维拉帕米与加温对MCF-7/Adm细胞株多药耐药的实验研究

目的观察维拉帕米(Vrp)与加温对人乳腺癌细胞敏感株和耐药株耐药的逆转作用和协同作用。方法以人乳腺癌细胞株MCF-7为对照,以人乳腺癌耐药细胞株MCF-7/Adm为研究对象,观察阿霉素(Adm)浓度为3μg.ml-1、37℃~43℃条件下,分别加入不同浓度的Vrp(0~5μmol.L-1)时,MCF-7与MCF-7/Adm亚细胞内阿霉素的荧光强度、温度、浓度分别与荧光强度间的变化趋势。结果MCF-7/Adm细胞对于阿霉素的敏感性明显低于MCF-7细胞,MCF-7组的亚细胞内阿霉素荧光强度较高,细胞凋亡率亦高;MCF-7/Adm随温度和Vrp浓度的增加,Adm荧光强度向胞核增加,细胞凋亡率也增加。结论加温联合耐药逆转剂Vrp能协同增强Vrp对MCF-7/Adm的耐药逆转作用。     

BP1在乳腺癌组织中的临床表达及其与Bcl-2表达相关性

目的：探讨BP1在乳腺癌组织中的临床表达及其与Bcl-2表达的相关性。方法资料随机选自2012年5月～2013年5月在本院行手术治疗的乳腺癌患者72例的乳腺癌组织标本作研究组,资料选择同一患者癌旁组织标本72例作对照组,对两组予以Westemblot和免疫组织的化学法行阳性检测,分析两组BP1蛋白的阳性表达,研究组BP1与Bcr-2表达相关性,以及BP1和乳腺癌病理因素相关性。结果研究组BP1阳性表达,比对照组BP1阳性表达高,比较差异具统计学上的意义（P＜0.05）;研究组Bcl-2的蛋白阳性表达和BP1阳性表达具正相关性（P＜0.05）。研究组BP1的阳性表达和乳腺癌病理因素中PR、ER和C-erb-B-2具相关性（P＜0.05）。结论乳腺癌组织内的BP1蛋白阳性表达,可同乳腺癌的发生、发展相关,且可与Bcl-2的上升表达的促进具相关性,具有一定临床研究和应用价值     

Antitumor effects of berberine against EGFR,ERK1/2, P38 and AKT in MDA-MB231 and MCF-7 breast cancer cells using molecular modelling and in vitro study

Background

Berberine is an alkaloid plant-based DNA intercalator that affects gene regulation, particularly expression of oncogenic and tumor suppressor proteins. The effects of berberine on different signaling proteins remains to be elucidated. The present study aimed to identify the effects of berberine against key oncogenic proteins in breast cancer cells.

乳腺癌石蜡肿瘤组织和相应血液细胞BRCA1、ERCC1、TSmRNA的表达相关性研究

目的 探讨乳腺癌石蜡肿瘤组织和相应血液细胞中乳腺癌易感基因1（BRCA1）与切除修复交叉互补基因1（ERCC1）及胸苷酸合成酶（TS）基因表达的相关性,同时探讨BRCA1、ERCC1、TS在乳腺癌肿瘤组织与对应患者血液细胞中表达的关系。方法 收集53例乳腺癌肿瘤石蜡标本及其对应的血液样本,利用实时荧光定量PCR技术检测肿瘤石蜡组织和对应血液样本中BRCA1、ERCC1、TS mRNA的表达水平;利用Pearson相关性分析方法分析肿瘤石蜡组织和相应血液细胞BRCA1、ERCC1、TS mRNA表达的相关性以及肿瘤石蜡组织细胞BRCA1、ERCC1、TS mRNA之间表达的相关性。结果 乳腺癌石蜡组织中BRCA1 mRNA表达的ΔCT为（7.516±2.257）,对应的血液组织中为（10.374±2.519）。乳腺癌石蜡组织中ERCC1和TS mRNA表达的ΔCT分别为（6.114±2.944）、（5.950±2.604）,对应的血液组织分别为（8.801±2.581）、（10.078±1.731）。Pearson相关性分析显示,BRCA1、ERCC1在肿瘤石蜡组织与血液组织中的表达均呈正相关（r=0.607、0.537,P〈0.05）。肿瘤石蜡组织与血液组织中的TS表达无相关性（r=0.074,P〉0.05）。BRCA1与ERCC1在肿瘤石蜡组织中的表达无相关性（r=0.250,P〉0.05）。BRCA1与TS在肿瘤石蜡组织中的表达无相关性（r=0.256,P〉0.05）。ERCC1与TS在肿瘤石蜡组织中的表达无相关性（r=0.169,P〉0.05）。结论 乳腺癌患者的血液标本也许可以替代其肿瘤石蜡组织检测BRCA1和ERCC1 mRNA的表达;BRCA1与ERCC1、TS的表达无相关性。     

