Caveolin-1促进乳癌Hs578T耐药细胞株侵袭和转移

通过基因转染技术培育caveolin-1过表达乳癌HS578T耐药细胞株Hs578T/Dox cav-1和空载体细胞株Hs578T/Dox vector,探讨caveolin-1蛋白对耐药肿瘤细胞体内和体外侵袭转移能力的影响.由于caveolin-1表达的增加,Hs578T/Dox cav-1细胞形态变为长梭形,伪足更长而伸展,其粘附能力高于空载体Hs578T/Dox vector细胞株((0.897±0.163)vs(0.633±0.053),P<0.01).侵袭小室试验发现,Hs578T/Dox cav-1侵袭转移能力增强,培养6h迁徙进入微孔膜的细胞数比Hs578T/Dox vector细胞株明显增多((107.8±10.9)vs(80.8±8.07),P<0.01),侵袭破坏matrigel基质蛋白胶进入膜内的细胞数也明显增多((68.8±9.88)vs(25.6±5.41),P<0.01).悬浮培养的Hs578T/Dox cav-1细胞比Hs578T/Dox vector细胞更容易聚集,形成相对较致密的细胞团块,24h检测流式细胞凋亡指数下降((8.79±1.54)%vs(16.42±1.42)%,P<0.01).这表明其具有更强的抗失巢凋亡和继续生存的能力,为循环转移的肿瘤细胞定植前取了更多时间.Hs578T/Dox vector细胞在裸鼠皮下种植成瘤试验中不能成瘤,而Hs578T/Dox cav-1细胞接种15只裸鼠,全部形成肿瘤,平均直径(0.8±0.45)cm.发现一只成瘤裸鼠双肺可见瘤样肿块转移.HE病理组织染色见肿瘤细胞弥漫性分布,细胞核异型性明显.上述结果表明,caveolin-1对Hs578T耐药肿瘤细胞侵袭、转移和成瘤性具有明显的促进作用.     

Rab11a通过PI3K/AKT和Ras/MEK/ERK信号通路调节胰腺癌细胞凋亡和侵袭

目的:探究Rab11a在胰腺癌中的表达模式及其对肿瘤生长和转移的影响。方法:通过免疫组织化学法、RT-PCR和Western blot检测60例胰腺癌患者的癌组织和癌旁组织中Rab11a的表达。通过对人胰腺癌细胞系PANC1转染靶向Rab11a的小干扰RNA或过表达Rab11a的pcDNA3.1质粒考察Rab11a对细胞增殖、凋亡、迁移和侵袭的影响。通过Western blot检测PANC1细胞中PI3K、AKT、Ras、MEK、ERK1/2和GSK3β的磷酸化水平。结果:胰腺癌组织中Rab11a的表达水平均高于癌旁组织(P<0.05)。Rab11a的表达水平与TNM分期和淋巴结转移有关(P<0.05)。CCK-8测试和细胞集落形成实验显示,下调Rab11a抑制了PANC1细胞的增殖(P<0.05)。流式细胞术显示,下调Rab11a促进了PANC1细胞的凋亡(P<0.05)。细胞划痕实验显示,下调Rab11a抑制了PANC1细胞的迁移能力(P<0.05)。Matrigel Transwell实验显示,下调Rab11a抑制了PANC1细胞的侵袭能力(P<0.05)。然而,上调Rab11a则促进了PANC1细胞的增殖、迁移和侵袭,并抑制了细胞凋亡(P<0.05)。蛋白质印迹分析显示,下调Rab11a抑制了PANC1细胞中PI3K/AKT和Ras/MEK/ERK信号通路的活化(P<0.05)。此外,应用PI3K/AKT和Ras/MEK/ERK信号通路的选择性抑制剂处理PANC1细胞可阻断Rab11a对细胞增殖的促进作用(P<0.05)。结论:Rab11a的高表达是胰腺癌预后恶化的潜在生物标志物。靶向抑制Rab11a可通过抑制PI3K/AKT和Ras/MEK/ERK信号通路来降低胰腺癌的生长和转移能力。     

