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医药导报, 2017, 36(8): 869-874
doi: 10.3870/j.issn.1004-0781.2017.08.007
唑来膦酸对食管鳞癌细胞转移的抑制作用*
Inhibition Effect of Zoledronic Acid on Metastasis of Esophagus Squamous Cell Carcinoma
辛淑波1,, 游颜杰2,, 林灿峰3

摘要:

目的 分析唑来膦酸(ZOL)对食管鳞癌细胞转移能力的抑制效应并探讨其分子机制。方法 食管鳞癌细胞株EC9706与EC109细胞经ZOL处理后,以噻唑蓝(MTT)比色法测定细胞增殖能力,细胞侵袭与划痕愈合实验检测细胞侵袭与转移能力,免疫印迹(Western blotting)、逆转录-聚合酶链反应(RT-PCR)与细胞免疫荧光实验检测转移相关蛋白表达变化。结果 不同浓度ZOL处理可显著抑制EC9706与EC109细胞的增殖、侵袭与迁移能力,同未加药对照组比较,均差异有统计学意义(P<0.05);ZOL处理通过抑制转录因子Slug转录上调紧密连接蛋白表达水平,转染因子Slug可逆转ZOL的抗细胞转移效应。结论 ZOL通过Slug调控紧密连接蛋白表达可能是其干预食管鳞癌细胞转移的分子机制。

关键词: 唑来膦酸 ; 食管鳞癌 ; 细胞增殖 ; 肿瘤侵润 ; 紧密连接蛋白

Abstract:

Objective To examine the inhibition effect of zoledronic acid (ZOL) on malignant metastasis of human esophagus squamous cell carcinoma (ESCC) cells and to analyze its molecular mechanisms. Methods EC9706 and EC109 cells were treated with ZOL,and then MTT assay,adhesion and invasion assay were performed to observe the inhibitory effect of As2O3 on proliferation and metastasis of esophagus carcinoma cells.The expression of metastasis-related proteins was detected by Western blotting. Results Exposure to ZOL significantly presented suppressive functions on growth and metastasis of both kinds of cancer cells,in a dose-dependent manner(P<0.05).Additionally,the expression level of occludin was increased after ZOL treatment by suppressing transcriptional factor Slug.Transfection of Slug could reverse anti-metastasis of ZOL. Conclusion ZOL possesses a significant anti-metastasis function on ESCC cells,mainly through repressing Slug to restore occludin expression.

Key words: Zoledronic acid ; Esophagus squamous cell carcinoma ; Cell proliferation ; Neoplasm invasiveness ; Occludin

我国是世界食管癌高发国家之一,以鳞状上皮细胞癌为主,其发病率和死亡率逐年上升[1-3]。研究表明远处转移是导致食管鳞癌治疗失败和患者死亡的主要原因之一[1-3]。唑来膦酸(zoledronic acid,ZOL)属于第3代的双膦酸盐类药物。一系列研究表明ZOL不仅直接抑制肿瘤细胞的增殖分化、诱导细胞凋亡,还可显著降低肿瘤细胞的迁移、侵袭能力[4-6]。有研究证实低浓度的ZOL作用于骨肉瘤细胞系导致细胞周期停滞在S期并伴随细胞周期蛋白(cyclin)的表达降低,尤其是cyclin E和cyclin D1的明显改变,从而阻滞DNA合成和细胞周期的进展[7-8]。随后在乳腺癌、肺癌、肾癌等的研究中进一步阐明ZOL通过提高P-ATR、P-chk1、Wee1、P-cdc2以及降低cdc25c的水平来激活S期DNA损伤检验点通路,将细胞周期阻滞在S和G2/M期[9-11]。然而目前在食管鳞癌中笔者尚未查到相关报道。本实验旨在分析ZOL处理对食管鳞癌细胞转移能力的影响并探讨其相关的分子机制,现报道如下。

