PI3K p85α表达缺失对大肠癌细胞的抗生存作用及其信号机制研究
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摘要
大肠癌是常见的恶性肿瘤之一,严重威胁人类的生命健康,其发病率在世界不同地区差异很大,以北美、大洋州最高,欧洲居中,亚非地区较低。我国南方,特别是东南沿海明显高于北方。近20多年来,世界上多数国家大肠癌发病率呈上升趋势,我国大肠癌发病率上升趋势亦十分明显,其发病率仅次于胃癌和食管癌。目前大肠癌的治疗方法以手术切除为主,辅以化疗和放疗,虽然短期内对患者有较好的效果,但其副作用大,对机体的伤害大,复发率高等缺陷不容忽视,特别是对已转移者很难达到良好的远期疗效。因此发展新的针对大肠癌发生发展机制方面的治疗策略将有望对大肠癌的治疗带来突破性进展。恶性肿瘤的生物学特性之一是细胞无限制的增殖和凋亡减少。肿瘤过快的增长率是导致大肠癌患者生存率降低的主要原因,因此,抑制大肠癌细胞的增殖促进其凋亡可达到降低死亡率的目的。近年来,随着分子生物学研究的迅速发展,对肿瘤相关生存信号通路的研究受到了越来越多的重视,正确认识参与大肠癌发生发展的各种信号通路的调节作用以及参与细胞增生的基因调控,有助于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、粘附、肿瘤血管生成以及细胞外基质的降解等相关。IA型PI3K是由p85调节亚基和p110催化亚基组成的异二聚体,p85调节亚基对于p110催化亚基的稳定、聚集以及IA型PI3K的激活是必须的,p85α是PI3K家族中表达量最多的调节亚基,由PIK3R1基因编码,自从有研究表明p85α突变可以引起PI3K/Akt通路的激活,从而证明PI3K p85α是一种癌基因以来,便引起许多研究者的兴趣。目前对PI3Kp85α的研究,主要集中在肿瘤中PI3K p85α所发生的突变及突变形式,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,则报道较少。
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引起的细胞恶性增殖密切相关,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PI3K p85α表达变化引起大肠癌细胞相关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,尚未见报道。
本研究在系统地检测PI3K p85α在大肠粘膜正常组织-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PI3K p85αshRNA干扰载体,转染大肠癌LoVo细胞和SW480细胞,观察下调大肠癌细胞中PI3Kp85α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PI3K p85α可能参与的大肠癌细胞的相关生存信号通路,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、PI3K p85α在大肠癌癌变过程中的表达及临床意义
用免疫组织化学法检测PI3K p85α在大肠粘膜正常组织、大肠良性息肉、大肠腺瘤以及原发性大肠癌组织中的表达情况,并分析PI3K p85α表达与大肠癌临床病理特征的关系。
二、PI3K p85α表达缺失对大肠癌细胞增殖、细胞周期与细胞凋亡的影响
1、PI3K p85αshRNA干扰载体的构建从GenBank中选定人类PI3K p85α亚单位编码基因PIK3R1 mRNA序列(NM 181504),依照shRNA的设计原则,设计4条shRNA片段,并设计一对非相关核苷酸序列作为阴性对照,利用BLAST在EST数据库查询,均未发现与另外任何基因同源,PGPU6/Neo shRNA表达载体的制备交由上海吉玛制药有限公司完成。
2、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24 h培养基中加入1000μg/mlG418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。
3、PI3K p85α干扰载体转染后效应检测Western-Blot检测稳定转染后LoVo细胞和SW480细胞PI3K p85α蛋白表达水平的改变,从4个干扰载体中选择干扰效率最高的一组细胞株进行后续实验。
4、细胞增殖实验稳定细胞株建立后,各组细胞连续培养0 h-96 h,CCK-8法检测PI3K p85α表达缺失对大肠癌细胞LoVo和SW480增殖的影响。
5细胞周期实验流式细胞术检测PI3K p85α表达缺失对大肠癌细胞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PI3K p85α表达缺失对5-FU诱导的大肠癌细胞凋亡的影响。
三、PI3K p85α表达缺失参与大肠癌细胞相关生存信号通路的调节
Western-Blot检测PI3K p85α表达缺失后总蛋白中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/Kip1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。免疫组化结果采用秩和检验(Kruskal-Wallis test),其中PI3K p85α表达水平与大肠组织类型及大肠癌Dukes分期之间的相关性采用等级相关系数(Spearman相关系数)检验。其它数据两组间比较采用t检验,多组间比较采用单因素方差分析(one-way ANOVA),结果以均数土标准差((?)±s)表示,以P＜0.05为差异有统计学意义。
结果
一、PI3K p85α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PI3K p85α表达主要定位于大肠病变组织上皮细胞的细胞浆,胞核中不表达,在间质炎症细胞中也有少量表达。PI3K p85α蛋白在正常大肠粘膜和大肠良性息肉中呈阴性或低表达,在大肠腺瘤组织中PI3K p85α蛋白表达相对于正常大肠粘膜和息肉开始升高,而在大肠癌组织中显著升高。PI3Kp85α蛋白在大肠癌癌变过程中阳性表达的增加有显著性意义(P＜0.001)。大肠癌高、中、低分化各组的PI3K p85α蛋白表达阳性趋势的差异无显著性意义(P＞0.050),但是在不同Dukes分期的大肠癌中PI3K p85α蛋白表达阳性趋势的增加有显著性意义(P＜0.050),其中Dukes B和Dukes C期的表达水平高于DukesA期,而伴有远处转移的Dukes D期大肠癌PI3K p85α蛋白表达水平最高。
二、PI3K p85α表达缺失对大肠癌细胞增殖、细胞周期及细胞凋亡的影响
1、PI3K p85αshRNA真核表达载体经酶切和测序证实插入序列与设计序列完全一致。
2、4个干扰载体分别转染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 shRNA/89;LoVo shRNA/324;LoVoshRNA/1073;LoVo shRNA/1123;SW480 shRNA/89;SW480 shRNA/324;SW480shRNA/1073;SW480 shRNA/1123,阴性对照载体分别转染LoVo细胞和SW480细胞后得到的稳定细胞克隆株分别命名为LoVo shRNA/N和SW480 shRNA/N。
