs is poor,due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually,amounting to492 000 related deaths.With the many changes having taken place in people's diet and lifestyle,CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing.The overall incidence is identical in men and women,with the risk beginning at age 40 and increasing with age.Thus colorectal cancer ranks as the frequent cause of cancer deaths among China.Despite the rate of improvements in surgery,radiotherapy and chemotherapy,unfortunately,the prognosis of CRCs has not been gained progress over the past decades,with an overall five-year survival rate of around 40%-50%.Therefore,novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium.In recent years,molecular biology has applied to the study of colonic carcinoma,both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.
Phosphatidylinositol-3-kinase(PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2,to generate PI(3,4,5) P3,a potent second-messenger required for fundamental cellular functions such as transcription,translation,proliferation,growth,and survival.There are three members in PI3K family.Signaling pathway composed of Class-IA PI3K and serine/threonine kinase Akt/PKB has close relationship with tumor progression.This pathway regulates proliferation and survival of cancer cells.The disturbed activation of PI3K signaling leads to not only neoplastic transformation of normal cells,but also correlation with tumor cell migration,adhesion,tumor angiogenesis,as well as the degradation of extracellular matrix.PTEN is a tumor-suppressor gene located on human chromosome 10q23.3.PTEN is both a lipid phosphatase and a protein phosphatase.As a lipid phosphatase,PTEN classically converts phosphatidylinositio-3, 4,5-trisphosphate(PIP3) in thecytoplasm to phosphatidylinositol-4,5-bisphosphate (PIP2),thereby directly antagonizing the activity of PI3 kinase(PI3K).Its inactivation results in constitutive activation of the PI3K/AKT pathway and in subsequent increases in protein synthesis,proliferation,migration and survival.It has been reporetd that deletion and mutation of PTENcould activate PI3K/Akt signaling pathway.Most studies focused on the mutation and the mutation form of PTEN in cancer.Reports about the amplification of PTEN in tumor and the biological changes result from the amplification are rare.
FoxO transcription factors belong to the Forkhead family of proteins,a family characterized by a conserved DNA binding domain termed the Forkhead box(Fox). The family is primarily regulated by PI3K pathway.Direct phosphorylation by PKB/Akt inhibits transcriptional activation by FoxO factors,causing their displacement from the nucleus into the cytoplasm.Deregulation of cell cycle and cell apoptosis are colosely related with tumor progression.FoxO transcription factors may be involved and play an important role in regulating cell cycle and cell apoptosis:①Inactivation of FoxO transcription factors leads to cell cycle progression which contributes to the development of tumor.②Inactivation of FoxO transcription factors leads to impaired ability to repair DNA damage which results in genomic instability.③Depletion of FoxO transcription proteins leads to inhibition of apoptosis which may contributes to tumor progression.④FoxO transcription proteins are found in human translocation mutational tumors.At present,the relationship between biological changes resulted from the changes of PTEN expression levels and the changes of FoxO transcription factors is not reported.
In this study,we investigate the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct pc-DNA3.1-PTEN expressional vector to transfect LoVo and SW480 cells.After stable transfection,we observed the effect of PTEN expression on colorectal cancer cell proliferation,cell cycle and cell apoptosis.We also investigated the expression changes of PI3K signaling pathway proteins including Akt,FoxO transcription proteins and associated cell cycle proteins.We aim not only to investigate the molecular mechanism of tumor proliferation and apoptosis,but also to search new effective target for gene therapy of CRC.
Materials and methods
The expression and significance of PTEN in progression of colorectal cancer
Immunohistochemical staining was used to detect the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal mucosa,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PTEN protein and clinicopathological factors was also analyzed.
The effect of PTEN expression on proliferation and apoptosis in colorectal cancer cells
1.Transfection and selection of stable transfected cell clones The pcDNA3.1-PTEN expressional vector and control vector were transfected into Lovo and SW480 cells using Lipofectamine 2000(Invitrogen) according to the manufacturer's protocol and cultured for 24 h without antibiotic selection.Then the cells were cultured in medium containing 1000μg/ml G418 until all of the cells in the nontransfected control culture were killed and antibiotic-resistant clones were formed in the transfected cells.Visible clones were picked up and expanded for another 4 weeks. Then the antib(?)tic-resistant cells were passaged in medium containing 500μg/ml G418 as needed.
