骨唾液酸蛋白与乳腺癌骨转移相关性的研究进展

多项研究已证实骨唾液酸蛋白(BSP)与乳腺癌骨转移具有相关性,而目前研究的热点是BSP诱导乳腺癌骨转移的具体作用机制及参与BSP基因表达调控的因子种类,这将为今后研究治疗骨转移相应靶向药物提供直接理论依据。同时将BSP与其他骨代谢指标如OPN、TRACP5b、NTx、PTHrP等联合检测可提高乳癌骨转移高风险人群筛选的精确性,并且这些因子可以作为乳腺癌的独立预后因素。     

骨唾液蛋白诊断乳腺癌骨转移的临床意义

文献报道,约80%的乳腺癌患者尸检发现有骨转移,而且在I期乳腺癌患者中就检出23%临床骨 转移隐匿病变。临床中这些乳腺癌骨转移患者在发 病早期大多无骨痛症状,X线亦无骨破坏征象,一旦发现多数已属晚期,治疗很困难。有文献报道,骨唾液蛋白(bonesialoproteinBSP)主要来源于肿瘤细胞,而且在转移瘤细胞靶向转移至骨的过程中发挥了重要作用,故在众多乳腺癌骨转移标志物中,显示了较高的灵敏性及特异性。     

骨涎蛋白单克隆抗体抑制亲骨转移人乳腺癌细胞血管侵袭机制的探讨

目的:探讨骨涎蛋白(BSP)单克隆抗体对亲骨转移人乳腺癌细胞株MDA-MB-23180血管侵袭能力的影响.方法:MTT法检测BSP抗体对MDA-MB-231BO细胞的增殖作用;鸡胚尿囊绒膜(CAM)实验检测BSP抗体对MDA-MB-231BO细胞血管侵袭能力的影响.结果:BSP抗体对MDA-MB-231BO细胞的生长具有特异抑制作用,其抑制率和剂量呈正相关,BSP抗体浓度为100 μg/mL时,抑制率为35.18%.BSP抗体能特异性抑制MDA-MB-231BO细胞穿透鸡胚血管内皮细胞和基底膜,抑制效应与抗体浓度呈正相关.100μg/mL的BSP抗体可以明显抑制MDA-MB-231BO细胞侵入鸡胚血管.结论:BSP抗体对MDA-MB-231BO细胞的血管侵袭能力具有特异抑制作用,提示BSP抗体可抑制乳腺癌靶向骨转移的进程.     

骨唾液酸蛋白与恶性肿瘤骨转移

骨骼是恶性肿瘤常见的转移部位之一。骨转移的诊断意义重大,它决定着患者的治疗方案和预后。临床上主要以影像学来评价有无骨转移发生,但其价格昂贵而且准确度差。临床医师正在寻找花费较低、可早期预示骨转移的标志物。骨唾液酸蛋白(bone sialoprotein,BSP)作为一种溶骨性标志物在恶性肿瘤骨转移过程中起着重要作用,可作为评价是否出现骨转移,评估肿瘤有无进展,监测疗效的一项指标。本文对BSP结构、功能及其在恶性肿瘤骨转移中的作用进行综述。     

骨代谢指标在乳腺癌骨转移的临床应用

乳腺癌骨转移在复发转移乳腺癌中的发生率为65%～75%。骨痛、骨损伤、骨相关事件(skeletal related event,SRE)的发生及生活质量的降低是乳腺癌骨转移的常见并发症,其将进一步降低癌症患者的生活质量并缩短生存期。随着疗效的增加,生存期的延长,骨转移在临床上出现的频率亦在增加,因此,乳腺癌骨转移的诊治日益成为乳腺癌治疗的     

骨唾液酸蛋白与乳腺癌

骨唾液酲蛋白(BSP)是细胞外基质中的一种高度磷酸化和糖基化分泌性蛋白,BSP的组织分布相对局限,分布在骨骼、牙齿等矿化组织以及钙化的软骨组织与骨的交界区。BSP还在多种骨转移瘤中表达,例如乳腺癌、前列腺癌、甲状腺癌等。现就BSP与乳腺癌的研究情况作一综述。     

