乳腺癌细胞体外摄取2-脱氧葡萄糖荧光类似物

目的 观察2-脱氧葡萄糖(2-DG)荧光类似物2-N[7-硝基苯-2-乙二酸,34羟氨基]-2-脱氧葡萄糖(2-NBDG)被高表达葡萄糖转运蛋白1(GLUT-1)的乳腺癌细胞靶向摄取的情况.方法 应用逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测乳腺癌MDA-MB-231细胞GLUT-1 mRNA和蛋白的表达,采用Western blot法比较乳腺癌MDA-MB-231细胞和MCF-7细胞GLUT-1的蛋白表达量.应用2-NBDG孵育人乳腺癌MDA-MB-231细胞,采用荧光显微镜及流式细胞仪观察、分析对2-NBDG的摄取情况,比较MDA-MB-231和MCF-7细胞吸收2-NBDG量的差异.结果 RT-PCR和免疫组化检测结果显示,MDA-MB-231细胞高表达GLUT-1;Western blot检测结果进一步显示,MDA-MB-231细胞的GLUT-1表达(0.946 4±0.007)高于MCF-7(0.833±0.010).荧光成像及流式细胞仪分析结果显示,MDA-MB-231细胞能快速摄取2-NBDG,且加入50 mmol/L D-葡萄糖后,荧光强度降低了46.0%.2-NBDG孵育乳腺癌细胞20 min后,MDA-MB-231细胞荧光强度(25.10±0.57)明显高于MCF-7细胞(10.12±0.62).结论 2-NBDG能迅速被高表达GLUT-1的乳腺癌MDA-MB-231细胞靶向吸收.     

Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells

Stromal cell-derived factor-1 (SDF-1), via its receptor CXCR4, has been implicated in metastasis of cancer, including breast cancer. Exogenous SDF-1 is known to regulate locomotion, chemotaxis and adhesion. The knowledge regarding the effect of autocrine SDF-1 on breast cancer cells is not available. The current study evaluated the effects of SDF-1 on the biological behaviour of breast cancer cells by genetically modifying the expression of SDF-1 in breast cancer cells. Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and a human fetal lung fibroblast cell line (MRC5) were used. The expression of SDF-1 and the SDF-1 receptor, CXCR4 in the cell lines were studied. Expression cassettes of human SDF-1 and hammerhead ribozyme transgenes specifically targeting human SDF-1 were constructed and used to over-express SDF-1 or to knockout the expression of SDF-1 in cancer cells, respectively. Invasiveness, migration and growth of the genetically modified cells were assessed. SDF-1 was expressed in wild-type human breast cancer cell line MDA-MB-435s and fibroblast cell line MRC5, but not in MDA-MB-231 cell line. In contrast, CXCR4 expression was observed in all three cell lines tested. The ability of invasion and migration was significantly reduced in SDF-1 knockout MDA-MB-435s cells, compared with wild-type and vector control cells (p<0.01). On the other hand, SDF-1 transfected MDA-MB-231epsilonSDF1+/+ cells that stably expressed SDF-1 showed a different behaviour from MDA-MB-231SDF1+/- (plasmid control) and wild-type MDA-MB-231 cells, both being SDF-1 negative. MDA-MB-231epsilonSDF1+/+ cells displayed a higher degree of invasiveness and migration, compared with wild-type and MDA-MB-231SDF+/- cells (p<0.01). Furthermore, SDF1-knockout MDA-MB-435s cells showed a slower growth rate over a 7-day period compared with the respective control and wild-type MDA-MB-435s cells. In contrast, the growth of the SDF-1 transfected MDA-MB-231SDF1+/+ cells was markedly enhanced when compared with wild-type and vector control cells. Breast cancer cell lines, when equipped with the autocrine SDF-1-CXCR4 signal pathway, display aggressive behaviour, including an increase in invasiveness, migration together with faster growth. SDF-1, together with its receptor CXCR4 may provide important information for predicting the aggressive nature and constitute important therapeutic targets in human breast cancer.     

