抑癌基因PTEN对乳腺癌ZR-75-1细胞增殖和转移的抑制作用

背景与目的:研究表明,抑癌基因PTEN不仅能抑制肿瘤细胞的增殖,还能抑制其转移,但其机理还不甚明了.本文研究抑癌基因PTEN对人乳腺癌ZR-75-1细胞增殖和转移的作用.方法:以脂质体介导法分别将野生型PTEN质粒和磷酸酶缺陷的PTEN质粒转染人乳腺癌ZR-75-1细胞,用MTT法测定细胞增殖抑制率:转染后用嘌呤霉素筛选阳性克隆.用Western blot法检测细胞中PTEN蛋白的表达.通过细胞-基质粘附实验和人工重组基底膜侵袭实验,检测细胞粘附抑制率与侵袭抑制率.结果:野生型PTEN质粒转染的ZR-75-1细胞增殖明显被抑制,并伴有部分细胞凋亡;该细胞与未经转染的和磷酸酶缺陷的PTEN质粒转染的ZR-75-1细胞比较.细胞增殖抑制率差异均有统计学意义(42.7% vs.0%及2.7%,P＜0.01),细胞增殖抑制效应随细胞培养时间与质粒浓度的增加而增强.而磷酸酶缺陷的PTEN质粒转染的与未经质粒转染的ZR-75-1细胞比较,细胞增殖抑制率差异无统计学意义(2.7%vs.0%,P＞0.05).在两种PTEN质粒转染的ZR-75-1细胞中PTEN蛋白均明显表达,其中转染野生型PTEN质粒的细胞的粘附抑制率与侵袭抑制率分别达65.7%和70.4%,而转染磷酸酶缺陷的PTEN质粒的ZR-75-1细胞的粘附抑制率与侵袭抑制率分别只有8.8%和6.9%(P＜0.05).结论:具有双特异磷酸酶活性的野生型PTEN基因对ZR-75-1细胞的增殖和转移有一定的抑制作用.     

抑癌基因PTEN抑制乳腺癌ZR-75-1细胞转移作用机制的探讨

目的 探讨抑癌基因PTEN抑制乳腺癌细胞ZR-75-1转移的作用机制.方法 用脂质体介导法分别将野生型PTEN质粒(wt)、磷酸酶活性缺失的PTEN质粒(G129R)和只具蛋白磷酸酶活性的PTEN质粒(G129E)转染PTEN基因缺失的乳腺癌细胞ZR-75-1.转染细胞以嘌呤霉素筛选后,用PCR和Western-blot分别检测PTEN基因及其蛋白;通过黏附、侵袭实验,比较3种质粒转染细胞和未转染细胞之间的黏附、侵袭能力的差异;用Western-blot法检测各组转染细胞总的黏着斑激酶(FAK)和磷酸化黏着斑激酶(P397-FAK)的表达水平;以免疫组化法检测基质金属蛋白酶-2(MMP-2)和E-钙连素(E-Cd)的表达,并用RT-PCR检测各组转染细胞的p53mRNA水平.结果 3种质粒均成功转染ZR-75-1细胞,并证实3种转染细胞内均有PTEN基因存在及PTEN蛋白表达;wt、G129R、G129E等 3种质粒转染的细胞黏附抑制率分别为65.7%、8.8%和43.5%;侵袭抑制率分别为70.4%、6.9% 和63.5%.将wt或G129E转染细胞的黏附抑制率及侵袭抑制率与G129R转染细胞的比较,均有显著性差异(P＜0.05);但wt与 G129E转染细胞比较,均无显著性差异(P＞0.05 ).3种转染细胞总FAK水平虽无显著性差异(P＞0.05 ),但wt和G129E转染细胞其P397-FAK和 MMP-2水平都显著低于G129R转染细胞(P＜0.05).3种转染细胞间的E-Cd水平未见显著差异.RT-PCR分析显示,wt、G129E和G129R 3种转染细胞p53 mRNA水平无显著性差异,但均显著高于未转染细胞.结论 野生型PTEN基因所表达的蛋白具脂质和蛋白双特异磷酸酶活性,对乳腺癌细胞ZR-75-1的转移具有抑制作用,其机制与磷酸酶活性有关,其中蛋白磷酸酶活性可能起主要作用.     

