磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶信号通路参与核因子-κB受体活化因子配体诱导的MDA-MB-231乳腺癌细胞迁移

目的探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号通路在核因子-κB受体活化因子配体(RANKL)诱导的乳腺癌细胞迁移中的作用。方法 Transwell法测定RANKL刺激后MDA-MB-231细胞迁移能力的改变。蛋白印迹法检测MDA-MB-231细胞表面RANK蛋白的表达及RANKL刺激后pAkt及Akt的表达;检测数据用x珚±s表示,采用SPSS 16.0软件进行t检验。结果 MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强,RANKL的圈套受体骨保护素(OPG)可阻断RANKL诱导的细胞迁移(P<0.01)。RANKL刺激后MDA-MB-231细胞p-Akt表达升高,PI3K抑制剂LY294002抑制RANKL诱导的细胞迁移(P<0.01)。结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞MDA-MB-231迁移。     

细胞外调节蛋白激酶参与RANKL诱导的SGC-7901胃癌细胞迁移

目的检测RANK在胃癌细胞系SGC-7901细胞中的表达,并进一步探讨细胞外调节蛋白激酶(ERK1/2)信号通路在RANKL诱导的胃癌细胞迁移中的作用。方法流式细胞术检测SGC-7901细胞表面RANK蛋白的表达;western-blot检测RANKL刺激后磷酸化ERK(p-ERK1/2)及ERK1/2的表达;Transwell法测定RANKL刺激后细胞迁移能力的改变。检测数据用x±s表示,采用SPSS16.0软件分析实验数据。结果 SGC-7901细胞表达RANK蛋白。RANKL诱导SGC-7901细胞迁移能力增强,RANK抑制剂rOPG抑制RANKL诱导的细胞迁移,RANKL刺激后,SGC-7901细胞p-ERK1/2表达升高,应用MEK/ERK的抑制剂PD98059显著抑制RANKL诱导的胃癌细胞SGC-7901迁移。结论胃癌细胞系SGC-7901细胞表达受体RANK,ERK1/2信号通路参与RANKL诱导的SGC-7901细胞迁移。     

Gab2通过PI3K/Akt/ARK5/MMP途径影响乳腺癌的侵袭和转移

目的探讨Gab2在乳腺浸润性导管癌侵袭和转移中的分子机制,为临床预防乳腺癌的侵袭和转移提供理论依据。方法采用免疫组织化学SP法检测80例乳腺浸润性导管癌组织及癌旁相对正常组织(>5 cm)中Gab2蛋白表达情况,并分析其表达与乳腺浸润性导管癌临床病理参数的相关性,分析浸润性导管癌中Gab2、MMP-2及MMP-9蛋白表达的相关性。采用Western blot检测乳腺癌细胞系MCF-7、MDA-MB-231中Gab2蛋白的表达情况。采用RNAi技术将小RNA干扰质粒瞬时转染乳腺癌细胞系MDA-MB-231,应用Western blot检测转染成功后各组细胞中MMP-2和MMP-9蛋白的表达情况,应用Transwell体外侵袭实验检测各组细胞的侵袭性,用EGF刺激细胞后Western blot检测Akt及ARK5的磷酸化情况。结果浸润性导管癌组织中Gab2蛋白的阳性表达率明显高于癌旁正常组织(P<0.01),浸润性导管癌中Gab2蛋白的表达与ER、组织学分期及淋巴结转移密切相关(P<0.05),且其表达与MMP-2及MMP-9蛋白的表达呈正相关;MDA-MB-231细胞系中Gab2蛋白表达量高于MCF-7细胞系中表达量;转染干扰质粒24 h后,与转染空载细胞组相比,SiG ab2/MDA-MB-231细胞组中Gab2蛋白的表达量明显降低,结果显示转染成功。同时,SiG ab2/MDA-MB-231细胞组穿过Transwell小室人工基膜数量明显减少(P<0.05),并伴有MMP-2、MMP-9蛋白表达降低(P<0.05)。用EGF刺激各组细胞,结果显示:在SiG ab2/MDAMB-231细胞组Akt及ARK5的磷酸化明显受到抑制。结论Gab2通过PI3K/Akt/ARK5信号通路影响MMP-2、MMP-9的表达从而促进乳腺癌的侵袭与转移。     