2.PTEN expression detecting The interference effect was evaluated by RT-PCR and Western blot analysis.Selected stable cell lines transfected with PTEN that had the highest interference effect for the following experiments.
4.Cell proliferation assays Cells were cultured for 96h after stable transfection. Cell proliferation was determined using Cell Counting Kit-8(CCK-8) solution.
5.Cell cycle analysis FACSC alibur Flow Cytometer was used to determine the cell cycle after stable transfection.
6.Apoptosis Assays Lovo and SW480 cells that expressing pc-DNA3.1-PTEN expressional vector and the control cells were treated with 5-FU for 48 h at final concentration of 0.01μmol/ml and 0.03μmol/ml,respectively.Annexin V-FITC Kit was used to determine the apoptosis.
Research of cell proliferation and apoptosis signaling pathway PTEN involved in colorectal cancer cells
Western blot was used to analyzed expression changes of signaling pathway proteins after expression of PTEN.The cell proliferation and apoptosis signaling pathway proteins include Akt、p-Akt,cytoplasmic proteins p-FoxO1a(FKHR, Ser~(256))、p-FoxO3a(FKHRL1,Ser~253))、p-FoxO4(AFX,Ser~(193)),nuclear proteins FoxO1a(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX),and cell cycle associated proteins cyclinD1、cdk4、cdk6 and p27/Kipl.
Statistical Analysis
All experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.For other results, one-way analysis of variance(ANOVA) and Student's t test were used in group comparison,repeatedly measured data analysis of variance were cell proliferation, LSD analysis method was used in group comparison.Dates are expressed as the mean±SD.A value of P＜0.05 was considered statistically significant.
Results
The expression and significance of PTEN in progression of colorectal cancer
Immunohistochemistry was performed to examine PTEN expression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PTEN expression was highest in surface epithelium of paraffin-embeded tissue from colorecta mucosa.The diffierence for the expression of PTEN protein in normal colorectal tissue,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma was significant(P＜0.05).No obvious correlation was found between expression of PTEN and pathological diagnosis (P＞0.05).
The effect of up-regulating PTEN expression on proliferation and apoptosis in colorectal cancer cells
1.Constructed human pc-DNA3.1-PTEN expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.
2.After pc-DNA3.1-PTEN expression vector and pc-DNA3.1 vector were transfected into LoVo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as LoVo pc-DNA3.1-PTEN;LoVo pc-DNA3.1; SW480 pc-DNA3.1-PTEN;SW480 pc-DNA3.1.
3.After stable transfection,western blot analysis was used to investigate the expression changes of PTEN.The expression levels were significantly enhanced in LoVo pc-DNA3.1-PTEN and SW480 pc-DNA3.1-PTEN cells compared LoVo pc-DNA3.1 and SW480 pc-DNA3.1cells.
4.To determine the effect of PTEN expression on proliferation of colorectal cancer cells,CCK-8 assay was performed at Oh,24h,48h,72h and 96h,respectively. Compared with the control cells,LoVo pc-DNA3.1-PTEN andSW480 pc-DNA3.1-PTEN cells grew much slowly at 24h,48h,72h and 96h(P＜0.05).The results indicate up-regulating PTEN expression could inhibit coloredtal cell proliferation.
5.To examine if expression of PTEN has an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that expression of PTEN cause a significant increase in the proportion of LoVo and SW480 cells at the G0/G1 phase(P＜0.05).The observed increase in G0/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.05).The findings suggest that expression of PTEN could obviously induce cell cycle arrest.
6.In order to evaluate the effect of up-regulating PTEN expression on the induction of apoptosis,colorectal cancer cells expressing pc-DNA3.1-PTEN and pc-DNA3.1 were treated with 5-Fu at final concentration of 0.01μmol/ml(LoVo) or 0.03μmol/ml(SW480) for 48h,followed by assessing early apoptotic rate by means of flow cytometric analysis.Results showed that the proportion of positive cells for Annexin V was significantly higher in up-regulating PTEN expression LoVo and SW480 cells than in control cells(P＜0.05).The results indicate that expression of PTEN sensitized colorectal cancer cells to 5-Fu induced apoptosis.