1 材料与方法
1.1 试剂与材料

ZOL注射液(100 mL:5 mg/支,批号:GS110)购自瑞士诺华公司;DMEM细胞培养液(批号:31600034)与胎牛血清(批号:10099141)购自美国Gibco公司,Matrigel(批号:356234)购自美国BD Biosciences公司,Transwell小室(批号:PIHT30R48)购自美国Millipore公司,兔抗人Vimentin(批号:ab45939)、N-钙粘蛋白(批号:ab18203)与GAPDH(批号:ab9485)多克隆抗体购自英国Abcam公司,兔抗人occludin(批号:sc-5562)与E-钙粘蛋白(批号:sc-393153)、山羊抗人Slug(批号:sc-10437)与Snail(批号:sc-7862)多克隆抗体购自美国Santa Cruz公司,FITC荧光标记羊抗兔IgG(批号:ZF-0315)、HRP标记羊抗兔IgG(批号:ZDR-5306)购自北京中杉金桥公司,ECL Kit(批号:28-9068-37)购自美国Amersham公司,Trizol试剂(批号:9108Q)、逆转录试剂盒(批号:639536)、Taq酶(批号:R300S)与PCR引物购自大连TaKaRa公司,噻唑蓝(MTT,批号:M2128)购自美国Sigma公司,其他常规化学试剂均为进口或国产分析纯。pCMV-myc-Slug载体由白求恩医务士官学校冉永刚硕士惠赠。

1.2 主要仪器

二氧化碳(CO2)恒温细胞培养箱(美国Thermo公司)、倒置显微镜(美国Sigma公司)、分光光度计(德国Eppendorf公司)、荧光显微镜(德国Carl Zeiss公司)。

1.3 细胞培养与转染

人食管鳞癌细胞株EC9706与EC109由汕头大学医学院附属肿瘤医院中心实验室提供,置含10%胎牛血清的DMEM培养液,于37 ℃、5%CO2条件下常规培养,定时换液、传代。细胞转染按照LipofectamineTM 2000试剂说明书操作。

1.4 噻唑蓝(MTT)比色法检测细胞增殖能力

细胞以每孔5 × 103个·mL-1接种于96孔板中,设4个复孔,培养24 h;更换含有不同浓度(0~64 μmol·L-1)ZOL的培养液继续培养48或72 h,加入5 mg·mL-1MTT 20 μL作用4 h;弃去培养液加入DMSO 150 μL,振荡10 min,测定各孔490 nm吸光度值(A490)。实验重复3次取平均值。按下式计算相对细胞活力:细胞活力(%)=(实验组A490/对照组A490)×100%。

1.5 细胞侵袭实验

细胞经ZOL(0,2,4,8 μmol·L-1)处理6 h,以无血清DMEM培养液调整细胞密度为2 × 106个·mL-1,于每个Transwell上室内加入100 μL;将整个Transwell小室放入含10%FBS的DMEM培养液的24孔板内培养24 h;上室甲醇固定,结晶紫染色,擦掉位于上室面未穿膜细胞后封片,光镜下计数5个200倍视野的迁移细胞数。

1.6 划痕愈合实验检测细胞迁移能力

细胞经ZOL(0,2,4,8 μmol·L-1)处理6 h后接入12孔板,待细胞铺满板底后用200 μL枪头垂直于孔板制造划痕,冲洗去除划痕产生的细胞碎片,加入无血清培养基后继续常规培养,每隔6~12 h取出观察并采集图片。

1.7 免疫荧光

细胞以适当浓度铺与盖玻片上继续培养12 h,加入E-cadherin抗体,37 ℃作用1 h;充分洗涤后加入1:1 000稀释的FITC荧光标记的羊抗兔IgG,37 ℃作用30 min,荧光显微镜下摄相分析。

1.8 免疫印迹(Western blotting)与逆转录-聚合酶链反应(RT-PCR)检测转移相关分子表达

Western blotting:EC109细胞经8 μmol·L-1 ZOL处理24 h后,裂解细胞取50 μg总蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)后转印至PVDF膜上,含5%BSA的TBST中4 ℃封闭过夜;分别加入适当稀释的抗体,37 ℃作用1 h;充分洗涤后加入1:1 000稀释的HRP标记羊抗兔IgG,37 ℃作用30 min,以ECL Kit进行化学发光反应,胶片随后进行显影、定影,并以GAPDH作为半定量内参。RT-PCR:以Trizol试剂与逆转录试剂盒提取组织或细胞总RNA并合成cDNA,操作均按试剂说明书进行。反应条件:95 ℃预变性5 min;94 ℃变性60 s,55 ℃退火30 s,72 ℃延伸60 s,共30个循环;最后72 ℃延伸10 min。取适量产物经2%琼脂糖凝胶电泳分离,成像系统进行拍照分析。PCR扩增引物序列见表1。