骨改良药物在乳腺癌骨转移治疗中的应用进展

骨改良药物包括在临床上应用的双膦酸盐及2010年FDA批准上市的新药地诺单抗,是目前治疗恶性肿瘤骨转移、高钙血症及骨质疏松症的主要药物。该类药物在临床上应用广泛,种类较多,近年来在药物的应用时机、用法及毒副反应方面有一定进展和争议。本文就骨改良药物在乳腺癌骨转移治疗中的临床应用进行探讨。     

BSP在原发性乳腺癌组织中的表达对预测术后骨转移的临床意义

目的:探讨BSP在原发性乳腺癌组织中的表达对预测术后骨转移的临床意义.方法:用免疫组织化学SP法测定70例原发性乳腺癌石蜡标本中BSP的表达,及ER、PR、C-erb-B2表达情况.结果:1)BSP在骨转移组表达明显高于无骨转移组(无转移组 骨外转移组)(P＜0.01),且强阳性表达高于后者(P＜0.05);同时,骨转移组与无转移组和骨外转移组之间阳性表达均有差别(P＜0.05;P＜0.01),而后两组间阳性表达无统计学差异(P＞0.05).2)BSP的表达在Ⅱ期及Ⅲ期患者组间有差异(P＜0.05),但与患者年龄及ER、PR、C-erb-B2状态,肿块大小,病理类型及淋巴结转移数目均无相关(P＞0.05).结论:在原发性乳腺癌组织水平上,BSP是早期预测术后骨转移的良好标志物.     

抑制肿瘤细胞骨保护素表达对乳腺癌细胞致骨转移能力的影响

目的观察抑制肿瘤细胞骨保护素（0steoprotegerin,OPG）表达对乳腺癌MDA～MB-231细胞株致骨转移的影响。方法32只4～6周龄雌性裸鼠被随机分为A、B、C、D4组,每组8只,A、C组的每只裸鼠按分组分别以5×10。个MDA—MB-231细胞注射入左心室（A组）或左胫骨骨髓腔（C组）。B、D组的每只裸鼠按分组分别以5×10。个MDA—MB-231i细胞（OPG表达抑制）注射人左心室（B组）或左胫骨骨髓腔（D组）,42d后行病理检查,比较A、B组骨转移发生率和骨转移灶数量,以及C、D组的骨肿瘤体积。定量资料的比较采用t检验或秩和检验,定性资料比较采用Fisher确切概率检验。结果A组有6只发生骨转移,全组共检出不连续性骨转移灶23处;B组有3只发生骨转移,全组共检出不连续性骨转移灶8处。A组的骨转移发生率和转移灶数量虽高于B组,但两者差异并无统计学意义（P〉0．05）。C组肿瘤平均体积为（66．29±41．01）mm3,D组肿瘤平均体积为（23．70±16．14）mm3,C组骨肿瘤体积大于D组,两组间差异有统计学意义（P=0．02）。结论乳腺癌细胞致骨转移能力与其OPG表达水平密切相关,抑制OPG表达可以降低乳腺癌细胞致骨转移的能力。     

抗骨形成蛋白单克隆抗体抑制骨肉瘤细胞生长的体外、体内实验

应用单克隆抗体治疗恶性肿瘤是近年来一个新的研究课题，并且在许多方面取得了一定的成绩．本实验成功的制备了抗骨肉瘤形成蛋白的单克隆抗体(BMP-McAb)，以此单抗检测人骨肉瘤、人成骨肉瘤OS-732细胞及其裸鼠移植瘤中BMP的分布情况，观察BMP-McAb对BMP的括性阻断作用，用于探讨BMP-McAb治疗骨肉瘤的可行性并为临床应用提供实验依据。结果发现：     

Treatment of bone metastasis induced by MDA-MB-231 breast cancer cells with an antibody against bone sialoprotein