3、干扰载体稳定转染LoVo细胞和SW480细胞后,经Western-Blot检测转染后PI3K p85α蛋白表达变化情况。与对照组相比,LoVo shRNA/324和SW480shRNA/324均能够显著抑制PI3K p85α蛋白的表达,因此选取该干扰载体转染的细胞株进行后续实验。
4、应用CCK-8法连续检测各组细胞0 h-96 h的增值情况,结果显示,与阴性对照组细胞相比,24 h、48 h、72 h、96 h时转染干扰载体的LoVo细胞增殖速度的减慢有显著性意义(P＜0.005);与阴性对照组细胞相比,48 h、72 h、96 h时转染干扰载体的SW480细胞增殖速度的减慢有显著性意义(P＜0.050),说明下调PI3K p85α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,转染干扰载体的LoVo细胞和SW480细胞出现G1期细胞阻滞(P＜0.050),同时伴有S期细胞数量减少(P＜0.050)。说明下调PI3K p85α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48 h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/ml5-FU和0.03μmol/ml 5-FU处理48 h后,阴性对照组细胞均未出现明显的细胞凋亡,而干扰组LoVo细胞和SW480细胞均出现Annexin V阳性凋亡细胞,增多有显著性意义(P＜0.050),说明下调PI3K p85α蛋白表达可以增加大肠癌细胞对化疗药物5-FU的敏感性。
三、PI3K p85α表达缺失参与大肠癌细胞相关生存信号通路的研究
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,转染干扰载体的LoVo细胞和SW480细胞的Akt和p-Akt的表达量降低有显著性意义(P＜0.050),说明下调PI3K p85α蛋白表达可以明显降低有活性的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细胞凋亡的调节,Western-Blot结果提示,与阴性对照组细胞相比,转染干扰载体的LoVo细胞和SW480细胞的胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)表达量的增加有显著性意义(P＜0.050),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))表达量的降低有显著性意义(P＜0.050),说明下调PI3K p85α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,转染干扰载体的LoVo细胞和SW480细胞的细胞周期相关蛋白cyclinD1、cdk4、cdk6表达降低有显著性意义(P＜0.050),而细胞周期素依赖性激酶抑制剂p27/Kip1表达水平的增加有显著性意义(P＜0.050)。这些结果提示下调PI3K p85α蛋白表达可以通过激活下游的FoxO转录因子家族成员,引起细胞周期相关蛋白的改变,从而参与调节了大肠癌细胞增殖减慢及细胞周期阻滞,并可能由于FoxO转录因子的激活而参与了对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PI3K p85α表达从大肠正常粘膜-大肠息肉-大肠腺瘤-大肠癌依次增加,提示PI3K p85α在大肠癌的发生发展中起重要作用。
2、抑制PI3K p85α蛋白表达可以抑制大肠癌LoVo细胞和SW480细胞的增殖。
3、抑制PI3K p85α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,抑制抑制大肠癌细胞的恶性增殖。
4、抑制PI3K p85α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,提示抑制PI3K p85α蛋白的表达与5-FU联合应用可能是治疗大肠癌的一种有效策略。
5、抑制PI3K p85α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转录因子家族活性的增加抑制了促细胞周期进展相关蛋白的表达。提示PI3K p85α蛋白表达缺失引起的大肠癌细胞增殖减慢、周期阻滞与下游FoxO转录因子家族的激活密切相关,并且FoxO转录因子家族的激活可能参与了对5-FU诱导的大肠癌细胞凋亡敏感性增加的调节。
Colorectal cancer(CRC) is one of the major malignancies which has high attack rate and death rate in the world.The morbidity is higher in North America and Oceania than in Europe and Asia.In China,the morbidity is higher in South than in North.In recent years,the morbidity of CRC has increased year by year. Conventional chemotherapy regiments for the treatment of colorectal cancer have limited efficacy and are associated with significant toxicity.Therefore,a great deal of effort and resources have been put into better understanding the mechanism of colorectal cancer development and developing novel targeted approaches for the management of this disease.Unlimited proliferation and resistance to apoptosis are major biological characters of CRC and are major causes lead to poor prognosis.With the rapid development of molecular biology recently,signaling pathways that involved in tumor progression have been payed more and more attention.Truly,an increasing knowledge about the genetic control of cellular proliferation and the modulation of the signaling pathways that are aberrant in colorectal cancer has the potential to provide an effective and better approach for its management.
