Rab27A对人乳腺癌细胞生物学特性的影响

背景与目的：Rab蛋白属Ras超家族中有超过60个成员的最大的一个亚家族。Rab27A是这一家族中唯一一个明确涉及到人类遗传性疾病的成员。本研究探讨Rab27A对人乳腺癌细胞生物学特性的影响及其机制。方法：构建含有Rab27A开放阅读框的真核表达质粒，通过脂质体转染的方法将其导入乳腺癌细胞MDA-MB-231，同时采用RNA干扰的方法下调Rab27A的表达。测定Rab27A上调及下调表达后乳腺癌细胞生长曲线、细胞周期分布、凋亡细胞比例、体外侵袭等生物学特性的变化，并采用RT．PCR方法来探讨Rab27A发挥作用的分子机制。结果：①Rab27A的表达水平随乳腺癌细胞侵袭转移能力的增强而增高。②上调Rab27A表达后导致乳腺癌细胞生长加速、s期细胞比例增多、凋亡细胞比例下降、体外侵袭能力增强。③Cyclin D1、MMP-7和MMP-9基因表达水平与Rab27A表达呈正相关，而p16的表达与Rab27A的表达呈负相关。结论：Rab27A通过调节Cyclin D1、MMP-7、MMP-9和p16基因的表达，从而在乳腺癌细胞增殖、细胞周期分布、凋亡和侵袭中发挥作用。     

结缔组织生长因子与乳腺癌及其血管形成的关系

结缔组织生长因子是即刻早期反应基因的家族成员之一，CCN家族中富含半胱氨酸的外分泌型多肽，是转化生长因子β1的下游效应子，参与了血管平滑肌细胞和内皮细胞的生长、迁移，在创伤修复中参与了血管生成，但在肿瘤及其血管形成过程中的作用机制至今未明。乳腺组织中含有丰富的纤维结缔组织基质，尤其在乳腺癌中结缔组织生长因子含量明显增高，故它很可能参与了乳腺癌的各种生物学全过程。     

结缔组织生长因子与乳腺癌的研究进展

乳腺癌是女性肿瘤中发病率最高的 1种 ,全世界共约有乳腺癌患者 5 7万例[1] 。大多数乳腺癌为实体癌 ,可分为单纯癌(实质与间质量大致相等 )、硬癌 (癌巢小而少、间质结缔组织多 )和癌髓样癌 (癌巢大而多、结缔组织相对较少 ) 3种类型。纤维结缔组织在正常乳腺组织中较丰富 ,在病变乳腺组织中的含量则明显增高 ,故围绕纤维结缔组织改变的研究对乳腺肿瘤的发生可能有重要意义。结缔组织生长因子 (connectivetissue growthfactor ,CT GF)属于CCN家族 (其他成员还包括Cef10 /ctr61、Cyr6WISP 1、Cyr61、Nxov、NOVH等 )。该因子在肿瘤发…     

结缔组织生长因子对胰腺癌细胞增殖和转移的影响

目的 探讨结缔组织生长因子(CTGF)在胰腺癌中的表达及其对胰腺癌细胞增殖和转移的影响.方法 采用实时定量聚合酶链反应(PCR)和免疫组化方法,分别检测胰腺癌细胞株PANC-1和50例胰腺癌组织中CTGF的表达.采用重组腺病毒介导的CTGF转染PANC-1细胞使CTGF高表达,以RNA干扰方法使CTGF表达缺失,分别通过四甲基偶氮唑蓝(MMT)法、划痕实验、体外侵袭实验观察PANC-1细胞增殖和转移能力的变化.结果 实时定量PCR和免疫组化检测均显示,CTGF在PANC-1细胞及胰腺癌组织中高表达.CTGF转染后,CTGF转染组PANC-1细胞比空载体对照组增殖能力和修补划痕能力明显增强,200倍显微镜视野下的穿膜细胞数为34个,比空载体对照组(11个)明显增多.干扰CTGF后,siCTGF转染组细胞比空载体对照组增殖能力和修补划痕能力明显降低,200倍显微镜视野下的穿膜细胞数为6个,比空载体对照组(15个)明显减少.结论 CTGF在胰腺癌中高表达,其表达的高低对胰腺癌细胞的增殖和转移有显著影响.     

