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医药导报, 2017, 36(1): 28-31
doi: 10.3870/j.issn.1004-0781.2017.01.006
熊果酸对人卵巢癌耐药SKOV3/DDP裸鼠移植瘤的抑制作用*
Inhibitory Effect of Ursolic Acid on Drug-resistant SKOV3/DDP Ovarian Carcinoma Xenografts in Nude Mice
杨茗钫1, 程晓华2, 刘丝荪1, 黄欧平3

摘要:

目的 观察熊果酸对人卵巢癌耐顺铂SKOV3/DDP裸鼠移植瘤生长的影响,探讨其作用途径。方法 建立人卵巢癌耐顺铂SKOV3/DDP细胞裸鼠异位移植瘤模型,将24只荷瘤鼠随机分为4组:空白对照(0.9%氯化钠溶液)组、顺铂(4 mg·kg-1·d-1)组和熊果酸小剂量 (30 mg·kg-1·d-1)、大剂量( 60 mg·kg-1·d-1)组,每组6只。腹腔注射给药,注射量均为10 mL·kg-1,每天1次,共15 d。给药后每3 d测量肿瘤体积,并绘制移植瘤生长曲线,计算各组抑瘤率,采用逆转录-聚合酶链反应(RT-PCR)、Western blotting 法分别检测肿瘤组织Bcl-2、Bax mRNA及其蛋白表达水平。结果 顺铂组和熊果酸小剂量、大剂量组抑瘤率分别为33.3%,43.4%,71.0%。移植瘤中Bcl-2表达均有下降,而Bax表达上调。结论 熊果酸对人卵巢癌耐顺铂SKOV3/DDP细胞裸鼠移植瘤具有一定的抗瘤活性,可抑制瘤体生长,并呈现浓度依赖性,其机制可能与抑制抗凋亡因子Bcl-2的表达、增强凋亡促进因子Bax 的表达有关。

关键词: 熊果酸 ; ; 卵巢 ; 移植瘤 ; 抗肿瘤活性

Abstract:

Objective To explore the effects and mechanisms of ursolic acid on drug-resistant SKOV3/DDP ovarian carcinoma xenografts in nude mice. Methods The models of drug-resistant SKOV3/DDP ovarian carcinoma on athymic mouse were established and randomly divided into four groups with intraperitoneal injection of different drugs: blank control (0.9% sodium chloride solution), cisplatin (4 mg·kg-1·d-1), ursolic acid low dose (30 mg·kg-1·d-1), and high dose (60 mg·kg-1·d-1).All drugs were injected at volumes of 10 mL·kg-1 perday for 15 days. The tumor volumes were measured during the process of drug treatment every three days. After 14 days, The tumorigenic rate and tumor inhibition rate were calculated. RT-PCR and Western blotting were performed to detect the expression of Bcl-2 and Bax.Results Anti-tumor rates of cisplatin group , low dose ursolic acid group, and high dose ursolic acid group was 33.3%, 43.3%, and 71.0%, respectively. Bcl-2 expressions were down-regulated, while Bax expressions were up-regulated in all three groups. Conclusion Ursolic acid has some anti-tumor activity on cisplatin-resistant human ovarian cancer SKOV3 /DDP cell in nude mice.It can inhibit tumor growth with dose-effect relationship. The mechanism may be to suppress the expression of anti-apoptotic factor Bcl-2 and to increase the expression of apoptosis-promoting factors Bax.

Key words: Ursolic acid ; Cancer ; ovarian ; Xenografts ; Anti-tumor activity

卵巢癌是妇科常见的恶性肿瘤之一,发病率逐年上升,在全世界范围内,卵巢癌的病死率目前位居妇科恶性肿瘤的第5位[1]。目前,卵巢癌的化学治疗(化疗)占据重要地位,尽管许多肿瘤患者最初对标准的组合手术和细胞毒性治疗表现出较好的化疗敏感性,但是因为肿瘤细胞易对化疗药物产生耐药性,使化疗药物的疗效受到限制,近90%会再次复发[2]

熊果酸是广泛存在于天然植物中的一种五环三萜类化合物。多年来,熊果酸在抗炎、抗病毒、抗肿瘤、抗动脉粥样硬化等显示的多种生物学效应而成为研究热点[3-5]。 熊果酸能抑制顺铂耐药人卵巢癌SKOV3/DDP细胞增殖,促进其凋亡[6],但其作用机制及在体内抑制情况尚不明确。笔者通过建立人卵巢癌耐顺铂SKOV3/DDP裸鼠移植瘤模型,观察和探讨熊果酸在其体内对人卵巢癌细胞的抑制作用及其作用机制。

