姜类萜类化合物激活P53信号通路诱导子宫内膜细胞凋亡研究
摘要
背景：
子宫内膜癌是常见的妇产科恶性肿瘤之一。2011年美国约有8,010名妇女死于子宫内膜癌,有近47,130名患者新诊断为子宫内膜癌。在所有子宫内膜癌患者中,约70%的患者其子宫内膜癌病灶局限于宫体,患者5年生存率高达85%。美国妇科肿瘤组单药或联合化疗药物临床试验(包括铂类,紫杉醇类及蒽环类抗生素类等),晚期(FIGO分期Ⅲ、Ⅳ期)和复发性子宫内膜癌患者中仅有少数患者表现为对化疗药物有较好反应,患者临床症状、体征,实验室检查以及影像学检查达到临床完全缓解。虽然子宫内膜癌患者经联合化疗后缓解率提高,但患者无病进展生存期(PFS)较短,一般为5-7个月,而且病死率较高。这些临床数据表明目前亟待开发新型有效子宫内膜癌化疗预防和治疗药物。
天然食品是众多化疗药物有效活性成份的重要来源之一,并对子宫内膜癌和其他类型肿瘤起化疗预防和治疗作用。已有研究发现姜干粉或可溶性提取液能够诱导皮肤癌、乳腺癌、前列腺癌、结肠癌以及卵巢癌细胞系细胞周期停滞和凋亡。在SENCAR小鼠肿瘤模型中,通过皮肤局部涂抹姜的乙醇萃取液可以降低DMBA/TPA诱导的皮肤肿瘤的发病率以及肿瘤大小和数量。
已有研究显示,姜干粉和可溶性提取液中起抗癌作用的主要生物活性成份是多聚酚类化合物,如4-、6-、8-、10-gingerols, paradol和shogaol。对不同肿瘤的体外和体内研究显示,多聚酚类化合物尤其是gingerols具有有效抗肿瘤细胞增生和抗肿瘤血管形成的特性。研究显示,6-gingerol可以通过诱导CyclinD蛋白降解及激活β-catenin、PKC delta和GSK3beta等信号通路的机制抑制人结直肠肿瘤细胞增生,诱导肿瘤细胞凋亡和G1期细胞周期停滞。姜有效活性成份shagaol可以通过抑制NF-κB激活以及降低VEGF(?)IL-8分泌的机制显著抑制上皮性卵巢癌细胞系细胞增生。6-gingerol可以通过增加凋亡相关p53蛋白及其下游促凋亡蛋白Bax表达,同时降低抗凋亡蛋白Bcl-2表达等机制诱导前列腺癌细胞系LnCap细胞凋亡。
提取姜有效活性成份的途径主要有两种。一种是通过干燥生姜的方法获得姜干粉,另一种是通过加热生姜的方法获得姜的可溶性提取液。也有研究发现通过蒸汽蒸馏生姜根茎的方法也能够获得姜的有效生物活性成分。到目前为止对姜蒸汽蒸馏提取物抗癌特性的研究甚少。之前研究表明姜干粉和可溶性提取物中起抗癌作用的主要活性成份是多聚酚类化合物,而姜蒸气蒸馏提取物的化学分析显示姜蒸馏提取物中多聚酚类化合物含量极少。姜蒸汽蒸馏提取物对有效抗癌成份及其作用机制有待研究。
研究目的：
本研究旨在探讨姜蒸汽蒸馏提取物(SDGE)的化学组成,有效抗肿瘤化学成份；姜蒸汽蒸馏提取物(SDGE)对子宫内膜癌细胞增生的影响以及其抗癌作用机制。
1.方法：
1.1提取姜蒸汽蒸馏产物(SDGE)
从供应商处购得生姜,用蒸馏水洗净后将生姜切成0.5厘米大小块状。将250-300克姜转移到1000毫升Clevenger水蒸汽蒸馏装置的圆底烧瓶中,注入500毫升去离子水(18MOhm-cm)浸没姜。持续加热圆底烧瓶4-6小时。从clevenger装置中分离的油状混合物比水轻,可通过定期引流分离管中的液体来进行收集。通过离心分离得到上层淡黄色提取物(SDGE)后立即分装在离心管中,-80℃冰箱保存实验前取出。通过称量SDGE质量和体积计算出SDGE密度为0.87g/ml,这个数值用于换算提取物的浓度,方便之后生物学实验的进行。
1.2细胞增殖实验(MTT实验)
1.21四甲基偶氮唑盐摄取法检测SDGE. citral和6-gingerol对子宫内膜癌细胞系Ishikawa和ECC-1细胞增生的影响。
用培养基重悬细胞以5000个／孔的密度接种于96孔板。用不同浓度梯度SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml、6.25μ/ml、12.5μg/ml), citral (1.5μM、15μM、150μM)和6-gingerol (1.5μM、15μM、150μM)处理细胞,细胞置于5%CO2,37℃细胞培养箱中培养24、48和72小时后,每孔加入20μl3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT),细胞培养板置于5%CO2,37℃细胞培养箱中再孵育3小时。3小时后每孔中加入100μl DMSO溶液溶解甲瓒结晶。细胞培养板置于水平摇床上低速震荡5分钟。紫外分光光度计波长570nm处读取吸光值OD。
1.22SDGE联合铯-137γ放射线照射或顺铂溶液处理细胞
四甲基偶氮唑盐摄取法(MTT实验)检测SDGE是否能够加强放射线或化疗试剂对子宫内膜癌细胞的增生抑制作用。实验第一天将Ishikawa或ECC-1细胞以5000个/孔的密度接种于96孔细胞培养板。细胞贴壁后,在细胞培养板中加入单纯细胞培养基或2.5μg/ml SDGE溶液,细胞培养板置于5%CO2,37℃细胞培养箱中培养24小时。实验第三天,在一部分细胞培养孔中加入顺铂溶液(5μM)；另一部分细胞培养孔用铯-137放射仪进行照射,单次照射剂量为4Gy。细胞培养板置于5%CO2,37℃细胞培养箱中继续培养72小时。用四甲基偶氮唑盐摄取法检测不同处理条件对子宫内膜癌细胞增生的影响,实验步骤如上。
1.3气相色谱-质谱(GC-MS)技术分析SDGE化学组成
用GC-17A气相色谱仪及QP-5000四极质谱分析仪(Shimadzu公司)分析SDGE化学组成。分析前将20μl新鲜解冻的SDGE样品加入到1000μl戊烷中进行稀释。吸取1μl稀释后的SDGE样品注入气相色谱仪。设置GC-MS参数,分流比：1：50；氦气流速：1.4ml/min。设置GC-MS内非极性RTX-5MS柱(长30米,内径0.25毫米,膜厚0.25微米)柱起始温度为70℃,以4℃/min速度加热至180℃后维持。设置GC-MS电离检测方式为全扫描,阳离子模式质荷比(m/z)范围：41-300。实验结束后通过搜索美国国家标准技术研究所(NIST图书馆)将获得的数据与Adams算术保留指数值进行对比进而鉴别SDGE的化合物组成。
1.4细胞凋亡实验
用FITC-Annexin V流式细胞实验方法(BD公司凋亡检测试剂盒)检测细胞凋亡。方法如下：在2x106个细胞中加入0.25μg/ml SDGE和或100μM Pifithrin-α,细胞培养瓶置于5%CO2,37℃细胞培养箱中培养0-16小时。用胰酶进行消化,收集细胞后用冷PBS缓冲液冲洗2遍,用l×结合缓冲液(10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaCl2)重悬制成1×106个/ml细胞悬液。将100μl细胞悬液(1x105个细胞)转移至5ml流式管中,加入5μlFITC-Annexin V和5μl propidium iodide (PI)进行染色,轻轻涡旋震荡后,室温避光孵育15分钟。l×结合缓冲液洗细胞一次,用500μl1×结合缓冲液重悬。将细胞置于冰上避光,用FACSCalibur流式细胞仪进行检测。流式细胞实验结果用FlowJo软件进行分析。
1.5细胞周期实验
用不同浓度SDGE (250ng/ml或2.5μg/ml)处理子宫内膜癌细胞24、48和72小时后,胰酶消化收集细胞。PBS缓冲液洗后重悬,涡旋震荡同时逐滴加入75%乙醇进行固定。PBS缓冲液洗细胞后加入propidium iodide (PI)进行染色。流式细胞仪检测细胞周期状态,步骤如上,流式细胞实验结果用FlowJo软件进行分析。
1.6蛋白质印迹实验
用250ng/ml SDGE处理细胞特定时间后收集细胞,冷PBS缓冲液冲洗,离心去掉上清,往细胞沉淀中加入RIPA缓冲液(Pierce公司)进行细胞裂解,裂解液中加入蛋白酶抑制剂cocktail (Thermo公司)。超声裂解细胞后离心收集上清,BCA实验方法测定蛋白质浓度。取25μg蛋白质样品加入聚丙烯酰胺凝胶上样孔中。7.5或12%变性聚丙烯酰胺凝胶进行电泳分离,电泳结束后将分离蛋白转到PVDF膜上。PVDF膜用含5%脱脂奶粉的TBST缓冲液室温封闭一小时,特异性一抗孵育过夜。第二天用TBST缓冲液洗膜3次后,过氧化物酶结合二抗孵育1小时。TBST缓冲液洗膜3次后用West Dura或VVest Femto化学发光试剂盒(Thermo公司)进行显色。应用数字凝胶成像系统扫描胶片。实验结果用Image J软件进行定量分析。
1.7线粒体膜电位分析
将子宫内膜癌细胞接种在T25细胞培养瓶中。细胞指数生长期内,在细胞培养基中加入0.025μg/ml或025ug/ml SDGE;对照组加入等量DMSO。细胞培养瓶置于5%CO2,37℃细胞培养箱中培养24小时。用PBS缓冲液洗细胞后胰酶消化收集细胞,制成1×106个/ml细胞悬液。将100μl细胞悬液(1×106个细胞)转移至5ml流式管中,加入40nM DiOC637℃孵育30分钟。洗细胞后用含2%FBS PBS缓冲液400μl重悬。FACSCALIBUR流式细胞仪检测线粒体膜电位变化。实验结果用FlowJo软件进行分析。
1.8钙离子内流实验分析
胰酶消化收集对数生长期子宫内膜癌Ishikawa细胞。用PBS缓冲液洗涤3次后,用含0.5%BSA培养基重悬,使细胞密度为1×10Vml。向1×107/ml细胞悬液中加入2mM Indo1-AM和4mM probenecid,置于5%CO2,37℃细胞培养箱中孵育30分钟。用PBS缓冲液洗涤3次,用含1mM CaCl2的0.5%BSA DPBS缓冲液重悬,使细胞密度为2×106/ml。用35微米滤器过滤细胞,并保持细胞在37℃。流式细胞仪进行分析时,初始3分钟检测细胞内基础钙离子浓度,然后各试管中分别加入0.025μg/ml、0.25μg/ml.2.5μg/ml SDGE或1μM Ionomycin继续上机检测7分钟,观察记录钙离子流量变化。实验结果用FlowJo软件进行分析。
1.9统计分析
采用单样本T检验,P≤0.05被认为有显著性差异。数据分析用GraphPadPrizm软件。
2结果
2.1蒸汽蒸馏实验从姜中提取姜蒸汽蒸馏产物(SDGE)
通过改良Clevenger水蒸汽蒸馏装置我们可以方便地从姜中提取出SDGE,从250克姜中可以提取出大约300毫克精油。我们先后用五批姜提取SDGE。其中两批是从印度不巴内什瓦尔购买,在当地实验室通过蒸汽蒸馏方法提取SDGE。其余三批是从美国威斯康辛州购买,在威斯康辛大学实验室提取SDGE。这五批姜提取的SDGE产量大致相同,通过计算SDGE密度约为0.8g/L
2.2姜蒸汽蒸馏提取物(SDGE)是有效的子宫内膜癌细胞增生抑制剂
首先检测SDGE对子宫内膜癌细胞系ECC-1和Ishikawa细胞增生的影响。MTT实验发现,SDGE低浓度250ng/ml时两种子宫内膜癌细胞增生即受抑制(图1A、B)：SDGE高浓度2.5μg/ml时,两种子宫内膜癌细胞增生显著受到抑制(P<0.05)。图1A、B中实验结果是3次独立MTT实验数据的累计,实验用SDGE来自3个不同产地。在实验中SDGE每个浓度设有16个复孔。因此图1中每点是48个独立数据的平均值。实验结果仅有微小的差异,可重复性高,能清楚地说明SDGE对子宫内膜癌细胞系ECC-1和Ishikawa细胞增生的影响。MTT实验显示72小时,SDGE对两种细胞的半数抑制浓度IC50约为1.25μg/ml。
2.3SDGE联合铯-137γ放射线照射或顺铂试剂加强对子宫内膜癌细胞的增生抑制作用
晚期(FIGO分期Ⅲ、Ⅳ期)和复发性子宫内膜癌的处理原则是放疗和化疗。因此我们进一步分析SDGE联合铯-137γ放射线照射或顺铂试剂是否能够加强对子宫内膜癌细胞的增生抑制作用。MTT实验结果显示：SDGE对两种子宫内膜癌细胞系的增生抑制率为40%；SDGE对ECC-1细胞增生抑制率与铯-137γ放射线照射对两种细胞的增生抑制率相同(图1C、D)；而Ishikawa细胞,SDGE对Ishikawa细胞的增生抑制率比铯-137γ放射线照射对Ishikawa田胞的增生抑制率高10%(图1C),SDGE比铯-137γ放射线照射更有效抑制Ishikawa细胞增生；对两种子宫内膜癌细胞系ECC-1和Ishikawa细胞,SDGE联合铯-137γ放射线照射对细胞增生抑制率比单独铯-137γ放射线照射对细胞的增生抑制率高23-25%(图1C、D),说明SDGE加强铯-137γ放射线照射对子宫内膜癌细胞的增生抑制作用。
我们进一步分析了SDGE联合顺铂试剂对Ishikawa和ECC1细胞增生的影响。MTT实验结果显示：Ishikawa细胞,72小时SDGE联合顺铂试剂对细胞的增生抑制率比单独顺铂处理对细胞增生抑制率高26%(图1E)；而ECC-1细胞,SDGE联合顺铂试剂与顺铂单独对细胞的增生抑制率相同(图1F)
通过MTT实验我们还比较了SDGE与顺铂的细胞毒性。与对照组相比,顺铂(5μM;1.5μg/ml)对Ishikawa(?)田胞和ECC-1细胞的增生抑制率分别为59%和61%(图1E、F)。而SDGE (2.5μg/ml)对Ishikawa和ECC-1细胞的增生抑制率分别为37%和42%(图1E、F)。这些数据提示,SDGE与顺铂有相同有效抑制子宫内膜癌细胞增生的作用,为进一步研究SDGE抗癌机制提供有力的依据。
2.4SDGE诱导子宫内膜癌细胞凋亡