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.Class-IA PI3K are heterodimers composed of a catalytic subunit(p110) and an adapter/regulatory subunit(p85).p85 regulatory subunit is essential for stabilization and collection of p110 catalytic subunit and for activation of Class-IA PI3K.p85αis the most abundant regulatory subunit of PI3K family and is encoded by PIK3R1 gene.It has been reporetd that mutation of PI3K p85αcould activate PI3K/Akt signaling pathway.Evidences have showed that PI3K p85αis a kind of oncogene.Most studies focused on the mutation and the mutation form of PI3K p85αin cancer.Reports about the amplification of PI3K p85αin 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 PI3K p85αexpression levels and the changes of FoxO transcription factors is not reported.
In this study,we investigate the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct shRNA vector to transfect Lovo and SW480 cells.After stable transfection,we observed the effect of PI3K p85αdepletion 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 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 PI3K p85αin progression of colorectal cancer
Immunohistochemical staining was used to detect the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal polyp,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PI3K p85αprotein and clinicopathological factors was also analyzed.
The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells
1.Construction of human PI3K p85αshRNA vectors The oligonucleotides encoding four 21-mer hairpin sequences were designed of PI3K p85αmRNA (Genebank:NM_181504).A scramble siRNA with the same nucleotide composition as the siRNA but which lacks significant sequence homology to the genome was also designed.The oligonucleotides are synthesized,annealed,and ligated into the linearized pGPU6/Neo siRNA Expression Vector by GnenPharma company.
2.Transfection and selection of stable transfected cell clones The shRNA vectors 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 antibiotic-resistant cells were passaged in medium containing 500μg/ml G418 as needed.
3.PI3K p85αexpression detecting The interference effect was evaluated by Western-blot analysis.Selected stable cell lines transfected with the shRNA that had the highest imerference effect for the following experiments.
4.Cell proliferation assays Cells were cultured for 96 h 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 shRNA 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 survival signaling pathway PI3K p85αinvolved in coloreetal cancer cells
Western-blot was used to analyzed expression changes of signaling pathway proteins after depletion of PI3K p85α.The cell proliferation and apoptosis signaling pathway proteins include Akt、p-Akt,cytoplasmic proteins p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,8er~(253)),p-FoxO4(AFX,Ser~(193)),nuclear proteins Foxo1 (FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX),and cell cycle associated proteins cyclinD1、cdk4、cdk6 and p27/Kip1.