延龄草总皂苷抑制乳腺癌细胞MDA-MB-231转移的作用及机制研究

目的观察延龄草总皂苷对乳腺癌细胞MDA-MB-231侵袭与迁移的影响及相关机制。方法培养乳腺癌细胞MDA-MB-231,加入延龄草总皂苷(1.5、3 mg·L~(-1))24 h后,采用细胞侵袭实验、划痕实验观察延龄草总皂苷对MDA-MB-231细胞侵袭与迁移的影响,以Western blot方法检测细胞中MMP-2、MMP-9蛋白的表达。结果延龄草总皂苷能够有效地抑制MDA-MB-231细胞的侵袭与迁移,降低MMP-2与MMP-9的蛋白表达并呈现浓度依赖性。结论延龄草总皂苷能够有效地抑制乳腺癌细胞MDA-MB-231侵袭与迁移,其可能机制与降低MMP-2、MMP-9的表达水平有关。     

孕激素及雌激素对乳腺癌细胞RANKL及其受体的调节作用

目的 观察孕激素及雌激素对不同乳腺癌细胞系中核因子kB受体活化因子配体(RANKL)及其受体(RANK、OPG)表达的调节作用.方法 取对数生长期的乳腺癌细胞系T47D、MCF-7及MDA-MB-231,分别给予孕激素及雌激素刺激.采用RT-PCR测定各乳腺癌细胞系中RANKL、RANK、OPG以及孕激素受体(PR)的表达.结果 孕激素可以明显提高T47D及MCF-7细胞中RANKL的表达.孕激素和雌激素作用后,RANK在乳腺癌细胞系T47D及MCF-7中的表达明显下降.然而孕激素及雌激素对于OPG在T47D及MCF-7细胞中表达的调节作用却不明显.结论 RANKL及其受体在T47D与MCF-7细胞系中的表达受到孕激素及雌激素的调节,提示孕激素和雌激素有可能通过RANKL及其受体影响乳腺癌细胞的骨转移.     

NF-κB和MAPK信号通路参与金雀异黄酮诱导乳腺癌MDA-MB-231细胞凋亡的体外研究

目的探讨金雀异黄酮(genistein)诱导人乳腺癌MDA-MB-231细胞凋亡的可能机制。方法采用MTT法观察金雀异黄酮对乳腺癌MDA-MB-231细胞增殖的抑制作用;集落形成法观察金雀异黄酮对MDA-MB-231细胞集落形成能力的影响;金雀异黄酮干预MDA-MB-231细胞36 h后,Western blot检测凋亡相关蛋白Bcl-2、Bax、caspase-3及NF-κB、ERK、p-ERK、JNK、p-JNK蛋白的表达。结果 MTT结果显示,金雀异黄酮呈时间、浓度依赖性抑制乳腺癌MDAMB-231细胞增殖;集落形成实验结果显示,金雀异黄酮能明显抑制乳腺癌MDA-MB-231细胞的集落形成(P<0.05);Western blot结果显示,金雀异黄酮干预乳腺癌MDA-MB-231细胞36 h后,与对照组相比,Bcl-2、NF-κB、p-ERK蛋白表达水平明显下调(P<0.05),Bax、caspase-3、p-JNK蛋白表达水平明显上调(P<0.05)。结论金雀异黄酮能抑制乳腺癌MDA-MB-231细胞生长,且能诱导其凋亡,其机制可能与抑制NF-κB、ERK,激活JNK信号转导通路有关。     