表1 PCR引物序列特征
Tab.1 PCR primer sequences
引物 序列 规格/bp
vimentin-forward 5'-AATGGCTCGTCA CCTTCGTGAAT-3' 160
vimentin-reverse 5'-CAGATTAGTTTCCCTCAGGTTCAG-3'
N-cadherin-forward 5'-CGCCAATCAACTTGCCAGAA-3' 110
N-cadherin-reverse 5'-TGGCCCAGTGACGCTGTATC-3'
E-cadherin-forward 5'-TTAAGGGGTCTGTCATGGAAGGT-3' 106
E-cadherin-reverse 5'-GTGTAAGCGATGGCGGCATTGTA-3'
occludin-forward 5'-AAGGGAAGAGCAGGAAGGTC-3' 397
occludin-reverse 5'-CTCCAACCATCTTCTTGATGTG-3'
Snail-forward 5'-TTGGATACAGCTGCTTTGAG-3' 150
Snail-reverse 5'-ATTGCATAGTTAGTCACACCTC-3'
Slug-forward 5'-GAGTCTGTAATAGGATTTCCCATAG-3' 122
Slug-reverse 5'-CTTTAGTTCAACAATGGCAAC-3'
GAPDH-forward 5'-GACCCCTTCATTGACCTCAAC-3' 219
GAPDH-reverse 5'-CTTCT CCATGGTGGTGAAGAC-3'

表1 PCR引物序列特征

Tab.1 PCR primer sequences

1.9 统计学方法

上述实验均重复3次。采用SPSS 11.0版统计软件包,计量资料以均数±标准差( x ¯ ±s)表示,采用单因素方差分析,以P<0.05为差异有统计学意义。

2 结果
2.1 ZOL抑制食管鳞癌细胞增殖能力

EC109与EC9706细胞经不同浓度ZOL处理72 h后,其增殖能力相对于未处理(0 μmol·L-1)对照组显著降低,抑制效应呈剂量依赖性(图1),均差异有统计学意义(F=45.31,71.21,均P<0.05)。

图1 ZOL对ESCC细胞增殖能力的抑制作用比较(x¯±s,n=5)
A.EC109细胞;B.EC9706细胞;与0 μmol·L-1组比较,*1P<0.05,*2P<0.01

Fig.1 Inhibition effect of ZOL on the proliferation of ESCC cells(x¯±s,n=5)
A.EC109 cells;B.EC9706 cells;compared with 0 μmol·L-1group,*1P<0.05,*2P<0.01

2.2 ZOL干预食管鳞癌细胞侵袭能力

EC109与EC9706细胞经较低浓度ZOL(2,4,8 μmol·L-1)处理后,细胞侵袭能力较未处理(0 μmol·L-1)对照组明显减低(F=175.87,125.93,均P<0.05),抑制效应呈剂量依赖性(图2)。

图2 ZOL对ESCC细胞侵袭能力的抑制作用
A.切片图;B.矩形图;与0 μmol·L-1组比较,*1P<0.05,*2P<0.01

Fig.2 Inhibition effect of ZOL on invasiveness of ESCC cells
A.slice image;B.histogram;compared with 0 μmol·L-1group,*1P<0.05,*2P<0.01

2.3 ZOL抑制食管鳞癌细胞迁移能力

EC109与EC9706细胞经较低浓度ZOL(2,4,8 μmol·L-1)处理后,细胞划痕愈合(迁移)能力较未处理(0 μmol·L-1)对照组明显降低(F=175.87,125.93,均P<0.05),抑制效应呈剂量依赖性。见图3。