Statistical Analysis
All experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.Spearman correlations test was used for analyzing the relationship between PI3K p85αexpression and colorectal tissue type and Dukes stage.For other results,one-way analysis of variance (ANOVA) and Student's t test were used in group comparison.Dates are expressed as the(?)+s.A value of P＜0.05 was considered statistically significant.
Results
The expression and significance of PI3K p85αin progression of colorectal cancer
Immunohistochemistry was performed to examine PI3K p85αexpression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PI3K p85αexpression was highest in surface epithelium of colorectal mucosa,and expression in the stroma was limited to inflammatory cells, with a predominantly cytoplasmic distribution.Representative shows that PI3K p85αexpression is negative in normal colorectal mucosa and polyp,begins to increase in adenoma and over-expressed in primary colorectal adenocarcinoma(P＜0.001). Furthermore,there was a significant difference in the positive rates of the PI3K p85αexpression duiring different Dukes' stage(P＜0.050).No obvious correlation was found between expression of PI3 K p85αand pathological diagnosis(P＞0.050).
The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells
1.Constructed human PI3K p85αshRNA expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.
2.After four shRNA vectors were transfected into Lovo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as Lovo shRNA/89;Lovo shRNA/324;Lovo shRNA/1073;Lovo shRNA/1123;SW480 shRNA/89;SW480 shRNA/324;SW480 shRNA/1073;SW480 shRNA/1123.Cells transfected with contro shRNA vectors were named as Lovo shRNA/N and SW480 shRNA/N.
3.After stable transfection,Western-blot analysis was used to investigate the expression changes of PI3K p85α.The expression levels were significantly inhibited in Lovo shRNA/324 and SW480 shRNA/324 cells compared to those of control cells. Thus,cells expressing shRNA/324 vectors were used for the following experiments.
4.To determine the effect of PI3K p85αknockdown on proliferation of colorectal cancer cells,CCK-8 assay was performed at 0h,24h,48h,72h and 96h, respectively.Compared with the control cells,Lovo shRNA/324 cells grew much slowly at 24h,48h,72h and 96h(P＜0.005),SW480 shRNA/324 cells grew much slowly at 48h,72h and 96h(P＜0.050).The results indicate that knockdown of PI3K p85αcould inhibit coloredtal cell proliferation.
5.To examine if RNA interference against PI3K p85αhas an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that depletion of PI3K p85αcause a significant increase in the proportion of Lovo and SW480 cells at the G1 phase(P＜0.050).The observed increase in/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.050).The findings suggest that depletion of PI3K p85αcould obviously induce cell cycle arrest.
6.In order to evaluate the effect of PI3K p85αknockdown on the induction of apoptosis,colorectal cancer cells expressing PI3K p85αshRNA and control shRNA 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 PI3K p85αknockdown Lovo and SW480 cells than in control cells(P＜0.050).The results indicate that depletion of PI3K p85αsensitized colorectal cancer cells to 5-FU induced apoptosis.
PI3K p85αdepletion 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 silencing of PI3K p85α,we examined expression levels of AKT,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that PI3K p85αdepletion led to substantial reduction in the levels of AKT and phospho-AKT(P＜0.050).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 PI3K p85α-knocked down cells(P＜0.050),the prominent accumulation of FoxO1 (FKHR),FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed(P＜0.050).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 PI3K p85αdepletion cells(P＜0.050),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.050).
Conclusions
1.Immunohistochemical results show that the expression levels of PI3K p85αgradually increases from normal colorectal mucosa,adenoma to primary colorectal adenocarcinoma,indicating that PI3K p85αplays an important role in the progression of colorectal cancer.
2.Depletion of PI3K p85αcould inhibit LoVo and SW480 cell proliferation.
3.Depletion of PI3K p85αcould induce G1 phase cell cycle arrest and reduction of S phase cells.
4.Depletion of PI3K p85αcould sensitize LoVo and SW480 cells to 5-FU induced apoptosis,indicating that combination of 5-FU and PI3K p85αdepletion may be an effective approach in treating colorectal cancer cells.
5.Depletion of PI3K p85αcould 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 inhibited cell proliferation and cell cycle arrest of colorectal cancer cells result from PI3K p85αdepletion are closely related with activation of FoxO transcription factors,and the activation of FoxO transcription factors maybe involved in the enhanced sensibility to 5-FU induced apoptosis.