MTT实验显示SDGE是一种有效的子宫内膜癌细胞增生抑制剂。细胞凋亡实验发现子宫内膜癌细胞增生降低是SDGE直接诱导子宫内膜癌细胞凋亡的结果。流式细胞实验显示,用低浓度SDGE (250μg/ml)处理细胞后,细胞表面AnnexinV和PI染色增加(图2A)。在细胞培养基中加入SDGE后,最早在30分钟即能观察到FITC-AnnexinV染色阳性细胞数增加(图2A)。蛋白印迹实验证实,用SDGE (250ng/ml)处理ECC-1(数据未显示)和Ishikawa细胞24,48和72小时后,裂解caspase-3蛋白表达增加(图2B)。我们进一步检测SDGE对子宫内膜癌细胞周期状态的影响。用SDGE (250ng/ml或2.5μg/ml)处理Ishikawa细胞24,48和72小时后,细胞用PI进行染色,流式细胞仪检测细胞周期状态改变(图2C)。流式细胞实验分析显示,SDGE对细胞周期状态没有造成显著的影响,仅观察到细胞周期中S期细胞比例轻微降低。而且仅在高浓度SDGE2.5μg/ml处理Ishikawa (?)田胞时观察到。而低浓度250ng/ml SDGE处理细胞时,细胞周期状态没有任何改变,即便这个浓度已经能诱导子宫内膜癌细胞凋亡。
2.5SDGE的化学组成
SDGE诱导子宫内膜癌细胞凋亡的特性促使我们进一步研究SDGE的化学组成以及其中起抗癌作用的有效生物活性成份。我们用气象色谱-质谱技术(GC-MS)对三批不同产地SDGE进行分析。戊烷稀释SDGE后,挥发成分在通过非极性RTX-5MS色谱柱时得到分离(图3),色谱流出曲线上的各个峰所代表的成分通过电子离子质谱仪进行鉴定。通过分析色谱流出曲线上每个峰的滞留时间,峰面积或峰高值,可以鉴定SDGE的化学组成及各成份所占比例。我们总共从SDGE中鉴别出22种化合物及其相对含量(表1)。数据分析显示从不同产地姜提取的SDGE化学组成基本一致。
气象色谱-质谱技术分析结果显示,SDGE中不含多聚酚类化合物,如gingerol, shogaol和paradol等。而在之前的研究中已经报道,姜干粉和可溶性提取液中的有效抗癌活性成份正是这些多聚酚类化合物[20,24,25,36]。因此我们用纯化的6-gingerol进行MTT实验检测其对子宫内膜癌细胞增生的影响。MTT实验显示即便在高浓度150μM时,6-gingerol仍不能有效抑制子宫内膜癌细胞增生(图4A和4B)。6-gingerol的MTT实验结果进一步支持我们的GC-MS分析。
气象色谱-质谱分析结果显示,SDGE的主要组成成份是类萜类化合物。而citral,是Neral和geranial两种类萜异构体的混合物,占到SDGE化合物组成的35-45%(表1)。MTT实验发现,citral能显著降低Ishikawa和ECC-1田胞增生。Citral对两种细胞的半数抑制摩尔浓度IC50约为15-25μM(图4C和4D)。Citral的半数抑制浓度IC50约为2.28-3.8μg/ml。而SDGE有效的IC5。为1.25μg/ml,因neral和geranial仅占SDGE组成的35-45%,因此SDGE的IC50相当于citral2.8-3.7μM。这个浓度要显著低于MTT实验得出的citral半数抑制浓度15-25μM(图4C和4D)。因此我们得出以下结论,在SDGE中除外citral,还有其他的类萜类化合物也贡献了显著的抗癌作用。在接下来的研究中,我们仍把SDGE而非citral作为机制研究的主要对象。
2.6SDGE诱导细胞内钙离子流量增加,线粒体膜电位下降
用SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml)处理Ishikawa细胞后,细胞内钙离子流量显著增加(图5A)。在Ishikawa细胞中加入SDGE初始3分钟后即可以观察到细胞内钙离子流量增加。钙离子通量的时间动力学曲线形态与ionomycin (1μM)的时间动力学曲线形态相似。细胞内钙离子流量与SDGE浓度成正比。实验显示SDGE (2.5μg/ml)诱导的细胞内钙离子流量峰值是ionomycin (1μM)钙流量峰值的60-70%。在细胞中加入Ionomycin后细胞内钙离子流量急剧增加,在一分钟内迅速降低。而SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml)诱导的细胞内钙离子流量增加以及下降相对缓慢(图5A)。细胞中加入SDGE之后5-6分钟细胞内钙离子流量下降但仍高于初始3分钟基线钙离子水平(图5A)。
在子宫内膜癌细胞Ishikawa培养基中加入SDGE后,细胞内钙离子流量增加,细胞凋亡诱导增加,说明线粒体功能发生缺陷。我们进一步用流式细胞实验检测线粒体膜电位改变,实验结果显示用SDGE (25ng/ml或250ng/ml)处理Ishikawa细胞后,细胞线粒体膜电位降低两倍(图5B)
2.7SDGE诱导Bax/Bcl-2比值增加
Bcl-2是一种线粒体外膜相关抗凋亡蛋白。实验显示SDGE处理Ishikawa细胞后,细胞线粒体膜电位下降。促使我们进一步研究SDGE对子宫内膜癌细胞Bcl-2蛋白表达的影响。我们用SDGE (250ng/ml)处理子Ishikawa细胞特定时间,实验结果显示24,48和72小时后Ishikawa细胞Bcl-2蛋白表达降低,而促凋亡Bax蛋白表达没有明显改变,Bax/Bcl-2比值增加1.3-2.0倍(图6A),ECC-1数据未显示。
2.8SDGE激活p53信号通路
p53肿瘤抑癌基因是一个转录因子,在调控细胞死亡和各种细胞程序中起关键作用。p53参与调控的细胞事件包括细胞周期、细胞凋亡、DNA损伤修复以及衰老等。实验显示SDGE能够诱导子宫内膜癌细胞凋亡,因此我们进一步研究SDGE对p53蛋白的影响。蛋白质印迹实验显示,SDGE (250ng/ml)处理Ishikawa细胞后,p53蛋白15位丝氨酸磷酸化迅速增加(图6B),说明p53被激活。
pifithrin-a是一种特异性p53蛋白抑制剂[37]。用pifithrin-a预处理Ishikawa田胞后,流式细胞实验显示SDGE (250ng/ml)诱导的子宫内膜癌细胞凋亡完全被抑制(图6C)。对照组中细胞FITC-Annexin V染色阳性率约为11%(细胞培养基中不加入SDGE或pifithrin-a); SDGE处理组细胞FITC-Annexin V染色阳性率约为39.5%；pifithrin-a预处理组细胞FITC-Annexin V染色阳性率约为10%。细胞凋亡实验进一步证实,SDGE诱导子宫内膜癌细胞凋亡是通过激活p53信号通路。
2.9SDGE不能诱导p53neg SKOV3细胞凋亡
有研究报道卵巢癌SKOV-3细胞系不表达p53蛋白。因此我们采用SKOV3细胞研究在无p53蛋白存在情况下,SDGE对细胞凋亡的影响。流式细胞实验显示,用SDGE (250ng/ml)处理SKOV-3细胞30分钟,2、4和16小时后,细胞表面FITC-Annexin V染色率没有改变,SKOV3细胞无凋亡发生(图6D)我们进一步用蛋白质印迹实验证实SDGE处理SKOV-3细胞未发生凋亡,实验显示SDGE处理后细胞无裂解caspase3蛋白表达(图6E)
3.结论：
(1)SDGE主要组成成份是22种类萜类化合物。SDGE可通过激活p53信号通路诱导子宫内膜细胞凋亡。
(2) SDGE及其纯化类萜类化合物可作为子宫内膜癌治疗剂值得进一步研究。
Introduction
In the year2011, approximately8,010women succumbed to endometrial cancer and nearly47,130patients were newly diagnosed with this cancer. In about70%of the women with a diagnosis of endometrial cancer, the disease is found localized to the corpus and five year survival is as high as85%. Advanced and recurrent endometrial cancer patients, enrolled in several gynecologic oncology group (GOG) trials for agents including platinum, taxanes and anthracyclines, rarely have complete responses to therapy. Combination regimens show higher response rates, but the progression free period with these therapies is relatively low (5-7months) with higher morbidity and continued lack of cure.These statistics highlight the need for the development of novel and effective chemopreventive and chemotherapeutic agents for endometrial cancer.
Naturally occurring dietary components provide an important source of bioactive compounds that can serve as both chemopreventive as well as chemotherapeutic agents against endometrial and other types of cancers. Our lab is currently investigating the anti-cancer properties of compounds present in the rhizomes of ginger (Zingiber officinale). These studies are supported by previous investigations demonstrating that dry ginger powder or solvent extracts of ginger roots induce cell cycle arrest and apoptosis in skin, breast, prostate, colon, and ovarian cancer cells. Topical application of the ethanolic extract of ginger decreased the incidence, size, and the number of DMBA/TPA induced tumors in SENCAR mice.
The majority of the previous studies have concluded that the bioactive components of the dry powder and solvent extract of ginger rhizomes responsible for the anti-cancer activities are the phenolic compounds4-,6-,8-and10-gingerols, paradol, and shogaol, a product formed after drying or heating of the roots. These phenolic compounds, and especially the gingerols exhibit anti-proliferative and anti-angiogenic properties as demonstrated by in vitro and in vivo studies in various cancer models. Human colorectal cancer cells when treated with6-gingerol, inhibited cell proliferation by inducing G1cell cycle arrest and apoptosis. Gingerols exhibit these anti-cancer effects via multiple mechanisms, which include protein degradation as well as β-catenin, PKC delta, and GSK3beta pathways. Studies in the ovarian cancer model have demonstrated that6-shogaol inhibits the secretion of VEGF by the cancer cells.6-gingerol induces apoptosis in the prostate cancer cell line LnCaP by increasing the expression of p53and Bax and simultaneously decreasing the experssion of Bcl-2.