图3 ZOL对ESCC细胞迁移能力的抑制作用
A.切片图;B.矩形图;与0 μmol·L-1组比较,*1P<0.05,*2P<0.01

Fig.3 Inhibition effect of ZOL on the migration of ESCC cells
A.slice image;B.histogram;compared with 0 μmol·L-1group,*1P<0.05,*2P<0.01

2.4 ZOL对转移相关蛋白表达的调节

EC109细胞经8 μmol·L-1 ZOL处理24 h后,以RT-PCR与Western Blotting检测转移相关分子表达状态。结果显示,间质细胞标志物波形蛋白、N-钙粘蛋白表达相对于未加药(0 μmol·L-1)对照组明显减弱,而上皮细胞标志物中紧密连接蛋白表达水平明显上调,E-钙粘蛋白表达未见明显变化;转移相关转录因子Slug表达水平相对于对照组显著减弱,而Snail未见明显变化(图4A)。细胞免疫荧光实验进一步证实ZOL处理增强紧密连接蛋白表达水平,而未改变E-钙粘蛋白表达(二者均定位与细胞膜)(图4B)。

图4 ZOL对转移相关蛋白表达的调节
A.电泳图;B.免疫荧光图

Fig.4 Regulation of ZOL on the expression of metastasis-related proteins
A.electrophotogram;B.immunofluorescence

2.5 ZOL通过干预Slug转录上调紧密连接蛋白表达

EC109细胞经8 μmol·L-1 ZOL处理24 h后,转染Slug表达载体(pCMV-myc-Slug),以RT-PCR与Western blotting检测紧密连接蛋白表达状态。结果显示,过表达Slug可以明显逆转ZOL处理对紧密连接蛋白表达(蛋白与mRNA水平)的上调效应(图5A)。细胞实验证实,过表达Slug部分抵消ZOL处理对EC109细胞侵袭迁移能力的抑制作用(图5B,5C)。

图5 ZOL通过调控Slug与紧密连接蛋白表达干预转移
A.Western blotting与RT-PCR检测;B.细胞侵袭实验;C.划痕愈合实验;与对照组比较,*1P<0.01,*2P<0.05

Fig.5 Inhibition of ZOL on cell metastasis by regulating the expression of Slug and Occludin
A.western blotting and RT-PCR detection;B.cell invasion test;C.wounding heal assay;compared with control group,*1P<0.01,*2P<0.05

3 讨论

ZOL是目前抗骨吸收作用最强的双膦盐类药物,临床上主要应用于治疗骨质疏松症、骨更新代谢异常加快以及恶性肿瘤骨转移引起的骨痛症和高钙血症等疾病[5-6]。研究显示ZOL具有直接的抗肿瘤作用,一方面可以通过干扰诸如Ras、Rho和Rac等小分子结合蛋白的翻译后修饰从而抑制肿瘤细胞生长并促进其凋亡,另一方面可以活化γ/δT细胞增强其对肿瘤细胞的杀伤效应[4-6]。笔者在本研究中发现ZOL呈剂量依赖性抑制食管鳞癌细胞的增殖能力。为了避免高浓度药物产生的细胞毒作用对后续实验的影响,本研究选择较低药物浓度(2~8 μmol·L-1),进一步观察ZOL对肿瘤细胞侵袭迁移的调节作用并深入探讨其分子机制。

肿瘤转移是一个多因素、多阶段参与的复杂过程。已知上皮-间质细胞转化(epithelial-mesenchymal transition,EMT)发生在肿瘤转移的起始阶段,是形成肿瘤转移的早期关键步骤[12-13]。EMT的主要分子特征是E-钙粘蛋白、紧密连接蛋白等上皮标志物表达和功能缺失,同时N-钙粘蛋白、波形蛋白等间质细胞的标志物过量表达。本研究亦显示,低剂量ZOL处理可下调波形蛋白与N-钙粘蛋白表达,增强上皮细胞标志物紧密连接蛋白表达。值得注意的是,另一个重要的细胞黏附分子E-钙粘蛋白表达水平未见明显变化。