保乳手术与全乳切除治疗T1N0期乳腺癌的疗效对比分析

目的对比分析保乳手术与全乳切除治疗T1N0期乳腺癌的临床疗效。方法将本院收治的220例T1N0期乳腺癌患者临床资料进行回顾性分析,分为对照组和治疗组,其中对照组行全乳切除手术,治疗组行保乳手术治疗。观察患者的评价外观优良率、局部复发率、生存率等指标。结果治疗组患者评价外观优良率为94.55%,而5年内局部复发率和生存率两组患者相当,无显著差异。结论早期乳腺癌患者行保乳手术治疗,在保证临床疗效的基础上保留乳房,显著提高外观优良率,是值得推广的常规术式。     

Induction of G1 cell cycle arrest and cyclin D1 down-regulation in response to pericarp extract of Baneh in human breast cancer T47D cells

Background and the purpose of the study

Natural products from plants have an important role in the development and production of new drugs mainly for cancer therapy. More recently, we have shown that the pericarp methanolic extract of Pistacia atlantica sub kurdica (with local name of Baneh) as a rich source of active biological components with high antioxidant and radical scavenging activities, has ability to cease proliferation and induce apoptosis in T47D human breast cancer cells. The present study aimed to clarify whether Baneh extract able to alter cell cycle progression of T47D cells or not.

Methods

In order to study the possible effect of Baneh extract on cell cycle of T47D cells, we evaluated cell cycle distribution and its regulatory proteins by flow cytometry and western blot analysis respectively.

Results

Baneh extract induced G₀/G₁ cell cycle arrest in conjunction with a marked decrease in expression of cyclin D1 and cdk4 that was strongly dependent on time of exposure. In parallel, Dox-treated T47D cells in early time points were accumulated on S phase, but after 48 h cell cycle progression was inhibited on G₂/M. Dox promoted striking accumulation of cyclin B1 rapidly and enhanced cyclin A abundance.

Conclusion

Taken together, our results establish that the antitumor activity of the pericarp extract of Baneh partly is mediated via cell cycle arrest and downregulation of cyclin D1 and cdk4 expression. These findings warrant further evaluation regarding the mechanism(s) of action of this promising anticancer agent.     

乳腺癌HPA、ET-1表达和肿瘤血管生成关系

目的研究乳腺癌患者内皮素-1（ET-1）和乙酰肝素酶（HPA）的表达及其与肿瘤血管生成的关系。方法应用免疫组化方法检测60例乳腺癌患者的ET-1和HPA的表达,同时检测肿瘤微血管密度（MVD）,并分析与临床病理之间的关系。结果①60例MVD平均计数为（25．40±8．91）,59例表达ET-1平均阳性率为（67．53±18．98）％,57例表达HPA平均阳性率为（56．30±24．16）％。②MVD与HPA呈正相关（r=0．3961、t=3．2899、P〈0．005）;MVD与ET-1呈正相关（r=013170、t=2．5462、P〈0．05）。③MVD与有无转移有关,与分期及肿块大小无关。④HPA和ET-1均与分期及转移有关,但与肿块大小均无关。结论乳腺癌患者高表达HPA和ET-1,并与MVD呈正相关关系。HPA、ET-1和MVD均与转移有关,而与肿块大小无关;HPA和ET-1与分期有关。     

SMARCAD1 knockdown uncovers its role in breast cancer cell migration,invasion, and metastasis

Objective: Breast cancer is the most common cancer seen in women worldwide and breast cancer patients are at high risk of recurrence in the form of metastatic disease. Identification of genes associated with invasion and metastasis is crucial in order to develop novel anti-metastasis targeted therapy. It has been demonstrated that the DEAD-BOX helicase DP103 was implicated in breast cancer invasion and metastasis. SMARCAD1 is also a DEAD/H box-containing helicase, suggested to play a role in genetic instability. However, its involvement in cancer migration, invasion, and metastasis has never been explored.