In addition to the powdered ginger and the solvent extraction, bioactive compounds can also be isolated by steam distillation of this rhizomes. To the best of our knowledge, only limited studies have been conducted to demonstrate the anti-cancer properties of the steam distilled extracts of ginger. Chemical analysis of the steam distilled extract of ginger indicates that the previously identified bioactive phenolic compounds are present at very low concentration in the steam distilled extracts of ginger. In the current study we demonstrate that the steam distilled extracts of ginger are potent mediators of apoptosis in endometrial cancer cells. Our studies suggest that one of the major bioactive components of the steam distilled extract of ginger is citral (a mixture of two terpenoid isomers, neral and geranial). We demonstrate that treatment of the endometrial cancer cells with the steam distilled extract of ginger results in significant increase in intracellular calcium, decrease in the mitochondrial membrane potential, increase in the expression of caspase3, phosphorylation of P53, and a significant decrease in the expression of Bcl-2. The observations outlined in our studies demonstrate that the steam distilled extract of ginger and its bioactive components have the potential to be developed as chemopreventive and chemotherapeutic agents for endometrial cancer.
The purpose of our study is to discover the bioactive components in the steam distilled extract of ginger and to study the underlying mechanisms of its anti-cancer property.
Methods
Steam distillation of ginger rhizomes. Ginger rhizomes were obtained from local vendors, cleaned with distilled water and cut into0.5cm pieces. Approximately250-300g of the cut ginger pieces were transferred to the1000ml round bottom flask of the Clevenger steam distillation apparatus. The ginger roots were submerged in500ml of deionized water (18MOhm-cm) and steam distillation was carried out for4-6hours by heating the flask. The oil separating in the Clevenger apparatus was lighter than water and was separated by periodically draining the liquid accumulating in the separation tube of the unit. The oil was immediately aliquoted in microfuge tubes and frozen until used in assays. The density of the oil was calculated to be0.87g/ml and this measurement was used to calculate the concentration of the extract used to conduct the biological assays.
Cell proliferation assays. Effect of steam distilled ginger extracts, citral, and6-gingerol on the proliferation of the cancer cell lines was determined by the3-(4.5-dimethythiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) uptake method [32,33]. Briefly, the cancer cells were plated in96-well plate at a density of5000cells/well in their respective medium. The cells were then treated with various concentrations of ginger extract (0.025μg,0.25μg,2.5μg,6.25μg and12.50μg/ml) and incubated at370C in a5%CO2environment for24h,48h and72h. After the designated time period,20μl3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide was added to each well and the plates were incubated at370C for additional3h. The formazan crystals formed in the wells were dissolved in100μl DMSO. The absorbance was measured at570nm using a Spectra MAX190(Molecular Devices, Sunnyvale, CA).
Combined treatment of cancer cells with SDGE and radiation or chemotherapy. MTT assays were conducted to determine if SDGE enhanced the anti-proliferation effect of radiation or chemotherapy in the endometrial cancer cells. Ishikawa or ECC-1cells were plated in multiple96well plates (5X103cells/well) on day1of the experiment. After allowing the cells to stabilize, media or SDGE were added to the wells containing the endometrial cancer cells on Day2. Cells in some of the wells were also treated with cisplatin (5μM) while others were irradiated with a single dose of4Gy using a Cesium-137radiator. Following these treatments, the cells were cultured for72h at37℃in5%CO2environment. Effect of the treatment on proliferation of the endometrial cancer cells was determined by conducting the MTT assays as described above.
Gas Chromatography-Mass Spectrometry of SDGE. Separation and identification of compounds in SDGE samples used a Shimadzu GC-17A gas chromatograph equipped with a QP-5000quadrupole mass analyzer (Shimadzu Scientific Instruments, Columbia, MD). Prior to analysis,20μL of freshly defrosted SDGE was dissolved in1000μL of pentane.1μl of this dissolved extract was injected manually to the gas chromatograph using a1:50inlet split ratio and helium as the carrier gas at a flow rate of1.4-ml/min. The gas chromatograph contained a nonpolar RTX-5MS column (30m length,0.25mm ID,0.25μm film thickness; Restek, Bellefonte, PA.) Column temperature was initially70℃followed by a ramp at4℃/min to180℃. Electron ionization detection was in full-scan, positive ion mode over a mass-to-charge ratio (m/z) range of41to300. Compounds were tentatively identified by searching a NIST library and by comparison of arithmetic retention indices to values reported by Adams.
Measurement of Apoptosis by flow cytometry. Apoptosis was measured using the FITC-Annexin V Apoptosis Detection kit (BD Pharmingen, San Diego, CA). Briefly,2×106cells were treated with0.25μg/ml ginger extract with or without100μM Pifithrin-a. After incubation at370C for0-16h, the cells were washed twice with cold PBS and resuspended in1×binding buffer,(10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaC12) at a concentration of1×106cells/ml. Then1×105cells in100μl binding buffer, were transferred to5ml tubes and stained with5μl of FITC-Annexin V and5μl propidium iodide (PI). The cells were gently vortexed and incubated at room temperature for15min. After washing the cells with lx binding buffer to remove the excess FITC-Annexin V and PI, the cells were analyzed on a FACSCalibur flow cytometer. The data were analyzed using FlowJo software.