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、粘附、肿瘤血管生成以及细胞外基质的降解等相关。IA型PI3K是由p85调节亚基和p110催化亚基组成的异二聚体,p85调节亚基对于p110催化亚基的稳定、聚集以及IA型PI3K的激活是必须的,p85α是PI3K家族中表达量最多的调节亚基,由PIK3R1基因编码,自从有研究表明p85α突变可以引起PI3K/Akt通路的激活,从而证明PI3K p85α是一种癌基因以来,便引起许多研究者的兴趣。目前对PI3Kp85α的研究,主要集中在肿瘤中PI3K p85α所发生的突变及突变形式,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,则报道较少。
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引起的细胞恶性增殖密切相关,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PI3K p85α表达变化引起大肠癌细胞相关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,尚未见报道。
本研究在系统地检测PI3K p85α在大肠粘膜正常组织-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PI3K p85αshRNA干扰载体,转染大肠癌LoVo细胞和SW480细胞,观察下调大肠癌细胞中PI3Kp85α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PI3K p85α可能参与的大肠癌细胞的相关生存信号通路,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、PI3K p85α在大肠癌癌变过程中的表达及临床意义
用免疫组织化学法检测PI3K p85α在大肠粘膜正常组织、大肠良性息肉、大肠腺瘤以及原发性大肠癌组织中的表达情况,并分析PI3K p85α表达与大肠癌临床病理特征的关系。
二、PI3K p85α表达缺失对大肠癌细胞增殖、细胞周期与细胞凋亡的影响
1、PI3K p85αshRNA干扰载体的构建从GenBank中选定人类PI3K p85α亚单位编码基因PIK3R1 mRNA序列(NM 181504),依照shRNA的设计原则,设计4条shRNA片段,并设计一对非相关核苷酸序列作为阴性对照,利用BLAST在EST数据库查询,均未发现与另外任何基因同源,PGPU6/Neo shRNA表达载体的制备交由上海吉玛制药有限公司完成。
2、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24 h培养基中加入1000μg/mlG418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。
3、PI3K p85α干扰载体转染后效应检测Western-Blot检测稳定转染后LoVo细胞和SW480细胞PI3K p85α蛋白表达水平的改变,从4个干扰载体中选择干扰效率最高的一组细胞株进行后续实验。
4、细胞增殖实验稳定细胞株建立后,各组细胞连续培养0 h-96 h,CCK-8法检测PI3K p85α表达缺失对大肠癌细胞LoVo和SW480增殖的影响。
5细胞周期实验流式细胞术检测PI3K p85α表达缺失对大肠癌细胞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PI3K p85α表达缺失对5-FU诱导的大肠癌细胞凋亡的影响。
三、PI3K p85α表达缺失参与大肠癌细胞相关生存信号通路的调节
Western-Blot检测PI3K p85α表达缺失后总蛋白中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/Kip1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。免疫组化结果采用秩和检验(Kruskal-Wallis test),其中PI3K p85α表达水平与大肠组织类型及大肠癌Dukes分期之间的相关性采用等级相关系数(Spearman相关系数)检验。其它数据两组间比较采用t检验,多组间比较采用单因素方差分析(one-way ANOVA),结果以均数土标准差((?)±s)表示,以P＜0.05为差异有统计学意义。
结果
一、PI3K p85α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PI3K p85α表达主要定位于大肠病变组织上皮细胞的细胞浆,胞核中不表达,在间质炎症细胞中也有少量表达。PI3K p85α蛋白在正常大肠粘膜和大肠良性息肉中呈阴性或低表达,在大肠腺瘤组织中PI3K p85α蛋白表达相对于正常大肠粘膜和息肉开始升高,而在大肠癌组织中显著升高。PI3Kp85α蛋白在大肠癌癌变过程中阳性表达的增加有显著性意义(P＜0.001)。大肠癌高、中、低分化各组的PI3K p85α蛋白表达阳性趋势的差异无显著性意义(P＞0.050),但是在不同Dukes分期的大肠癌中PI3K p85α蛋白表达阳性趋势的增加有显著性意义(P＜0.050),其中Dukes B和Dukes C期的表达水平高于DukesA期,而伴有远处转移的Dukes D期大肠癌PI3K p85α蛋白表达水平最高。
二、PI3K p85α表达缺失对大肠癌细胞增殖、细胞周期及细胞凋亡的影响
1、PI3K p85αshRNA真核表达载体经酶切和测序证实插入序列与设计序列完全一致。
2、4个干扰载体分别转染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 shRNA/89;LoVo shRNA/324;LoVoshRNA/1073;LoVo shRNA/1123;SW480 shRNA/89;SW480 shRNA/324;SW480shRNA/1073;SW480 shRNA/1123,阴性对照载体分别转染LoVo细胞和SW480细胞后得到的稳定细胞克隆株分别命名为LoVo shRNA/N和SW480 shRNA/N。
3、干扰载体稳定转染LoVo细胞和SW480细胞后,经Western-Blot检测转染后PI3K p85α蛋白表达变化情况。与对照组相比,LoVo shRNA/324和SW480shRNA/324均能够显著抑制PI3K p85α蛋白的表达,因此选取该干扰载体转染的细胞株进行后续实验。