Up-regulating PTEN expression activated FoxO transcription factors associated with regulating cell cycle-associated protein expression in colorectal cancer cells
To further explore the mechanism underlying the enhancement of cell cycle arrest and 5-FU-induced apoptosis by the expression of PTEN,we examined expression levels of Akt,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that up-regulating PTEN expression led to substantial reduction in the levels of Akt and phospho-Akt(P＜0.05).Consistent with this reduction in the phospho-Akt level, Western blot analysis showed significantly decreased expression of phospho-FoxO1 (FKHR),phospho-FoxO3a(FKHRL1) and phospho-FoxO4(AFX) in cytoplasm of PTEN expresion cells(P＜0.05),the prominent accumulation of FoxO1(FKHR), FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed (P＜0.05).As potential downstream targets of FoxO transcription factors,the expression levels of cell cycle-associated protein were also determined.Results showed that the expression levels of cyclin D1,cdk4 and cdk6 were significantly decreased in up-regulating PTEN expression cells(P＜0.05),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.05).
Conclusions
1.Immunohistochemical results show that the expression levels of PTEN gradually decreases from normal colorectal mucosa,colorectal proliferational plopy,adenoma to primary colorectal adenocarcinoma,indicating that PTEN plays an important role in the progression of colorectal cancer.
2.Up-regulating PTEN expression could inhibit LoVo and SW480 cell proliferation.
3.Up-regulating PTEN expression could induce G1 phase cell cycle arrest and reduction of S phase cells.
4.Up-regulating PTEN expression could sensitize LoVo and SW480 cells to 5-Fu induced apoptosis.
5.Up-regulating PTEN expression could inhibit the activity of Akt and activate FoxO transcription factors,which,in turn,activated transcription of target genes such as those involved in cell cycle regulation and apoptosis.Thus,the colorectal cancer cell cycle arrest and enhanced sensibility to 5-Fu induced apoptosis result from expression of PTEN are closely related with activation of FoxO transcription factors.
磷脂酰肌醇3—激酶(phosphatidylinositol 3-kinase,PI3K)是一类特异的催化磷脂酰肌醇脂物质的激酶。正常情况下,由其活化而产生的类脂产物3,4-二磷酸磷脂酞肌醇[PI(3,4)P2]和3,4,5-三磷酸磷脂酞肌醇[PI(3,4,5)P3]作为第二信使结合并激活多种细胞内的靶蛋白,形成一个信号级联复合物,最终调节细胞的增殖、分化、存活和迁移等。哺乳动物的PI3K家族主要有三型,在PI3K家族中,IA型PI3K和其下游分子丝氨酸/苏氨酸蛋白激酶Akt(或PKB)所组成的信号通路因其与肿瘤发生发展的相关性,近年来备受瞩目。该通路调节肿瘤细胞的增殖和存活,其活性异常不仅能导致细胞恶性转化,而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关。PTEN是一种定位于10q23.3的抑癌基因,同时具有脂质磷酸酶活性和蛋白磷酸酶活性。作为脂质磷酸酶,PTEN可以使3,4,5-三磷酸磷脂酞肌醇[PI(3,4,5)P3]转化为,4,5-二磷酸磷脂酞肌醇[PI(4,5)P3],从而抑制磷脂酰肌醇3—激酶的活性,减弱PI3K/AKT通路增强蛋白合成、增殖、侵袭和存活的作用。研究表明PTEN突变或表达缺失可以引起PI3K/Akt通路的激活,目前对PTEN的研究,主要集中在肿瘤中PTEN所发生的突变及突变形式,但是对于其在肿瘤中的扩增现象及引起的肿瘤相关生物学特性的变化,则较少报道。