1 材料与方法
1.1 实验动物及细胞株

健康Balb/c雌性裸鼠,24只,4~6周龄,体质量(20±3) g,购于南昌大学医学部实验动物中心,实验动物生产许可证号:SCXK(赣)2014-0001,饲养于无特定病原体级(SFP)医学动物实验中心。人卵巢癌顺铂耐药SKOV3/DDP细胞株来源于中国医学科学院肿瘤医院。

1.2 试剂与药物

熊果酸对照品(江西省宜春学院生物工程开发中心,含量约99.8%,批号:20100103);RPMI 1640培养液干粉(Hy-Clone 批号:117859);胎牛血清(杭州四季青公司,批号:141012);青霉素(华北制药股份有限公司,批号:20130226)、链霉素(华北制药股份有限公司,批号:20130226);二喹啉甲酸(bicinchoninic acid,BCA)试剂盒(Pierce);增强化学发光剂(electro-chemilumine scence,ECL)(Amersham);噻唑蓝(MTT)、兔抗人β-actin (Sigma公司);鼠抗人Bcl-2单抗(批号:D1410)、鼠抗人Bax单抗(批号:D1310-1)、兔抗人Bcl-2多抗(批号:C1411)、兔抗人bax多抗(SantaCruz公司)。

1.3 细胞培养

人卵巢癌耐顺铂SKOV3/DDP细胞培养于含有10%胎牛血清、100 μg·mL-1青霉素和100 μg·mL-1链霉素的RPMI 1640培养液中,并于37 ℃、5%二氧化碳(CO2)、饱和湿度培养箱中培养,每3~5 d传代一次,取对数生长期的细胞进行实验。

1.4 裸鼠移植瘤模型的建立

取对数生长期的人卵巢癌耐顺铂SKOV3/DDP细胞,0.25%胰酶常规消化细胞后,800 r·min-1(r=15 cm)离心5 min,弃上清液,0.9%氯化钠溶液重悬,计数并调整细胞密度为1×107·mL-1,接种于裸鼠右前肢背部皮下,每只0.2 mL,接种后,观察并记录肿瘤发生的时间,用游标卡尺测量肿瘤的大小,待移植瘤直径约5.0 mm,表明荷瘤裸鼠模型制备成功,实测移植瘤直径8~10 cm,备实验用。

1.5 实验分组及药物处理

以移植瘤的体积为分组因素,将24只成瘤裸鼠按体质量随机分为4组 ,每组6只。空白对照组:0.9%氯化钠溶液10 mL·kg-1;顺铂组(4 mg·kg-1·d-1);熊果酸小剂量组(30 mg·kg-1·d-1);熊果酸大剂量组(60 mg·kg-1·d-1)。各组均为腹腔注射,注射量均为10 mL·kg-1,每天给药1次,持续15 d,每3 d测量肿瘤长、短径。用药结束2 d后处死动物,剥离肿瘤组织,称质量。肿瘤体积计算公式:体积=长径(a)×短径(b)2/2;抑瘤率(%)=(对照组瘤质量-实验组瘤质量)/对照组瘤质量×100 %。

1.6 反转录-聚合酶链反应(RT -PCR)检测移植瘤组织中Bax、Bcl-2 mRNA的表达

按Trizol试剂说明步骤提取移植瘤组织的总RNA,按反转录试剂盒说明进行逆转后,进行RT-PCR。β-Actin引物:上游5'-GCATTGTAACCA ACTGGGAC,下游5'-GCGTAACCCTCATAGATGGGC-3';Bax上游引物序列为5'-ATGGACGGGT CCGGGGAGCA-3',下游引物序列为5'-TGCTCGATCCTGGATGAAACCCT-3';Bcl-2上游引物序列为5'-TCGCC- CTGTGGATGACTGAG-3',下游引物序列为5'-CAGAGTCTTCAGAGACAGCCAGGA-3'。RT-PCR 反应体系:10×PCR buffer 2 μL,25 mmol·L-1 氯化镁 2 μL,5 U·μL-1 Taq 酶 0.1 μL,ddH2O 11.3 μL,10 mmol ·L-1 dNTPs 2 μL,cDNA 1 μL,上、下游引物各1 μL。反应条件:95 ℃预变性3 min,94 ℃ 30 s,56 ℃ 30 s,72 ℃ 30 s,30次循环;最后72 ℃延伸5 min。取RT-PCR反应产物进行琼脂糖凝胶电泳成像,采用Bio-Rad凝胶扫描成像分析系统进行成像及Image pro plus 6.0进行图像分析,分别测定目的基因(Bcl-2、Bax)和β-Actin条带吸光度值,计算Bcl-2、Bax mRNA 的相对表达量,相对表达量=目的基因条带吸光度值/β-Actin条带吸光度值。