吞噬细胞运动蛋白1在IL-8诱导的乳腺癌细胞的侵袭和转移中发挥重要作用

目的探讨吞噬细胞运动蛋白1(engulfment and cell motility 1,ELMO1)在IL-8诱导的乳腺癌细胞侵袭和转移过程中的作用。方法采用趋化运动实验检测不同浓度IL-8刺激下乳腺癌细胞的趋化运动能力;采用Western blot检测乳腺癌细胞中ELMO1的表达情况;利用小RNA干扰技术,瞬时转染乳腺癌细胞MDA-MB-231,用过表达质粒上调乳腺癌细胞MCF-7中ELMO1的表达;应用趋化运动实验和Transwell侵袭实验检测各组转染细胞的趋化和侵袭能力。结果趋化运动实验结果显示,在IL-8刺激下,乳腺癌细胞MDA-MB-231和MCF-7的运动能力明显增强,具有剂量依赖性;Western blot结果显示ELMO1在si ELMO1/MDA-MB-231细胞中的表达明显降低,而在MCF-7/ELMO1细胞中的表达明显增高;趋化运动实验结果显示在IL-8刺激下,Si ELMO1/MDA-MB-231细胞组的趋化运动能力明显降低,MCF-7/ELMO1细胞组的趋化运动能力明显增强;Transwell侵袭实验结果显示在IL-8刺激下,敲除ELMO1明显降低MDA-MB-231细胞的侵袭能力,过表达ELMO1明显增强MCF-7细胞的侵袭能力。结论 IL-8能促进MDA-MB-231和MCF-7细胞的趋化运动和侵袭能力,而ELMO1在IL-8诱导的乳腺癌细胞趋化和侵袭作用中发挥重要作用。     

金雀异黄酮对三阴乳腺癌MDA-MB-231细胞凋亡及EGFR/PI3K/Akt通路的影响

目的研究金雀异黄酮(genistein)诱导三阴性乳腺癌MDA-MB-231细胞凋亡及其机制。方法 MTT法观察金雀异黄酮对乳腺癌MDA-MB-231细胞增殖的抑制作用;Hoechst 33258染色观察金雀异黄酮对MDA-MB-231细胞核凋亡形态学的影响;qRT-PCR法观察金雀异黄酮干预MDAMB-231细胞36 h后,EGFR mRNA表达水平的变化;金雀异黄酮干预MDA-MB-231细胞36 h后,Western blot检测凋亡相关蛋白Bcl-2、Bax、caspase-3,EGFR、Akt、p-Akt蛋白的变化;Akt激活剂胰岛素(insulin)、金雀异黄酮单独及联合胰岛素干预乳腺癌MDA-MB-231细胞后,Western blot检测Akt和p-Akt蛋白表达量的变化。结果 MTT结果显示,金雀异黄酮呈时间浓度依赖性抑制乳腺癌MDA-MB-231细胞增殖;Hoechst 33258染色结果显示,金雀异黄酮干预乳腺癌MDAMB-231细胞36 h后细胞核呈现典型凋亡形态学改变;qRTPCR结果显示,经金雀异黄酮干预MDA-MB-231细胞36 h后,EGFR的mRNA表达水平明显下降(P<0.01);Western blot结果显示金雀异黄酮干预乳腺癌MDA-MB-231细胞36 h后,与对照组对比,Bcl-2、EGFR、Akt、p-Akt蛋白表达水平明显下调(P<0.01),Bax、caspase-3蛋白表达水平明显上调(P<0.01),Akt激活剂胰岛素可以明显激活p-Akt(P<0.01),金雀异黄酮可以明显下调被激活的p-Akt(P<0.01)。结论金雀异黄酮能抑制三阴乳腺癌MDA-MB-231细胞生长并诱导其凋亡,其机制可能与抑制EGFR/PI3K/Akt信号通路有关。     