克霉唑对结肠癌细胞系CCL229生物学行为的影响

目的 研究克霉唑对结肠癌细胞生长,粘附和侵袭转移中的作用,并初步探讨其机制。方法用不同浓度的克霉唑加入培养中的结肠癌细胞株ＣＣＬ２２９中,观察加药前后细胞生长速度的变化,检测细胞与基质的粘附能力,用细胞分离试验测定细胞的同质粘附性。Ｂｏｙｄｅｎ小室法检测细胞的侵袭能力;流式细胞光度术检测细胞尿激酶型纤溶原激活剂（Ｕｒｏｋｉｎａｓｅ－ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ＵＰＡ）表达。结     

结缔组织生长因子在肿瘤中的作用及胃癌中研究进展

结缔组织生长因子(CTGF)是CCN家族分泌蛋白成员之一,通过多种机制参与肿瘤的发生发展、浸润转移、血管生成等过程。全文就近年来CTGF在肿瘤发生发展中的作用,及其在胃癌中的研究进展作一简要综述。     

上皮钙黏着蛋白对人炎性乳腺癌细胞系生物学特性的影响

目的 研究上皮钙黏着蛋白（E-cadherin）对人炎性乳腺癌细胞系SUM149的生长、侵袭等生物学特性的影响。方法 应用脂质体法基因转染技术,将编码E-cadherin基因显性负调控突变体（H-2k^d-E-cadherin）质粒导入SUM149中,应用RT-PCR、流式细胞分析法筛选、鉴定出E-cadherin基因显性负调控突变体高表达的阳性克隆。观察转染前后人炎性乳腺癌细胞系SUM149生长、侵袭能力等特性的变化以及相关分子的改变。结果 RT-PCR及Western印迹法结果均显示,与对照组（未转染及空质粒组）相比,转染后高表达小鼠H-2k^d的阳性克隆细胞的内源性上皮钙黏着蛋白表达明显下调,其细胞生长速度增殖无明显变化,而体外侵袭能力分析显示其侵袭能力明显下降,进一步研究发现,基质金属蛋白酶MMP-1、MMP-9在mRNA水平明显下调;明胶酶谱分析也显示,MMP-9明显下调。结论 在E-cadherin高表达的人炎性乳腺癌细胞系SUM149中,其表达的下调可明显抑制其侵袭能力。     

不同HER-2表达水平对乳腺癌细胞生物学行为的影响

背景与目的:人表皮生长因子受体-2(human epidermal growth factor receptor-2,HER-2)属于受体酪氨酸激酶中的生长因子受体家族,其表达差异在乳腺癌靶向药物的临床使用中起决定作用.该研究旨在筛选出HER-2不同表达水平的乳腺癌细胞株,研究HER-2表达差异对肿瘤细胞生物学行为的影响.方法:对乳腺癌细胞系SK-BR-3进行克隆纯化,利用德国西门子公司ADVIA Centuar CP系统电化学发光技术检测细胞培养上清液中可溶性HER-2(soluble HER-2,sHER-2)的表达水平,筛选出sHER-2高表达细胞株(大于50.0 ng/mL)、中表达细胞株(15.8~50.0 ng/mL)及低表达细胞株(小于15.8 ng/mL).通过细胞培养观察以上3种乳腺癌细胞株的形态学变化,并通过一系列体外实验比较细胞的生物学特性,包括克隆形成实验、划痕实验和Transwell检测等.结果:与正常乳腺上皮细胞相比,乳腺癌细胞系SK-BR-3表达的sHER-2水平明显增高.其中,sHER-2高表达细胞株的克隆形成率为(51.3±3.4)%,明显高于中表达细胞株[(42.0±3.7)%]和低表达细胞株[(26.7±2.9)%];sHER-2高表达细胞株的迁移率为(50.0%±0.6)%,明显高于sHER-2中表达细胞株[(19.5±3.4)%]和sHER-2低表达细胞株[(13.6±1.0)%];sHER-2高表达细胞株的侵袭能力为(53.5±4.2)%也明显比sHER-2中表达细胞株[(33.2±3.9)%]和低表达细胞株[(28.9±5.4)%]细胞株强,差异均有统计学意义(P<0.05).结论:乳腺癌sHER-2高表达细胞株具有促增殖、促细胞动力等生物学效应,因此能为临床合理使用乳腺癌靶向药物提供参考依据.     