三阴性乳腺癌中程序性死亡配体1的表达及其与PTEN基因的关系

目的探讨程序性死亡配体1(PD-L1)在三阴性乳腺癌(TNBC)中的表达情况及其与PTEN基因的关系。 方法通过癌症基因组图谱(TCGA)数据库查询PD-L1 mRNA在浸润性乳腺癌数据集(包括115例TNBC和702例非TNBC)中的表达情况。收集2012年1月至2016年12月解放军第180医院收治的182例浸润性乳腺癌术后石蜡包埋组织标本,包括62例TNBC和120例非TNBC,并用免疫组织化学方法检测PD-L1和PTEN的表达情况。用t检验比较TCGA数据库中2组的PD-L1 mRNA表达量,率的比较用χ²检验,Mann-Whitney U秩和检验比较石蜡标本中2组PD-L1表达强度,并用χ²检验和Spearman秩相关检验分析PD-L1与PTEN表达的相关性。 结果TCGA数据库分析显示,浸润性乳腺癌中3.8%(31/817)有PD-L1 mRNA上调,其中TNBC组的表达上调率为8.7%(10/115),高于非TNBC组的3.0%(21/702)(χ²＝7.314,P＝0.007),TNBC组的PD-L1 mRNA表达量为8.05±0.91,高于非TNBC组的7.38±0.73 (t＝7.510,P<0.001)。石蜡标本的免疫组织化学结果显示,TNBC组的PD-L1阳性表达率为14.5%(9/62),高于非TNBC组的5.0% (6/120)(χ²＝4.895,P＝0.027),而且PD-L1阳性表达强度也高于非TNBC组(Z＝－2.291,P＝0.022)。TNBC石蜡标本中PD-L1与PTEN蛋白表达呈负相关(χ²＝6.913,P＝0.009; r＝－0.382,P＝0.002)。 结论TNBC中PD-L1表达高于非TNBC,并与PTEN负相关,其可能成为TNBC患者的免疫治疗靶点。     

乳腺癌细胞转染野生型PTEN质粒后转移能力的变化

 目的 研究抑癌基因PTEN对乳腺癌细胞转移的影响。方法 脂质体介导法将外源野生型抑癌基因PTEN转染入该基因缺陷的ZR-75-1乳腺癌细胞中，用嘌呤霉素筛选阳性克隆,Western- blot法检测PTEN蛋白表达；重组人工基底膜上检测黏附与侵袭能力。结果 转染后ZR-75-1乳腺癌细胞PTEN蛋白有明显表达；转染后ZR-75-1乳腺癌细胞侵袭抑制率与黏附抑制率分别达70.4 %和60.0 %。结论 PTEN基因对乳腺癌细胞转移具有一定的抑制作用；PTEN基因的缺失与否在一定程度上可以评价乳腺癌患者发生转移的危险程度。     