锌指转录因子Snail与Slug是重要的转录抑制因子,可通过识别E-钙粘蛋白基因启动子区域特异性DNA结合序列5’-CACCTG-3’(E-box元件)直接抑制其转录,从而引发EMT和肿瘤转移[14]。已有文献表明,紧密连接蛋白基因启动子区域含有转录抑制因子Snail与Slug识别序列[15]。本研究显示,ZOL处理后Slug表达水平相对于对照组显著减弱,而Snail未见明显变化。过表达Slug逆转ZOL处理对紧密连接蛋白表达的影响,并进一步抵消ZOL处理对食管鳞癌细胞侵袭迁移能力的抑制效应。

有研究证实,ZOL处理显著抑制三阴性乳腺癌细胞增殖并造成细胞周期停滞[14]。此外,ZOL处理可减低N-钙粘蛋白和Snail表达水平,同时增强E-钙粘蛋白表达,这是通过干扰NF-κB通路重要因子RelA磷酸化并影响其细胞内定位产生的[16]。上述在乳腺癌中的相关报道同本研究结果有所不同,这可能与肿瘤类型的不同和肿瘤细胞的异质性差异有关。

目前ZOL抗肿瘤作用的大规模多中心临床研究仍不成熟。由于双膦酸盐特殊的药代动力学特点,使用较高剂量可能是临床中达到抗肿瘤作用所需要的,可能也会增加药物的毒副作用[5,7]。因此在以后的研究中,需要进一步解决双膦酸盐的最适剂量和给药方案及如何与现有的肿瘤治疗手段进行联合应用的问题。本实验结果显示鳞癌细胞的转移能力,为进一步拓宽ZOL的临床应用范围和抑制食管鳞癌转移提供了新的研究思路。

The authors have declared that no competing interests exist.