FoxO转录因子是Forkhead转录因子家族成员之一,该家族的共同特征是具有一个长110个氨基酸的保守的DNA结合结构域,称为Fox(Forkhead box)结构域。该家族成员主要受PI3K/Akt通路的磷酸化/去磷酸化修饰,磷酸化/去磷酸化改变FoxO蛋白的亚细胞定位。磷酸化的FoxO蛋白被排出细胞核,其转录活性受到抑制,这是FoxO转录活性受到负性调节的主要机制。肿瘤的发生与细胞周期失控及细胞凋亡机制异常密切相关,其中FoxO蛋白分子可能参与并发挥重要作用:①FoxO活性降低,细胞周期停止减弱,有助于肿瘤的发展;②FoxO活性降低,DNA损伤修复能力受损,可能导致基因组的不稳定;③FoxO蛋白缺乏,细胞凋亡减弱,可能导致肿瘤的扩展;④在人类染色体移位突变的肿瘤中发现有FoxO蛋白分子。目前关于PTEN表达变化引起大肠癌细胞生物学特性的变化与下游通路中FoxO转录因子家族变化的关系,未见报道。
本研究在系统地检测PTEN在大肠黏膜正常组织-大肠息肉-大肠腺瘤-原发性大肠癌组织中的表达差异和分布特点基础上,构建了pc-DNA3.1-PTEN表达质粒,转染大肠癌LoVo细胞和SW480细胞,观察上调大肠癌细胞PTEN表达后对大肠癌细胞增殖、细胞周期及细胞凋亡的影响,同时检测了PI3K通路下游的Akt蛋白、FoxO转录因子家族蛋白及相关细胞周期蛋白的表达变化,探讨PTEN可能参与大肠癌细胞增殖与凋亡的信号通路,旨在为发现大肠癌基因治疗新的靶点提供理论依据。
材料与方法
一、PTEN在大肠癌癌变过程中的表达及临床意义
应用免疫组织化学法检测PTEN在大肠黏膜正常组织、大肠增生性息肉、大肠腺瘤以及原发性大肠癌组织中的表达情况,并分析PTEN表达与大肠癌临床病理特征的关系。
二、PTEN表达对大肠癌细胞增殖与凋亡的影响
1、转染细胞和建立稳定转染细胞克隆转染实验参照Invitrogen公司的Lipofectamine 2000~(TM)产品说明书进行,转染后24h培养基中加入1000μg/ml G418进行筛选。形成阳性单细胞克隆群落后,用尖吸管吸取单克隆阳性细胞培养,连续培养4周后以含500μg/ml G418的培养基维持培养。
2、pc-DNA3.1-PTEN表达质粒转染后效应检测逆转录聚合酶链式反应(Reverse transcription polymerase chain reaction,RT-PCR)和免疫蛋白质印迹(western blot)检测稳定转染后LoVo细胞和SW480细胞PTEN mRNA和蛋白表达水平的改变。
3、细胞增殖实验稳定细胞株建立后,各组细胞连续培养0h-96h,CCK-8法检测上调PTEN表达对大肠癌细胞LoVo和SW480增殖的影响。
5细胞周期实验流式细胞术检测上调PTEN表达对大肠癌细胞LoVo和SW480细胞周期的影响。
6细胞凋亡实验采用Annexin V-FITC标记的流式细胞术检测上调PTEN表达对5-Fu诱导的大肠癌细胞凋亡的影响。
三、PTEN表达参与大肠癌细胞增殖与凋亡的信号通路研究
Western-Blot检测上调PTEN表达后总蛋白中Akt及p-Akt的表达水平的变化。作为PI3K/Akt信号通路的下游靶点,同时检测FoxO转录因子家族蛋白的表达水平变化,即胞浆蛋白p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,Ser~(253))、p-FoxO4(AFX,Ser~(193))和胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)的表达变化。FoxO转录因子家族参与调节细胞周期与细胞凋亡的变化,Western-Blot进一步检测了细胞周期相关蛋白cyclinD1、cdk4、cdk6、p27/Kipl的表达水平变化。
四、统计学分析
所有结果均经SPSS13.0统计软件统计。免疫组化半定量结果采用等级相关的秩和检验(Kruskal-Wallis test);数据以均数±标准差((?)±s)表示,两组间比较采用t检验,多组间比较采用单因素方差分析(one-way ANOVA),细胞增殖采用重复测量数据的方差分析,组间多重比较LSD方法进行分析,以P＜0.05为差异有统计学意义。
结果
一、PTEN在大肠癌癌变过程中的表达及临床意义
免疫组化结果显示PTEN表达主要定位于大肠病变组织上皮细胞的细胞浆。在大肠正常黏膜、大肠增生性息肉、大肠腺瘤和大肠癌组织中的PTEN蛋白表达有显著性差异(P＜0.05)。统计分析显示在大肠癌中PTEN蛋白与患者的肿瘤分化程度无关(P＞0.05)。
二、上调PTEN表达对大肠癌细胞增殖与凋亡的影响
1、PTEN真核表达质粒pc-DNA3.1-PTEN经酶切鉴定,所插入的片段与预期目的片段大小一致,并经测序证实,BLAST序列对比证实是PTEN基因mRNACDS序列。
2、pc-DNA3.1-PTEN表达质粒和pc-DNA3.1质粒分别转染LoVo细胞和SW480细胞后,经G418抗性筛选得到稳定细胞克隆株,分别命名为:LoVo pc-DNA3.1-PTEN;LoVo pc-DNA3.1;SW480 pc-DNA3.1-PTEN;SW480 pc-DNA3.1。
3、表达质粒稳定转染LoVo细胞和SW480细胞后,经RT-PCR和Western-Blot检测转染后PTEN mRNA和蛋白表达变化情况。与LoVo pc-DNA3.1、SW480pc-DNA3.1相比,LoVo pc-DNA3.1-PTEN和SW480 pc-DNA3.1-PTEN均能够显著增强了PTEN蛋白的表达。