重组人TNF-α诱导乳腺癌细胞ZR75-1凋亡及其机制的初步研究

背景与目的:肿瘤坏死因子α(TNF-α)是抗肿瘤活性较强的细胞因子,是重要的抗肿瘤生物治疗制剂之一,但由于其对人体有较严重的副作用,临床应用受到了限制。为了降低其毒副作用,同时保留其抗肿瘤能力,TNF-α的突变体——突变型471TNF-α应运而生。本研究对重组人突变型471rhTNF-α与野生型rhTNF-α蛋白的抗肿瘤能力进行了比较,并对突变型471rhTNF-α诱导ZR75-1细胞发生凋亡的作用机制进行初步研究。方法:以乳腺癌细胞系ZR75-1为靶细胞,以基因组DNA琼脂糖凝胶电泳及流式细胞技术分析100"g/L突变体471rhTNF-α与100"g/L野生型rhTNF-α作用ZR75-1细胞12h,肿瘤细胞发生凋亡的情况;利用以ELISA为基础的TransAMTMNF-κBp65试剂盒检测10μg/L、50μg/L和100μg/L突变体471rhTNF-α或野生型rhTNF-α作用ZR75-1细胞4h后核因子NF-κB的活化情况。结果:2%琼脂糖凝胶电泳显示,突变体471rhTNF-α处理组的ZR75-1细胞基因组DNA呈现明显的“ladder”状分布,野生型rhTNF-α处理组的“ladder”条带明显减弱;流式细胞仪分析显示,突变型471rhTNF-α诱导的凋亡峰面积为(44.6±3.6)%,野生型rhTNF-α诱导凋亡峰面积占(24.7±2.7)%,两组差异具有显著性(P<0.05);从细胞周期结果来看,突变型471rhTNF-α处理组G1期细胞占(66.6±4.2)%,比例明显高于对照组[(34.8±3.1)%]与野生型hTNF-α[(46.2±3.8)%],S期细胞占(33.2±2.9)%,比例明显低于对照组[(50.1±3.8)%]与野生型hTNF-α处理组[(45.7±2.9)%],G2期细胞比例为(0.2±0.1)%,明显低于对照组[(15.1±2.2)%]和野生型hTNF-α处理组[(8.1±3.1)%],而野生型hTNF-α处理组与对照组相比均无明显变化(P>0.05)。NF-κB活化检测结果显示,50"g/L野生型rhTNF-α处理ZR75-1细胞4h后NF-κB的活化量(A值为3.27±0.1)明显高于突变型471rhTNF-α处理组(A值为1.51±0.2)。结论:突变型471rhTNF-α比野生型rhTNF-α具有更强的诱导ZR75-1细胞凋亡的能力;ZR75-1细胞内NF-κB活化明显受限是突变型471rhTNF-α诱导凋亡能力增强的主要原因之一。     

乳腺癌转移抑制基因1对乳腺癌细胞运动能力的影响

目的：研究乳腺癌转移抑制基因1（breast cancer metastasis suppressor gene1，BRMS1）对人乳腺癌细胞运动能力的影响。方法：构建BRMS1真核表达载体，利用Lipofectin2000转染肺高转移潜能乳腺癌细胞MDA-MB-231-HM，荧光实时定量PCR检测目的基因表达，Transwell实验检测细胞运动能力。同时，设计3对针对BRMS1的特异小干扰RNA（smallinterferingRNA，siRNA），通过荧光实时定量PCR筛选出最有效的siRNA，干扰人乳腺癌细胞MDA-MB-231，观察干扰后细胞运动能力的变化。结果：与转染空载体的细胞相比，稳定转染BRMS1的MDA-MB-231-HM细胞穿过膜的数量减少51．9％。筛选出1对有效siRNA能明显下调BRMS1，用此siRNA干扰MDA-MB-231细胞后，其穿过膜的细胞数量增加17．9％。结论：BRMS1能抑制人乳腺癌细胞的运动，提示其可能通过抑制人乳腺癌细胞运动而抑制肿瘤远处转移。     

5-Aza-CdR诱导抑癌基因PTEN重新表达对乳腺癌细胞的影响及机制

目的观察5-氮杂-2’-脱氧胞苷(5-Aza-CdR)对人乳腺癌细胞系MCF-7增殖、凋亡及体外侵袭能力的影响并初步探讨其机制。方法体外培养MCF-7细胞,采用不同浓度的5-Aza-CdR(1×10-6、2×10-6、5×10-6和1×10-5mol/L)分别作用24h、48h和72h,分别采用MTT法检测细胞增殖抑制率、流式细胞仪检测细胞凋亡、Transwell法检测细胞侵袭能力、Western-blot检测PTEN和VEGF-C蛋白表达的变化。结果经不同浓度的5-Aza-CdR作用后,MCF-7细胞的增殖受到不同程度的抑制并发生凋亡,细胞侵袭能力也发生不同程度的降低,且其作用随浓度增加和时间延长而增强(P<0.05)。在5-Aza-CdR作用后,MCF-7细胞PTEN的表达逐渐增强,而VEGF-C的表达逐渐减弱。结论 5-Aza-CdR可抑制乳腺癌细胞增殖、诱导其发生凋亡、降低其体外侵袭能力,其可能是通过去甲基化作用使抑癌基因PTEN重新表达并下调VEGF-C的表达而发挥作用的。     