4、应用CCK-8法连续检测各组细胞0 h-96 h的增值情况,结果显示,与阴性对照组细胞相比,24 h、48 h、72 h、96 h时转染干扰载体的LoVo细胞增殖速度的减慢有显著性意义(P＜0.005);与阴性对照组细胞相比,48 h、72 h、96 h时转染干扰载体的SW480细胞增殖速度的减慢有显著性意义(P＜0.050),说明下调PI3K p85α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,转染干扰载体的LoVo细胞和SW480细胞出现G1期细胞阻滞(P＜0.050),同时伴有S期细胞数量减少(P＜0.050)。说明下调PI3K p85α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48 h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/ml5-FU和0.03μmol/ml 5-FU处理48 h后,阴性对照组细胞均未出现明显的细胞凋亡,而干扰组LoVo细胞和SW480细胞均出现Annexin V阳性凋亡细胞,增多有显著性意义(P＜0.050),说明下调PI3K p85α蛋白表达可以增加大肠癌细胞对化疗药物5-FU的敏感性。
三、PI3K p85α表达缺失参与大肠癌细胞相关生存信号通路的研究
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,转染干扰载体的LoVo细胞和SW480细胞的Akt和p-Akt的表达量降低有显著性意义(P＜0.050),说明下调PI3K p85α蛋白表达可以明显降低有活性的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细胞凋亡的调节,Western-Blot结果提示,与阴性对照组细胞相比,转染干扰载体的LoVo细胞和SW480细胞的胞核蛋白FoxO1(FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX)表达量的增加有显著性意义(P＜0.050),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))表达量的降低有显著性意义(P＜0.050),说明下调PI3K p85α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,转染干扰载体的LoVo细胞和SW480细胞的细胞周期相关蛋白cyclinD1、cdk4、cdk6表达降低有显著性意义(P＜0.050),而细胞周期素依赖性激酶抑制剂p27/Kip1表达水平的增加有显著性意义(P＜0.050)。这些结果提示下调PI3K p85α蛋白表达可以通过激活下游的FoxO转录因子家族成员,引起细胞周期相关蛋白的改变,从而参与调节了大肠癌细胞增殖减慢及细胞周期阻滞,并可能由于FoxO转录因子的激活而参与了对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PI3K p85α表达从大肠正常粘膜-大肠息肉-大肠腺瘤-大肠癌依次增加,提示PI3K p85α在大肠癌的发生发展中起重要作用。
2、抑制PI3K p85α蛋白表达可以抑制大肠癌LoVo细胞和SW480细胞的增殖。
3、抑制PI3K p85α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,抑制抑制大肠癌细胞的恶性增殖。
4、抑制PI3K p85α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,提示抑制PI3K p85α蛋白的表达与5-FU联合应用可能是治疗大肠癌的一种有效策略。
5、抑制PI3K p85α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转录因子家族活性的增加抑制了促细胞周期进展相关蛋白的表达。提示PI3K p85α蛋白表达缺失引起的大肠癌细胞增殖减慢、周期阻滞与下游FoxO转录因子家族的激活密切相关,并且FoxO转录因子家族的激活可能参与了对5-FU诱导的大肠癌细胞凋亡敏感性增加的调节。
Colorectal cancer(CRC) is one of the major malignancies which has high attack rate and death rate in the world.The morbidity is higher in North America and Oceania than in Europe and Asia.In China,the morbidity is higher in South than in North.In recent years,the morbidity of CRC has increased year by year. Conventional chemotherapy regiments for the treatment of colorectal cancer have limited efficacy and are associated with significant toxicity.Therefore,a great deal of effort and resources have been put into better understanding the mechanism of colorectal cancer development and developing novel targeted approaches for the management of this disease.Unlimited proliferation and resistance to apoptosis are major biological characters of CRC and are major causes lead to poor prognosis.With the rapid development of molecular biology recently,signaling pathways that involved in tumor progression have been payed more and more attention.Truly,an increasing knowledge about the genetic control of cellular proliferation and the modulation of the signaling pathways that are aberrant in colorectal cancer has the potential to provide an effective and better approach for its management.