4、应用CCK-8法连续检测各组细胞0h-96h的增值情况,结果显示,与阴性对照组细胞相比,24h、48h、72h、96h时转染pc-DNA3.1-PTEN表达质粒的LoVo、SW480细胞增殖速度显著减慢(P＜0.05),说明上调PTEN蛋白表达可显著减慢LoVo细胞和SW480细胞的恶性增值。
5、稳定转染后行流式细胞术检测细胞周期,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞出现显著的G1期细胞阻滞(P＜0.05),同时伴有显著的S期细胞数量减少(P＜0.05)。说明上调PTEN蛋白表达可显著抑制大肠癌细胞周期进展,减慢大肠癌细胞的恶性增殖速度。
6、细胞凋亡实验显示,未用5-Fu处理的各组细胞培养48h均未检测到明显的Annexin V阳性凋亡细胞,LoVo细胞和SW480细胞分别用0.01μmol/ml 5-Fu和0.03μmol/ml 5-Fu处理48h后,阴性对照组细胞均未出现明显的细胞凋亡,而转染pc-DNA3.1-PTEN的LoVo细胞和SW480细胞组均出现显著的Annexin V阳性凋亡细胞(P＜0.05),说明上调PTEN蛋白表达可以显著增加大肠癌细胞对化疗药物5-Fu的敏感性。
三、PTEN表达参与大肠癌细胞增殖与凋亡的信号通路研究
通过Western-Blot检测PI3K下游参与细胞增殖与凋亡的信号通路蛋白的变化。结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的Akt和p-Akt的表达量明显降低(P＜0.05),说明上调PTEN蛋白表达可以显著降低有活性的Akt蛋白的表达。作为Akt的下游靶蛋白,FoxO转录因子家族参与细胞增殖与凋亡的调节,Western-Blot结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)表达量显著增加(P＜0.05),而相应的胞浆磷酸化蛋白p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,Ser~(253))、p-FoxO4(AFX,Ser~(193))表达量显著降低(P＜0.05),说明上调PTEN蛋白表达可以促进FoxO转录因子家族的胞浆胞核移位,使其胞核表达增加,从而促进下游参与细胞增殖与细胞凋亡的靶基因的转录。Western-Blot进一步检测了FoxO转录因子家族蛋白表达变化对细胞周期相关蛋白的影响,结果提示,与阴性对照组细胞相比,转染pc-DNA3.1-PTEN表达质粒的LoVo细胞和SW480细胞的细胞周期相关蛋白cyclinD1、cdk4、cdk6表达显著降低(P＜0.05),而细胞周期素依赖性激酶抑制剂p27/Kipl表达水平显著增加(P＜0.05)。这些结果提示上调PTEN蛋白表达可以通过激活下游的FoxO转录因子家族成员,并引起细胞周期相关蛋白的改变,从而参与调节大肠癌细胞周期阻滞和对5-Fu诱导的细胞凋亡敏感性增加等生物学行为的改变。
结论
1、免疫组织化学检测结果显示PTEN表达从大肠正常黏膜-大肠增生性息肉-大肠腺瘤-大肠癌依次降低,提示PTEN在大肠癌的发生发展中起重要作用。
2、上调PTEN蛋白表达可以抑制大肠癌LoVo细胞和SW480细胞的增殖。
3、上调PTEN蛋白表达可以引起大肠癌细胞G1期阻滞,S期细胞数量减少,抑制恶性增殖。
4、上调PTEN蛋白表达可以增强大肠癌LoVo细胞和SW480细胞对5-Fu的敏感性。
5、上调PTEN蛋白表达可以降低Akt的活性,进而增强了FoxO转录因子家族的活性。FoxO转录因子家族的活性的增加抑制了促细胞周期进展相关蛋白的表达。提示上调PTEN蛋白表达引起的大肠癌细胞周期阻滞和对5-Fu诱导凋亡敏感性的增加是与下游FoxO转录因子家族的激活密切相关的。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world.The prognosis of CRCs is poor,due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually,amounting to492 000 related deaths.With the many changes having taken place in people's diet and lifestyle,CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing.The overall incidence is identical in men and women,with the risk beginning at age 40 and increasing with age.Thus colorectal cancer ranks as the frequent cause of cancer deaths among China.Despite the rate of improvements in surgery,radiotherapy and chemotherapy,unfortunately,the prognosis of CRCs has not been gained progress over the past decades,with an overall five-year survival rate of around 40%-50%.Therefore,novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium.In recent years,molecular biology has applied to the study of colonic carcinoma,both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.