DKK1对乳腺癌细胞迁移侵袭能力影响的研究

  目的  探讨Dikkopf 1（DKK1）对乳腺癌细胞迁移侵袭能力的影响。  方法  采用免疫组织化学方法检测DKK1在乳腺癌组织中的表达,并通过免疫荧光对DKK1在细胞中的定位进行分析;DKK1过表达的真核表达载体转染乳腺癌细胞MDA231,应用Western blot检测转染后乳腺癌细胞MDA231中DKK1蛋白表达水平。划痕实验检测转染后MDA231细胞的迁移能力,Transwell实验检测MDA231细胞侵袭能力的改变。  结果  免疫组织化学检测到乳腺癌组织原发病灶及转移病灶DKK1表达明显高于对应的癌旁组织（P < 0.05）。DKK1表达的阳性率在有淋巴结转移组织中明显比无淋巴结转移组织中低。免疫荧光结果显示DKK1主要呈细颗粒状散在分布于细胞质中。Western blot检测表明DKK1过表达明显。过表达DKK1的MDA231细胞迁移和侵袭能力明显比阴性对照组和空白组低。  结论  DKK1的表达与乳腺癌细胞迁移和侵袭能力呈负相关。      

弥漫大B细胞淋巴瘤中PTEN蛋白表达的临床意义

 目的　研究PTEN蛋白在弥漫大B细胞淋巴瘤（DLBCL）中的表达情况,探讨PTEN蛋白表达与DLBCL预后的关系。方法　采用免疫组化方法检测40例原发结内DLBCL患者肿瘤组织中PTEN蛋白的表达情况。应用Kaplan-Merie生存分析、Log-Rank差异显著性检验和Logistic回归分析进行统计学分析。结果　PTEN蛋白阳性组16例,阴性组24例。PTEN蛋白阳性组和阴性组患者的2年总体生存率差异无统计学意义（57.7 %比62.5 %,P >0.05）,两组患者的生存率随时间的延长均逐渐减低,PTEN阳性组的生存率低于阴性组,但两组的生存曲线差异无统计学意义（P >0.05）。PTEN蛋白表达与DLBCL分子分型无相关性（P >0.05）。PTEN蛋白表达与患者年龄、体力状态、病情分期、乳酸脱氢酶（LDH）及结外受侵无相关性（P >0.05）,回归分析表明,DLBCL中PTEN蛋白表达不是疾病的保护因素,国际预后指数（IPI）是疾病的危险因素（OR＞1）。此外,PTEN蛋白细胞核表达占15 %（6／40）,核表达的例数在PTEN蛋白表达阳性组和阴性组间差异无统计学意义（1∶1）。结论　DLBCL中PTEN蛋白表达高低与预后无相关性,IPI仍是判断其预后的指标。     

Combined effects of 1,25-dihydroxyvitamin D3 and tamoxifen on the growth of MCF-7 and ZR-75-1 human breast cancer cells

Summary In the present study we assessed the effect of combined treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) and tamoxifen (TAM) on the growth of estrogen-responsive (MCF-7) and estrogen-dependent (ZR-75-1) human breast cancer cells. Both basal and 17-estradiol (17-E₂)-stimulated growth were studied. 1,25-(OH)₂D₃ (10^–10 – 10^–7 M) time- and dose-dependently inhibited basal growth of MCF-7 cells, with growth arrest at 10^–7 M. Also, 17-E₂-stimulated growth of MCF-7 and ZR-75-1 cells was inhibited by 1,25-(OH)₂D₃ in a time- and dose-dependent manner. TAM inhibited 17-E₂-stimulated growth of both cell lines and at high concentration (10^–6 M) it also inhibited basal growth of MCF-7 cells. 10^–6 M TAM together with 1,25-(OH)₂D₃ resulted in a further inhibition of basal (MCF-7 cells) as well as 17-E₂-stimulated proliferation (MCF-7 and ZR-75-1 cells) compared to the inhibition by these agents alone. TAM in combination with 10^–7 M 1,25-(OH)₂D₃ resulted in growth arrest of 17-E₂-stimulated growth of MCF-7 cells. The inhibition of basal and 17-E₂-stimulated growth of MCF-7 cells was additive at early time points (4 days), but less than additive at later time points (8–10 days). It was demonstrated that with co-treatment of MCF-7 cells an equipotent inhibition of basal growth could be reached with lower concentrations of 1,25-(OH)₂D₃, compared to treatment with 1,25-(OH)₂D₃ alone. Studies with low concentrations (< 10^–7 M) of TAM revealed a partial estrogenic effect, i.e. stimulation of MCF-7 proliferation in the absence of 17-E₂. This effect, which may resemble TAM-induced tumor flare, was completely prevented by co-treatment with a low concentration of 1,25-(OH)₂D₃ (10^–9 M). Together, these results demonstrate the potent inhibition of breast cancer cell proliferation by 1,25-(OH)₂D₃ combined with TAM and indicate a potential benefit of combining these agents for the treatment of breast cancer.     