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.Class-IA PI3K are heterodimers composed of a catalytic subunit(p110) and an adapter/regulatory subunit(p85).p85 regulatory subunit is essential for stabilization and collection of p110 catalytic subunit and for activation of Class-IA PI3K.p85αis the most abundant regulatory subunit of PI3K family and is encoded by PIK3R1 gene.It has been reporetd that mutation of PI3K p85αcould activate PI3K/Akt signaling pathway.Evidences have showed that PI3K p85αis a kind of oncogene.Most studies focused on the mutation and the mutation form of PI3K p85αin cancer.Reports about the amplification of PI3K p85αin 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 PI3K p85αexpression levels and the changes of FoxO transcription factors is not reported.
In this study,we investigate the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal adenoma and primary colorectal carcinoma.Then we construct shRNA vector to transfect Lovo and SW480 cells.After stable transfection,we observed the effect of PI3K p85αdepletion 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 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 PI3K p85αin progression of colorectal cancer
Immunohistochemical staining was used to detect the expression and significance of PI3K p85αin the progression of colorectal cancer,including normal colorectal tissue,colorectal polyp,colorectal adenoma and primary colorectal carcinoma.The relationship between the expression of PI3K p85αprotein and clinicopathological factors was also analyzed.
The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells
1.Construction of human PI3K p85αshRNA vectors The oligonucleotides encoding four 21-mer hairpin sequences were designed of PI3K p85αmRNA (Genebank:NM_181504).A scramble siRNA with the same nucleotide composition as the siRNA but which lacks significant sequence homology to the genome was also designed.The oligonucleotides are synthesized,annealed,and ligated into the linearized pGPU6/Neo siRNA Expression Vector by GnenPharma company.
2.Transfection and selection of stable transfected cell clones The shRNA vectors 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 antibiotic-resistant cells were passaged in medium containing 500μg/ml G418 as needed.
3.PI3K p85αexpression detecting The interference effect was evaluated by Western-blot analysis.Selected stable cell lines transfected with the shRNA that had the highest imerference effect for the following experiments.
4.Cell proliferation assays Cells were cultured for 96 h 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 shRNA 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 survival signaling pathway PI3K p85αinvolved in coloreetal cancer cells
Western-blot was used to analyzed expression changes of signaling pathway proteins after depletion of PI3K p85α.The cell proliferation and apoptosis signaling pathway proteins include Akt、p-Akt,cytoplasmic proteins p-FoxO1(FKHR,Ser~(256))、p-FoxO3a(FKHRL1,8er~(253)),p-FoxO4(AFX,Ser~(193)),nuclear proteins Foxo1 (FKHR)、FoxO3a(FKHRL1)、FoxO4(AFX),and cell cycle associated proteins cyclinD1、cdk4、cdk6 and p27/Kip1.
Statistical Analysis
All experiments results were from at least three separate experiments.For immunohistochemistry results,Kruskal-Wallis test was used.Spearman correlations test was used for analyzing the relationship between PI3K p85αexpression and colorectal tissue type and Dukes stage.For other results,one-way analysis of variance (ANOVA) and Student's t test were used in group comparison.Dates are expressed as the(?)+s.A value of P＜0.05 was considered statistically significant.
Results
The expression and significance of PI3K p85αin progression of colorectal cancer
Immunohistochemistry was performed to examine PI3K p85αexpression levels in paraffin-embeded tissue from colorecta mucosa,benign polypi and adenomas to primary colorectal cancers.PI3K p85αexpression was highest in surface epithelium of colorectal mucosa,and expression in the stroma was limited to inflammatory cells, with a predominantly cytoplasmic distribution.Representative shows that PI3K p85αexpression is negative in normal colorectal mucosa and polyp,begins to increase in adenoma and over-expressed in primary colorectal adenocarcinoma(P＜0.001). Furthermore,there was a significant difference in the positive rates of the PI3K p85αexpression duiring different Dukes' stage(P＜0.050).No obvious correlation was found between expression of PI3 K p85αand pathological diagnosis(P＞0.050).