Vitamin E succinate (VES) induces Fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis

Fas (CD95/APO-1) is an important mediator of apoptosis. We show that Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 human breast cancer cells become responsive to anti-Fas (CD95) agonistic antibody-triggered apoptosis after pretreatment or cotreatment with vitamin E succinate (VES; RRR-alpha-tocopheryl succinate). In contrast, no enhancement of anti-Fas agonistic antibody-triggered apoptosis was observed following VES pretreatment or cotreatment with Fas-sensitive primary cultures of human mammary epithelial cells, immortalized MCF-10A cells, or T47D human breast cancer cells. Although VES is itself a potent apoptotic triggering agent, the 6-h pretreatment procedure for Fas sensitization did not initiate VES-mediated apoptosis. The combination of VES plus anti-Fas in pretreatment protocols was synergistic, inducing 2.8-, 3.0-, and 6.3-fold enhanced apoptosis in Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells, respectively. Likewise, cotreatment of Fas-resistant MCF-7, MDA-MB-231, and MDA-MB-435 cells with VES plus anti-Fas enhanced apoptosis 1.9-, 2.0-, and 2.6-fold, respectively. Functional knockout of Fas-mediated signaling with either Fas-neutralizing antibody (MCF-7-, MDA-MB-231-, and MDA-MB-435-treated cells) or Fas antisense oligomers (MDA-MB-435-treated cells only), reduced VES-triggered apoptosis by approximately 50%. Analyses of whole cell extracts from Fas-sensitive cells revealed high constitutive expression of Mr 43,000 Fas, whereas Fas-resistant cells expressed low levels that were confined to the cytosolic fraction. VES treatment of the Fas-resistant cells caused a depletion of cytosolic Mr 43,000 Fas with a concomitant increase in Mr 43,000 membrane Fas. These data show that VES can convert Fas-resistant human breast cancer cells to a Fas-sensitive phenotype, perhaps by translocation of cytosolic Mr 43,000 Fas to the membrane and show that VES-mediated apoptosis involves Mr 43,000 Fas signaling.     

Semaphorin4C 在乳腺癌子宫内膜癌及前列腺癌中的表达及意义*

目的:研究轴突导向分子Semap horin4C（Sema4C）在乳腺癌、子宫内膜癌及前列腺癌组织和细胞水平的表达情况及其与上述三种恶性肿瘤转移的关系。方法:应用免疫组化技术检测45例乳腺癌、42例子宫内膜癌及49例前列腺癌组织中Sema4C的表达情况,分别采用蛋白免疫印迹法Western Blot及细胞免疫荧光方法检测Sema4C 在三种乳腺癌细胞（MDA-MB-435S、MDA-MB-231、MCF-7）、两种子宫内膜癌细胞株（AN3CA、HEC-1-B）和两种前列腺癌细胞株（PC- 3M-1E8、PC- 3M-2B4）的蛋白表达及其亚细胞定位情况。结果:乳腺癌和子宫内膜癌中,其分别在有淋巴结转移的乳腺癌组和子宫内膜癌组的原发灶中的表达高于无淋巴结转移组,差异有统计学意义（P<0.05）。 前列腺癌中,Sema4C 的阳性表达率随着Gleason评分增加而升高（P<0.05）。 Western blot在七株肿瘤细胞均检测到Sema4C 的表达,且提示Sema4C 的表达与细胞的转移潜能呈正相关。荧光显微镜下观察到Sema4C 主要定位在细胞膜和细胞浆。结论:Sema4C 在乳腺癌、子宫内膜癌和前列腺癌组织和细胞中表达具有普遍性,且与上述三种恶性肿瘤转移关系密切,有望成为上述三种恶性肿瘤治疗的一个新的分子靶点。      

HOXD3在乳腺癌细胞干性和化疗耐药性中的作用及机制研究

  目的  探讨HOXD3表达对乳腺癌细胞干性的影响,并研究HOXD3表达与乳腺癌细胞化疗耐药的关系。  方法  收集2006年1月至2008年12月哈尔滨医科大学附属肿瘤医院87例乳腺癌患者组织标本。采用免疫组织化学染色法检测乳腺癌细胞和组织中HOXD3表达;采用RT-PCR、Western blot和免疫荧光染色法检测HOXD3在顺铂或阿霉素耐药细胞系MDA-MB-231和MDAMB-435中的表达水平,分析HOXD3过表达对乳腺癌细胞系MDA-MB-231和MDA-MB-435的干细胞生物标志物表达水平的影响;采用MTT法和集落形成实验分析HOXD3在乳腺癌细胞化疗耐药中的作用。  结果  乳腺癌组织中HOXD3 mRNA相对表达量显著高于癌旁正常组织,乳腺癌细胞系MDA-MB-231、MDA-MB-435和MCF-7的HOXD3 mRNA相对表达量均高于正常乳腺上皮细胞系MCF-10A（均P < 0.05）。顺铂或阿霉素耐药的细胞系MDA-MB-231和MDA-MB-435的半抑制浓度（half maximal inhibitory concentration,IC₅₀）分别为（20.82±0.05）μmol/L和（19.69±0.47）μmol/L,或（32.26±0.23）mmol/L和（26.08±0.55）mmol/L,均高于对应原始细胞系（均P < 0.05）;耐药倍数分别为2.47和3.10倍,或1.86和2.08倍。HOXD3过表达MDA-MB-231、MDA-MB-435的肿瘤球体数目、干细胞生物标志物的表达水平均明显增加（均P < 0.05）。  结论  HOXD3过表达对乳腺癌细胞干性的维持及化疗耐药性的发生发挥重要的作用,为制定针对肿瘤干细胞的分子靶向治疗提供理论参考。      

Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.     