4-氨基-2-三氟甲基苯基维甲酸酯对人乳腺癌细胞株MDA-MB-231诱导分化作用及可能的机制研究

目的观察4-氨基-2-三氟甲基苯基维甲酸酯(4-anino-2-trifluoromethyl-phenyl retimate,ATPR)对人乳腺癌细胞株MDA-MB-231抑制增殖诱导分化作用,探讨其可能的作用机制。方法体外培养人乳腺癌细胞株MDA-MB-231,MTT检测细胞增殖,绘制细胞生长曲线,瑞氏-吉姆萨染色观察细胞形态变化,酶联免疫法检测粘蛋白MUC-1活性,流式细胞术检测细胞周期,实时荧光定量PCR法和Western blot法检测维甲酸受体(retinoic acid receptors,RAR)RARα、RARβ、RARγ和维甲类受体(retinoid X receptors,RXR)RXRα、RXRβ、RXRγ基因和蛋白的表达。结果 ATPR能够抑制MDA-MB-231细胞的增殖,具有浓度-时间依赖性,染色后镜下观察MDA-MB-231细胞生长密度降低,形态趋于正常。ELISA结果显示,ATPR作用后明显降低MDA-MB-231细胞培养上清中MUC-1的浓度;流式细胞术结果显示,MDA-MB-231细胞中G0/G1期表达量增加,S期表达量减少,细胞阻滞在G0/G1期比例增加。q-RT-PCR和Western blot结果显示,ATPR作用后,RARγ的mRNA和蛋白表达水平降低,RXRs mRNA和蛋白水平无明显变化。结论 ATPR可以抑制人乳腺癌细胞株MDA-MB-231增殖并诱导其分化,其机制可能与RARγ的表达有关。     

刺五加皂苷B/E对乳腺癌MDA-MB-231细胞迁移能力的影响及作用机制研究

目的：探讨刺五加皂苷B/E对乳腺癌MDA-MB-231细胞迁移能力的影响及作用机制。方法：CCK-8检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞增殖能力的影响;细胞划痕实验检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞迁移的影响;Transwell实验检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞侵袭的影响;试剂盒检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞乳酸分泌的影响;PCR检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞HK、PFK和Glut4基因表达的影响;Western Blot法检测刺五加皂苷B/E对乳腺癌MDA-MB-231细胞HK、PFK和Glut4蛋白表达的影响。结果：刺五加皂苷B/E 25、50、100、200 μmol·L-1剂量不抑制MDA-MB-231细胞增殖;刺五加皂苷B/E 100、200 μmol·L-1能够抑制MDA-MB-231细胞迁移;刺五加皂苷B/E 50、100、200 μmol·L-1能够抑制MDA-MB-231细胞侵袭和乳酸分泌,也能够降低MDA-MB-231细胞有氧糖酵解中相关蛋白HK、PFK、Glut4的表达。结论：刺五加皂苷B/E能够抑制乳腺癌MDA-MB-231细胞的迁移、侵袭,其机制可能与抑制肿瘤细胞糖酵解有关。     

RANKL/RANK/OPG: key therapeutic target in bone oncology

Cancer is one of the major leading causes of death all over the world. Primary and secondary bone tumors can significantly deteriorate the quality of life (QOL) and the activity of daily living (ADL) of the patients. These unwelcome diseases become a social and economic burden seriously. Thus, more effective therapies for both primary and secondary bone tumors are actually required. Bone homeostasis depends on the strictly balanced activities between bone formation by osteoblasts and bone resorption by osteoclasts. Imbalance of bone formation and resorption results in various bone diseases. Both primary and secondary bone tumors develop in the unique environment bone, it is therefore necessary to understand bone cell biology in tumoral bone environment. Recent findings strongly revealed the significant involvement of the receptor activator of nuclear factor kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) triad, the key regulators of bone remodeling in bone oncology. Indeed, RANKL/RANK blocking successfully prevented the development of bone metastases. Furthermore, some cancer cells express RANK which is involved in tumor cell migration. Thus, the regulation of this triad will be a rational, encouraged therapeutic hot spot in bone oncology. In this review, we summarize the accumulating knowledge of the RANKL/RANK/OPG triad and discuss about its therapeutic capability in primary and secondary bone tumors.     