The effect of PI3K p85αdepletion on proliferation,cell cycle and apoptosis in colorectal cancer cells
1.Constructed human PI3K p85αshRNA expression vetors successfully,which were identified by restriction enzyme digestion analysis and DNA sequencing.
2.After four shRNA vectors were transfected into Lovo and SW480 cells,stable colnes were formed after G418 selection.Then the cells were name as Lovo shRNA/89;Lovo shRNA/324;Lovo shRNA/1073;Lovo shRNA/1123;SW480 shRNA/89;SW480 shRNA/324;SW480 shRNA/1073;SW480 shRNA/1123.Cells transfected with contro shRNA vectors were named as Lovo shRNA/N and SW480 shRNA/N.
3.After stable transfection,Western-blot analysis was used to investigate the expression changes of PI3K p85α.The expression levels were significantly inhibited in Lovo shRNA/324 and SW480 shRNA/324 cells compared to those of control cells. Thus,cells expressing shRNA/324 vectors were used for the following experiments.
4.To determine the effect of PI3K p85αknockdown on proliferation of colorectal cancer cells,CCK-8 assay was performed at 0h,24h,48h,72h and 96h, respectively.Compared with the control cells,Lovo shRNA/324 cells grew much slowly at 24h,48h,72h and 96h(P＜0.005),SW480 shRNA/324 cells grew much slowly at 48h,72h and 96h(P＜0.050).The results indicate that knockdown of PI3K p85αcould inhibit coloredtal cell proliferation.
5.To examine if RNA interference against PI3K p85αhas an impact on cell cycle of colorectal cancer cells,flow cytometry analyses were performed and the results showed that depletion of PI3K p85αcause a significant increase in the proportion of Lovo and SW480 cells at the G1 phase(P＜0.050).The observed increase in/G1 cell population in Lovo and SW480 cells was accompanied by a reduction of cells in the S phase(P＜0.050).The findings suggest that depletion of PI3K p85αcould obviously induce cell cycle arrest.
6.In order to evaluate the effect of PI3K p85αknockdown on the induction of apoptosis,colorectal cancer cells expressing PI3K p85αshRNA and control shRNA 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 PI3K p85αknockdown Lovo and SW480 cells than in control cells(P＜0.050).The results indicate that depletion of PI3K p85αsensitized colorectal cancer cells to 5-FU induced apoptosis.
PI3K p85αdepletion 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 silencing of PI3K p85α,we examined expression levels of AKT,phospho-Akt,FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm.The results showed that PI3K p85αdepletion led to substantial reduction in the levels of AKT and phospho-AKT(P＜0.050).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 PI3K p85α-knocked down cells(P＜0.050),the prominent accumulation of FoxO1 (FKHR),FoxO3a(FKHRL1) and FoxO4(AFX) in nucleus was simultaneously observed(P＜0.050).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 PI3K p85αdepletion cells(P＜0.050),while the expression level of cyclin kinase inhibitor p27/Kip1 was markedly induced(P＜0.050).
Conclusions
1.Immunohistochemical results show that the expression levels of PI3K p85αgradually increases from normal colorectal mucosa,adenoma to primary colorectal adenocarcinoma,indicating that PI3K p85αplays an important role in the progression of colorectal cancer.
2.Depletion of PI3K p85αcould inhibit LoVo and SW480 cell proliferation.
3.Depletion of PI3K p85αcould induce G1 phase cell cycle arrest and reduction of S phase cells.
4.Depletion of PI3K p85αcould sensitize LoVo and SW480 cells to 5-FU induced apoptosis,indicating that combination of 5-FU and PI3K p85αdepletion may be an effective approach in treating colorectal cancer cells.
5.Depletion of PI3K p85αcould 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 inhibited cell proliferation and cell cycle arrest of colorectal cancer cells result from PI3K p85αdepletion are closely related with activation of FoxO transcription factors,and the activation of FoxO transcription factors maybe involved in the enhanced sensibility to 5-FU induced apoptosis.
引文
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