The expression of growth hormone-releasing hormone (GHRH) and its receptor splice variants in human breast cancer lines; the evaluation of signaling mechanisms in the stimulation of cell proliferation

Antagonists of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers including breast cancer, xenografted into nude mice or cultured in vitro. Splice variants (SVs) of receptors for GHRH have been found in several human cancers and cancer cell lines. The antiproliferative actions of GHRH antagonists could be mediated in part through these SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and SVs of its receptors in human breast cancer cell lines MCF-7, MCF-7MIII, MDA-MB-231, MDA-MB-435, MDA-MB-468, and T47D. mRNA for GHRH was present in all lines tested. mRNA for SV₁ isoform of GHRH receptors was found in MCF-7MIII, MDA-MB-468, and T47D; and for SV₂ isoform in MCF-7MIII and T47D cell lines. In proliferation studies in vitro, the growth of T47D cells was stimulated by GHRH and dose-dependently inhibited by GHRH antagonist JV-1-38. H89 (protein kinase A inhibitor), bisindolylmaleimide I (protein kinase C [PKC] inhibitor) and verapamil (voltage-dependent calcium channel blocker) inhibited the GHRH-stimulated proliferation of T47D cells. The GHRH antagonist JV-1-38 suppressed the T47D cell growth in vitro stimulated by PKC activator (phorbol-12-myristate-13-acetate). The stimulation of T47D cells by GHRH was followed by an increase in cAMP production and GHRH antagonist JV-1-38 competitively inhibited this effect. Our results suggest that SVs of GHRH receptors could mediate the responses to GHRH and GHRH antagonists in breast cancer through Ca²⁺-, cAMP- and PKC-dependent mechanisms. The presence of SV₁ of GHRH receptors in human cancers provides a rationale for antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.     

FGF13 promotes metastasis of triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents 10–20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.     

Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules

Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.     

上皮性肿瘤细胞中Snail与E-cadherin的表达及其与体外侵袭性的关系

目的 研究上皮性肿瘤细胞Snail与E-cadherin的表达以及与瘤细胞表型、转移潜能的关系。方法 采用Northern blot、共聚焦激光显微镜研究6株不同组织来源、不同分化程度及转移潜能的上皮性肿瘤细胞、1株正常上皮细胞、1株成纤维细胞中Snail与E-cadherin mRNA和蛋白表达与定位;Boyden小室体外侵袭实验反映细胞的转移潜能。结果 在分化程度较高的癌细胞和对照正常上皮细胞中,E-cadherin mRNA与蛋白表达较强,而Snail表达缺如;在分化程度低、转移潜能高的癌细胞和对照成纤维细胞中,E-cadherin、Snail mRNA及蛋白表达与上述情况相反。E-cadherin多定位于细胞胞浆和胞膜,Snail主要定位于胞核和胞浆。结论 在上皮性肿瘤细胞中E-cadherin和Snail在mRNA与蛋白表达水平上存在逆反关系,且与细胞分化、转移潜能相关。     

miR-100抑制乳腺癌细胞迁移的作用和机制

目的:探究miR-100对乳腺癌细胞株MDA-MB-231迁移能力的调节与机制.方法:Real time-PCR检测人正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-100的基础表达水平.应用脂质体法将 miR-100 mimic及阴性对照分别转染乳腺癌细胞株MDA-MB-231,通过real time-PCR检测转染后miR-100的表达水平,细胞划痕实验检测过表达miR-100对MDA-MB-231细胞迁移能力的影响,Western blot方法检测slug、snail和E-cadherin等EMT蛋白表达水平的变化.结果:miR-100在乳腺癌细胞株MDA-MB-231中的表达明显低于人正常乳腺上皮细胞MCF-10A.转染miR-100 mimic的乳腺癌细胞株MDA-MB-231的miR-100表达水平明显增高,细胞划痕实验显示过表达miR-100的MDA-MB-231细胞划痕愈合速度明显减慢.过表达miR-100的MDA-MB-231细胞E-cadherin蛋白表达水平明显增加,而slug和snail蛋白表达水平明显降低.结论:miR-100抑制乳腺癌细胞株MDA-MB-231的迁移能力与其上调E-cadherin,下调slug、snail蛋白表达,抑制EMT有关.     