The extracellular bone matrix protein bone sialoprotein (BSP) is considered to play an important role in the pathogenesis of lytic skeletal lesions which are associated with severe morbidity in breast, prostate or lung cancer patients. In addition to in vitro studies, nude rats were implanted with 10(5) MDA-MB-231 cells transfected with GFP into a small branch of the femoral artery. Osteolytic lesions of the respective hind leg were detected by X-ray and CT analysis as well as by immunohistochemistry. Exposure of MDA-MB-231GFP cells in vitro to an antibody against BSP (0-400 microg/ml) decreased proliferation, colony formation and migration of these cells by up to 95, 83 and 89 T/C%, respectively. In nude rats, pre-incubation of MDA-MB-231GFP cells prior to inoculation (25-100 microg/ml) reduced the mean osteolytic lesion size to 22 T/C% after 90 days of observation (p<0.05). Treatment of overt lytic metastasis with the anti-BSP antibody (10 mg/kg) resulted in a significantly smaller mean lesion size of 57 T/C% at the end of the observation period (p<0.05) as well as in new bone formation. Immunohistochemical analysis revealed the presence of BSP in MDA-MB-231GFP cells and in vessel endothelium cells during processes such as migration and invasion. In conclusion, an anti-BSP antibody decreased proliferation, colony formation and migration of MDA-MB-231GFP cells in vitro and reduced osteolysis besides inducing bone formation in a nude rat model.     

抗人骨唾液蛋白单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人骨唾液蛋白（BSP）单克隆抗体（mAb）,并对其生物学特性进行鉴定,为BSP作为乳腺癌骨转移临床诊断靶点的研究奠定基础。方法重组BSP蛋白免疫BALB／c小鼠,取其脾细胞与Sp2／0细胞融合,经筛选建立可稳定分泌抗BSPmAb细胞株,制备腹腔积液,ProteinG纯化。ELISA检测抗体的效价和亲和力,并用亚型鉴定试纸条鉴定mAb的亚型。应用Western blotting检测mAb的特异性,进一步利用纯化的mAb经免疫组织化学法检测乳腺癌细胞MDA-MB-231中BSP的表达情况。结果获得9株抗BSPmAb细胞株,选择其中2株（D001和D002）进一步鉴定,其上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200。2株抗体亚型均为IgG1型,轻链均为K型。Western blotting结果均在预期位置出现阳性条带。利用此2株细胞株所得抗体均可在乳腺癌细胞MDA-MB-231中检测到BSP的表达。结论成功制备能特异性识别BSP的mAb,为进一步研究BSP的生物学功能以及对BSP作为乳腺癌骨转移的标志物进行临床评价奠定基础。     

Characterization of a rat model with site-specific bone metastasis induced by MDA-MB-231 breast cancer cells and its application to the effects of an antibody against bone sialoprotein

Metastasis into the skeleton is a serious complication of certain neoplastic diseases such as breast, prostate and lung cancer, but the reasons for this osteotropism are poorly understood. Our aim was to establish a physiologically relevant animal model that is characterized by osteolytic lesions confined to the hind leg of nude rats. For this purpose, we injected 1x10(5) MDA-MB-231 human breast cancer cells transfected with GFP into the superficial epigastric artery, which is an anastomosing vessel between the femoral and iliac arteries. As assessed with the aid of X-rays, computed tomography and immunohistochemisty, osteolytic lesions occurred exclusively in the femur, tibia and fibula of the animals. The tumor take rate was 93% in a series of 96 rats and the increase in lesion size was observed up to 110 days after tumor cell inoculation. When applying this animal model to the effects of an antibody against bone sialoprotein (BSP), a significantly reduced osteolytic lesion size was observed after preincubation of cells (2 hr, 600 microg/ml anti-BSP) prior to intra-arterial tumor cell injection resulting in 19 T/C% at day 60 after tumor implantation (p < 0.05). In addition, the osteolytic lesion size was also significantly reduced after s.c. treatment of the animals with the antibody (20 mg/kg anti-BSPx3 within 5 days after tumor implantation), resulting in 30 T/C% at day 60 after tumor cell implantation (p < 0.05). In conclusion, the novel rat model for site-specific osteolytic lesions provides in vivo evidence that preincubation of MDA-MB-231GFP cells and treatment of rats after tumor implantation with an antibody against BSP significantly reduces the size of lytic lesions in bone.     

Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model

Bone sialoprotein (BSP) is a major non-collagenous protein found almost exclusively in bone and other mineralized tissues including enamel, dentin and cementum. Although a role for BSP in mineralization has been indicated, BSP also appears to function in patho-physiological processes, including the metastasis of breast and prostate cancer cells to bone. The purpose of this study was to determine the role of BSP in the homing of cancer cells and to provide insights into the role of BSP in physiological as well as pathological processes. We established cultures of MDA-231 breast cancer cells stably transfected with DNA constructs of pIRES2-EGFP (green fluorescent protein) expressing human BSP (hBSP) cDNA (231BSP) under a CMV promoter, or with an antisense sequence of hBSP cDNA (231BSPAS), or with an empty vector as a control (231EV). These 3 cell groups were selected for neomycin resistance using G418 and analyzed by flow cytometry for GFP expression. The resultant cultured cells expressed different levels of hBSP as detected by RT-PCR and Western blot. Among the three, 231BSP expressed the highest levels of hBSP while 231BSPAS expressed the lowest. The capacity of the tumor cells to metastasize to bone was determined in nude mice (5 in each group) by intra-cardiac injection of the cells from the 3 different groups. Four weeks after inoculation, radiological examination revealed that all the 5 mice in the 231BSP cell group had developed osteolytic bone metastases. In the 231BSPAS group only 1 mouse demonstrated metastatic bone lesions while 3 out of 5 mice in the control group (231EV) developed metastatic lesions in the bone. These results strongly suggest that BSP over-expression in human tumor cells can enhance bone metastasis of MDA-231 cells whereas repressed expression of BSP, using antisense BSP cDNA, inhibits this effect in a mouse model.     

乳腺癌细胞对成骨细胞增殖和分化功能的抑制作用

背景与目的：乳腺癌细胞对成骨细胞（osteoblast,OB）正常功能的影响作用尚不明确。本研究探讨人乳腺癌细胞MCF-7或MDA．MB．231条件培养基对OB正常功能的影响。方法：源于大鼠颅盖骨的原代OB与雌激素受体阴性（ER-）或阳性（ER＋）的MDA-MB-231细胞或MCF-7细胞的条件培养基共同培养,MTT法观察条件培养基对OB增殖的影响,对硝基苯磷酸盐偶氮法观察条件培养基对0B碱性磷酸酶（alkaline phosphatase,ALP）活性的影响,茜素红s进行矿化结节染色并计算面积以观察条件培养基对OB矿化能力的影响。结果：MDA—MB．231细胞和MCF．7细胞条件培养基使OB发生纤维细胞形态改变．使细胞突起和突起连接均减少,并显著抑制OB的增殖。与培养基对照组比较,加入50％来自MDA-MB-231S细胞或MCF-7细胞的条件培养基后1d、3d、5d和7d,OB的增殖抑制率分别为18．1％、13．0％、19．2％、19．3％和15．8％、20_8％、33．9％、28．7％,两组之间的差异均有统计学意义（P〈0．01）。条件培养基可明显下调OB的ALP活性,并呈剂量．效应关系,50％的条件培养基使ALP活性分别下降31．9％和47．5％（p〈O．01）。条件培养基对OB的矿化结节形成能力也呈明显抑制作用：加入50％MDA．MB．231细胞或MCF．7细胞的条件培养基培养后,OB形成矿化结节面积分别减少89％和74％（P〈0．01）。结论：乳腺癌细胞可抑制OB的增殖能力和矿化形成能力,下调其AKP活性。     

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells

Background:

Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC.

Methods:

We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines.

Results:

D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells.

Conclusion:

Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.     

A murine monoclonal antibody to human breast cancer cells associated with DNA ploidy status

A murine monoclonal antibody, 5D10, raised against the human breast cancer cell line MCF7 reacted preferably with mammary carcinomas and weakly with normal epithelial cells. The antigens recognised by the antibody had molecular weights of about 28 and 90 kD. The reactivity of the antibody to human breast carcinomas correlated with the DNA ploidy status of the tumour cells. Upon analysis of 54 breast carcinoma specimens, the percentage of antibody positive cells was significantly higher in tumours with an aneuploid stemline than in those with a diploid DNA content (P < 0.001). This antibody therefore could be a useful tool in evaluating the prognosis of breast carcinomas.