Cell cycle assay. The endometrial cancer cells were treated with SDGE (250ng/ml or2.5μg/ml) for24,48. and72h. Following treatment, the cells were harvested, washed with PBS and fixed in75%ethanol, washed with PBS, and stained with propidium iodide. Flow cytometry was then performed to analyze the samples for both apoptosis and cell cycle status as described earlier [35].
Western blot analysis. After treatment of the cells with the steam distilled extracts of ginger, the cancer cells were washed with ice cold phosphate buffered saline (PBS) and lysed with RIPA buffer (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Thermo Scientific, Rockford. IL). The total amount of protein in the lysate was determined by using the BCA assay (Pierce). Cell lysates were loaded at25μg/well onto a7.5or12%resolving polyacrylamide gel and separated by electrophoresis, after which, the proteins were transferred to PVDF membranes. The membranes were blocked with5%milk in Tris buffered saline and probed with the appropriate primary antibodies. Horseradish peroxidase conjugated secondary antibodies and SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL) were used for detection of the proteins on the blots. The films were scanned using FLUORCHEM890and Image J software was used to quantify the intensities of the bands.
Mitochondrial membrane potential assay. The endometrial cancer cells were grown in T25tissue culture flasks. Exponentially growing cells were treated with0.025μg/ml or0.25μg/ml of ginger extract for24hrs. The cells were then washed and harvested,1×106cells were added to each flow tube from untreated,0.025μg/ml and0.25μg/ml ginger extract treated cells. The cells were treated with40nM DiOC6at37℃for30min. The cells were then washed, resuspended in400μl of PBS containing2%FBS and analyzed by FACSCALIBUR flowcytometer to assess the mitochondrial membrane potential.The data were analyzed using FlowJo software.
Calcium flux measurements. The Ishikawa cells in the log phase of growth were harvested using trypsin. The cells (1.2×107) were washed three times and suspended in1ml of0.5%bovine serum albumin (BSA) containing Hanks buffered saline that did not contain any divalent cations. The cells were loaded with Indo1-AM (2μM) in the presence of4mM probenecid for30min at37℃in5%CO2environment. The cells were then washed and resuspended in Dulbecco's phosphate buffered saline containing0.5%BSA and1mM CaCl2to a final concentration of2X106cells/ml. The cells were filtered through a35micron membrane filter prior to flow cytometry on LSR-Ⅱ cytometer. Cells were initially analyzed for3min to determine the baseline intracellular calcium concentration. SDGE (0.025.0.25, or2.5μg/ml) or Ionomycin (1μM used as a positive control) were subsequently added to the cells and the change in the Indo-1fluorescence was determined by continuously streaming the cells through the flow cytometer for approximately7mins. The data obtained were analyzed using FlowJo software.
Statistical analysis. Statistical analysis was done using the GraphPad Prizm software. The threshold for statistical significance is a probability of0.05. The data was analyzed using unpaired T-test.
Result
Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. At concentrations as low as0.25mg/ml. SDGE, isolated from six different batches of ginger rhizomes, consistently showed40%inhibition in the proliferation of the endometrial cancer cell lines Ishikawa and ECC-1. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1cells was a direct result of SDGE-induced apoptosis as demonstrated by annexin V staining and expression of cleaved caspase3. GC/MS analysis identified a total of22different terpenoid compounds in SDGE. with the isomers neral and geranial constituting30-40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1cells at an IC5010μM (2.3μg/ml). Phenolic compounds such as gingerol, shogaol were not detected in SDGE and6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a20-40%decrease in the mitochondrial membrane potential. Ser-15of p53was phosphorylated after15min treatment of the cancer cells with SDGE. This increase in p53was associated with90%decrease in Bcl2whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE. Our studies demonstrate that terpenoids from SDGE activate p53, resulting in decreased expression of the anti-apoptotic Bcl-2. The studies indicate that terpenoids from ginger rhizomes should be investigated as therapeutic agents for endometrial cancer.
子宫内膜癌是常见的妇产科恶性肿瘤之一。2011年美国约有8,010名妇女死于子宫内膜癌,有近47,130名患者新诊断为子宫内膜癌。在所有子宫内膜癌患者中,约70%的患者其子宫内膜癌病灶局限于宫体,患者5年生存率高达85%。美国妇科肿瘤组单药或联合化疗药物临床试验(包括铂类,紫杉醇类及蒽环类抗生素类等),晚期(FIGO分期Ⅲ、Ⅳ期)和复发性子宫内膜癌患者中仅有少数患者表现为对化疗药物有较好反应,患者临床症状、体征,实验室检查以及影像学检查达到临床完全缓解。虽然子宫内膜癌患者经联合化疗后缓解率提高,但患者无病进展生存期(PFS)较短,一般为5-7个月,而且病死率较高。这些临床数据表明目前亟待开发新型有效子宫内膜癌化疗预防和治疗药物。
天然食品是众多化疗药物有效活性成份的重要来源之一,并对子宫内膜癌和其他类型肿瘤起化疗预防和治疗作用。已有研究发现姜干粉或可溶性提取液能够诱导皮肤癌、乳腺癌、前列腺癌、结肠癌以及卵巢癌细胞系细胞周期停滞和凋亡。在SENCAR小鼠肿瘤模型中,通过皮肤局部涂抹姜的乙醇萃取液可以降低DMBA/TPA诱导的皮肤肿瘤的发病率以及肿瘤大小和数量。
已有研究显示,姜干粉和可溶性提取液中起抗癌作用的主要生物活性成份是多聚酚类化合物,如4-、6-、8-、10-gingerols, paradol和shogaol。对不同肿瘤的体外和体内研究显示,多聚酚类化合物尤其是gingerols具有有效抗肿瘤细胞增生和抗肿瘤血管形成的特性。研究显示,6-gingerol可以通过诱导CyclinD蛋白降解及激活β-catenin、PKC delta和GSK3beta等信号通路的机制抑制人结直肠肿瘤细胞增生,诱导肿瘤细胞凋亡和G1期细胞周期停滞。姜有效活性成份shagaol可以通过抑制NF-κB激活以及降低VEGF(?)IL-8分泌的机制显著抑制上皮性卵巢癌细胞系细胞增生。6-gingerol可以通过增加凋亡相关p53蛋白及其下游促凋亡蛋白Bax表达,同时降低抗凋亡蛋白Bcl-2表达等机制诱导前列腺癌细胞系LnCap细胞凋亡。
提取姜有效活性成份的途径主要有两种。一种是通过干燥生姜的方法获得姜干粉,另一种是通过加热生姜的方法获得姜的可溶性提取液。也有研究发现通过蒸汽蒸馏生姜根茎的方法也能够获得姜的有效生物活性成分。到目前为止对姜蒸汽蒸馏提取物抗癌特性的研究甚少。之前研究表明姜干粉和可溶性提取物中起抗癌作用的主要活性成份是多聚酚类化合物,而姜蒸气蒸馏提取物的化学分析显示姜蒸馏提取物中多聚酚类化合物含量极少。姜蒸汽蒸馏提取物对有效抗癌成份及其作用机制有待研究。
研究目的：
本研究旨在探讨姜蒸汽蒸馏提取物(SDGE)的化学组成,有效抗肿瘤化学成份；姜蒸汽蒸馏提取物(SDGE)对子宫内膜癌细胞增生的影响以及其抗癌作用机制。
1.方法：
1.1提取姜蒸汽蒸馏产物(SDGE)