肝细胞生长因子对人结肠癌细胞系内皮生长因子表达影响的观察

目的:探讨人重组肝细胞生长因子(rhHGF)对结肠癌细胞SW480和SW620中3种内皮生长因子VEGF-A、VEGF-C和VEGF-D表达的影响.方法:实验分对照组(5%胎牛血清)和HGF组(80 ng/mL的HGF).采用MTT法分析HGF对肿瘤细胞增殖的作用,流式细胞术检测HGF对细胞周期的影响.48 h后进行蛋白质印迹法检测,并对信号强度进行相对定量分析.结果:HGF能促进SW480和SW620增殖.流式细胞术结果显示,与对照组相比,SW480的HGF组中的G0/G1期细胞减少,S期细胞增多,G2/M期细胞增多,但HGF对SW620细胞周期的影响不明显.SW480细胞HGF组的VEGF-A表达量是对照组的1.59倍,SW620细胞HGF组的VEGF-A表达量是对照组的2.08倍(P＜0.01);SW480细胞HGF组的VEGF-C表达量是对照组的1.37倍,SW620细胞HGF组的VEGF-C表达量是对照组的1.27倍(P＜0.01);SW480细胞HGF组的VEGF-D表达量是对照组的1.46倍,SW620细胞HGF组的VEGF-D表达量是对照组的1.38倍(P＜0.01).结论:HGF通过上调结直肠癌细胞VEGF-A、VEGF-C和VEGF-D的表达而促进肿瘤血管和淋巴管的新生,可能是潜在的结直肠癌治疗的新靶点.     

肝细胞生长因子基因转染对阿霉素诱导胃癌细胞调亡的影响

目的研究肝细胞生长因子（HGF）基因转染对阿霉素诱导的胃癌细胞凋亡的影响。方法先构建HGF基因的真核表达质粒pIRES2-EGFP-HGF,应用pIRES2-EGFP-HGF重组质粒和pIRES2-EGFP空质粒转染人胃癌MKN-45细胞,以未转染组为对照。用转染细胞的培养液培养MDCK细胞后,以细胞形态学改变来分析目的基因mRNA、蛋白的表达,分别用RT-PCR、Western blot测定其功能。MTT法测定阿霉素对细胞生长的抑制作用,DNA凋亡条带法和PI染色法检测细胞凋亡。结果稳定转染HGF基因的MKN-45细胞株可表达HGF mRNA,其分泌的HGF蛋白具有正常功能。MTT检测表明,HGF质粒转染组活细胞数高于空质粒转染和未转染组。0．1μg／ml阿霉素作用细胞后DNA凋亡条带分析发现,空质粒转染及未转染组的MKN-45细胞出现典型阶梯状条带, HGF转染组细胞凋亡条带不显著。流式细胞术结果显示,HGF质粒转染组细胞凋亡率显著低于未转染及空质粒转染组。结论HGF基因稳定转染可显著抑制阿霉素诱导的胃癌细胞凋亡。     

Bisphosphonates antagonise bone growth factors' effects on human breast cancer cells survival

Bone tissue constitutes a fertile 'soil' for metastatic tumours, notably breast cancer. High concentrations of growth factors in bone matrix favour cancer cell proliferation and survival, and a vicious cycle settles between bone matrix, osteoclasts and cancer cells. Classically, bisphosphonates interrupt this vicious cycle by inhibiting osteoclast-mediated bone resorption. We and others recently reported that bisphosphonates can also induce human breast cancer cell death in vitro, which could contribute to their beneficial clinical effects. We hypothesised that bisphosphonates could inhibit the favourable effects of 'bone-derived' growth factors, and indeed found that bisphosphonates reduced or abolished the stimulatory effects of growth factors (IGFs, FGF-2) on MCF-7 and T47D cell proliferation and inhibited their protective effects on apoptotic cell death in vitro under serum-free conditions. This could happen through an interaction with growth factors' intracellular phosphorylation transduction pathways, such as ERK1/2-MAPK. In conclusion, we report that bisphosphonates antagonised the stimulatory effects of growth factors on human breast cancer cell survival and reduced their protective effects against apoptotic cell death. Bisphosphonates and growth factors thus appear to be concurrent compounds for tumour cell growth and survival in bone tissue. This could represent a new mechanism of action of bisphosphonates in their protective effects against breast cancer-induced osteolysis.     

Deregulated expression of connective tissue growth factor (CTGF/CCN2) is linked to poor outcome in human cancer

Connective tissue growth factor (CTGF/CCN2) has long been associated with human cancers. The role it plays in these neoplasms is diverse and tumour specific. Recurring patterns in clinical outcome, histological desmoplasia and mechanisms of action have been found. When CTGF is overexpressed compared to low‐expressing normal tissue or is underexpressed compared to high‐expressing normal tissue, the functional outcome favours tumour survival and disease progression. CTGF acts by altering proliferation, drug resistance, angiogenesis, adhesion and migration contributing to metastasis. The pattern of CTGF expression and tumour response helps to clarify the role of this matricellular protein across a multitude of human cancers.     