参考文献

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Background Cervical cancer ranks second among all cancers reported in Sri Lankan women. This study assessed the prevalence and type-distribution of human papillomavirus (HPV) among Sri Lankan women with invasive cervical cancer (ICC) and pre-cancerous lesions. Methods 114 women aged 21 years and above, hospitalized in the National Cancer Institute, Sri Lanka with a diagnosis of ICC or cervical intraepithelial neoplasia (CIN) 2/3 were prospectively enrolled between October 2009 and September 2010 (110430/NCT01221987). The cervical biopsy or excision specimens collected during routine clinical procedures were subjected to histopathological review. DNA was extracted from samples with a confirmed histological diagnosis and was amplified using polymerase chain reaction and HPV DNA was detected using Enzyme Immuno Assay. HPV positive samples were typed using reverse hybridization Line Probe Assay. Results Of the cervical samples collected, 93.0% (106/114) had a histologically confirmed diagnosis of either ICC (98/106) or CIN 2/3 (8/106). Among all ICC cases, squamous cell carcinoma was diagnosed in the majority of women (81.6% [80/98]). HPV prevalence among ICC cases was 84.7% (83/98). The HPV types most commonly detected in ICC cases with single HPV infection (98.8% [82/83]) were HPV-16 (67.3%) and HPV-18 (9.2%). Infection with multiple HPV types was recorded in a single case (co-infection of HPV-16 and HPV-59). Conclusions HPV was prevalent in most women with ICC in Sri Lanka; HPV-16 and HPV-18 were the predominantly detected HPV types. An effective prophylactic vaccine against the most prevalent HPV types may help to reduce the burden of ICC disease.
DOI:10.1186/1471-2407-14-116      PMID:3936905      URL    
[本文引用:0]
[3] YOU Y,CHEN Y,ZHENG X,et al.Aberrant methylation of the PTPRO gene in peripheral blood as a potential biomarker in esophageal squamous cell carcinoma patients[J].Cancer Lett,2012,315(2):138-144.
Epigenetic inactivation of protein tyrosine phosphatase receptor-type O (PTPRO), a new member of the PTP family, has been described in several forms of cancer. We evaluated PTPRO promoter hypermethylation as a potential biomarker in esophageal squamous cell carcinoma (ESCC). This alteration was observed in 27 (75%) of 36 primary tumors and correlated significantly with depth of invasion (T-stage, P = 0.013). Among matched peripheral blood samples from ESCC patients, 13 (36.1%) of 36 exhibited detectable methylated PTPRO in plasma, while 15 (41.7%) of 36 had this abnormality in buffy coat. No methylated PTPRO was observed in normal peripheral blood samples from 10 healthy individuals. In addition, demethylation by 5-aza-dC treatment led to gene reactivation in PTPRO-methylated and -silenced ESCC cell lines. To our knowledge, this is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood. These findings suggest that hypermethylated PTPRO occurs frequently in ESCC. Further, detection in peripheral blood of ESCC patients suggests potential clinical application for noninvasive diagnosis and disease monitoring.
DOI:10.1016/j.canlet.2011.08.032      PMID:22099875      Magsci     URL    
[本文引用:2]
[4] YOU Y,LIU J,WANG Z,et al.The enhancement of radios-ensitivity in human esophageal squamous cell carcinoma cells by zoledronic acid and its potential mechanism[J].Cytotechnology,2014,66(1):17-25.
Esophageal squamous cell carcinoma (ESCC) has a low 5-year patient survival rate. Radiotherapy, as a preoperative or postoperative treatment of surgery, has a crucial role in improving local control and survival of ESCC. Various chemotherapeutic and biologic agents have been used as radio-sensitizers in combination with radiotherapy. Here, we demonstrate that zoledronic acid (ZOL) has a radio-sensitizing effect on ESCC cells. Exposure of ESCC cancer cells to ZOL plus radiation resulted in increased cell death through arresting the cell cycle between S and G2/M phases. ZOL appeared to inhibit proliferation, tube formation and invasion of endothelial cells. These anti-angiogenetic effects were more marked concurrently with irradiation. In addition, synergistic suppressive effects on VEGF expression were observed after combined treatment. Our data suggest that the combination of ZOL and radiation is a promising therapeutic strategy to enhance radiation therapy for ESCC patients.
DOI:10.1007/s10616-012-9532-4      PMID:23334334      Magsci     URL    
[本文引用:2]
[5] LIN C,XIN S,QIN X,et al.Zoledronic acid suppresses me-tastasis of esophageal squamous cell carcinoma cells through upregulating the tight junction protein occludin[J].Cytotechnology,2016,68(4):1233-1241.
We have previously demonstrated the radio-sensitizing effect of zoledronic acid (ZOL), a third generation bisphosphonate, on human esophageal squamous cell carcinoma (ESCC) cells. Here we show that ZO
DOI:10.1007/s10616-015-9884-7      PMID:26204820      URL    
[本文引用:2]
[6] BANYS M,SOLOMAYER E F,GEBAUER G,et al.Influen-ce of zoledronic acid on disseminated tumor cells in bone marrow and survival:results of a prospective clinical trial[J].BMC Cancer,2013,13:480.