1.7 Western blotting法检测Bcl-2和Bax蛋白的表达

液氮中取适量肿瘤组织,加入RIPA裂解液后进行超声裂解,14 000 r·min-1(r=10 cm)离心15 min,取上清液,采用BCA法测定蛋白浓度,加入适量蛋白上样缓冲液,煮沸10 min,进行Western blotting 实验,将制备好的电泳凝胶进行拔梳上样,电泳结束后进行转膜,膜于含5% 脱脂牛奶的TBST 封闭液室温封闭1 h,一抗4 ℃孵育过夜,0.1%TBST洗膜3次,每次10 min;二抗室温孵育1 h,同样TBST洗膜3次,每次10 min,采用增强ECL法 Bio-Rad显影仪进行显色成像。

1.8 统计学方法

采用SPSS 17.0版统计软件进行统计分析,计量资料均采用均数±标准差( x ̅ ±s)表示,各组间治疗前后瘤体积、体质量、抑瘤率等均采用单因素方差分析,两组间均数比较采用LSD-t检验,以P<0.05为差异有统计学意义。

2 结果
2.1 瘤质量、瘤体积的比较

24只裸鼠皮下接种人卵巢癌肿瘤细胞均有瘤长出,接种成功率100%。给药期间观测裸鼠瘤体积、肿瘤生长情况,绘制肿瘤生长曲线,其体质量变化趋势相同,组间差异无统计学意义,由肿瘤生长曲线可看出,空白对照组移植瘤随生长时间延长呈递增性增长,而顺铂组和熊果酸小、大剂量组肿瘤生长明显减慢,大剂量熊果酸对肿瘤的生长抑制作用更强(图1);各组取出瘤体的质量也显著小于空白对照组 (t=8.512,10.021,12.135,均P<0.05)。根据公式计算各实验组的肿瘤生长抑制率,结果见表1。

图1 4组荷瘤裸鼠移植瘤生长曲线

Fig.1 Growth curve of transplantation tumor in four groups of nude mice

表1 4组裸鼠移植瘤的体积、质量及抑瘤率
Tab.1 Volume, quality and inhibitory rate of transplantation tumor in four groups of nude mice x¯±s,n=6
组别 剂量/
(mg·kg-1·d-1)
瘤体积/mm3 瘤质量/g 抑瘤率/%
空白对照组 - 780.5±48.3 0.69±0.08 -
顺铂组 4 524.4±52.8 0.46±0.10 33.3
熊果酸
小剂量组 30 421.8±46.2 0.39±0.06 43.4
大剂量组 60 156.8±38.4 0.20±0.04 71.0

表1 4组裸鼠移植瘤的体积、质量及抑瘤率

Tab.1 Volume, quality and inhibitory rate of transplantation tumor in four groups of nude mice x¯±s,n=6

2.2 RT-PCR检测移植瘤组织中Bax、Bcl-2 mRNA表达

RT-PCR检测结果:空白对照组、顺铂组和熊果酸小、大剂量组Bcl-2 mRNA表达量分别为2.05±0.37,1.14±0.12,1.05±0.21,0.48±0.08。顺铂组和熊果酸小、大剂量组的Bcl-2 mRNA转录强度与空白对照组比较均差异有统计学意义(t=5.187,5.407,9.432,均P<0. 01);熊果酸大剂量组分别与顺铂组和熊果酸小剂量组比较差异有统计学意义(t=6.710,8.250,均P<0.01)。Bax mRNA的表达量分别为0.29±0.03,0.97±0.06,1.01±0.15,1.52±0.25。顺铂组和熊果酸小、大剂量组的Bax mRNA转录强度与空白对照组比较均差异有统计学意义(t=13.670,10.722,8.733,均P<0.01),熊果酸大剂量组分别与顺铂组和熊果酸小剂量组比较差异有统计学意义(t=3.929,3.409,均P<0.05)。见图2。