结缔组织生长因子在卵巢癌中的表达及其临床意义

目的:探讨结缔组织生长因子(connective tissue growth factor,CTGF)在卵巢癌组织中的表达及临床意义.方法:采用免疫组化和逆转录聚合酶链反应(RT-PCR)方法检测105例卵巢癌、20例正常卵巢组织和30例良性卵巢肿瘤组织中CTGF的表达,并分析CTGF与卵巢癌临床病理参数的关系.结果:免疫组化检测观察到CTGF主要定位于细胞质内,卵巢癌、正常卵巢组织和良性卵巢肿瘤中CTGF的阳性表达率分别为53.33％ (56/105)、95.00％ (19/20)、93.33％ (28/30),卵巢癌中CTGF阳性表达率低于正常卵巢(x2=12.15,P=0.00)及良性卵巢肿瘤组织(x2=15.88,P=0.00),正常卵巢与良性卵巢肿瘤组织相比较差异无统计学意义(x2=0.05,P=0.80),RT-PCR检测结果显示卵巢癌中CTGF mRNA的表达明显低于正常卵巢组织和良性卵巢肿瘤组织(F=3.39,P=0.039).进一步采用x2检验统计分析得出CTGF与卵巢癌的临床分期、淋巴转移相关(P＜0.05),而与年龄、病理分级、病理类型及腹水癌细胞阳性率无相关性(P＞0.05).结论:CTGF在卵巢癌中低表达,与卵巢癌的发生、发展相关.     

Direct effects of bisphosphonates on breast cancer cells

In addition to inhibiting bone resorption, bisphosphonates have also been shown to exhibit antitumour effects. In vitro, bisphosphonates inhibit proliferation and induce apoptosis in cultured human breast cancer cells. In addition, bisphosphonate treatment interferes with breast cancer cell adhesion to bone matrix, and inhibits cell migration and invasion. The combination of bisphosphonates with other anticancer drugs such as the taxoids markedly enhances these effects. These newly recognized direct actions of bisphosphonates on breast cancer cells indicate that these agents may have a greater role to play in treatment of patients suffering from cancers with a propensity to metastasize to bone.     

P38 pathway as a key downstream signal of connective tissue growth factor to regulate metastatic potential in non‐small‐cell lung cancer

Although the secretory matricellular protein connective tissue growth factor (CTGF) has been reported to be related to lung cancer metastasis, the precise mechanism by which CTGF regulates lung cancer metastasis has not been elucidated. In the present study, we show the molecular link between CTGF secretion and the p38 pathway in the invasive and metastatic potential of non‐small‐cell lung cancer (NSCLC). Among three different human NSCLC cell lines (PC‐14, A549, and PC‐9), their in vitro invasiveness was inversely correlated with the level of CTGF secretion. By supplementing or reducing CTGF secretion in NSCLC culture, dysregulation of the invasive and metastatic potential of NSCLC cell lines was largely compensated. By focusing on the protein kinases that are known to be regulated by CTGF, we found that the p38 pathway is a key downstream signal of CTGF to regulate the metastatic potential of NSCLC. Importantly, a negative correlation between CTGF and phosphorylation status of p38 was identified in The Cancer Genome Atlas lung adenocarcinoma dataset. In the context of the clinical importance of our findings, we showed that p38 inhibitor, SB203580, reduced the metastatic potential of NSCLC secreting low levels of CTGF. Collectively, our present findings indicate that the CTGF/p38 axis is a novel therapeutic target of NSCLC metastasis, particularly NSCLC secreting low levels of CTGF.     

LKB1基因对乳腺癌细胞促进微血管生成的因子基因表达的影响

目的 研究LKB1基因对乳腺癌细胞促进微血管生成的因子基因表达的影响及机理。方法筛选LKB1基因转染的MDA-MB-435乳腺癌细胞，随机取样一株高表达细胞株和一株低表达细胞株，同时与未转染细胞株和空质粒转染细胞株比较。用RT-PCR观察促进微血管生成的因子的基因表达和分泌情况，并用Western blot对其蛋白定量分析。并采用Tramswell试验(Matrigel胶侵袭力检测法)对其进行膜穿透能力检测。结果 无论从mRNA还是蛋白水平上分析LKB1转染的乳癌细胞的促进微血管生成的因子基因表达VEGF和bFGF较未转染细胞和空质粒细胞为低，且高表达LKBl的细胞较低表达者为低。相似的结果在Tramswell试验得到证实。结论 LKB1基因作为一种抑癌基因在乳腺癌细胞的侵袭和转移中起到重要作用，特别对促进微血管生成的因子作用不容忽视。     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.     

Epidermal growth factor receptors in human breast cancer

Summary The capacity for specific binding of¹²⁵I-epidermal growth factor (EGF) was studied in crude membrane fractions from 95 human breast carcinomas. About 42% of the samples showed saturable, high affinity, specific binding of EGF. In 21% of the tumors we were able to demostrate high (above 10 fmoles/mg protein) binding capacity. Moreover, high EGF receptor values were associated with a low content of estradiol receptor. These studies are related to the definition of new biochemical markers in human breast cancer.