RASSF1A对食管癌细胞增殖影响的研究

目的:观察外源性RASSF1A基因对食管癌细胞的增殖作用并探讨机制.方法:将包含RASSF1A基因的质粒转染食管癌细胞EC9706,建立稳定转染细胞克隆,Western blot检测RASSF1A基因表达,通过细胞生长曲线检测细胞生长活性和增殖能力、流式细胞仪检测对细胞周期的影响、裸鼠移植瘤实验检测转染细胞的体内成瘤特性.结果:稳定表达RASSF1A基因的EC9706细胞中RASSF1A蛋白表达增加;细胞生长速度明显减慢;细胞周期中G1/G0期比例明显增加,S期比例减少;EC9706细胞的裸鼠致瘤能力被抑制.结论:RASSF1A基因能显著抑制食管癌细胞在体内外的生长,其机制可能与该基因诱导凋亡、抑制增殖作用有关.     

多胺类似物BENS和TBP对胰腺癌PANC-1细胞增殖、凋亡和迁移能力的影响

为探讨多胺类似物BENS和TBP对胰腺癌PANC-1细胞生长的影响、促进细胞凋亡的机制以及迁移能力的影响,运用MTT法检测药物对细胞生长的影响;亚凋亡峰流式细胞分析法及TUNEL法检测细胞凋亡;Western blotting法测定Bax蛋白、细胞色素C蛋白的表达变化;Ranswell技术检测细胞迁移能力的改变。分析发现,两种多胺类似物均可显著抑制PANC1癌细胞增殖,抑制作用随药物浓度加大而增加,其细胞毒性大小依次为BENSTBP。细胞凋亡分析发现,BENS和TBP均可诱导PANC1发生细胞程序性凋亡,导致亚凋亡峰出现和明显的DNA断裂现象。胞浆中Bax蛋白和细胞色素C含量显著增加,同时显著性抑制PANC1细胞的迁移能力。表明BENS和TBP能够抑制人胰腺癌PANC1细胞增殖;阻止PANC1细胞迁移的能力;同时诱导细胞发生凋亡,其作用机制可能与改变肿瘤细胞正常的细胞周期,活化线粒体参与的细胞凋亡途径相关。     

TGF-β信号通路抑制剂LY364947对急性髓系白血病细胞增殖、凋亡和侵袭的影响

该文旨在探讨抑制TGF-β信号通路对人急性髓系白血病(acute myeloid leukemia,AML)细胞体外增殖、凋亡和侵袭能力的影响。使用不同浓度TGF-β信号通路抑制剂LY364947处理AML细胞系(KG1a、OCI-AML3)后,采用CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期分布及凋亡情况; Western blot检测细胞周期调控因子Cyclin D1/p21、凋亡相关蛋白Bcl-2/Bax以及上皮细胞间质转化相关蛋白E-cadherin、N-cadherin和vimentin的表达; Transwell实验测定AML细胞迁移及侵袭能力的变化。结果显示:LY364947作用后,白血病细胞生长明显受抑制;细胞周期阻滞在G1期,伴有Cyclin D1表达下调和p21表达上调;细胞凋亡率增加,同时细胞抗凋亡蛋白Bcl-2的表达水平下降,促凋亡蛋白Bax表达增高;细胞体外迁移和侵袭能力减弱。此外, E-cadherin表达增高, N-cadherin和vimentin表达下降。该研究结果提示,抑制TGF-β信号通路能够抑制白血病细胞的体外增殖,诱导细胞凋亡,降低细胞迁移及侵袭能力。     