图2 4组荷瘤裸鼠移植瘤组织Bcl-2 mRNA和Bax mRNA表达
A.空白对照组;B.顺铂组;C.熊果酸小剂量组;D. 熊果酸大剂量组;与空白对照组比较,*1P<0.01;与顺铂组、熊果酸小剂量组比较,均*2P<0.05

Fig.2 The mRNA expression of Bcl-2 and Bax in transplantation tumor of four groups of nude mice
A. blank control group; B. cisplatin group; C. low-dose ursolic acid group; D.high-dose ursolic acid group; Compared with blank control group, * 1P< 0.01; compared with cisplatin group and low-dose ursolic acid group, * 2P<0.05

2.3 Western blotting检测移植瘤组织中Bax、Bcl-2蛋白表达

结果显示,与空白对照组比较,顺铂组和熊果酸小、大剂量组Bcl-2蛋白表达下调; Bax蛋白表达上调,其中以熊果酸大剂量组上调最为显著。见图3。

3 讨论

肿瘤的发生、发展、消退与细胞凋亡异常有较密切的联系,肿瘤细胞的增殖和凋亡的失衡是肿瘤发生发展的重要环节。Bcl 作为一种抑癌基因,可以通过抑制线粒体膜电位下降,阻止线粒体细胞色素C的释放抑制半胱氨酰天冬氨酸酶-3 (cysteinyl aspartate specificproteases-3 ,caspase-3)的激活;此外,Bcl-2亦可直接抑制线粒体细胞色素C 的释放减少caspase-3激活[7]。Caspase家族在细胞凋亡的信号传导中具有关键作用,而caspase-3是caspase家族中最重要的细胞凋亡执行者之一,在蛋白酶级联切割过程中处于核心

位置,发挥着重要的作用,一旦caspase 被激活就会导致细胞死亡。同时,Bcl-2家族蛋白质表达在肿瘤细胞凋亡中亦扮演着非常重要的角色,Bcl-2和Bax同属Bcl-2家族,而Bax为Bcl-2家族中非常重要的成员,Bax能与Bcl-2结合形成二聚体,从而抑制细胞增殖或促进细胞凋亡。研究表明,Bax表达增加、Bcl-2表达减少促进细胞凋亡的主要途径是通过激活caspase-3而实现的。卵巢癌和其他许多肿瘤已被证明过度表达了Bcl-2和(或)其家庭成员[8-10]。熊果酸作为五环三萜类天然化合物的代表,以其毒副作用低、生物活性高而成为备受关注的研究热点。亦有研究显示熊果酸可以通过激活Bcl-2相关X蛋白和蛋白水解激活caspase-3,从而下调Bcl-2、上调Bax的表达抑制SNU-484胃癌细胞的增殖,有效诱导其细胞凋亡[11]

图3 4组荷瘤裸鼠移植瘤组织Bax和Bcl-2蛋白表达
A.空白对照组;B.顺铂组;C.熊果酸小剂量组;D. 熊果酸大剂量组

Fig.3 Protein expression of Bax and Bcl-2 in transplantation tumor of four groups of nude mice
A.blank control group; B.cisplatin group; C.low-dose ursolic acid group; D.high-dose ursolic acid group

本研究成功建立了人卵巢癌耐药SKOV3/DDP裸鼠移植瘤模型,观察了熊果酸对移植瘤生长及凋亡相关基因表达的影响。结果显示,熊果酸能明显抑制移植瘤的生长,且呈剂量相关性;同时,可上调移植瘤组织Bax mRNA和蛋白表达,下调Bcl-2 mRNA和蛋白表达,说明熊果酸能改变人卵巢癌耐药SKOV3/DDP细胞裸鼠移植瘤组织中凋亡相关基因的表达,可能是通过诱导凋亡抑制人卵巢癌裸鼠耐药SKOV3/DDP移植瘤

的生长,发挥抗肿瘤作用。研究中也发现大剂量熊果酸抑制肿瘤生长作用明显强于顺铂,其是否能逆转人耐顺铂卵巢癌对顺铂的耐药性,为耐顺铂卵巢癌的治疗提供新思路还有待于进一步研究。

The authors have declared that no competing interests exist.