Phosphatidylinositol-3-kinase(PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2,to generate PI(3,4,5) P3,a potent second-messenger required for fundamental cellular functions such as transcription,translation,proliferation,growth,and survival.There are three members in PI3K family.Signaling pathway composed of Class-IA PI3K and serine/threonine kinase Akt/PKB has close relationship with tumor progression.This pathway regulates proliferation and survival of cancer cells.The disturbed activation of PI3K signaling leads to not only neoplastic transformation of normal cells,but also correlation with tumor cell migration,adhesion,tumor angiogenesis,as well as the degradation of extracellular matrix.PTEN is a tumor-suppressor gene located on human chromosome 10q23.3.PTEN is both a lipid phosphatase and a protein phosphatase.As a lipid phosphatase,PTEN classically converts phosphatidylinositio-3, 4,5-trisphosphate(PIP3) in thecytoplasm to phosphatidylinositol-4,5-bisphosphate (PIP2),thereby directly antagonizing the activity of PI3 kinase(PI3K).Its inactivation results in constitutive activation of the PI3K/AKT pathway and in subsequent increases in protein synthesis,proliferation,migration and survival.It has been reporetd that deletion and mutation of PTENcould activate PI3K/Akt signaling pathway.Most studies focused on the mutation and the mutation form of PTEN in cancer.Reports about the amplification of PTEN in tumor and the biological changes result from the amplification are rare.
FoxO transcription factors belong to the Forkhead family of proteins,a family characterized by a conserved DNA binding domain termed the Forkhead box(Fox). The family is primarily regulated by PI3K pathway.Direct phosphorylation by PKB/Akt inhibits transcriptional activation by FoxO factors,causing their displacement from the nucleus into the cytoplasm.Deregulation of cell cycle and cell apoptosis are colosely related with tumor progression.FoxO transcription factors may be involved and play an important role in regulating cell cycle and cell apoptosis:①Inactivation of FoxO transcription factors leads to cell cycle progression which contributes to the development of tumor.②Inactivation of FoxO transcription factors leads to impaired ability to repair DNA damage which results in genomic instability.③Depletion of FoxO transcription proteins leads to inhibition of apoptosis which may contributes to tumor progression.④FoxO transcription proteins are found in human translocation mutational tumors.At present,the relationship between biological changes resulted from the changes of PTEN expression levels and the changes of FoxO transcription factors is not reported.
In this study,we investigate the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct pc-DNA3.1-PTEN expressional vector to transfect LoVo and SW480 cells.After stable transfection,we observed the effect of PTEN expression on colorectal cancer cell proliferation,cell cycle and cell apoptosis.We also investigated the expression changes of PI3K signaling pathway proteins including Akt,FoxO transcription proteins and associated cell cycle proteins.We aim not only to investigate the molecular mechanism of tumor proliferation and apoptosis,but also to search new effective target for gene therapy of CRC.
Materials and methods
The expression and significance of PTEN in progression of colorectal cancer
Immunohistochemical staining was used to detect the expression and significance of PTEN in the progression of colorectal cancer,including normal colorectal mucosa,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PTEN protein and clinicopathological factors was also analyzed.
The effect of PTEN expression on proliferation and apoptosis in colorectal cancer cells
1.Transfection and selection of stable transfected cell clones The pcDNA3.1-PTEN expressional vector and control vector were transfected into Lovo and SW480 cells using Lipofectamine 2000(Invitrogen) according to the manufacturer's protocol and cultured for 24 h without antibiotic selection.Then the cells were cultured in medium containing 1000μg/ml G418 until all of the cells in the nontransfected control culture were killed and antibiotic-resistant clones were formed in the transfected cells.Visible clones were picked up and expanded for another 4 weeks. Then the antib(?)tic-resistant cells were passaged in medium containing 500μg/ml G418 as needed.