弱碱性消癌液抑制乳腺癌细胞生长、转移并介导其消亡

为了研究新的肿瘤治疗方法,设计了1种弱碱性消癌液（weak alkaline cancer-eliminating liquid,WACEL）,测试WACEL是否抑制癌细胞的生长转移及消亡.在体外,检测WACEL对3种细胞株,即子宫颈鳞癌细胞SiHa、非小细胞肺鳞癌细胞H1299、人乳腺癌细胞MDA-MB-231的生长增殖、细胞周期、细胞凋亡、侵袭转移的影响. 在体内,建立人乳腺癌裸鼠颈背皮下移植瘤模型,观察WACEL对裸鼠皮下肿瘤生长转移的作用. 研究结果发现,WACEL明显抑制3种细胞系的生长增殖,G2/M期细胞增多,出现G2/M期阻滞,阻止细胞周期的进程. 细胞形态呈圆状,细胞核浓缩,caspase-3活性检测增加,线粒体的膜电位降低,细胞凋亡;活细胞数目减少,细胞膜破裂,发生消亡现象,癌细胞迁移明显减少（P﹤0.001）.目标基因SCCA1、cyclinB1、MMP-2以及MMP-9 的mRNA表达水平下调,caspase-3的mRNA表达水平上调;SCCA1、cyclinB1和MMP-2蛋白表达下调,caspase-3蛋白表达上调.体内动物实验发现,处理组的肿瘤生长较对照组明显缓慢,肺组织HE染色未见明显癌细胞转移,而对照组可见癌细胞转移. WACEL能抑制乳腺癌细胞的生长、转移并介导其消亡.本研究系统分析WACEL与癌细胞本身及其pH微环境之间的相互作用机制,发现其改变癌细胞所处的微环境的重要性.     

IscU2对非小细胞肺癌增殖、迁移、侵袭能力的影响及其作用机制的研究

该文通过shRNA干扰技术敲低IscU2干扰细胞IscU2的表达,研究了干扰IscU2对非小细胞肺癌(NSCLC)细胞NCI-H520增殖、迁移及侵袭能力的影响。构建了稳定低表达IscU2的非小细胞肺癌细胞系NCI-H520;采用CCK-8和平板克隆实验检测细胞的增殖能力;流式细胞仪检测细胞周期、凋亡、ROS、线粒体膜电位变化情况;Transwell实验检测细胞迁移及侵袭能力;Western blot检测相关蛋白的表达。结果表明,干扰IscU2后,非小细胞肺癌细胞的增殖及克隆形成能力降低;细胞周期停滞在G1/G0期,同时伴随有p-AKT和Cyclin D1蛋白含量的下降;细胞晚期凋亡率明显增加,凋亡蛋白Cleaved-caspase3和Cleaved-PARP表达上调;细胞迁移和侵袭能力降低,上皮标志物E-Cadherin表达上调,间质标志物N-Cadherin和Snail表达下调;细胞ROS积累和线粒体膜电位下降。该研究结果表明,干扰IscU2显著抑制非小细胞肺癌的增殖、迁移、侵袭能力和上皮–间质转化,这为非小细胞肺癌的诊断和治疗提供了新的潜在靶点和视角。     

下调窖蛋白-1表达抑制小鼠肝癌H22细胞侵袭能力

窖蛋白-1在不同肿瘤中发挥作用不同. 本研究以小鼠肝癌细胞H22为研究对象 ,观察下调窖蛋白-1表达对H22细胞侵袭能力的影响,并探讨其可能的分子机制. 利用RT－PCR和Western印迹法检测了窖蛋白-1在H22及小鼠正常肝细胞IAR20中的 表达.结果显示,窖蛋白 1在H22中的表达高于其在IAR20中的表达,提示窖蛋白 -1高表达可能与H22细胞恶性表型有关. RNA干扰和凝集素印记实验结果显示,窖 蛋白-1－siRNA能够有效抑制窖蛋白－1mRNA和蛋白表达,并抑制细胞表面N－聚糖 β1,6GlcNAc分支形成. Transwell细胞迁移和侵袭实验结果显示,与未转染组和 siRNA 对照组比较,转染窖蛋白-1 siRNA的H22细胞迁移和侵袭数目明显减少. 本研究证明,下调窖蛋白－1表达可抑制H22细胞表面N 聚糖β1,6GlcNAc分支形 成,从而抑制细胞迁移和侵袭能力.     