柚皮素对破骨细胞特异性基因及OPG/RANKL/RANK表达的影响

目的 观察柚皮素对破骨细胞特异性基因组织蛋白酶-K(CATK)、基质金属蛋白酶-9(MMP-9)、抗酒石酸酸性磷酸酶(TRAP)及骨保护素/核因子-κB受体活化因子配体/核因子-κB受体活化因子(OPG/RANKL/RANK)表达的影响.方法 采用全骨髓细胞诱导法培养破骨细胞,细胞分5组:空白对照组、HG-DMEM诱导组、HG-DMEM+0构.1μmol/L柚皮素组、HG-DMEM+1μmol/L柚皮素组、HG-DMEM+10μmol/L柚皮素组.HE染色、TRAP染色观察破骨细胞形态;分光光度计检测TRAP阳性细胞数;Real time-PCR和Western-blot检测CATK、MMP-9、TRAP及OPG/RANKL/RANK mRNA和蛋白表达.结果 与空白对照组比较,HG-DMEM诱导组TRAP阳性细胞数、TRAP、MMP-9、CATK、RANKL、RANK mRNA和蛋白表达明显增加,OPG mRNA和蛋白表达明显降低(P<0.05).与HG-DMEM诱导组比较,柚皮素预处理24 h能够明显降低TRAP阳性细胞数、CATK、MMP-9、TRAP、RANKL、RANK mRNA和蛋白表达,增加OPG mRNA和蛋白表达(P<0.05),呈剂量依赖性.结论 柚皮素能够抑制破骨细胞的生成和分化,该作用可能是通过OPG/RANKL/RANK信号通路抑制TRAP、MMP-9、CATK表达实现的.     

Zoledronic acid inhibits RANK expression and migration of osteoclast precursors during osteoclastogenesis

Bisphosphonates have been known to directly inhibit bone resorption and promote apoptosis in mature osteoclasts. Although bisphosphonates have been recognized as the most effective drugs to treat osteoporosis and bone cancer metastasis, the exact effects and mechanism(s) of bisphosphonates on osteoclastogenesis are unclear. The aim of this study was to clarify whether nitrogen-containing bisphosphonates affect recruitment and differentiation in osteoclasts. We examined the effects of zoledronic acid on receptor activator of NF-κB (RANK) expression and cell migration during osteoclastogenesis in two types of osteoclast precursors, RAW264.7 cells and Bone marrow cells (BMCs). Tumor necrosis factor-α (TNF-α) and RANK ligand (RANKL) upregulated RANK expression in RAW264.7 and BMCs in the presence of macrophage colony stimulating factor in a time-dependent manner. Zoledronic acid (30 and 50 μM) had no effect on cell viability in osteoclast precursors after 36 h of cultivation. Zoledronic acid (10 and 30 μM) strongly inhibited TNF-α- and RANKL-induced upregulation of RANK in a dose-dependent manner. The inhibitory effects on RANK expression were likely to be associated with the suppression of the NF-κB pathway, but not other downstream signaling pathways. Zoledronic acid (30 μM) also suppressed the TNF-α- and RANKL-induced migration of precursors by inhibiting the mevalonic acid pathway. Our results suggest that nitrogen-containing bisphosphonates not only inhibit mature osteoclasts but also prevent osteoclast precursors from differentiating and migrating towards inflammatory osteolysis lesions.     

Gab2 antisense oligonucleotide blocks rat basophilic leukemic cell functions

Adapter molecule Grb2-associated binder-like protein 2 (Gab2) plays a critical role in FcepsilonRI-induced mast cell degranulation and activation. The present study aimed to investigate the pharmacological effects of an antisense oligonucleotide (ASO) targeted at Gab2 on the immune responses of rat basophilic leukemic (RBL)-2H3 cells. Gab2 ASOs were rationally designed and transfected into RBL-2H3 cells. Gab2 mRNA and protein knockdown was confirmed by real-time RT-PCR and immunoblotting, respectively. Effects of Gab2 ASO on FcepsilonRI-induced release of histamine and beta-hexosaminidase was measured by EIA and an enzymatic assay, respectively; signaling events by immunoblotting; and cytokine mRNA expression by RT-PCR. Effects of Gab2 ASO on cell adhesion and migration were performed on fibronectin-coated 96-well plate and transwells cell culture chambers, respectively. We have characterized a phosphorothioate-modified ASO targeted at Gab2 mRNA that was able to knockdown Gab2 mRNA and protein in RBL-2H3 cells. Gab2 ASO significantly blocked IgE-mediated mast cell release of beta-hexosaminidase and histamine; phosphorylation of Akt, p38 mitogen-activated protein kinase and PKCdelta; and up-regulation of cytokine mRNA levels (e.g. IL-4, -6, -9 and -13, and TNF-alpha). In addition, Gab2 ASO markedly prevented mast cell adhesion to fibronectin-coated plates and restrained random migration of RBL-2H3 cells in cell culture chambers. Our findings show that Gab2 knockdown in RBL-2H3 cells by ASO strategy can suppress many aspects of the mast cell functions and, therefore, a selective Gab2 ASO may have therapeutic potential for mast cell-dependent allergic disorders.     