从供应商处购得生姜,用蒸馏水洗净后将生姜切成0.5厘米大小块状。将250-300克姜转移到1000毫升Clevenger水蒸汽蒸馏装置的圆底烧瓶中,注入500毫升去离子水(18MOhm-cm)浸没姜。持续加热圆底烧瓶4-6小时。从clevenger装置中分离的油状混合物比水轻,可通过定期引流分离管中的液体来进行收集。通过离心分离得到上层淡黄色提取物(SDGE)后立即分装在离心管中,-80℃冰箱保存实验前取出。通过称量SDGE质量和体积计算出SDGE密度为0.87g/ml,这个数值用于换算提取物的浓度,方便之后生物学实验的进行。
1.2细胞增殖实验(MTT实验)
1.21四甲基偶氮唑盐摄取法检测SDGE. citral和6-gingerol对子宫内膜癌细胞系Ishikawa和ECC-1细胞增生的影响。
用培养基重悬细胞以5000个／孔的密度接种于96孔板。用不同浓度梯度SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml、6.25μ/ml、12.5μg/ml), citral (1.5μM、15μM、150μM)和6-gingerol (1.5μM、15μM、150μM)处理细胞,细胞置于5%CO2,37℃细胞培养箱中培养24、48和72小时后,每孔加入20μl3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT),细胞培养板置于5%CO2,37℃细胞培养箱中再孵育3小时。3小时后每孔中加入100μl DMSO溶液溶解甲瓒结晶。细胞培养板置于水平摇床上低速震荡5分钟。紫外分光光度计波长570nm处读取吸光值OD。
1.22SDGE联合铯-137γ放射线照射或顺铂溶液处理细胞
四甲基偶氮唑盐摄取法(MTT实验)检测SDGE是否能够加强放射线或化疗试剂对子宫内膜癌细胞的增生抑制作用。实验第一天将Ishikawa或ECC-1细胞以5000个/孔的密度接种于96孔细胞培养板。细胞贴壁后,在细胞培养板中加入单纯细胞培养基或2.5μg/ml SDGE溶液,细胞培养板置于5%CO2,37℃细胞培养箱中培养24小时。实验第三天,在一部分细胞培养孔中加入顺铂溶液(5μM)；另一部分细胞培养孔用铯-137放射仪进行照射,单次照射剂量为4Gy。细胞培养板置于5%CO2,37℃细胞培养箱中继续培养72小时。用四甲基偶氮唑盐摄取法检测不同处理条件对子宫内膜癌细胞增生的影响,实验步骤如上。
1.3气相色谱-质谱(GC-MS)技术分析SDGE化学组成
用GC-17A气相色谱仪及QP-5000四极质谱分析仪(Shimadzu公司)分析SDGE化学组成。分析前将20μl新鲜解冻的SDGE样品加入到1000μl戊烷中进行稀释。吸取1μl稀释后的SDGE样品注入气相色谱仪。设置GC-MS参数,分流比：1：50；氦气流速：1.4ml/min。设置GC-MS内非极性RTX-5MS柱(长30米,内径0.25毫米,膜厚0.25微米)柱起始温度为70℃,以4℃/min速度加热至180℃后维持。设置GC-MS电离检测方式为全扫描,阳离子模式质荷比(m/z)范围：41-300。实验结束后通过搜索美国国家标准技术研究所(NIST图书馆)将获得的数据与Adams算术保留指数值进行对比进而鉴别SDGE的化合物组成。
1.4细胞凋亡实验
用FITC-Annexin V流式细胞实验方法(BD公司凋亡检测试剂盒)检测细胞凋亡。方法如下：在2x106个细胞中加入0.25μg/ml SDGE和或100μM Pifithrin-α,细胞培养瓶置于5%CO2,37℃细胞培养箱中培养0-16小时。用胰酶进行消化,收集细胞后用冷PBS缓冲液冲洗2遍,用l×结合缓冲液(10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaCl2)重悬制成1×106个/ml细胞悬液。将100μl细胞悬液(1x105个细胞)转移至5ml流式管中,加入5μlFITC-Annexin V和5μl propidium iodide (PI)进行染色,轻轻涡旋震荡后,室温避光孵育15分钟。l×结合缓冲液洗细胞一次,用500μl1×结合缓冲液重悬。将细胞置于冰上避光,用FACSCalibur流式细胞仪进行检测。流式细胞实验结果用FlowJo软件进行分析。
1.5细胞周期实验
用不同浓度SDGE (250ng/ml或2.5μg/ml)处理子宫内膜癌细胞24、48和72小时后,胰酶消化收集细胞。PBS缓冲液洗后重悬,涡旋震荡同时逐滴加入75%乙醇进行固定。PBS缓冲液洗细胞后加入propidium iodide (PI)进行染色。流式细胞仪检测细胞周期状态,步骤如上,流式细胞实验结果用FlowJo软件进行分析。
1.6蛋白质印迹实验
用250ng/ml SDGE处理细胞特定时间后收集细胞,冷PBS缓冲液冲洗,离心去掉上清,往细胞沉淀中加入RIPA缓冲液(Pierce公司)进行细胞裂解,裂解液中加入蛋白酶抑制剂cocktail (Thermo公司)。超声裂解细胞后离心收集上清,BCA实验方法测定蛋白质浓度。取25μg蛋白质样品加入聚丙烯酰胺凝胶上样孔中。7.5或12%变性聚丙烯酰胺凝胶进行电泳分离,电泳结束后将分离蛋白转到PVDF膜上。PVDF膜用含5%脱脂奶粉的TBST缓冲液室温封闭一小时,特异性一抗孵育过夜。第二天用TBST缓冲液洗膜3次后,过氧化物酶结合二抗孵育1小时。TBST缓冲液洗膜3次后用West Dura或VVest Femto化学发光试剂盒(Thermo公司)进行显色。应用数字凝胶成像系统扫描胶片。实验结果用Image J软件进行定量分析。
1.7线粒体膜电位分析
将子宫内膜癌细胞接种在T25细胞培养瓶中。细胞指数生长期内,在细胞培养基中加入0.025μg/ml或025ug/ml SDGE;对照组加入等量DMSO。细胞培养瓶置于5%CO2,37℃细胞培养箱中培养24小时。用PBS缓冲液洗细胞后胰酶消化收集细胞,制成1×106个/ml细胞悬液。将100μl细胞悬液(1×106个细胞)转移至5ml流式管中,加入40nM DiOC637℃孵育30分钟。洗细胞后用含2%FBS PBS缓冲液400μl重悬。FACSCALIBUR流式细胞仪检测线粒体膜电位变化。实验结果用FlowJo软件进行分析。
1.8钙离子内流实验分析
胰酶消化收集对数生长期子宫内膜癌Ishikawa细胞。用PBS缓冲液洗涤3次后,用含0.5%BSA培养基重悬,使细胞密度为1×10Vml。向1×107/ml细胞悬液中加入2mM Indo1-AM和4mM probenecid,置于5%CO2,37℃细胞培养箱中孵育30分钟。用PBS缓冲液洗涤3次,用含1mM CaCl2的0.5%BSA DPBS缓冲液重悬,使细胞密度为2×106/ml。用35微米滤器过滤细胞,并保持细胞在37℃。流式细胞仪进行分析时,初始3分钟检测细胞内基础钙离子浓度,然后各试管中分别加入0.025μg/ml、0.25μg/ml.2.5μg/ml SDGE或1μM Ionomycin继续上机检测7分钟,观察记录钙离子流量变化。实验结果用FlowJo软件进行分析。
1.9统计分析
采用单样本T检验,P≤0.05被认为有显著性差异。数据分析用GraphPadPrizm软件。
2结果
2.1蒸汽蒸馏实验从姜中提取姜蒸汽蒸馏产物(SDGE)
通过改良Clevenger水蒸汽蒸馏装置我们可以方便地从姜中提取出SDGE,从250克姜中可以提取出大约300毫克精油。我们先后用五批姜提取SDGE。其中两批是从印度不巴内什瓦尔购买,在当地实验室通过蒸汽蒸馏方法提取SDGE。其余三批是从美国威斯康辛州购买,在威斯康辛大学实验室提取SDGE。这五批姜提取的SDGE产量大致相同,通过计算SDGE密度约为0.8g/L
2.2姜蒸汽蒸馏提取物(SDGE)是有效的子宫内膜癌细胞增生抑制剂
首先检测SDGE对子宫内膜癌细胞系ECC-1和Ishikawa细胞增生的影响。MTT实验发现,SDGE低浓度250ng/ml时两种子宫内膜癌细胞增生即受抑制(图1A、B)：SDGE高浓度2.5μg/ml时,两种子宫内膜癌细胞增生显著受到抑制(P<0.05)。图1A、B中实验结果是3次独立MTT实验数据的累计,实验用SDGE来自3个不同产地。在实验中SDGE每个浓度设有16个复孔。因此图1中每点是48个独立数据的平均值。实验结果仅有微小的差异,可重复性高,能清楚地说明SDGE对子宫内膜癌细胞系ECC-1和Ishikawa细胞增生的影响。MTT实验显示72小时,SDGE对两种细胞的半数抑制浓度IC50约为1.25μg/ml。
2.3SDGE联合铯-137γ放射线照射或顺铂试剂加强对子宫内膜癌细胞的增生抑制作用
晚期(FIGO分期Ⅲ、Ⅳ期)和复发性子宫内膜癌的处理原则是放疗和化疗。因此我们进一步分析SDGE联合铯-137γ放射线照射或顺铂试剂是否能够加强对子宫内膜癌细胞的增生抑制作用。MTT实验结果显示：SDGE对两种子宫内膜癌细胞系的增生抑制率为40%；SDGE对ECC-1细胞增生抑制率与铯-137γ放射线照射对两种细胞的增生抑制率相同(图1C、D)；而Ishikawa细胞,SDGE对Ishikawa细胞的增生抑制率比铯-137γ放射线照射对Ishikawa田胞的增生抑制率高10%(图1C),SDGE比铯-137γ放射线照射更有效抑制Ishikawa细胞增生；对两种子宫内膜癌细胞系ECC-1和Ishikawa细胞,SDGE联合铯-137γ放射线照射对细胞增生抑制率比单独铯-137γ放射线照射对细胞的增生抑制率高23-25%(图1C、D),说明SDGE加强铯-137γ放射线照射对子宫内膜癌细胞的增生抑制作用。
我们进一步分析了SDGE联合顺铂试剂对Ishikawa和ECC1细胞增生的影响。MTT实验结果显示：Ishikawa细胞,72小时SDGE联合顺铂试剂对细胞的增生抑制率比单独顺铂处理对细胞增生抑制率高26%(图1E)；而ECC-1细胞,SDGE联合顺铂试剂与顺铂单独对细胞的增生抑制率相同(图1F)
通过MTT实验我们还比较了SDGE与顺铂的细胞毒性。与对照组相比,顺铂(5μM;1.5μg/ml)对Ishikawa(?)田胞和ECC-1细胞的增生抑制率分别为59%和61%(图1E、F)。而SDGE (2.5μg/ml)对Ishikawa和ECC-1细胞的增生抑制率分别为37%和42%(图1E、F)。这些数据提示,SDGE与顺铂有相同有效抑制子宫内膜癌细胞增生的作用,为进一步研究SDGE抗癌机制提供有力的依据。
2.4SDGE诱导子宫内膜癌细胞凋亡
MTT实验显示SDGE是一种有效的子宫内膜癌细胞增生抑制剂。细胞凋亡实验发现子宫内膜癌细胞增生降低是SDGE直接诱导子宫内膜癌细胞凋亡的结果。流式细胞实验显示,用低浓度SDGE (250μg/ml)处理细胞后,细胞表面AnnexinV和PI染色增加(图2A)。在细胞培养基中加入SDGE后,最早在30分钟即能观察到FITC-AnnexinV染色阳性细胞数增加(图2A)。蛋白印迹实验证实,用SDGE (250ng/ml)处理ECC-1(数据未显示)和Ishikawa细胞24,48和72小时后,裂解caspase-3蛋白表达增加(图2B)。我们进一步检测SDGE对子宫内膜癌细胞周期状态的影响。用SDGE (250ng/ml或2.5μg/ml)处理Ishikawa细胞24,48和72小时后,细胞用PI进行染色,流式细胞仪检测细胞周期状态改变(图2C)。流式细胞实验分析显示,SDGE对细胞周期状态没有造成显著的影响,仅观察到细胞周期中S期细胞比例轻微降低。而且仅在高浓度SDGE2.5μg/ml处理Ishikawa (?)田胞时观察到。而低浓度250ng/ml SDGE处理细胞时,细胞周期状态没有任何改变,即便这个浓度已经能诱导子宫内膜癌细胞凋亡。
2.5SDGE的化学组成
SDGE诱导子宫内膜癌细胞凋亡的特性促使我们进一步研究SDGE的化学组成以及其中起抗癌作用的有效生物活性成份。我们用气象色谱-质谱技术(GC-MS)对三批不同产地SDGE进行分析。戊烷稀释SDGE后,挥发成分在通过非极性RTX-5MS色谱柱时得到分离(图3),色谱流出曲线上的各个峰所代表的成分通过电子离子质谱仪进行鉴定。通过分析色谱流出曲线上每个峰的滞留时间,峰面积或峰高值,可以鉴定SDGE的化学组成及各成份所占比例。我们总共从SDGE中鉴别出22种化合物及其相对含量(表1)。数据分析显示从不同产地姜提取的SDGE化学组成基本一致。
气象色谱-质谱技术分析结果显示,SDGE中不含多聚酚类化合物,如gingerol, shogaol和paradol等。而在之前的研究中已经报道,姜干粉和可溶性提取液中的有效抗癌活性成份正是这些多聚酚类化合物[20,24,25,36]。因此我们用纯化的6-gingerol进行MTT实验检测其对子宫内膜癌细胞增生的影响。MTT实验显示即便在高浓度150μM时,6-gingerol仍不能有效抑制子宫内膜癌细胞增生(图4A和4B)。6-gingerol的MTT实验结果进一步支持我们的GC-MS分析。
气象色谱-质谱分析结果显示,SDGE的主要组成成份是类萜类化合物。而citral,是Neral和geranial两种类萜异构体的混合物,占到SDGE化合物组成的35-45%(表1)。MTT实验发现,citral能显著降低Ishikawa和ECC-1田胞增生。Citral对两种细胞的半数抑制摩尔浓度IC50约为15-25μM(图4C和4D)。Citral的半数抑制浓度IC50约为2.28-3.8μg/ml。而SDGE有效的IC5。为1.25μg/ml,因neral和geranial仅占SDGE组成的35-45%,因此SDGE的IC50相当于citral2.8-3.7μM。这个浓度要显著低于MTT实验得出的citral半数抑制浓度15-25μM(图4C和4D)。因此我们得出以下结论,在SDGE中除外citral,还有其他的类萜类化合物也贡献了显著的抗癌作用。在接下来的研究中,我们仍把SDGE而非citral作为机制研究的主要对象。