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[7] LI Y Y,CHANG J W,LIU Y C,et al.Zoledronic acid indu-ces cell-cycle prolongation in murine lung cancer cells by perturbing cyclin and Ras expression[J].Anticancer Drugs,2011,22(1):89-98.
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[8] KUBISTA B,TRIEB K,SEVELDA F,et al.Anticancer effe-cts of zoledronic acid against human osteosarcoma cells[J].J Orthop Res,2006,24(6):1145-1152.
[本文引用:1]
[9] KIJIMA T,KOGA F,FUJII Y,et al.Zoledronic acid sensiti-zes renal cell carcinoma cells to radiation by downregulating STAT1[J].PLoS One,2013,8(5):e64615.
[本文引用:1]
[10] LAN Y C,CHANG C L,SUNG M T,et al.Zoledronic acid-induced cytotoxicity through endoplasmic reticulum stress triggered REDD1-mTOR pathway in breast cancer cells[J].Anticancer Res,2013,33(9):3807-3814.
Zoledronic acid (ZOL) used for the prevention/treatment of osteopathic complications has been reported to have antitumor effects in breast cancer treatment. However, little is known about the exact molecular mechanisms for antitumor actions of ZOL. In this study, two breast cancer cell lines were used to investigate the antitumor efficacy of ZOL and the underlying molecular mechanisms.The growth of two breast cancer cell lines was markedly decreased following treatment with ZOL. Compared with MCF-7 cells, MDA-MB-231 cells were more sensitive to ZOL treatment. Western blot analysis showed that the inhibitory effect of zoledronic acid on growth was related to the extent of inhibition of phosphorylated-protein kinase B (p-AKT), and phosphorylated-mammalian target of rapamycin (p-mTOR). Moreover, the expression of the stress-responsive protein regulated in development and DNA damage response 1 (REDD1), an inhibitor of mTOR, was induced markedly to various degrees in different breast cancer cell lines after ZOL treatment. Interestingly, by examining the upstream signaling pathway of REDD1, we found that ZOL can induce endoplasmic reticulum stress responses through activating the protein kinase R (PKR)-related ER kinase-eukaryotic initiation factor 2 alpha-CCAAT/enhancer binding protein homologous protein (PERK-eIF2 -CHOP) pathway.Taken together, these results indicated that ZOL-induced cell death was caused by endoplasmic reticulum stress activating PERK-eIF2 -CHOP pathway to induce REDD1 expression and inhibit the mTOR pathway.
DOI:10.1007/s11839-013-0432-4      PMID:24023313      Magsci     URL    
[本文引用:0]
[11] DO SALVATORE M,ORLANGI A,BAGALA C,et al.Anti-tumour and anti-angiogenetic effects of zoledronic acid on human non-small-cell lung cancercell line[J].Cell Prolif,2011,44(8):139-146
Objectives: Although emerging data suggest that zoledronic acid (Zol) may have different anti-tumour activities against a broad range of cancers, its effects on lung cancer remain largely unknown. The aim of this study was to evaluate in vitro the anti-tumoural and anti-angiogenetic effect of zoledronic acid in non-small-cell lung cancer (NSCLC) cells.Material and methods: We treated A549 NSCLC cells with zoledronic acid to investigate survival, cell cycle activity, anti-angiogenic activity and apoptotic responses to it.Results: We observed that highest Zol concentration (100 μm) caused arrest in G1 phase of the cell cycle and also induced different percentages of apoptosis in presence (0.9% versus 4.4%) or absence (2.4% versus 28.5%) of serum (P = 0.0001). Zol concentration from 5 to 100 μm for 2 days induced significant concentration-dependent cell death in adherent cells. Furthermore, Zol (10–100 μm) induced dose-dependent reduction both of mRNA and protein expression of VEGF associated with parallel decrease in VEGF secretion in the culture medium.Conclusion: Taken together, these results support a possible anti-cancer and anti-angiogenetic activity of Zol. Our data may not only provide a basis for the clinical use of this drug as preventive agent of bone metastases but also suggest that Zol deserves attention as an anti-cancer agent in non-small-cell lung cancer.
DOI:10.1111/j.1365-2184.2011.00745.x      PMID:21401755      URL    
[本文引用:1]
[12] CHRISTIANSEN J J,RAJASEKARAN A K.Reassessing e-pithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J].Cancer Res,2006,66(17):8319-8326.
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[13] RAN Y,WU S,YOU Y.Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy[J].J Biochem,2011,149(1):49-54.