Research design and methods: Using two different designs of shRNA targeting SMARCAD1, we investigated the impact of SMARCAD1 knockdown on the migration, invasion, and metastasis potential of the breast cancer cells MDA-MB-231 and T47D.

Results: We observed that SMARCAD1 knockdown in the invasive breast cancer cells MDA-MB-231, unlike in the non-invasive breast cancer cells T47D, was associated with an increased cell-cell adhesion and a significant decrease in cell migration, invasion, and metastasis due at least in part to a strong inhibition of STAT3 phosphorylation.

Conclusions: These results indicate that SMARCAD1 is involved in breast cancer metastasis and can be a promising target for metastatic breast cancer therapy.     

Dual inhibition of STAT1 and STAT3 activation downregulates expression of PD-L1 in human breast cancer cells

Objectives: Breast cancer is the most commonly diagnosed cancer, and it is a leading cause of cancer-related deaths in females worldwide. Triple-negative breast cancer (TNBC) constitutes 15% of breast cancer and shows distinct metastasis profiles with poor prognosis. Strong PD-L1 expression has been observed in some tumors, supporting their escape from immune surveillance. Targeting PD-L1 could be a promising therapeutic approach in breast cancer patients. We investigated potential molecular mechanisms for constitutive expression of PD-L1 by inhibiting upstream STAT1 and STAT3 signals.

Methods: PD-L1 expression in three breast cancer cell lines was measured using quantitative PCR and western blotting. Activation of STAT1 and STAT3 was blocked using pharmacological inhibitors and siRNA. The mechanism underlying the constitutive expression of PD-L1 was investigated using ChIP and co-immunoprecipitation assays.

Results: We found that individual inhibition of STAT1 and STAT3 activation partially downregulated PD-L1, while combined inhibition completely downregulated PD-L1 expression. Moreover, our results suggest that pSTAT1-pSTAT3 dimerize in cytosol and translocate to the nucleus, where they bind to PD-L1 promoter and induce PD-L1 expression.

Conclusion: These findings provide a rationale for combined targeting of STAT1 and STAT3 for the development of immune-based cancer therapies for down regulation of PD-L1 expression.     

Antiproliferative and receptor binding properties of α- and β-casomorphins in the T47D human breast cancer cell line

In previous studies, we have shown that opioid agonists ([ -Ala², -Leu⁵]enkephalin (DADLE), [ -Ser²,Leu⁵]enkephalin-Thr⁶ (DSLET), ethylketocyclazocine and etorphine) bind to opioid binding sites and decrease cell proliferation of human T47D breast cancer cells. Furthermore, we provided evidence about a cross-reaction, also in the T47D human breast cancer cell line, of μ-acting opioids with type-II somatostatin receptors. Since a potential source of opioid activity in the breast might be casomorphin peptides (produced by the enzymatic degradation of α-casein and β-casein), we investigated the antiproliferative action of five different casomorphin peptides: α-casein-(90-95), α-casein-(90-96), β-casomorphin, β-casomorphin-(1-5) and morphiceptin. We show that all five peptides decreased, in a dose-dependent manner, cell proliferation. The general antagonist diprenorphine produced only a partial reversal of their action. Furthermore, we provide evidence that all peptides (except for morphiceptin) bind to δ- and κ-opioid binding sites of T47D cells with different selectivity. Finally, we show that these peptides are also partial competitors at the somatostatin receptors present in the same cell line.     