FSIP1促进乳腺癌细胞迁移和侵袭并导致患者不良预后

目的 探讨乳腺癌组织中纤维鞘相互作用蛋白1(FSIP1)表达对乳腺癌细胞侵袭和迁移能力的影响及其与乳腺癌患者预后的关系,从而为乳腺癌的诊断和治疗提供一定的理论参考。方法 收集2004年1月—2018年12月于哈尔滨医科大学附属肿瘤医院确诊的404例乳腺癌患者的乳腺组织样本和病例资料,对收集的乳腺癌患者资料进行回顾性分析并采用Kaplan-Meier方法绘制生存曲线,采用免疫组织化学方法分析FSIP1在乳腺癌和癌旁组织中的表达情况,取乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435、SK-BR-3、T-47D及正常乳腺上皮细胞(HMECs)MCF-10A进行细胞培养,采用CRISPR/CAS9技术敲除乳腺癌细胞系MDA-MB-231和SK-BR-3中的FSIP1基因,通过Western blot实验检测各乳腺癌细胞系中FSIP1蛋白的表达情况并对FSIP1基因敲除结果进行检测,通过细胞迁移和侵袭实验评估FSIP1蛋白敲除对乳腺癌细胞迁移和侵袭能力的影响。结果 与正常乳腺上皮细胞(MCF-10A)相比,乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435、S...     

Influence of antiestrogens on the invasiveness and laminin attachment of breast cancer cells

Metastatic spread of breast cancer is responsible for most of the morbidity and mortality associated with this disease. Thus, it is important to identify agents with antimetastatic activity. Because invasiveness and tumor cell attachment are fundamental steps in the metastatic cascade, the major objective of the present study was to evaluate the antimetastatic potential of three antiestrogens, each with different chemical structure and mechanism of action, on breast cancer cell invasiveness and laminin attachment. The antiestrogens examined were tamoxifen, a mixed antagonist/agonist; Analog II, a cyclopropyl antiestrogen with pure antagonist activity; and ICI-182,780, a steroidal antiestrogen with pure antagonist activity. Our results indicate that MDA-MB-231 human breast cancer cells are much more invasive and have a higher affinity for laminin than do MCF-7 human breast cancer cells. All three antiestrogens, at a concentration of 10(-6) M, produced a reduction in MDA-MB-231 cell invasiveness, which was comparable in magnitude to their inhibition of MDA-MB-231 attachment to laminin. Evaluation of MDA-MB-231 cell morphology using scanning electron microscopy revealed the involvement of cellular pseudopodia and microvilli in the process of invasion. The results of this study suggest that antiestrogen-induced inhibition of breast cancer cell invasiveness could be due in part to a decrease in the attachment of tumor cells to laminin in the basement membrane.     

A molecular role for lysyl oxidase in breast cancer invasion

We identified previously an up-regulation in lysyl oxidase (LOX) expression,an extracellular matrix remodeling enzyme, in a highly invasive/metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this study, we demonstrate that the mRNA expression of LOX and other LOX family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and LOXL4] was observed only in breast cancer cells with a highly invasive/metastatic phenotype but not in poorly invasive/nonmetastatic breast cancer cells. LOX and LOXL2 showed the strongest association with invasive potential in both highly invasive/metastatic breast cancer cell lines tested (MDA-MB-231 and Hs578T). To determine whether LOX is directly involved in breast cancer invasion, LOX antisense oligonucleotides were transfected into MDA-MB-231 and Hs578T cells, and found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated controls. In addition, treatment of MDA-MB-231 and Hs578T cells with beta-aminopropionitrile (an irreversible inhibitor of LOX enzymatic activity) decreased invasive activity. Conversely, MCF-7 cells transfected with the murine LOX gene demonstrated a 2-fold increase in invasiveness that was reversible by the addition of beta-aminopropionitrile in a dose-dependent manner. In addition, endogenous LOX mRNA expression was induced when MCF-7 cells were cultured in the presence of fibroblast conditioned medium or conditioned matrix, suggesting a role for stromal fibroblasts in LOX regulation in breast cancer cells. Moreover, the correlation of LOX up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal melanoma cell lines. These results provide substantial new evidence that LOX is involved in cancer cell invasion.     