2.PTEN expression detecting The interference effect was evaluated by RT-PCR and Western blot analysis.Selected stable cell lines transfected with PTEN that had the highest interference effect for the following experiments.
4.Cell proliferation assays Cells were cultured for 96h after stable transfection. Cell proliferation was determined using Cell Counting Kit-8(CCK-8) solution.
5.Cell cycle analysis FACSC alibur Flow Cytometer was used to determine the cell cycle after stable transfection.
6.Apoptosis Assays Lovo and SW480 cells that expressing pc-DNA3.1-PTEN expressional vector and the control cells were treated with 5-FU for 48 h at final concentration of 0.01μmol/ml and 0.03μmol/ml,respectively.Annexin V-FITC Kit was used to determine the apoptosis.
Research of cell proliferation and apoptosis signaling pathway PTEN involved in colorectal cancer cells
Western blot was used to analyzed expression changes of signaling pathway proteins after expression of PTEN.The cell proliferation and apoptosis signaling pathway proteins include Akt、p-Akt,cytoplasmic proteins p-FoxO1a(FKHR, Ser~(256))、p-FoxO3a(FKHRL1,Ser~253))、p-FoxO4(AFX,Ser~(193)),nuclear proteins FoxO1a(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX),and cell cycle associated proteins cyclinD1、cdk4、cdk6 and p27/Kipl.
Statistical Analysis
All experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.For other results, one-way analysis of variance(ANOVA) and Student's t test were used in group comparison,repeatedly measured data analysis of variance were cell proliferation, LSD analysis method was used in group comparison.Dates are expressed as the mean±SD.A value of P＜0.05 was considered statistically significant.
Results
The expression and significance of PTEN in progression of colorectal cancer
Immunohistochemistry was performed to examine PTEN expression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PTEN expression was highest in surface epithelium of paraffin-embeded tissue from colorecta mucosa.The diffierence for the expression of PTEN protein in normal colorectal tissue,colorectal proliferational plopy,colorectal adenoma and primary colorectal carcinoma was significant(P＜0.05).No obvious correlation was found between expression of PTEN and pathological diagnosis (P＞0.05).
The effect of up-regulating PTEN expression on proliferation and apoptosis in colorectal cancer cells
1.Constructed human pc-DNA3.1-PTEN expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.
2.After pc-DNA3.1-PTEN expression vector and pc-DNA3.1 vector were transfected into LoVo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as LoVo pc-DNA3.1-PTEN;LoVo pc-DNA3.1; SW480 pc-DNA3.1-PTEN;SW480 pc-DNA3.1.
3.After stable transfection,western blot analysis was used to investigate the expression changes of PTEN.The expression levels were significantly enhanced in LoVo pc-DNA3.1-PTEN and SW480 pc-DNA3.1-PTEN cells compared LoVo pc-DNA3.1 and SW480 pc-DNA3.1cells.
4.To determine the effect of PTEN expression on proliferation of colorectal cancer cells,CCK-8 assay was performed at Oh,24h,48h,72h and 96h,respectively. Compared with the control cells,LoVo pc-DNA3.1-PTEN andSW480 pc-DNA3.1-PTEN cells grew much slowly at 24h,48h,72h and 96h(P＜0.05).The results indicate up-regulating PTEN expression could inhibit coloredtal cell proliferation.
5.To examine if expression of PTEN has an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that expression of PTEN cause a significant increase in the proportion of LoVo and SW480 cells at the G0/G1 phase(P＜0.05).The observed increase in G0/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.05).The findings suggest that expression of PTEN could obviously induce cell cycle arrest.
6.In order to evaluate the effect of up-regulating PTEN expression on the induction of apoptosis,colorectal cancer cells expressing pc-DNA3.1-PTEN and pc-DNA3.1 were treated with 5-Fu at final concentration of 0.01μmol/ml(LoVo) or 0.03μmol/ml(SW480) for 48h,followed by assessing early apoptotic rate by means of flow cytometric analysis.Results showed that the proportion of positive cells for Annexin V was significantly higher in up-regulating PTEN expression LoVo and SW480 cells than in control cells(P＜0.05).The results indicate that expression of PTEN sensitized colorectal cancer cells to 5-Fu induced apoptosis.