2.6SDGE诱导细胞内钙离子流量增加,线粒体膜电位下降
用SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml)处理Ishikawa细胞后,细胞内钙离子流量显著增加(图5A)。在Ishikawa细胞中加入SDGE初始3分钟后即可以观察到细胞内钙离子流量增加。钙离子通量的时间动力学曲线形态与ionomycin (1μM)的时间动力学曲线形态相似。细胞内钙离子流量与SDGE浓度成正比。实验显示SDGE (2.5μg/ml)诱导的细胞内钙离子流量峰值是ionomycin (1μM)钙流量峰值的60-70%。在细胞中加入Ionomycin后细胞内钙离子流量急剧增加,在一分钟内迅速降低。而SDGE (0.025μg/ml、0.25μg/ml、2.5μg/ml)诱导的细胞内钙离子流量增加以及下降相对缓慢(图5A)。细胞中加入SDGE之后5-6分钟细胞内钙离子流量下降但仍高于初始3分钟基线钙离子水平(图5A)。
在子宫内膜癌细胞Ishikawa培养基中加入SDGE后,细胞内钙离子流量增加,细胞凋亡诱导增加,说明线粒体功能发生缺陷。我们进一步用流式细胞实验检测线粒体膜电位改变,实验结果显示用SDGE (25ng/ml或250ng/ml)处理Ishikawa细胞后,细胞线粒体膜电位降低两倍(图5B)
2.7SDGE诱导Bax/Bcl-2比值增加
Bcl-2是一种线粒体外膜相关抗凋亡蛋白。实验显示SDGE处理Ishikawa细胞后,细胞线粒体膜电位下降。促使我们进一步研究SDGE对子宫内膜癌细胞Bcl-2蛋白表达的影响。我们用SDGE (250ng/ml)处理子Ishikawa细胞特定时间,实验结果显示24,48和72小时后Ishikawa细胞Bcl-2蛋白表达降低,而促凋亡Bax蛋白表达没有明显改变,Bax/Bcl-2比值增加1.3-2.0倍(图6A),ECC-1数据未显示。
2.8SDGE激活p53信号通路
p53肿瘤抑癌基因是一个转录因子,在调控细胞死亡和各种细胞程序中起关键作用。p53参与调控的细胞事件包括细胞周期、细胞凋亡、DNA损伤修复以及衰老等。实验显示SDGE能够诱导子宫内膜癌细胞凋亡,因此我们进一步研究SDGE对p53蛋白的影响。蛋白质印迹实验显示,SDGE (250ng/ml)处理Ishikawa细胞后,p53蛋白15位丝氨酸磷酸化迅速增加(图6B),说明p53被激活。
pifithrin-a是一种特异性p53蛋白抑制剂[37]。用pifithrin-a预处理Ishikawa田胞后,流式细胞实验显示SDGE (250ng/ml)诱导的子宫内膜癌细胞凋亡完全被抑制(图6C)。对照组中细胞FITC-Annexin V染色阳性率约为11%(细胞培养基中不加入SDGE或pifithrin-a); SDGE处理组细胞FITC-Annexin V染色阳性率约为39.5%；pifithrin-a预处理组细胞FITC-Annexin V染色阳性率约为10%。细胞凋亡实验进一步证实,SDGE诱导子宫内膜癌细胞凋亡是通过激活p53信号通路。
2.9SDGE不能诱导p53neg SKOV3细胞凋亡
有研究报道卵巢癌SKOV-3细胞系不表达p53蛋白。因此我们采用SKOV3细胞研究在无p53蛋白存在情况下,SDGE对细胞凋亡的影响。流式细胞实验显示,用SDGE (250ng/ml)处理SKOV-3细胞30分钟,2、4和16小时后,细胞表面FITC-Annexin V染色率没有改变,SKOV3细胞无凋亡发生(图6D)我们进一步用蛋白质印迹实验证实SDGE处理SKOV-3细胞未发生凋亡,实验显示SDGE处理后细胞无裂解caspase3蛋白表达(图6E)
3.结论：
(1)SDGE主要组成成份是22种类萜类化合物。SDGE可通过激活p53信号通路诱导子宫内膜细胞凋亡。
(2) SDGE及其纯化类萜类化合物可作为子宫内膜癌治疗剂值得进一步研究。
Introduction
In the year2011, approximately8,010women succumbed to endometrial cancer and nearly47,130patients were newly diagnosed with this cancer. In about70%of the women with a diagnosis of endometrial cancer, the disease is found localized to the corpus and five year survival is as high as85%. Advanced and recurrent endometrial cancer patients, enrolled in several gynecologic oncology group (GOG) trials for agents including platinum, taxanes and anthracyclines, rarely have complete responses to therapy. Combination regimens show higher response rates, but the progression free period with these therapies is relatively low (5-7months) with higher morbidity and continued lack of cure.These statistics highlight the need for the development of novel and effective chemopreventive and chemotherapeutic agents for endometrial cancer.
Naturally occurring dietary components provide an important source of bioactive compounds that can serve as both chemopreventive as well as chemotherapeutic agents against endometrial and other types of cancers. Our lab is currently investigating the anti-cancer properties of compounds present in the rhizomes of ginger (Zingiber officinale). These studies are supported by previous investigations demonstrating that dry ginger powder or solvent extracts of ginger roots induce cell cycle arrest and apoptosis in skin, breast, prostate, colon, and ovarian cancer cells. Topical application of the ethanolic extract of ginger decreased the incidence, size, and the number of DMBA/TPA induced tumors in SENCAR mice.
The majority of the previous studies have concluded that the bioactive components of the dry powder and solvent extract of ginger rhizomes responsible for the anti-cancer activities are the phenolic compounds4-,6-,8-and10-gingerols, paradol, and shogaol, a product formed after drying or heating of the roots. These phenolic compounds, and especially the gingerols exhibit anti-proliferative and anti-angiogenic properties as demonstrated by in vitro and in vivo studies in various cancer models. Human colorectal cancer cells when treated with6-gingerol, inhibited cell proliferation by inducing G1cell cycle arrest and apoptosis. Gingerols exhibit these anti-cancer effects via multiple mechanisms, which include protein degradation as well as β-catenin, PKC delta, and GSK3beta pathways. Studies in the ovarian cancer model have demonstrated that6-shogaol inhibits the secretion of VEGF by the cancer cells.6-gingerol induces apoptosis in the prostate cancer cell line LnCaP by increasing the expression of p53and Bax and simultaneously decreasing the experssion of Bcl-2.
In addition to the powdered ginger and the solvent extraction, bioactive compounds can also be isolated by steam distillation of this rhizomes. To the best of our knowledge, only limited studies have been conducted to demonstrate the anti-cancer properties of the steam distilled extracts of ginger. Chemical analysis of the steam distilled extract of ginger indicates that the previously identified bioactive phenolic compounds are present at very low concentration in the steam distilled extracts of ginger. In the current study we demonstrate that the steam distilled extracts of ginger are potent mediators of apoptosis in endometrial cancer cells. Our studies suggest that one of the major bioactive components of the steam distilled extract of ginger is citral (a mixture of two terpenoid isomers, neral and geranial). We demonstrate that treatment of the endometrial cancer cells with the steam distilled extract of ginger results in significant increase in intracellular calcium, decrease in the mitochondrial membrane potential, increase in the expression of caspase3, phosphorylation of P53, and a significant decrease in the expression of Bcl-2. The observations outlined in our studies demonstrate that the steam distilled extract of ginger and its bioactive components have the potential to be developed as chemopreventive and chemotherapeutic agents for endometrial cancer.