E-cadherin has been proven to be widely down-regulated and tightly associated with tumour invasion and metastasis in multiple human cancer types. Recent research demonstrated that aberrant methylation around gene promoter region attributes to E-cadherin silencing. However, the detailed information about this epigenetic inactivation in nasopharyngeal carcinoma (NPC) is rare. The aim of this study was to probe more into the basic mechanism of E-cadherin methylation in NPC and elucidate the application of demethylating agents to restore E-cadherin expression. To address this question, we initially studied E-cadherin methylation status in NPC primary tumours and cell lines by methylation-specific PCR, and compared it with E-cadherin expression. Methylated E-cadherin was detected in 13 of 20 (65%) NPC clinical specimens and 2 of 2 (100%) NPC cell lines (HNE-1 and CNE-2), which was inversely correlated with E-cadherin expression. The detailed methylation profile at individual CpGs within CpG island of E-cadherin promoter region was confirmed by bisulphite sequencing. E-cadherin gene could be demethylated and reactivated in HNE-1 and CNE-2 cells upon treatment with 5-aza-dC, a DNA demethylating agent. Our findings indicate that frequent aberrant methylation of E-cadherin may play an important role in downregulation of E-cadherin, and demethylation therapy could serve as a promising strategy for NPC patients. Furthermore, a high frequency of E-cadherin methylation (9/20, 45%) in peripheral blood of NPC patients suggests its potential clinical application as an early diagnostic or predictive marker.
DOI:10.1093/jb/mvq128      PMID:21059597      Magsci     URL    
[本文引用:1]
[14] UCHIKADO Y,NATSUGOE S,OKUMURA H,et al.Slug expression in the E-cadherin preserved tumors is related to prognosis in patients with esophageal squamous cell carcinoma[J].Clin Cancer Res,2005,11(3):1174-1180.
The expression of E-cadherin correlates with the development, progression, and metastasis of esophageal squamous cell carcinoma (ESCC). Slug, a member of the snail family of transcriptional factors, is a newly identified suppressive transcriptional factor of E-cadherin. The purpose of the present study was to evaluate the clinical significance of E-cadherin and Slug expression in ESCC.Immunohistochemistry was used to investigate the expression of E-cadherin and Slug proteins in 203 patients with ESCC. The relationships between expression of these proteins and clinicopathologic factors, including prognosis, were analyzed.Positive expression of E-cadherin and Slug was observed in 43% and 48% of cases, respectively. The tumors with reduced E-cadherin expression or positive Slug expression invaded deeper, had more lymph node metastasis, and had more lymphatic invasion than the tumors with preserved E-cadherin expression or negative Slug expression. Slug expression significantly correlated with reduced E-cadherin expression. Sixty-seven of the 98 (68.4%) tumors with positive Slug expression had reduced E-cadherin expression (P = 0.0011). Patients with reduced E-cadherin expression or positive Slug expression had poor clinical outcomes. In the preserved E-cadherin group, the 5-year survival rate was better for patients who were negative for Slug expression than for those who were positive for Slug expression (P = 0.035). Multivariate analysis indicated that E-cadherin expression and Slug expression were not independent prognostic factors.Evaluation of not only the expression of E-cadherin but also the co-expression of E-cadherin and Slug in preserved E-cadherin group is useful for predicting malignant properties of ESCC.
PMID:15709186      URL    
[本文引用:2]
[15] WANG Z,WADE P,MANDELL K J,et al.Raf 1 represses expression of the tight junction protein occluding via activation of the zinc-finger transcription factor slug[J].Oncogene,2007,26(8):1222-1230.
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[16] SCHECH A J,KAZI A A,et al.GILANI R A.Zoledronic ac-id reverses the epithelial-mesenchymal transition and inhi-bits self-renewal of breast cancercells through inacti-vation of NF-κB[J].Mol Cancer Ther,2013,12(7):1356-1366.
Zoledronic acid, a third-generation bisphosphonate, has been shown to reduce cell migration, invasion, and metastasis. However, the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT), a cellular process essential to the metastatic cascade, remain unclear. Therefore, the effects of zoledronic acid on EMT, using triple-negative breast cancer (TNBC) cells as a model system, were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers, N-cadherin, Twist, and Snail, and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability, induced cell-cycle arrest, and decreased the proliferative capacity of TNBC, suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype, the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins, BMI-1 and Oct-4, and both prevented and eliminated mammosphere formation. To understand the mechanism of these results, the effect of zoledronic acid on established EMT regulator NF-kappa B was investigated. Zoledronic acid inhibited phosphorylation of RelA, the active subunit of NF-kappa B, at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter, providing a direct link between inactivation of NF-kappa B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased, correlating modulation of EMT to decreased self-renewal. On the basis of these results, it is proposed that through inactivation of NF-kappa B, zoledronic acid reverses EMT, which leads to a decrease in self-renewal. (C) 2013 AACR.
DOI:10.1158/1535-7163.MCT-12-0304      PMID:23619300      Magsci     URL    
[本文引用:1]
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关键词(key words)
唑来膦酸
食管鳞癌
细胞增殖
肿瘤侵润
紧密连接蛋白

Zoledronic acid
Esophagus squamous cell c...
Cell proliferation
Neoplasm invasiveness
Occludin

作者
辛淑波
游颜杰
林灿峰

XIN Shubo
YOU Yanjie
LIN Canfeng