人乳腺癌B7-H3分子的高表达及其抑制T淋巴细胞活化增殖作用的机制

目的 探讨乳腺癌组织及细胞中高表达协同刺激分子B7-H3对T淋巴细胞作用的机制.方法 RT-PCR及流式细胞仪方法分析乳腺癌组织及细胞中B7-H3基因的高表达,以PGPU6/GFP/Neo质粒为载体,构建shRNA重组体,采用脂质体法转染乳腺癌细胞株MCF-7.采用免疫荧光标记和流式细胞术分析在MCF-7细胞上的表达.继而,利用CCK8法和酶联免疫吸附测定法(ELISA)分析B7-H3基因干扰细胞株对T细胞体外增殖和细胞因子的分泌.结果 乳腺癌组织和细胞中高表达B7-H3分子.成功通过RNAi技术构建低表达B7-H3基因乳腺癌细胞株.体外生物学功能分析表明,与转染空载体的MCF-7/mock细胞相比,B7-H3表达干扰细胞能有效增强T细胞增殖以及对IFN-γ的分泌.结论 乳腺癌中高表达协同刺激分子B7-H3能有效逃避T淋巴细胞对其的作用和杀伤.     

组织蛋白D和E-钙黏附蛋白与乳腺癌患者预后的关系

目的探讨E-Ca、Ca-D在乳腺癌预后中的作用及相互关系。方法对106例浸润性乳腺癌患者的肿瘤组织石蜡切片采用免疫组织化学染色技术标记E-Ca、Ca-D。结果肿瘤间质内Ca-D表达强度与复发呈正相关。E-Ca表达在导管癌内表达强于小叶癌。乳腺癌组织中nm23-H1蛋白表达明显降低,与生存期呈正相关,与复发期呈负相关(P<0.05)。MVD在复发组高于对照组。结论①Ca-D、MVD、nm23-H1在乳腺癌组织内共同参与癌组织的浸润和转移,可作为预后的判断指标。②E-Ca作为预后判断不理想,但可以作为差分化小叶癌和导管癌的鉴别诊断依据。     

In vitro mechanisms involved in the regulation of cell survival by lithium chloride and IGF-1 in human hormone-dependent breast cancer cells (MCF-7)

Lithium, the lightest of all solid elements, has been used for the treatment of bipolar disorder since 1970s and prescribed to millions of women worldwide. Lithium chloride (LiCl) has been considered to be a potent inhibitor of glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase that is involved in the control of cell proliferation, differentiation, and apoptosis. In addition, GSK-3β has been found to be inhibited endogenously by insulin-like growth factor-1 (IGF-1), a potent mitogen that plays an important role in the survival, growth, and differentiation of normal and neoplastic cells. Although both IGF-1 and LiCl have the ability to inhibit GSK-3β, the specific signaling difference that mediates the survival of breast cancer cells was not clear. Therefore, in the present study, MCF-7 cells (human breast cancer cells) were treated with or without IGF-1 or LiCl in the presence or absence of LY294002 or PD98059 (pharmacological inhibitors) for 24 h. As the expression of signaling proteins is crucial in the maintenance of cell survival and apoptosis, we analyzed the cells using immunoblotting procedure. In summary, our results have shown that LiCl and IGF-1 mediates cell survival by inhibiting GSK-3β but differ in their mechanisms. IGF-1 involves PI3K/Akt or MAPK pathways whereas LiCl is completely independent of these pathways. IGF-1 upregulates anti-apoptotic proteins whereas LiCl downregulates apoptotic proteins in order to maintain cell survival.     

乳腺癌患者外周血、骨髓斯钙素基因检测及其意义

目的 观察人类斯钙素-1(hSTC-1)基因在原发性乳腺癌患者外周血液及骨髓中的表达,研究将hSTC-1作为一种新的肿瘤分子标记用来检测乳腺癌患者血液及骨髓中的肿瘤微转移情况.方法 采用逆转录-聚合酶链反应(RT-PCR)方法分别检测51例乳腺癌病人的外周血(A组)、8例乳腺癌病人的骨髓(B组)以及30例乳腺癌病人的肿瘤组织(C组)中hSTC-1 mRNA的表达水平.结果 C组标本hSTC-1 mRNA的阳性率为93.3%,A组和B组hSTC-1 mRNA的阳性率分别为37.3%和37.5%.结论 乳腺癌患者外周血、骨髓中检测到hSTC-1基因的表达,说明乳腺癌已发牛血行微转移;hSTC-1可作为检测乳腺癌病人外周血、骨髓微转移新的分子标记.     