姜黄素对A549肺癌细胞增殖、侵袭和迁移的抑制作用及与Keap1/Nrf2信号通路的关系

目的探究姜黄素对肺癌A549细胞增殖、侵袭和迁移的抑制作用及其机制。方法通过MTT法检测姜黄素对A549细胞增殖能力的影响;通过流式细胞术测定姜黄素对A549细胞凋亡的调节作用;通过Transwell试验,观察姜黄素对肺癌A549细胞的侵袭和迁移能力的影响;采用Western blot实验和RT-PCR实验,观察姜黄素对其Keap1/Nrf2信号通路的调节作用。结果增殖实验、凋亡实验和Transwell实验显示,姜黄素能够显著性抑制癌细胞的增殖、侵袭和迁移,并能够促进其凋亡。Western blot检测显示姜黄素能够显著抑制Keap1和Nrf2表达;RT-PCR实验结果显示姜黄素能够显著抑制Keap1mRNA和Nrf2 mRNA表达。结论姜黄素可能通过抑制Keap1/Nrf2信号通路蛋白的表达而抑制肺癌A549细胞的增殖、迁移和侵袭,并促进其凋亡。     

人β防御素-2通过TGF-β/Smad信号通路对胃癌SGC7901细胞增殖、迁移和侵袭的影响

该文探讨了人β防御素-2(hBD2)对胃癌SGC7901细胞的增殖、迁移和侵袭的影响。将真核表达载体pCMV-hBD2转染于人胃癌SGC7901细胞。采用qPCR和免疫印迹法(Western blot)检测转染效率。Western blot检测TGF-β1、p-Smad2/3、Smad2/3、MMP9的蛋白表达水平。Transwell法检测SGC7901细胞迁移和侵袭能力。EdU法和流式细胞术分别检测增殖能力与细胞周期。结果显示,转染真核表达载体pCMV-hBD2的SGC7901细胞, hBD2的表达水平明显高于SGC7901和转染pCMV-Blank的SGC7901细胞。SGC7901-hBD2细胞中TGF-β1、p-Smad2/3和MMP9的蛋白表达水平均低于SGC7901和SGC7901-Blank细胞,而Smad2/3表达水平不变。同时,其迁移侵袭和增殖能力均受到抑制,细胞周期G0/G1期阻滞。该实验结果表明, hBD2可能通过下调TGF-β/Smad信号通路调控SGC7901细胞的迁移侵袭以及增殖能力。     

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.     

Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.     

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.     

Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.     

Caveolin一1真核表达载体的构建及其在Hepal-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用。旨在分析caveolin-l在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepal-6细胞。利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hca-F和Hepal-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达载体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepal-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株。结果显示,caveolin-l在Hepal-6细胞中表达呈阴性,在H22和Hca-F中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2／Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepal-6稳定细胞株Cl和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础。     

Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.     

Caveolin-1 is associated with VCAM-1 dependent adhesion of gastric cancer cells to endothelial cells.

BACKGROUND/AIMS: Cell adhesion molecules play a critical role in the invasion and metastasis of a variety of human tumors. Abnormal expression of VCAM-1 has been demonstrated to correlate with the malignant progression of gastric tumors, but the molecular mechanism underlying the VCAM-1-dependent metastasis has been rarely investigated. To explore the role for tumor cell-expressing adhesion molecules in the carcinoma-endothelium adhesion, we analyzed expression status of adhesion molecules in gastric cancer cells and its association with tumor cell capability of endothelial adhesion. METHODS: Endothelial adhesion ability of gastric tumor cells was tested using calcein AM staining assay. Expression of cell surface proteins was determined by Western blot, flow cytometry, and immunofluorescence assays. RNAi-mediated knockdown of gene expression and neutralization with specific antibodies were utilized for functional analysis. RESULTS: One of three cell lines tested was identified to be adhesive to endothelial cells and express VCAM-1. Adherence ability of the cells was dramatically decreased by neutralization of surface VCAM-1. VCAM-1 was co-localized with Caveolin-1 and siRNA-mediated knockdown of Caveolin-1 expression significantly blocked the VCAM-1-dependent cell adhesion. CONCLUSIONS: Our data imply important roles for VCAM-1 and Caveolin- 1 in the regulation of metastatic potential of gastric tumor cells.