RANK/RANKL/OPG系统及其相关疾病和抗RANKL疗法的研究进展

骨骼是一个动态组织,在生物的整个生命周期中不断地进行构建、吸收、重建,核因子(nuclear factor,NF)κB受体活化因子(receptor activator of NF-κB,RANK)/NF-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)/护骨素(osteoprotegerin,OPG)系统是调控这一过程的关键系统.RANK/RANKL/OPG属于肿瘤坏死因子及其受体超家族,三者通过调控破骨细胞的分化和活化来影响骨吸收和重建的过程.另外,免疫细胞也参与和调节RANK/RANKL的表达和分泌,而免疫细胞本身亦受到该系统的影响,因此,RANK/RANKL/OPG系统成为骨和免疫之间的重要关联.RANK/RANKL/OPG系统的失衡与多种骨代谢性疾病及免疫系统疾病引发的继发性骨病密切相关,该系统的发现为研究相关疾病的病理机制和治疗方法提供了基础.由此建立的抗RANKL疗法已经开始应用于临床试验和研究.此文对RANK/RANKL/OPG系统、系统相关疾病以及抗RANKL治疗的进展做一综述.     

Monensin inhibits proliferation,migration, and promotes apoptosis of breast cancer cells via downregulating UBA2

Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast gland, the morbidity, and mortality of which continue to increase. Therefore, it is crucial to find new drugs to treat breast cancer. Monensin is a carrier antibiotic that has been reported to inhibit the growth of cancer cells; however, its impacts on breast cancer cells have not been reported. In this article, the cell survival rate was measured by CCK-8. Colony formation assay was utilized to detect the level of cell proliferation. Transwell was used to measure the ability of cell invasion, and wound healing was used to measure the ability of cell migration. RT-qPCR and western blot were, respectively, used to detect the expression of related genes and proteins. The level of apoptosis was detected by flow cytometry. Cell transfection technique was used for overexpressing UBA2. We found that Monensin inhibited the proliferation and migration of breast cancer cells and inhibited the expression of MMP-2 and MMP-9. In addition, Monensin promoted the apoptosis accompanied by the increase of Bax, caspase3, caspase7, and caspase9 and the decreased of bcl-2 of breast cancer cells. Monensin was also found to inhibit UBA2 expression in breast cancer cells. Subsequently, after overexpression of UBA2, the impacts of Monensin on proliferation, migration, and apoptosis of breast cancer cells was inhibited. In conclusion, Monensin can inhibit the proliferation and migration and activate apoptosis of breast cancer cells via downregulating the expression of UBA2.     

miR-1307-3p通过靶向ISM1促进乳腺癌细胞的增殖和迁移

目的 探讨miR-1307-3p通过靶向ISM1促进乳腺癌细胞增殖和迁移的分子机制。方法 （1）利用TCGA 数据库分析miR-1307-3p在乳腺癌患者中的表达水平及其与临床指标的关系。（2）利用qPCR、CCK-8实验、细胞划 痕实验和流式细胞术检测过表达或沉默miR-1307-3p后乳腺癌MCF-7细胞miR-1307-3p表达、细胞增殖、迁移和凋 亡水平变化。（3）生物信息学分析软件预测 miR-1307-3p 的靶基因,同时采用双荧光素酶实验进行验证。结果 miR-1307-3p在乳腺癌细胞及乳腺癌组织中均显著上调。miR-1307-3p过表达可促进MCF-7细胞增殖和迁移;而 miR-1307-3p inhibitor则抑制细胞迁移,并诱导凋亡。ISM1可能是miR-1307-3p的靶基因。乳腺癌组织中ISM1表 达水平明显低于正常乳腺组织,且与临床病理分期有关。 结论miR-1307-3p可促进乳腺癌细胞增殖和迁移,可能 通过调控ISM1基因而影响乳腺癌的发生发展。