Tamoxifen induced apoptosis in ZR-75 breast cancer xenografts antedates tumour regression

The ZR-75-1 ER positive breast cancer cell line,xenografted in female nude mice, has been usedto determine the effect of tamoxifen on cellproliferation (as measured by mitosis) and cell death(as evidenced by apoptosis and necrosis). After 2days treatment, there was a significant rise inapoptosis (p < 0.05), whereas a fall inmitosis was not apparent until 7 days (p< 0.05). Furthermore there was an increase inthe apoptotic : mitotic ratio on day 7(p < 0.05). These changes antedated tumour regression,which did reach not significance until day 14.Tamoxifen did not increase necrosis (which significantly decreasedin treated tumours once they had regressed (p< 0.01)). In contrast tamoxifen treatment of xenograftedMDA-MB-231 ER-negative breast cancer cells produced no significanteffects on growth, apoptosis, or mitosis. This studypresents clear evidence for tamoxifen inducing apoptosis inZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changesin apoptosis and mitosis antedate tumour regression, theirassessment may provide the potential by which topredict tumour response to tamoxifen therapy.     

Characterisation of a tamoxifen-resistant variant of the ZR-75-1 human breast cancer cell line (ZR-75-9a1) and ability of the resistant phenotype

A 6-month exposure of ZR-75-1 human breast cancer cells to tamoxifen (1 microM rising to 2 microM). resulted in a fall in oestrogen receptor (ER) levels from 225 fmol mg protein-1 to 56 fmol mg protein-1 while progesterone receptor (PGR) concentration fell from 63 fmol mg protein-1 to undetectable levels. Sensitivity to the anti-proliferative effects of tamoxifen was unchanged. A further 6 months' exposure to 4 microM tamoxifen resulted in loss of detectable ER and PGR and development of resistance to tamoxifen. Resistant cells, designated ZR-75-9a1, displayed morphological changes consistent with the acquisition of a less well differentiated phenotype. Flow cytometric studies demonstrated that the cell cycle distribution pattern of the resistant variant growing in the presence of 8 microM tamoxifen was identical to that of the untreated parent line, which showed marked accumulation of cells in G0/G1 when exposed to 8 microM tamoxifen. The resistant phenotype was not stable if cells were transferred to complete drug-free medium, but remained stable for at least 3 months in the presence of medium lacking oestrogenic activity. ZR-75-9a1 cells differ from previously reported tamoxifen-resistant variants of the MCF-7 line which retain ER and may prove a valuable model for the study of the development and stability of tamoxifen resistance in human breast cancer.     

Androgen and glucocorticoid receptor-mediated inhibition of cell proliferation by medroxyprogesterone acetate in ZR-75-1 human breast cancer cells