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[3] 周蕾,刘卓刚.熊果酸抗肿瘤作用机制的研究进展[J].医药导报,2011,30(4):490-494.
根据近年的中外文献和研究成果,对熊果酸的抗肿瘤作用和作用机制等方面进行论述。大量的基础研究证实,熊果酸能在体内外抑制多种肿瘤细胞生长。熊果酸抗肿瘤的作用机制是多方面的,包括预防肿瘤形成、诱导肿瘤细胞分化和凋亡、抗氧化、抑制肿瘤血管新生、抑制肿瘤侵袭转移、增敏抗肿瘤化疗药物及逆转耐药,以及调节机体免疫功能等。
DOI:10.3870/yydb.2011.04.031      URL    
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[4] KIM S H, HONG J H, LEE Y C.Ursolic acid, a potential PPARγ agonist, suppresses ovalbumin induced airway inflammationand Penh by down-regulating IL-5, IL-13, and IL-17 in a mouse model of allergic asthma[J]. Eur J Pharmacol, 2013, 701(1/3):131-143.
Our data suggest the therapeutic mechanism of ursolic acid in asthma is based on reductions of Th2 cytokines (IL-5 and IL-13), ovalbumin-specific IgE production, and eosinophil infiltration via the Th2-GATA-3, STAT6, and IL-17-NF-κB pathways.
DOI:10.1016/j.ejphar.2012.11.033      PMID:23201068      URL    
[本文引用:0]
[5] 程晓华,熊玉卿.五环三萜皂苷的药理作用研究进展[J].中草药,2007,38(5):792-795.
五环三萜皂苷是天然产物中重要的成分之一,大多具有广泛的药理活性,临床应用前景十分诱人.随着五环三萜 皂苷研究的不断深入,对其药理学作用机制的阐明和新药研发已成为此类化合物研究的热点之一.就国内外学者对五环三萜皂苷抗肿瘤、护肝、抗炎、以及机体免疫 调节等药理学作用的研究进展做一综述.
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[6] 杨茗钫,刘丝荪.熊果酸对顺铂耐药人卵巢癌SKOV3/DDP细胞增殖与凋亡的影响[J].南昌大学学报(医学版),2011,51(5):15-18.
目的 探讨熊果酸对体外培养的顺铂耐药人卵巢癌SKOV3/DDP细胞增殖与凋亡的影响.方法 将熊果酸作用于体外培养的顺铂耐药人卵巢癌SKOV3/DDP细胞,采用MTT法检测不同浓度的熊果酸(0、10、20、40、50、60、80 μmol·L-1)对SKOV3/DDP细胞生长的抑制作用,采用流式细胞术检测SKOV3/DDP的细胞凋亡和细胞周期.结果不同浓度的熊果酸对体外培 养的SKOV3/DDP细胞均有明显的抑制增殖作用,并呈剂量、时间依赖效应(均P<0.05),熊果酸(60 μmol·L-1)作用于SKOV3/DDP细胞48 h后,达到最大细胞凋亡率(31.22±1.98)%;熊果酸可影响SKOV3/DDP细胞增殖周期中的G0/G1期,使细胞在此期停滞,诱导其发生凋 亡.结论熊果酸对体外培养的顺铂耐药人卵巢癌SKOV3/DDP细胞增殖有抑制作用,并能诱导细胞凋亡.
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[7] 胡瑾,张晓丽,张辉.牛痘疫苗致炎兔皮提取物注射液对大鼠脑挫伤皮质细胞凋亡基因Bcl-2 mRNA 表达的影响[J].医药导报,2013,32(4):435-437.
目的 探讨脑挫伤灶周围区凋亡基因Bcl-2 mRNA表达及牛痘疫苗致炎兔皮提取物注射液(恩再适)对其干预作用. 方法 采用Feeny’s自由落体法复制大鼠脑挫伤动物模型. 牛痘疫苗致炎兔皮提取物注射液治疗24, 72 h取脑切片, 采用原位杂交与医学图像分析技术检测Bcl-2 mRNA阳性细胞数目、平均灰度值. 结果 治疗组Bcl-2 mRNA阳性细胞数目和深度高于对照组和模型组(P〈0.05), 72 h组明显高于24 h治疗组(P〈0.05). 结论 牛痘疫苗致炎兔皮提取物注射液对脑挫伤皮质细胞有保护作用, 并能明显上调Bcl-2 mRNA的表达, 从而减轻神经元的损伤.
DOI:10.3870/yydb.2013.04.006      URL    
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[8] MARX D,MEDEN H.Differential expression of apoptosis-associated genes bax and bcl-2 in ovarian cancer[J].Methods Mol Med,2001,39(5):687-691.
The deregulation of the balance between proliferation and programmed cell death is considered one of the most important features of malignant tumors. The search for new markers, which may reflect the tumor progress and response to various therapy regimens, has recently focused on alterations of genes involved in regulation of programmed cell death and apoptosis. The bcl -2-family is a still growing family of genes, which play a major role in regulation of cell suicide, acting either as inhibitors (e.g., bcl-2, bcl-xl, mcl- 1) or promoters (e.g., bcl-xs, bax, bak, bad) of apoptosis ( 1 - 3 ). The chromosomal translocation t(14;18), leading to overexpression of the Bcl-2 protein was first described in human B-cell lymphoma ( 4 ). Later on, Bcl-2-overexpression without chromosomal translocations was also detected in various epithelial tumors ( 5 - 12 ). It has been suggested that Bcl-2 as the major inhibitor of apoptosis plays a role in tumor