Up-regulating PTEN expression activated FoxO transcription factors associated with regulating cell cycle-associated protein expression in colorectal cancer cells
To further explore the mechanism underlying the enhancement of cell cycle arrest and 5-FU-induced apoptosis by the expression of PTEN,we examined expression levels of Akt,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that up-regulating PTEN expression led to substantial reduction in the levels of Akt and phospho-Akt(P＜0.05).Consistent with this reduction in the phospho-Akt level, Western blot analysis showed significantly decreased expression of phospho-FoxO1 (FKHR),phospho-FoxO3a(FKHRL1) and phospho-FoxO4(AFX) in cytoplasm of PTEN expresion cells(P＜0.05),the prominent accumulation of FoxO1(FKHR), FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed (P＜0.05).As potential downstream targets of FoxO transcription factors,the expression levels of cell cycle-associated protein were also determined.Results showed that the expression levels of cyclin D1,cdk4 and cdk6 were significantly decreased in up-regulating PTEN expression cells(P＜0.05),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.05).
Conclusions
1.Immunohistochemical results show that the expression levels of PTEN gradually decreases from normal colorectal mucosa,colorectal proliferational plopy,adenoma to primary colorectal adenocarcinoma,indicating that PTEN plays an important role in the progression of colorectal cancer.
2.Up-regulating PTEN expression could inhibit LoVo and SW480 cell proliferation.
3.Up-regulating PTEN expression could induce G1 phase cell cycle arrest and reduction of S phase cells.
4.Up-regulating PTEN expression could sensitize LoVo and SW480 cells to 5-Fu induced apoptosis.
5.Up-regulating PTEN expression could inhibit the activity of Akt and activate FoxO transcription factors,which,in turn,activated transcription of target genes such as those involved in cell cycle regulation and apoptosis.Thus,the colorectal cancer cell cycle arrest and enhanced sensibility to 5-Fu induced apoptosis result from expression of PTEN are closely related with activation of FoxO transcription factors.
引文
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[13] Ward SG, Finan P. Isoform-specific phosphoinositide 3-kinase inhibitors as therapeutic agents [J]. Curr Opin Plzarrnacol, 2003, 3 (4): 426-434.
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[15]Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors [J]. Genes Dev, 2000, 14 (2): 142-146.
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[17] Zheng WH, Kar S, Quirion R. Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells [J]. J Biol Chem, 2000, 275 (50): 39152-39158.
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[21] Liang J, Slingerland JM. Multiple roles of the PI3K/PKB(Akt) pathway in cell cycle progession [J]. Cell Cycle, 2003, 2 (4): 339-345.
[22] Viglietto G, Motti ML, Bruru P, et al. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kipl) by PKB/Akt-mediated phosphorylation in breast cancer [J]. Nat Med, 2002, 8 (4): 1136-1144.
[23] Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy [J]. Leukemia, 2003, 17 (3): 590-603.
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[25] Philp AJ, Campbell IG, Leet C, et al. The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors [J]. Cancer Res, 2001, 61 (20): 7426-7429.
[26] Lee SH, Kim HS, Park WS, et al. Non-small cell lung cancers frequently express phosphorylated Akt; an immunohistochemical study [J]. APMIS, 2002, 110 (20): 587-592.
[27] Itoh N, Semba S, Ito M, et al. Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma [J]. Cancer, 2002, 94 (12): 3127-3134.
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[31] Gao N, Flynn DC, Zhang Z, et al. Gl cell cycle progression and the expression of Gl cyclins are regulated by PI3K/AKT/mTOR/p70S6Kl signaling in human ovarian cancer cells [J]. Am J Physiol Cell Physiol, 2004, 287 (1): C281-C291.
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[33] Levine DA, Bogomolniy F, Yee CJ, et al. Frequent mutation of the PIK3CA gene in ovarian and breast cancers [J]. Clin Cancer Res, 2005, 11 (8): 2875-2878.
[34] Hartmann C, Bartels G, Gehlhaar C, et al. PIK3CA mutations in glioblastoma cnultiforzne [J]. Acta Neuropathol, 2005, 109 (8): 639-642.
[35] Wu G, Mambo E, Guo Z, et al. Uncoxrunon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors [J]. J Clin Endocrinol Metab, 2005, 90 (8):4688-4693.
[36] Ikenoue T, Kanai F, Hikiba Y, et al. Functional analysis of PIK3CA gene mutations in human colorectal cancer [J]. Cancer Res, 2005, 65 (8): 4562-4567.
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