The purpose of our study is to discover the bioactive components in the steam distilled extract of ginger and to study the underlying mechanisms of its anti-cancer property.
Methods
Steam distillation of ginger rhizomes. Ginger rhizomes were obtained from local vendors, cleaned with distilled water and cut into0.5cm pieces. Approximately250-300g of the cut ginger pieces were transferred to the1000ml round bottom flask of the Clevenger steam distillation apparatus. The ginger roots were submerged in500ml of deionized water (18MOhm-cm) and steam distillation was carried out for4-6hours by heating the flask. The oil separating in the Clevenger apparatus was lighter than water and was separated by periodically draining the liquid accumulating in the separation tube of the unit. The oil was immediately aliquoted in microfuge tubes and frozen until used in assays. The density of the oil was calculated to be0.87g/ml and this measurement was used to calculate the concentration of the extract used to conduct the biological assays.
Cell proliferation assays. Effect of steam distilled ginger extracts, citral, and6-gingerol on the proliferation of the cancer cell lines was determined by the3-(4.5-dimethythiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) uptake method [32,33]. Briefly, the cancer cells were plated in96-well plate at a density of5000cells/well in their respective medium. The cells were then treated with various concentrations of ginger extract (0.025μg,0.25μg,2.5μg,6.25μg and12.50μg/ml) and incubated at370C in a5%CO2environment for24h,48h and72h. After the designated time period,20μl3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide was added to each well and the plates were incubated at370C for additional3h. The formazan crystals formed in the wells were dissolved in100μl DMSO. The absorbance was measured at570nm using a Spectra MAX190(Molecular Devices, Sunnyvale, CA).
Combined treatment of cancer cells with SDGE and radiation or chemotherapy. MTT assays were conducted to determine if SDGE enhanced the anti-proliferation effect of radiation or chemotherapy in the endometrial cancer cells. Ishikawa or ECC-1cells were plated in multiple96well plates (5X103cells/well) on day1of the experiment. After allowing the cells to stabilize, media or SDGE were added to the wells containing the endometrial cancer cells on Day2. Cells in some of the wells were also treated with cisplatin (5μM) while others were irradiated with a single dose of4Gy using a Cesium-137radiator. Following these treatments, the cells were cultured for72h at37℃in5%CO2environment. Effect of the treatment on proliferation of the endometrial cancer cells was determined by conducting the MTT assays as described above.
Gas Chromatography-Mass Spectrometry of SDGE. Separation and identification of compounds in SDGE samples used a Shimadzu GC-17A gas chromatograph equipped with a QP-5000quadrupole mass analyzer (Shimadzu Scientific Instruments, Columbia, MD). Prior to analysis,20μL of freshly defrosted SDGE was dissolved in1000μL of pentane.1μl of this dissolved extract was injected manually to the gas chromatograph using a1:50inlet split ratio and helium as the carrier gas at a flow rate of1.4-ml/min. The gas chromatograph contained a nonpolar RTX-5MS column (30m length,0.25mm ID,0.25μm film thickness; Restek, Bellefonte, PA.) Column temperature was initially70℃followed by a ramp at4℃/min to180℃. Electron ionization detection was in full-scan, positive ion mode over a mass-to-charge ratio (m/z) range of41to300. Compounds were tentatively identified by searching a NIST library and by comparison of arithmetic retention indices to values reported by Adams.
Measurement of Apoptosis by flow cytometry. Apoptosis was measured using the FITC-Annexin V Apoptosis Detection kit (BD Pharmingen, San Diego, CA). Briefly,2×106cells were treated with0.25μg/ml ginger extract with or without100μM Pifithrin-a. After incubation at370C for0-16h, the cells were washed twice with cold PBS and resuspended in1×binding buffer,(10mM HEPES/NaOH, pH7.4,140mM NaCl,2.5mM CaC12) at a concentration of1×106cells/ml. Then1×105cells in100μl binding buffer, were transferred to5ml tubes and stained with5μl of FITC-Annexin V and5μl propidium iodide (PI). The cells were gently vortexed and incubated at room temperature for15min. After washing the cells with lx binding buffer to remove the excess FITC-Annexin V and PI, the cells were analyzed on a FACSCalibur flow cytometer. The data were analyzed using FlowJo software.
Cell cycle assay. The endometrial cancer cells were treated with SDGE (250ng/ml or2.5μg/ml) for24,48. and72h. Following treatment, the cells were harvested, washed with PBS and fixed in75%ethanol, washed with PBS, and stained with propidium iodide. Flow cytometry was then performed to analyze the samples for both apoptosis and cell cycle status as described earlier [35].
Western blot analysis. After treatment of the cells with the steam distilled extracts of ginger, the cancer cells were washed with ice cold phosphate buffered saline (PBS) and lysed with RIPA buffer (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Thermo Scientific, Rockford. IL). The total amount of protein in the lysate was determined by using the BCA assay (Pierce). Cell lysates were loaded at25μg/well onto a7.5or12%resolving polyacrylamide gel and separated by electrophoresis, after which, the proteins were transferred to PVDF membranes. The membranes were blocked with5%milk in Tris buffered saline and probed with the appropriate primary antibodies. Horseradish peroxidase conjugated secondary antibodies and SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL) were used for detection of the proteins on the blots. The films were scanned using FLUORCHEM890and Image J software was used to quantify the intensities of the bands.
Mitochondrial membrane potential assay. The endometrial cancer cells were grown in T25tissue culture flasks. Exponentially growing cells were treated with0.025μg/ml or0.25μg/ml of ginger extract for24hrs. The cells were then washed and harvested,1×106cells were added to each flow tube from untreated,0.025μg/ml and0.25μg/ml ginger extract treated cells. The cells were treated with40nM DiOC6at37℃for30min. The cells were then washed, resuspended in400μl of PBS containing2%FBS and analyzed by FACSCALIBUR flowcytometer to assess the mitochondrial membrane potential.The data were analyzed using FlowJo software.
Calcium flux measurements. The Ishikawa cells in the log phase of growth were harvested using trypsin. The cells (1.2×107) were washed three times and suspended in1ml of0.5%bovine serum albumin (BSA) containing Hanks buffered saline that did not contain any divalent cations. The cells were loaded with Indo1-AM (2μM) in the presence of4mM probenecid for30min at37℃in5%CO2environment. The cells were then washed and resuspended in Dulbecco's phosphate buffered saline containing0.5%BSA and1mM CaCl2to a final concentration of2X106cells/ml. The cells were filtered through a35micron membrane filter prior to flow cytometry on LSR-Ⅱ cytometer. Cells were initially analyzed for3min to determine the baseline intracellular calcium concentration. SDGE (0.025.0.25, or2.5μg/ml) or Ionomycin (1μM used as a positive control) were subsequently added to the cells and the change in the Indo-1fluorescence was determined by continuously streaming the cells through the flow cytometer for approximately7mins. The data obtained were analyzed using FlowJo software.
Statistical analysis. Statistical analysis was done using the GraphPad Prizm software. The threshold for statistical significance is a probability of0.05. The data was analyzed using unpaired T-test.
Result
Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. At concentrations as low as0.25mg/ml. SDGE, isolated from six different batches of ginger rhizomes, consistently showed40%inhibition in the proliferation of the endometrial cancer cell lines Ishikawa and ECC-1. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1cells was a direct result of SDGE-induced apoptosis as demonstrated by annexin V staining and expression of cleaved caspase3. GC/MS analysis identified a total of22different terpenoid compounds in SDGE. with the isomers neral and geranial constituting30-40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1cells at an IC5010μM (2.3μg/ml). Phenolic compounds such as gingerol, shogaol were not detected in SDGE and6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a20-40%decrease in the mitochondrial membrane potential. Ser-15of p53was phosphorylated after15min treatment of the cancer cells with SDGE. This increase in p53was associated with90%decrease in Bcl2whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE. Our studies demonstrate that terpenoids from SDGE activate p53, resulting in decreased expression of the anti-apoptotic Bcl-2. The studies indicate that terpenoids from ginger rhizomes should be investigated as therapeutic agents for endometrial cancer.
引文
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5. Fleming GF, Filiaci VL. Bentley RC, Herzog T, Sorosky J, et al. (2004) Phase Ⅲ randomized trial of doxorubicin+cisplatin versus doxorubicin+24-h paclitaxel+ filgrastim in endometrial carcinoma:a Gynecologic Oncology Group study. Ann Oncol 15:1173-1178.
6. Fleming GF, Brunetto VL, Cella D, Look KY, Reid GC, et al. (2004) Phase Ⅲ trial of doxorubicin plus cisplatin with or without paclitaxel plus filgrastim in advanced endometrial carcinoma:a Gynecologic Oncology Group Study. J Clin Oncol 22:2159-2166.
7. Randall ME, Filiaci VL, Muss H, Spirtos NM, Mannel RS, et al. (2006) Randomized phase Ⅲ trial of whole-abdominal irradiation versus doxorubicin and cisplatin chemotherapy in advanced endometrial carcinoma:a Gynecologic Oncology Group Study. J Clin Oncol 24:36-44.
8. Thigpen JT, Brady MF, Homesley HD. Malfetano J. DuBeshter B, et al. (2004) Phase Ⅲ trial of doxorubicin with or without cisplatin in advanced endometrial carcinoma:a gynecologic oncology group study. J Clin Oncol 22:3902-3908.