TRAF4蛋白在乳腺癌组织中的表达及意义

背景与目的:对肿瘤坏死因子受体相关因子4(tumor necrosis factor receptor-associated factor 4,TRAF4)在乳腺癌中表达的研究存在争议.本研究探讨TRAF4在正常乳腺组织、乳腺癌组织及不同侵袭能力乳腺癌细胞系中的表达情况.方法:应用免疫组化方法检测TRAF4在70例乳腺癌组织和14例正常乳腺组织中的表达.应用Western blot方法检测TRAF4在乳腺癌细胞系MDA-MA-231(高侵袭)和MCF-7(低侵袭)中的表达.结果:TRAF4在正常乳腺组织中呈浆、核阳性表达.其浆阳性率在正常乳腺组织(78.57%)、非浸润性导管癌(88.57%)和浸润性导管癌(91.43%)中逐渐增高,但差异无统计学意义(P＞0.05).而核阳性率在正常乳腺组织(64.28%)中显著高于非浸润性导管癌(28.57%,P＜0.01),在非浸润性导管癌中高于浸润性导管癌(5.7%,P＜0.05).TRAF4总蛋白在乳腺癌高侵袭细胞系中的表达量高于低侵袭细胞系,但差异无统计学意义(P＞0.05).结论:TRAF4在乳腺癌细胞核中表达明显降低,与乳腺癌侵袭性相关.     

Estrogen receptor-α and sex steroid hormones regulate Toll-like receptor-9 expression and invasive function in human breast cancer cells

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor, which is widely expressed in cancer. Synthetic TLR9-ligands induce cancer cell invasion in vitro, but the role of TLR9 in cancer pathophysiology remains unclear. Increased TLR9 expression has been, however, detected in estrogen receptor negative (ER−) breast cancers. In this study, we investigated the effects of ERα expression and sex steroid hormones on TLR9 expression in human ER+ (MCF-7, T47-D) and ER− (MDA-MB-231) breast cancer cell lines in vitro. We also studied TLR9 mRNA expression in archival breast cancer specimens (n = 12) with qRT-PCR, using primer sets that detect only the TLR9A isoform or the isoforms A and B (TLR9A/B). The TLR9 mRNA expression was detected in 10/12 specimens with both primer sets, and in 1/12 with only the TLR9A or the TLR9A/B primer sets. The basal TLR9 mRNA expression levels were significantly lower in the ER+ cell lines as compared with the ER− MDA-MB-231 cells. The transfection of ERα cDNA into MDA-MB-231 cells also resulted in down-regulation of TLR9 expression. While sex steroids had no effect on TLR9 expression in MCF-7 cells, testosterone (10⁻⁸ M) induced TLR9 expression in MDA-MB-231 and T47-D cells. Although bicalutamide blocked this testosterone effect in MDA-MB-231 cells, in T47-D cells bicalutamide increased TLR9 expression and only partially blocked the testosterone effects. Estradiol (10⁻⁸ M) induced TLR9 expression in T47-D cells. The invasive effects of synthetic TLR9-ligands were augmented by testosterone in vitro. This effect was lost in TLR9 siRNA MDA-MB-231 cells and also decreased by over-expression of ERα, which also inhibited NF-κB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERα and sex steroid hormones regulate TLR9 expression and invasive effects in the breast cancer cells. Also, the commonly used hormonal cancer therapy bicalutamide affects TLR9 expression.     

人乳腺癌细胞株WAF1／GIP1／p21基因的表达及其与细胞生物学特性的关系

OBJECTIVE: To study the WAF1/CIP1 gene DNA status, mRNA and protein expressions in human breast cancer cell line and its significance. METHODS: Using cell culture, molecular biological techniques such as Southern blot, Northern blot and immunocytochemical methods, the WAF1/CIP1 gene DNA status, mRNA and protein expression levels in MCF-7 expressing wild type p53(wtp53) and MDA-MB-231 expressing mutant p53 (mtp53) human breast cancer cell lines were detected respectively. The p53 and mdm-2 protein expression levels and cytobiological features of the 2 cell lines were compared and correlated to their WAF1/CIP1 gene expression levels. RESULTS: (1) There was no difference in WAF1/CIP1 gene DNA status in the two breast cancer cell lines. Neither of them showed gene amplificatian or deletion. However, the WAF1/CIP1 mRNA and p21WAF1/CIP1 protein expression levels of MCF-7 cells were higer than those of MDA-MB-231 cells (P < 0.05). (2) The character and cellular distribution of p53 protein in the two cell lines were clearly different. The expression level of mdm-2 proteion was significantly higher in MCE-7 than in MDA-MB-231 cells (P < 0.05). (3) Compered to the other breast cancer cell line, MCF-7 cells were better differentiated, grew more slowly and adhered more closely with each other. CONCLUSION: The WAF1/CIP1 gene expression at mRNA and protein levels is associated with p53 phenotype and some cytobiological features of human breast cancer.