development and progress by prolonging the survival of malignant cells. Unexpectedly, expression of Bcl-2 has been shown to be connected with parameters of favorable prognosis and prolonged survival in nonsmall-cell lung cancer ( 6 ), breast ( 7 - 9 ), and, recently, in ovarian cancer ( 10 - 12 ). Bax-expression, in contrast, was associated with an unfavorable outcome, as well as negative histopathological features in breast ( 13 ) and ovarian cancer ( 12 ). Moreover, the association of Bax -expression with predictors of poor clinical outcome was strongly connected with concomitant downregulation of Bcl -2-expression ( 12 , 13 ). The unexpected effect of Bcl- 2- and Bax-expression on prognosis of ovarian cancer patients is underlined by the survival curves of patients. Especially, patients with exclusively Bax -positive tumors had a statistically significantly reduced survival as compared to patients with exclusively Bcl -2-positive tumors ( 12 ). This difference could be observed for patients with tumors of different stage and grade, as well as for patients with no evidence of disease or residual tumor after primary surgery ( 12 ). One explanation for these observations is that the apoptosis inhibiting or promoting effect of these homologous proteins depends partly on protein-protein interactions. Bax, for example, the main antagonist of Bcl-2, heterodimerizes with Bcl-2 or Bcl-Xl and homodimerizes with itself ( 1 - 3 ). The ratio of Bax-heterodimers to Bax-homodimers seems to be the critical determinant for regulating cell death ( 2 , 3 ). In cells in which 80% of Bax is found in homodimers, an apoptotic signal results in cell death ( 2 , 3 ), suggesting a crucial role of the Bax/Bcl-2 balance for the regulation of proliferation or cell suicide.
DOI:10.1385/1-59259-071-3:687      PMID:21340831      URL    
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[9] 颜明,李洪志,刘洁婷,.黄芩苷对SH-SY5Y 细胞损伤的Bcl-2 和Bcl-xL mRNA 基因表达的影响[J].医药导报,2012,31(7):843-845.
目的探讨黄芩苷对过氧化氢(H2O2)诱导的sH—SY5Y细胞损伤的Bcl-2和Bcl- xLmRNA表达的影响。方法建立人神经母细胞瘤SH—SY5Y细胞的体外H:O:损伤模型,采用噻唑蓝(MTT)法检测不同浓度黄芩苷对SH—SY5Y 细胞存活率的影响,采用Real—timePCR法检测各组细胞的Bel-2和Bcl—xL表达水平的变化。结果50,100,200μmol·L^-1 黄芩苷组细胞存活率分别为130.4%,89.9%和60.9%,H2O2损伤组细胞存活率为33.9%,50,100μmol·L^-1黄芩苷组与 H2O2损伤组比较,差异有统计学意义(P〈0.01)。50,100,200txmol·L^-1黄芩苷组Bcl-2 mRNA表达量分别为(11.48±0.48),(7.37±1.57),(7.39±2.01),H2O2损伤组为 (5.84±0.58);50,100,200μmol·L^-1黄芩苷组Bcl.xLmRNA表达量分别为(19.96±2.22), (11.36±3.94),(13.07±2.37),H202损伤组为(7.95±0.58),50μmol·L^-1纽Bcl-2和Bcl-xL mRNA与H202损伤组比较,差异均有统计学意义(均P〈0.05)。结论黄芩苷对H2O2诱导的SH.SY5Y细胞损伤的保护作用,可能与黄芩苷上调 Bcl-2和Bcl—xL的表达而起到抗凋亡的作用有关。
DOI:10.3870/yydb.2012.07.004      URL    
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[10] WANG H,ZHANG Z,WEI X,et al.Small-molecule inhibitor of Bcl-2 (TW-37) suppresses growth and enhances cisplatin-induced apoptosis in ovarian cancer cells[J].J Ovarian Res,2015,8(1):3-8.