Medroxyprogesterone acetate (MPA) is a synthetic progestin, currently used in the adjuvant treatment of advanced breast cancer, which induces remission rates (30–40%) comparable to those obtained with other types of endocrine therapies. Since, in addition to its progestin-like action, MPA exhibits androgen- and glucocorticoid-like activities in other tissues, the present study was designed to assess the relative contribution of the different steroid receptor systems in the direct action of MPA on breast cancer cell growth, using the ZR-75-1 human mammary carcinoma cell line as anin vitro model.Unlike pure progestins, MPA potently inhibited the proliferation of ZR-75-1 cells in a concentrationdependent manner either in the presence or in the absence of estrogens, and the addition of insulin had only marginal effects on its growth-inhibitory activity. On the other hand, both hydroxyflutamide (OHF, a non-steroidal monospecific antiandrogen) and RU486 (a potent antiglucocorticoid and antiprogestin also endowed with antiandrogenic activity) competitively reversed MPA antiproliferative effects. MPA further decreased the growth of ZR-75-1 cells co-incubated with maximally inhibitory concentrations of either 5-dihydrotestosterone (DHT) or dexamethasone (DEX), although at about 300-fold higher MPA concentrations with DHT-treated than with DEX-treated ZR-75-1 cells, thus demonstrating a highly predominant androgenic effect. However, MPA had no effect on the growth of ZR-75-1 cells co-incubated with DHT and DEX simultaneously, thus supporting the predominant role of androgen and glucocorticoid receptors in MPA action. A 12-day preincubation of ZR-75-1 cells with increasing concentrations of MPA (10^–12 to 3 × 10^–6M) decreased the specific uptake of [³H]estradiol (E₂) by intact cell monolayers to the same extent as 10 nM DHT, an effect which was competitively blocked by the addition of OHF (3 µM). MPA action on ZR-75-1 cell growth also significantly differed from that of progestins in being additive to the inhibition of E₂-stimulated growth by the steroidal antiestrogen ICI164384.The present data indicate that the main action of MPA on ZR-75-1 human breast cancer cell growth is due to its androgen receptor-mediated inhibitory action, while its glucocorticoid-like activity could play an additional role at high concentrations.     

缺氧对人乳腺癌细胞系MCF-7生物学行为的影响

目的:研究缺氧对人乳腺癌细胞系MCF-7的生物学行为,增殖、侵袭能力的影响。通过影响缺氧诱导因子1α(hypoxic-induciblefactor1α,HIF-1α)的表达,检验其在这一过程中的作用,并进一步探讨作用机制。方法:慢病毒感染细胞,干扰乳腺癌细胞系MCF-7中HIF-1α的表达,得到HIF-1α表达正常的乳腺癌细胞系MCF-7-NC和HIF-1α表达受干扰的细胞系MCF-7-HIF△。分别低氧培养及化学药物诱导缺氧,用MTT法检测2组细胞系在缺氧和正常培养条件下的增殖能力,ANOVA检验检测其增殖能力的区别。Transwell实验检验缺氧和正常培养条件下人乳腺癌细胞的侵袭能力,计数通过Transwell小室的细胞,ANOVA检验检测细胞侵袭能力的区别。共聚焦免疫荧光法检测缺氧后2组乳腺癌细胞的上皮和间质标记物的表达,检验细胞系是否发生了上皮-间质转化(epithelial-mesenchymaltransition,EMT)。结果:MTT实验结果:正常表达HIF-1α的MCF-7-NC的增殖能力在缺氧后与正常培养条件下相比明显减弱,其差别具有统计学意义(P<0.05)。HIF-1α表达受干扰的MCF-7-HIF△的增殖能力在缺氧和正常培养条件下相比无显著区别。Transwell实验检测细胞的侵袭能力,缺氧后正常表达HIF-1α的MCF-7-NC的侵袭能力较正常培养的细胞明显增强,其差别具有统计学意义(P<0.05)。HIF-1α表达受干扰的MCF-7-HIF△侵袭能力在缺氧和正常培养下无显著区别。缺氧后,正常表达HIF-1α的细胞系MCF-7-NC发生上皮-间质转化(epithelial-mesenchymaltransition,EMT)。共聚焦荧光染色显示:在诱导缺氧后,正常表达HIF-1α的MCF-7-NC细胞极性发生变化,呈长梭形改变,上皮标志物人广谱角蛋白(pan-cytokeratin,P-CK)表达下调,间质标志物人α平滑肌动蛋白(α-SMA)表达上调;而HIF-1α表达受干扰的MCF-7-HIF△无明显EMT变化。结论:在体外条件下,缺氧可以引起乳腺癌细胞系MCF-7增殖能力减弱、侵袭能力增强,可以诱导乳腺癌细胞MCF-7发生EMT,干扰缺氧诱导因子亚基HIF-1α的表达,则缺氧对乳腺癌细胞增殖、侵袭的影响消失。