9. Homesley HD, Filiaci V, Gibbons SK, Long HJ, Cella D, et al. (2009) A randomized phase Ⅲ trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel:A Gynecologic Oncology Group study. Gynecol Oncol 112:543-552.
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12. Brown AC, Shah C, Liu J, Pham JT, Zhang JG, et al. (2009) Ginger's (Zingiber officinale Roscoe) inhibition of rat colonic adenocarcinoma cells proliferation and angiogenesis in vitro. Phytother Res 23:640-645.
13. Ishiguro K, Ando T, Maeda O, Ohmiya N, Niwa Y, et al. (2007) Ginger ingredients reduce viability of gastric cancer cells via distinct mechanisms. Biochem Biophys Res Commun 362:218-223.
14. Jeong CH, Bode AM. Pugliese A, Cho YY, Kim HG. et al. (2009) [6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase. Cancer Res 69:5584-5591.
15. Shukla Y, Singh M (2007) Cancer preventive properties of ginger:a brief review. Food Chem Toxicol 45:683-690.
16. Rhode J, Fogoros S, Zick S, Wahl H, Griffith KA, et al. (2007) Ginger inhibits cell growth and modulates angiogenic factors in ovarian cancer cells. BMC Complement Altern Med 7:44.
17. Shukla Y, Prasad S, Tripathi C, Singh M, George J, et al. (2007) In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]-gingerol. Mol Nutr Food Res 51:1492-1502.
18. Katiyar SK, Agarwal R, Mukhtar H (1996) Inhibition of tumor promotion in SENCAR mouse skin by ethanol extract of Zingiber officinale rhizome. Cancer Res 56:1023-1030.
19. Krell J, Stebbing J (2012) Ginger:the root of cancer therapy? Lancet Oncol 13: 235-236.
20. Pan MH, Hsieh MC, Kuo JM, Lai CS, Wu H, et al. (2008) 6-Shogaol induces apoptosis in human colorectal carcinoma cells via ROS production, caspase activation, and GADD 153 expression. Mol Nutr Food Res 52:527-537.
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22. Weng CJ, Wu CF, Huang HW, Ho CT, Yen GC (2010) Anti-invasion effects of 6-shogaol and 6-gingerol, two active components in ginger, on human hepatocarcinoma cells. Mol Nutr Food Res 54:1618-1627.
23. Ju SA, Park SM, Lee YS, Bae JH, Yu R, et al. (2012) Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors. Int J Cancer 130:2618-2628.
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26. Lee SH, Cekanova M, Baek SJ (2008) Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells. Mol Carcinog 47:197-208.
27. Wohlmuth H, Smith MK, Brooks LO, Myers SP, Leach DN (2006) Essential oil composition of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe) grown in Australia. J Agric Food Chem 54:1414-1419.
28. Riyazi A, Hensel A, Bauer K, Geissler N, Schaaf S, et al. (2007) The effect of the volatile oil from ginger rhizomes (Zingiber officinale), its fractions and isolated compounds on the 5-HT3 receptor complex and the serotoninergic system of the rat ileum. Planta Med 73:355-362.
29. Mo B, Vendrov AE, Palomino WA, DuPont BR, Apparao KB, et al. (2006) ECC-1 cells:a well-differentiated steroid-responsive endometrial cell line with characteristics of luminal epithelium. Biol Reprod 75:387-394.
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2. Creasman WT, Odicino F, Maisonneuve P, Quinn MA, Beller U, et al. (2006) Carcinoma of the corpus uteri. FIGO 26th Annual Report on the Results of Treatment in Gynecological Cancer. Int J Gynaecol Obstet 95 Suppl 1:S105-143.
3. Cohen CJ, Bruckner HW, Deppe G, Blessing JA, Homesley H, et al. (1984) Multidrug treatment of advanced and recurrent endometrial carcinoma:a Gynecologic Oncology Group study. Obstet Gynecol 63:719-726.
4. Gallion HH, Brunetto VL, Cibull M, Lentz SS, Reid G, et al. (2003) Randomized phase Ⅲ trial of standard timed doxorubicin plus cisplatin versus circadian timed doxorubicin plus cisplatin in stage Ⅲ and IV or recurrent endometrial carcinoma: a Gynecologic Oncology Group Study. J Clin Oncol 21:3808-3813.
5. Fleming GF, Filiaci VL. Bentley RC, Herzog T, Sorosky J, et al. (2004) Phase Ⅲ randomized trial of doxorubicin+cisplatin versus doxorubicin+24-h paclitaxel+ filgrastim in endometrial carcinoma:a Gynecologic Oncology Group study. Ann Oncol 15:1173-1178.
6. Fleming GF, Brunetto VL, Cella D, Look KY, Reid GC, et al. (2004) Phase Ⅲ trial of doxorubicin plus cisplatin with or without paclitaxel plus filgrastim in advanced endometrial carcinoma:a Gynecologic Oncology Group Study. J Clin Oncol 22:2159-2166.
7. Randall ME, Filiaci VL, Muss H, Spirtos NM, Mannel RS, et al. (2006) Randomized phase Ⅲ trial of whole-abdominal irradiation versus doxorubicin and cisplatin chemotherapy in advanced endometrial carcinoma:a Gynecologic Oncology Group Study. J Clin Oncol 24:36-44.
8. Thigpen JT, Brady MF, Homesley HD. Malfetano J. DuBeshter B, et al. (2004) Phase Ⅲ trial of doxorubicin with or without cisplatin in advanced endometrial carcinoma:a gynecologic oncology group study. J Clin Oncol 22:3902-3908.
9. Homesley HD, Filiaci V, Gibbons SK, Long HJ, Cella D, et al. (2009) A randomized phase Ⅲ trial in advanced endometrial carcinoma of surgery and volume directed radiation followed by cisplatin and doxorubicin with or without paclitaxel:A Gynecologic Oncology Group study. Gynecol Oncol 112:543-552.
10. Gehrig PA, Bae-Jump VL Promising novel therapies for the treatment of endometrial cancer. Gynecol Oncol 116:187-194.
11. Park EJ, Pezzuto JM (2002) Botanicals in cancer chemoprevention. Cancer Metastasis Rev 21:231-255.
12. Brown AC, Shah C, Liu J, Pham JT, Zhang JG, et al. (2009) Ginger's (Zingiber officinale Roscoe) inhibition of rat colonic adenocarcinoma cells proliferation and angiogenesis in vitro. Phytother Res 23:640-645.
13. Ishiguro K, Ando T, Maeda O, Ohmiya N, Niwa Y, et al. (2007) Ginger ingredients reduce viability of gastric cancer cells via distinct mechanisms. Biochem Biophys Res Commun 362:218-223.
14. Jeong CH, Bode AM. Pugliese A, Cho YY, Kim HG. et al. (2009) [6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase. Cancer Res 69:5584-5591.
15. Shukla Y, Singh M (2007) Cancer preventive properties of ginger:a brief review. Food Chem Toxicol 45:683-690.
16. Rhode J, Fogoros S, Zick S, Wahl H, Griffith KA, et al. (2007) Ginger inhibits cell growth and modulates angiogenic factors in ovarian cancer cells. BMC Complement Altern Med 7:44.
17. Shukla Y, Prasad S, Tripathi C, Singh M, George J, et al. (2007) In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]-gingerol. Mol Nutr Food Res 51:1492-1502.
18. Katiyar SK, Agarwal R, Mukhtar H (1996) Inhibition of tumor promotion in SENCAR mouse skin by ethanol extract of Zingiber officinale rhizome. Cancer Res 56:1023-1030.
19. Krell J, Stebbing J (2012) Ginger:the root of cancer therapy? Lancet Oncol 13: 235-236.
20. Pan MH, Hsieh MC, Kuo JM, Lai CS, Wu H, et al. (2008) 6-Shogaol induces apoptosis in human colorectal carcinoma cells via ROS production, caspase activation, and GADD 153 expression. Mol Nutr Food Res 52:527-537.
21. Nigam N, George J, Srivastava S, Roy P, Bhui K, et al. (2010) Induction of apoptosis by [6]-gingerol associated with the modulation of p53 and involvement of mitochondrial signaling pathway in B[a]P-induced mouse skin tumorigenesis. Cancer Chemother Pharmacol 65:687-696.
22. Weng CJ, Wu CF, Huang HW, Ho CT, Yen GC (2010) Anti-invasion effects of 6-shogaol and 6-gingerol, two active components in ginger, on human hepatocarcinoma cells. Mol Nutr Food Res 54:1618-1627.
23. Ju SA, Park SM, Lee YS, Bae JH, Yu R, et al. (2012) Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors. Int J Cancer 130:2618-2628.
24. Chen CY, Chen CH. Kung CH, Kuo SH, Kuo SY (2008) [6]-gingerol induces Ca2+ mobilization in Madin-Darby canine kidney cells. J Nat Prod 71:137-140.
25. Lee HS, Seo EY, Kang NE, Kim WK (2008) [6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells. J Nutr Biochem 19:313-319.
26. Lee SH, Cekanova M, Baek SJ (2008) Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells. Mol Carcinog 47:197-208.
27. Wohlmuth H, Smith MK, Brooks LO, Myers SP, Leach DN (2006) Essential oil composition of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe) grown in Australia. J Agric Food Chem 54:1414-1419.
28. Riyazi A, Hensel A, Bauer K, Geissler N, Schaaf S, et al. (2007) The effect of the volatile oil from ginger rhizomes (Zingiber officinale), its fractions and isolated compounds on the 5-HT3 receptor complex and the serotoninergic system of the rat ileum. Planta Med 73:355-362.
29. Mo B, Vendrov AE, Palomino WA, DuPont BR, Apparao KB, et al. (2006) ECC-1 cells:a well-differentiated steroid-responsive endometrial cell line with characteristics of luminal epithelium. Biol Reprod 75:387-394.
30. Satyaswaroop PG, Tabibzadeh SS (1991) Extracellular matrix and the patterns of differentiation of human endometrial carcinomas in vitro and in vivo. Cancer Res 51:5661-5666.
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