Bcl-2 plays a major role in the pathobiology and drug resistance of ovarian cancer, and inhibition of bcl-2 was useful for OC therapy. It has previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in many cancer cells. In the present study,we investigate the effect of TW-37 or / and in combination with cisplain on several ovarian cancer (OC) cell lines with high bcl-2 expression.The bcl-2 mRNA and protein expression, and the cisplain (DDP) sensitivity of OC cell lines SKOV3, OVCAR3, OV-90 and 3AO and SKOV3DDP were determined by Quantitative real-time RT-PCR,Western blot, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting (MTT) assays. The effects of TW-37 alone or combined with cisplain on growth and apoptosis in bcl-2 overexpressed OVCAR3, OV-90 and SKOV3DDP cells was detected by MTT,clonogenic assay, ELISA and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay.The cell lines SKOV3 and 3AO were sensitive, whereas OVCAR3, OV-90 and SKOV3DDP were resistant to cisplain. Significant positive correlation was observed between basal bcl-2 mRNA and protein and cisplain sensitivity. Cisplain treatment did not activate bcl-2 in vitro. Treatment with TW-37 inhibited bcl-2 expression in bcl-2 overexpressed OVCAR3, OV-90 and SKOV3DDP cells , and inhibited growth and induced apoptosis ,and increased cisplain killing of the bcl-2 overexpressed cells in a does and time-dependant manner in vitro.Bcl-2 level positively correlated with sensitivity to cisplain. Treatment with TW-37 was effective alone and in combination with cisplain in bcl-2 overexpressed OC cell lines in vitro. Thus, TW-37 may be a useful therapeutic agent for OCs.
DOI:10.1186/s13048-015-0130-x      PMID:25823945      URL    
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[11] KIM E S,MOON A.Ursolic acid inhibits the invasive phenotype of SNU-484 human gastric cancer cells[J].Oncol Lett,2015, 9(2):897-902.
Metastasis is a major cause of cancerrelated mortality in patients with gastric cancer. Ursolic acid, a pentacyclic triterpenoid compound derived from medicinal herbs, has been demonstrated to exert anticancer effects in various cancer cell systems. However, to the best of our knowledge, the inhibitory effect of ursolic acid on the invasive phenotype of gastric cancer cells has yet to be reported. Therefore, the aim of the present study was to investigate the effect of ursolic acid on the invasiveness of SNU484 human gastric cancer cells. Ursolic acid efficiently induced apoptosis, possibly via the downregulation of Bcell lymphoma聽2 (Bcl2), the upregulation of Bcl2associated X聽protein and the proteolytic activation of caspase3. Furthermore, the activation of p38聽mitogenactivated protein kinase and cJun Nterminal kinase was increased by the administration of ursolic acid. In addition, ursolic acid significantly suppressed the invasive phenotype of the SNU484聽cells and significantly decreased the expression of matrix metalloproteinase (MMP)2, indicating that MMP2 may be responsible for the antiinvasive activity of ursolic acid. Taken together, the results of the present study demonstrate that ursolic acid induces apoptosis and inhibits the invasive phenotype of gastric cancer cells; therefore, ursolic acid may have a potential application as a chemopreventive agent to prevent the metastasis of gastric cancer or to alleviate the process of metastasis.
DOI:10.3892/ol.2014.2735      PMID:25621065484      URL    
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关键词(key words)
熊果酸
卵巢
移植瘤
抗肿瘤活性

Ursolic acid
Cancer
ovarian
Xenografts
Anti-tumor activity

作者
杨茗钫
程晓华
刘丝荪
黄欧平

YANG Mingfang
CHENG Xiaohua
LIU Sisun
HUANG Ouping