Enantiomerically pure [1, 2-diamino-1-(4-fluorophenyl)butane]platinum(II) complexes: synthesis and antitumor activity against MCF-7 and MDA-MB 231 breast cancer and LnCaP/FGC prostate cancer cell lines

Enantiomerically pure 1, 2-diamino-1-(4-fluorophenyl)butanes were synthesized by stereoselective procedures. The enantiomeric purity was determined by (1)H NMR spectroscopy after derivatization with (1R)-myrtenal. For the coordination to platinum, the diamines were reacted with K(2)PtI(4). Reaction with Ag(2)SO(4) yielded the respective sulfatoplatinum(II) complexes, which were converted into the dichloroplatinum(II) complexes by treatment with 2 N HCl. The influence of the configuration and the kind of leaving group on the antitumor activity was studied on the MCF-7 and MDA-MB 231 breast cancer cell lines, as well as on the LnCaP/FGC prostate cancer cell line. It was demonstrated that the dichloroplatinum(II) complexes were more active than the respective diiodoplatinum(II) derivatives. Conversion into the sulfatoplatinum(II) complexes further enhanced the antiproliferative effects. The configuration determined the antitumor effects, dependent on the cell line used: MCF-7: (R, R) > (S, S) > (R, S) > (S, R); MDA-MB 231: (S, S) > (R, R) > (R, S) = (S, R); LnCaP/FGC: (S, S) > (R, R) > (R, S) > (S, R).     

hnRNP A2/B1在乳腺癌组织中表达水平及其凋亡调控的研究

目的 检测乳腺癌组织标本中hnRNP A2/B1的表达水平;探讨hnRNP A2/B1基因对乳腺癌细胞株MCF-7凋亡的影响.方法 选取乳腺癌患者40例、乳腺纤维瘤患者20例(对照),采用免疫组化方法检测组织中hnRNP A2/B1的表达水平;体外培养乳腺癌细胞株MCF-7,并将hnRNP A2/B1 siRNA转染至MCF-7中,TUNEL法检测转染前后MCF-7细胞的凋亡情况.结果 40例乳腺癌组织标本中36例有hnRNP A2/B1阳性表达,而作为对照的乳腺纤维瘤组织中只有3例有hnRNP A2/B1阳性表达,乳腺纤维瘤组与乳腺癌组hnRNP A2/B1表达水平差异有统计学意义(P＜0.01).体外培养乳腺癌细胞MCF-7在转染hnRNP A2/B1 siRNA后,MCF-7细胞凋亡明显增加.结论 hnRNP A2/B1在乳腺癌组织中的表达较乳腺纤维瘤组织高,推测hnRNP A2/B1与乳腺癌的发生有关.通过siRNA干预、降低hnRNP A2/B1的表达水平可以促进乳腺癌细胞凋亡,提示hnRNP A2/B1可能通过抗肿瘤细胞失巢凋亡实现肿瘤的发生.     

Comparison of the global gene expression profiles produced by methylparaben, n-butylparaben and 17beta-oestradiol in MCF7 human breast cancer cells

Since the alkyl esters of p-hydroxybenzoic acid (parabens) can be measured intact in the human breast and possess oestrogenic properties, it has been suggested that they could contribute to an aberrant burden of oestrogen signalling in the human breast and so play a role in the rising incidence of breast cancer. However, although parabens have been shown to regulate a few single genes (reporter genes, pS2, progesterone receptor) in a manner similar to that of 17beta-oestradiol, the question remains as to the full extent of the similarity in the overall gene profile induced in response to parabens compared with 17beta-oestradiol. The GE-Amersham CodeLink 20 K human expression microarray system was used to profile the expression of 19881 genes in MCF7 human breast cancer cells following a 7-day exposure to 5 x 10(-4) M methylparaben, 10(-5) M n-butylparaben and 10(-8) M 17beta-oestradiol. At these concentrations, the parabens gave growth responses in MCF7 cells of similar magnitude to 17beta-oestradiol. The study identified genes which are upregulated or downregulated to a similar extent by methylparaben, n-butylparaben and 17beta-oestradiol. However, the majority of genes were not regulated in the same way by all three treatments. Some genes responded differently to parabens from 17beta-oestradiol, and furthermore, differences in expression of some genes could be detected even between the two individual parabens. Therefore, although parabens possess oestrogenic properties, their mimicry in terms of global gene expression patterns is not perfect and differences in gene expression profiles could result in consequences to the cells that are not identical to those following exposure to 17beta-oestradiol.     

Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines.