CN115282282B - 靶向pdk1调控糖代谢重编程联合二甲双胍在子宫内膜癌合并糖尿病患者治疗的应用 - Google Patents
靶向pdk1调控糖代谢重编程联合二甲双胍在子宫内膜癌合并糖尿病患者治疗的应用 Download PDFInfo
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Abstract
本发明公开了靶向PDK1调控糖代谢重编程联合二甲双胍在子宫内膜癌合并糖尿病患者治疗的应用。本发明提供了PDK1蛋白作为靶点在开发、筛选和/或制备治疗或辅助治疗子宫内膜癌的试剂中的应用。本发明的实验证明了通过联合JX06和二甲双胍的这种方式,实现了同时抑制糖酵解和氧化磷酸化两条途径,导致子宫内膜癌细胞凋亡的增加。因此,本研究创新性发现针对PDK1的抑制剂JX06联合二甲双胍在高糖状态下具有联合增强的抗肿瘤效果,为子宫内膜癌合并糖尿病的患者治疗提供全新的策略。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种靶向PDK1调控糖代谢重编程联合二甲双胍在子宫内膜癌合并糖尿病患者治疗的应用。
背景技术
子宫内膜癌(EC)是最常见的妇科恶性肿瘤之一,其发病与雌激素的失衡和代谢紊乱密切相关。近年来随着代谢性疾病发病率的增加,子宫内膜癌患者的发病率在全球范围内呈现明显上升化趋势。越来越多的研究表明,糖尿病是子宫内膜癌高危风险因素。与非糖尿病者相比,糖尿病患者的子宫内膜癌发病风险增加2倍。此外,子宫内膜癌合并糖尿病的患者,其死亡率增加41%。然而,目前临床上针对子宫内膜癌合并糖尿病的患者尚无标准化治疗方案。高血糖是糖尿病的主要临床特征,也被认为是糖尿病与癌症之间关联的关键因素。因此,控制血糖水平或干预高糖相关分子信号通路,有可能为子宫内膜癌合并糖尿病的患者的临床治疗提供新的思路。
二甲双胍是临床上最常见的控糖药物之一。近年来,研究还发现它能抑制乳腺癌,结肠癌和子宫内膜癌细胞的生长,其主要机制是通过作用于线粒体呼吸链复合体I,抑制氧化磷酸化,从而发挥抗肿瘤作用。此外,临床实践表明,二甲双胍辅助治疗有助于逆转子宫内膜非典型增生,降低肿瘤增殖标志物的表达,提高了子宫内膜癌患者总体生存率。然而,有研究表明,在高糖条件下,二甲双胍对子宫内膜癌细胞生长的抑制作用有所降低,这表明高血糖导致了子宫内膜癌细胞对二甲双胍产生抗性。文献研究表明,由于Warburg效应的存在,肿瘤细胞的糖代谢方式主要包含氧化磷酸化和糖酵解,但是以后者占主导。然而,高糖条件下肿瘤细胞对二甲双胍的抵抗机制可能与高糖条件下肿瘤细胞的代谢方式向糖酵解转变增加有关。
发明内容
本发明一个目的是提供PDK1蛋白的用途。
本发明提供了PDK1蛋白作为靶点在开发、筛选和/或制备治疗或辅助治疗子宫内膜癌的试剂中的应用。
或,本发明提供了PDK1蛋白作为靶点在开发、筛选和/或制备治疗或辅助治疗子宫内膜癌合并糖尿病的试剂中的应用。
本发明另一个目的是提供抑制或者干扰PDK1蛋白表达的物质的用途。
本发明提供了抑制或者干扰PDK1蛋白表达的物质在如下至少一种或制备具有如下至少一种功能的产品中的应用:
1)治疗或辅助治疗子宫内膜癌;
2)治疗或辅助治疗子宫内膜癌合并糖尿病;
3)治疗或辅助治疗由子宫内膜癌细胞的增殖和/或侵袭力引发的疾病;
4)治疗或辅助治疗由高糖诱导子宫内膜癌细胞的增殖和/或侵袭力引发的疾病;
5)治疗或辅助治疗由高糖诱导子宫内膜癌细胞的糖酵解引发的疾病;
6)治疗或辅助治疗由高糖诱导子宫内膜癌细胞的增长引发的疾病;
7)增强二甲双胍治疗子宫内膜癌合并糖尿病疗效;
8)联合二甲双胍治疗子宫内膜癌合并糖尿病。
本发明还提供了抑制或者干扰PDK1蛋白表达的物质和二甲双胍在如下至少一种或制备具有如下至少一种功能的产品中的应用:
1)治疗或辅助治疗子宫内膜癌;
2)治疗或辅助治疗子宫内膜癌合并糖尿病。
上述应用中,所述抑制或者干扰PDK1蛋白表达的物质为干扰PDK1蛋白表达的shRNA或PDK1抑制剂。
在本发明的实施例中,干扰PDK1蛋白表达的shRNA有序列1和序列2退火形成。
PDK1抑制剂为JX06。
本发明还有个目的是提供一种具有如下1)或2)至少一种功能的产品。
本发明提供的产品,其为如下a)或b):
a)抑制或者干扰PDK1蛋白表达的物质;
b)二甲双胍和所述抑制或者干扰PDK1蛋白表达的物质;
1)治疗或辅助治疗子宫内膜;
2)治疗或辅助治疗子宫内膜癌合并糖尿病。
PDK1蛋白或其基因作为标志物在开发、筛选和/或制备用于预测或辅助预测子宫内膜癌预后的试剂中的应用也是本发明保护的范围。
检测PDK1基因表达量的物质在如下至少一种或制备具有如下至少一种功能的产品中的应用也是本发明保护的范围:
(1)预测或辅助预测子宫内膜癌的预后;
(2)预测或辅助预测术后子宫内膜癌的总生存时间长短。
上述检测PDK1基因表达量的物质可以包括特异结合PDK1蛋白的抗体或特异结合PDK1基因的探针或扩增PDK1基因的引物。
在本研究中,首先建立了长期高糖培养的子宫内膜癌细胞模型,并以正常糖培养的子宫内膜癌细胞作为对照研究组。选择长期高糖培养的细胞模型的原因主要是先前的研究大多观察不同糖浓度短时间0~72h对子宫内膜癌细胞生物学行为的影响。然而,糖尿病是一种长期伴随高血糖的慢性代谢疾病,长期持续的高血糖刺激才是子宫内膜癌合并糖尿病患者的重要临床特征,才能更为准确地模拟高糖在癌症发生和发展过程中的作用模式。随后,通过运用该高糖子宫内膜癌细胞模型,发现:1)长期高糖培养加剧子宫内膜癌细胞糖代谢由氧化磷酸化向糖酵解转变。2)首次发现在高糖培养条件下子宫内膜癌细胞中糖酵解关键酶PDK1的表达显著升高,通过shRNA下调PDK1的表达能够显著抑制高糖导致子宫内膜癌细胞的增殖,侵袭和糖酵解;3)在高糖条件下,PDK1的小分子抑制剂JX06联合二甲双胍具有联合增强诱导子宫内膜癌细胞凋亡的作用。随后,为了充分模拟临床上子宫内膜癌合并糖尿病患者的生理病理特征,接下来通过给与小鼠腹腔注射STZ诱发了糖尿病,并于皮下接种了子宫内膜癌细胞,最终构建了小鼠子宫内膜癌合并糖尿病的模型。最后,一方面,将JX06通过尾静脉注射给药的方式注入子宫内膜癌合并糖尿病的小鼠体内,另外一方面,二甲双胍经过口服给药小鼠,起到两个方面的作用,一是降糖控糖;二是抑制线粒体复合物I抑制细胞氧化磷酸化。
本发明的实验证明,通过联合JX06和二甲双胍的这种方式,实现了同时抑制糖酵解和氧化磷酸化两条途径,导致子宫内膜癌细胞凋亡的增加。因此,本研究创新性发现针对PDK1的抑制剂JX06联合二甲双胍在高糖状态下具有联合增强的抗肿瘤效果,为子宫内膜癌合并糖尿病的患者治疗提供全新的策略。
附图说明
图1为高糖促进子宫内膜癌细胞生长和糖代谢重编程。
图2为高糖通过促进糖酵解关键酶PDK1表达来调控糖代谢重编程。
图3为组织水平和TCGA数据库验证PDK1在子宫内膜癌组织中的表达情况。
图4为干预PDK1的表达对子宫内膜癌细胞增殖,侵袭和糖酵解的影响。
图5为靶向PDK1小分子抑制剂JX06抑制PDK1的表达,抑制子宫内膜癌细胞增殖活性,促进子宫内膜癌细胞凋亡。
图6为靶向PDK1小分子抑制剂JX06联合二甲双胍具有联合抑制子宫内膜癌细胞增殖的作用。
图7为体内水平-靶向PDK1的JX06小分子抑制剂联合二甲双胍抑制子宫内膜癌生长。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中主要实验方案如下:
1、细胞培养
子宫内膜癌ishikawa细胞系由北京大学人民医院妇产科实验保存(北京,中国)。将子宫内膜癌ishikawa细胞培养于包含10%FBS(购自于Gibco)的DMEM培养基(含glucose1g/L和4.5g/L)-购自于中科迈晨公司,放置于含5%CO2的37℃培养箱中。当细胞融合度达到80-90%时,用胰酶消化细胞,传代或接种细胞板用于后续实验研究。
2、免疫组织化学染色
免疫组织化学染色在子宫内膜癌芯片上进行。子宫内膜癌组织芯片(EMC1351)购自于上海卓灏医药科技有限公司,将子宫内膜癌组织芯片脱蜡至水,抗原修复采用高温高压修复法,在高压锅中加入抗原修复液(PH6.0柠檬酸)并进行高温煮沸。将切片用3%H2O2浸泡30min,去除内源性过氧化物酶,PBS冲洗5min×3次。在切片上加入兔抗PDK1抗体(1:50,货号:#3820,Cell Signaling Technology),放入湿盒4℃过夜。第二天,进行复温30min后用PBS冲洗3次×5min。在切片上加入HRP标记的goat-anti-rabbit-IgG(abcam公司,货号:ab6721)后室温孵育30min,PBS洗5min×3次。滴加DAB显色剂于组织上,并于显微镜下观察显色情况,将片子浸入自来水中终止显色。采用苏木素染色液复染,盐酸酒精分化液分化,将芯片放入100%无水乙醇脱水,封片,显微镜观察并扫描拍照。
结果判定:采用Fromowitz综合计分法:①染色强度评分:无染色为0分,呈淡黄色颗粒、明显高于背景为1分,呈浅棕黄色颗粒为2分,出现大量深棕黄色颗粒为3分;②阳性细胞数评分:每张片随机计数500个细胞中染色阳性细胞数,<5%为0分,5%~25%为1分,26%~50%为2分,51%~75%为3分,>75%为4分。染色强度评分与阳性细胞数比例的评分之和<2分为阴性(-),2~3分为弱阳性(+),4~5分为中度阳性(++),6~7分为强阳性(+++),+~+++均为阳性。
根据免疫组织化学的评分结果将PDK1的表达分为强阳表达组(High),中度阳性组(Moderate)和阴性或者弱阳性组(Neg/poor)。
子宫内膜癌芯片共包含135例样本,进一步根据样本信息将样本分为癌旁正常组织组n=17,子宫内膜癌组织(n=118),非糖尿病的子宫内膜癌组织n=116,糖尿病的子宫内膜癌组织(n=19),比较以上各组PDK1的表达情况。
3、seahorse能量代谢分析实验
在探针板下方的板子中加入200μl/well的水化液(Seahorse XF校准液,103059-000,购自安捷伦公司),将探针板轻轻插入加好液体的板子中,放于37℃无CO2培养箱中过夜。取出细胞培养板(XFe96 Cell Culture Microplates),用胰蛋白酶消化细胞,制备细胞悬液。将细胞悬液稀释成所需浓度1.25×105/ml,以每孔80μl的体积加入培养板,每组设置8个复孔,接种完细胞后室温静置1h,然后放入培养箱中培养,16-24h后上机检测。配制糖酵解应激检测实验培养液(总体50ml=49ml基础培养基+1ml200mM谷氨酰胺水溶液,pH调至7.35±0.05,其中基础培养基购自安捷伦公司,货号:102353-100;)和线粒体应激检测实验培养液(55μl2.5mM葡萄糖水溶液/250μl 2.5mM葡萄糖水溶液+500μl 200mM谷氨酰胺水溶液+500μl 100mM丙酮酸钠水溶液,基础培养基至终体积25ml)。用配置好的培养液反复清洗细胞两遍,最后分别加入配置好的糖酵解培养基和线粒体应激检测实验培养基,使得每孔总体积是175μl,换液完成后,放入37℃无CO2培养箱中放置1h。在探针板上放上与孔位(Port)对应的加液板(A、B、C、D四个孔位),各加入25μl/well相应的药物。a.糖酵解应激检测实验上样顺序:A:葡萄糖;B:寡霉素;C:糖酵解抑制剂2-脱氧-D-葡萄糖2-DG b.线粒体应激检测实验上样顺序:A:寡霉素;B:羰基-氰-对-三氟甲氧基苯肼;C:鱼藤酮/抗霉素。按照程序设置矫正探针板,放入细胞板,检测细胞ECAR和OCR水平。
4、Label-free定量蛋白质组学技术
收集细胞沉淀,送样量为1×107或细胞沉淀不少于50μl。将收获的细胞沉淀在8M尿素溶液中悬浮,然后进行5分钟超声处理,得到细胞裂解物。将裂解物在14000×g和4℃下离心15分钟以去除任何不溶性物质。用BCA蛋白定量法测定所得溶液的蛋白浓度。用5mM三-(2-氯乙基)-磷酸(TCEP)(室温,30分钟)对含有50μg蛋白质的溶液进行二硫化物还原,并用10mM IAA(室温,黑暗中30分钟)进行烷基化。然后加入三氯乙酸(TCA)沉淀蛋白质。通过14000g离心30min分离沉淀的蛋白质,并且将颗粒在丙酮中洗涤三次以去除残余的TCA。然后在含有8M尿素溶液的50mM Tris-HCl(pH 8.2)中重组干燥的蛋白质。用50mM Tris-HCl将溶液稀释至2M尿素,并在37℃下以1/50(w/w)的酶蛋白比消化蛋白质过夜。用C18柱(基于德国达姆施塔特默克KGaA公司的单片技术)对等量的肽样品进行预清洗。将上述获得的肽样品装载到柱上。通过柱的液体被标记为流通(FT),其中含有不结合或弱结合到C18珠的肽。柱用300μl ddH2O洗涤,用200μl甲醇洗脱,真空干燥后进行LC-MS/MS分析。每个样品采用纳升流速的HPLC液相系统Easy nLC进行分离。样品经色谱分离后用Q-Exactive质谱仪进行质谱分析。数据分析:质谱分析原始数据为RAW文件,采用MaxQuant软件进行查库鉴定及定量分析。
实施例1、高糖促进子宫内膜癌细胞生长和糖代谢重编程
1、培养
为模拟正常生理水平和糖尿病患者体内的血糖水平,进行如下实验:
正常浓度葡萄糖培养(模拟正常生理水平):将子宫内膜癌细胞系ishikawa在包含10%FBS的DMEM培养基(含葡萄糖1g/L,5.5mM-正常糖)中培养8周,得到正常浓度葡萄糖培养子宫内膜癌细胞;
高糖浓度葡萄糖培养(模拟糖尿病患者体内的血糖水平):将子宫内膜癌细胞系ishikawa在包含10%FBS的DMEM培养基(含葡萄糖4.5g/L,25mM-高糖)中培养8周,得到高浓度葡萄糖培养子宫内膜癌细胞。
2、检测
1)、克隆形成实验
将上述1得到的不同组的子宫内膜癌细胞用含有0.25%EDTA的胰酶消化成单个细胞,并用1mL培养基重悬。细胞计数,接种800个细胞于六孔板中,轻轻晃动六孔板,将六孔板置于37℃,5%CO2培养箱中继续培养10天。从培养箱中取出六孔板,倒去培养基,用PBS洗3次,每次5min。用4%多聚甲醇固定细胞20min,PBS洗3次。将1%结晶紫工作液加入六孔板中以覆盖细胞为准,染色30min,PBS洗两次。将六孔板自然凉干后计数克隆形成的数目,并拍照保存。
2)、Transwell实验
将matrigel基质胶(BD,美国)和DMEM培养基以1:8的比例稀释,取50μl加入transwell小室中,培养箱中干燥5小时备用(用于细胞侵袭实验)。将处于对数期生长的上述1得到的不同组的子宫内膜癌细胞事先给予12小时饥饿处理,以排除血清的影响作用。胰酶消化细胞后加入培养基终止消化,离心后弃掉上清,PBS洗涤细胞两遍,用无血清培养基重悬细胞,并将细胞制成105个/mL悬液。反复吹打均匀后取200μl细胞悬液放入transwell小室中(注:细胞侵袭实验-带有基质胶的小室,细胞迁移实验-不带基质胶的小室)。24孔板中加入500μl含10%FBS的完全培养基,将小室放入24孔板中。将细胞培养板放入37℃含5%CO2培养箱中继续培养24h和48h。染色:将24孔板中的上室取出,用PBS洗去小室中的培养基,加入适量的4%多聚甲醛对细胞进行固定,用棉签轻轻将小室中的胶和细胞擦除干净,加入适量的结晶紫染色10min,PBS洗三遍,于倒置显微镜下对非细胞接种侧拍照,并记录穿过膜的细胞数目。
3)、划痕实验
培养板画线:使用marker笔在六孔板背后画均匀的直线,横穿过孔,每个孔三条线,注意画线不要太粗。接种细胞:胰酶消化上述1得到的不同组的子宫内膜癌细胞后加入培养基终止消化,离心后弃掉上清,培养基重悬细胞后,计数细胞,每个孔中大约接种5×105个细胞,过夜后细胞融合率达到100%。细胞划线:第二天用黄色枪头垂直于细胞平面,垂直于前一天在平板底面画的线在细胞上进行划痕,不同孔间使用同一只枪头。洗涤细胞:划痕完成后,用PBS洗涤细胞3次,洗去未贴壁的细胞,即随着划线脱落的细胞,留下清晰的划痕,然后更换含1%血清的培养基。细胞培养和观察:将细胞放入37℃含5%CO2培养箱中进行培养,然后在适当的时间点,如0h和24h,显微镜下观察划痕的宽度并拍照。结果分析:使用ImageJ软件打开拍照的图片后,计算划痕愈合的面积百分比。
上述克隆形成实验,Transwell实验和划痕实验的结果如图1A-1F,其中1A为正常糖和高糖培养培养条件下克隆形成的结果,1B为正常糖和高糖组克隆形成数目统计的结果,1C为正常糖和高糖培养条件下ishikawa细胞侵袭情况,1D为正常糖和高糖培养条件下细胞侵袭数目统计结果,1E为正常糖和高糖培养条件下0小时和24小时细胞划痕拍照结果,1F为正常糖和高糖培养条件下划痕实验面积统计的结果;可以看出,高糖培养促进子宫内膜癌细胞克隆形成,侵袭和迁移。
4)、SeahorseXF细胞能量代谢分析实验
为进一步研究高糖对细胞糖代谢过程的影响,通过SeahorseXF细胞能量代谢分析技术实时探测氧消耗速率(OCR-反应氧化磷酸化情况)和胞外酸化速率(ECAR-反应糖酵解情况)变化,来分别反应细胞内的线粒体有氧代谢和糖酵解能量代谢状态。
将上述1得到的正常浓度葡萄糖培养子宫内膜癌细胞和高浓度葡萄糖培养子宫内膜癌细胞进行seahorse能量代谢分析实验,结果发现高糖培养状态促进了子宫内膜癌ishikawa细胞糖酵解而抑制了氧化磷酸化,即促进子宫内膜癌细胞糖代谢重编程过程(图1G-1J,其中1G为正常糖和高糖培养组ishikawa细胞外酸化速率曲线,反应糖酵解功能,1H为正常糖和高糖培养组ishikawa细胞外酸化速率统计结果,1I为正常糖和高糖培养组ishikawa细胞耗氧率曲线,反应线粒体呼吸情况,1J为正常糖和高糖培养组ishikawa细胞耗氧率统计结果)。
实施例2、高糖通过促进糖酵解关键酶PDK1表达来调控糖代谢重编程
1、糖酵解关键酶PDK1标志物的发现
为进一步探索高糖导致子宫内膜癌细胞糖酵解增加的分子机制,利用质谱蛋白质组学Label free定量蛋白质组学技术来筛选实施例1中高糖培养组和正常糖培养两组子宫内膜癌ishikawa细胞的差异表达蛋白。共鉴定到216个蛋白在高糖和正常糖培养组存显著差异,其中92个表达上调,124个表达下调(图2A)。通过GO和KEGG富集分析,结果表明这216个差异表达的蛋白主要参与丙酮酸代谢调控,细胞骨架粘附,TCA循环和多种癌症相关的信号通路(图2B,图2C)。为进一步筛选出高糖参与调控子宫内膜癌进展的关键蛋白,对差异表达的蛋白进行蛋白互作通路网络分析,结果发现在TCA循环通路中,α-酮戊二酸到琥珀酸之间的多个重要的催化酶(DLST、SUCLG2,OGDH)以及氧化磷酸化通路中多个分子(NDUFC2,NDUFA1和NDUFS8)均发生显著下调,这表明了高糖培养组癌细胞TCA循环和氧化磷酸化受到了抑制。在糖酵解途径中,PDK1,PEKP和ENO2显著上调(图2D)。
值得注意的是,上述蛋白质组学结果分析还显示了与正常糖培养的癌细胞相比,PDK1(Gene ID:5163,提交日:2021年7月11日)是高糖组细胞的差异蛋白上调最为显著的前十位蛋白之一,具体表现为高糖组PDK1的蛋白表达是正常糖培养细胞的3.33倍。
Western blot和RT-PCR(PDK1引物:Forward:AACCGACACAATGATGTCATTC;Reverse:ATGCGACTCATGTAGAATCGAT)检测实施例1中高糖培养组和正常糖培养两组子宫内膜癌ishikawa细胞,结果提示,高糖水平促进PDK1在蛋白和mRNA水平的表达(图2E-2G)。
以上结果表明,高糖环境可能通过促进PDK1的表达从而促进糖酵解导致糖代谢重编程。
2、糖酵解关键酶PDK1标志物在预测子宫内膜癌合并糖尿病患者预后中的应用
1)免疫组织化学法
为进一步从临床上证实该结论,运用免疫组织化学法检测了118例子宫内膜癌组织和17例癌旁正常组织的PDK1的表达。
根据PDK1的表达评分,将样本分成阴性、弱阳性组、中度阳性组和强阳性组。
结果发现PDK1主要表达于癌细胞的胞浆中,且子宫内膜癌组织中PDK1中度阳性和强阳性比例均明显高于癌旁正常组织。子宫内膜癌合并糖尿病的患者组织中PDK1强阳性表达比例明显高于罹患子宫内膜癌而非糖尿病的患者(图3A-3C)。
2)TCGA数据库验证
进一步运用TCGA数据库(https://portal.gdc.cancer.gov/)分析了23例癌旁组织和23例子宫内膜癌组织中PDK1的表达,具体如下表1:
表1为TCGA数据库23例癌症和癌旁组织PDK1的表达的检测结果
结果发现子宫内膜癌组织中PDK1基因表达明显高于癌旁组织,且在子宫内膜癌组织中PDK1随着肿瘤分级越高其表达值呈现增高趋势(图3D,3E)。
3)Kaplan-Meier生存分析
TCGA数据库(https://portal.gdc.cancer.gov/)下载552例子宫内膜癌组织mRNA表达谱数据。根据对雌激素的依赖性,子宫内膜癌被分为I型和II型子宫内膜癌,其中I型子宫内膜癌占80%。为进一步分析PDK1的表达与子宫内膜癌患者预后的关系,首先从UCSCXena在线网站(https://xenabrowser.net/datapages/)下载与552例子宫内膜癌样本对应的临床信息,包括病理类型,生存时间和生存状态。其次,从552例子宫内膜癌患者中筛选包含完整生存状态和生存时间的I型子宫内膜癌患者共396例,具体信息如下:
表2为TCGA数据库396例I型子宫内膜癌患者生存情况及PDK1的表达结果
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上表中,第3列中生存状态的1表示第2列随访时间内死亡,0表示第2列随访时间内存活。
按照PDK1表达中位值(为1.4876355),将患者分为PDK1低表达(小于中位值)和PDK1高表达(大于等于中位值)。Kaplan-Meier生存分析显示,在10年生存期内,PDK1高表达与I型子宫内膜癌患者生存率呈负相关(图3F)。
PDK1高表达组的子宫内膜癌患者预后效果差于或候选差于PDK1低表达组的子宫内膜癌患者。
在同一随访时间内,PDK1高表达组的子宫内膜癌患者生存率低于或候选低于PDK1低表达组的子宫内膜癌患者。
综上所述,高糖促进PDK1高表达可能与子宫内膜癌合并糖尿病患者进展和不良预后密切相关。
实施例3、干预PDK1的表达对子宫内膜癌细胞增殖,侵袭和糖酵解的影响
一、干预PDK1的表达的子宫内膜癌细胞
为进一步证实PDK1在高糖调控子宫内膜癌进展中的作用,构建了稳定敲低PDK1的子宫内膜癌细胞ishikawa细胞株。
sh-PDK1组细胞:将表达PDK1-shRNA1的慢病毒感染ishikawa细胞,得到sh-PDK1组细胞;
提取细胞总RNA进行RT-PCR(PDK1引物:Forward:AACCGACACAATGATGTCATTC;Reverse:ATGCGACTCATGTAGAATCGAT),结果可以看出,与ishikawa细胞相比,sh-PDK1组细胞中PDK1的表达量降低,说明PDK1-shRNA1干扰PDK1的表达构建成功。
sh-NC组细胞:将表达PDK1-shNC的慢病毒感染ishikawa细胞,得到sh-PDK1组细胞;
上述表达PDK1-shRNA1的慢病毒由汉恒生物科技有限公司完成包装,其中PDK1-shRNA1的序列为表3的PDK1-shRNA1-F和PDK1-shRNA1-R构成;
上述表达PDK1-sh-NC的慢病毒由汉恒生物科技有限公司完成包装,其中PDK1-sh-NC的序列为表3的PDK1 shNC-F和PDK1-shNC-R构成。
针对PDK1分子设计阴性对照(Negative Control,NC)病毒载体shRNA序列(shNC)和三个目的基因shRNA序列(shRNA1序列,shRNA2序列和shRNA3序列),并进行慢病毒包装(由汉恒生物科技有限公司完成),序列如下:
表3为PDK1的sh-NC序列和sh-RNA序列
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慢病毒感染ishikawa细胞:①将生长状态良好的细胞消化离心后计数,接种于六孔板中,每个孔加入2.5×105个细胞,放入37℃,5%CO2培养箱中。②吸去六孔板中原来的培养基,加入1ml新鲜培养基,病毒感染前将病毒从冰箱中取出放于冰上慢慢融化,加入适量的病毒进行感染(每孔加入的病毒量=MOI(30)×细胞数/病毒滴度(TU/ml)×1000),同时加入慢病毒转染试剂浓度为4μg/ml,感染4h后补足培养基至2ml。感染72h后,提取不同组的总RNA进行RT-PCR筛选最佳转染shRNA序列,最佳转染shRNA序列为PDK1-shRNA1。
嘌呤霉素筛选稳转细胞株:慢病毒感染72h后,加入嘌呤霉素(4μg/ml),48h后更换新鲜培养基,之后根据细胞生长速度进行定期药物筛选。
二、干预PDK1的表达对子宫内膜癌细胞的影响
1、干预PDK1的表达抑制子宫内膜癌细胞的增殖和侵袭力
高糖培养克隆形成实验和Transwell实验
将上述稳定敲低PDK1的子宫内膜癌细胞ishikawa细胞株sh-PDK1组细胞和未敲除的ishikawa细胞株sh-NC组在含正常葡萄糖浓度和高浓度葡萄糖的条件下培养(培养方法同实施例1),并按照实施例1中实验方法进行克隆形成实验和Transwell实验。
结果如图4A-4D所示,A图为正常糖和高糖培养条件下,敲低-阴性对照(sh-NC)和敲低PDK1(sh-PDK1)对子宫内膜癌ishikawa细胞株克隆形成的影响,B图为A图的量化统计结果,C图为正常糖和高糖培养条件下,敲低-阴性对照(sh-NC)和敲低PDK1(sh-PDK1)对子宫内膜癌ishikawa细胞侵袭的影响情况,D图为C图的量化统计结果。发现在高糖组培养条件和正常糖培养条件下,与sh-NC组相比,sh-PDK1组处理的细胞,其克隆数目和细胞侵袭数目分别降低,这表明了无论是在正常糖状态下还是高糖状态下敲低PDK1均能够明显抑制子宫内膜癌细胞的增殖和侵袭力。
2、干预PDK1的表达抑制高糖对子宫内膜癌细胞糖酵解的促进作用
高糖培养的Seahorse细胞能量代谢分析技术检测
为了进一步探究PDK1在高糖诱导糖酵解水平增加中的作用,利用Seahorse细胞能量代谢分析技术检测高糖培养条件下sh-PDK1组及sh-NC组子宫内膜癌细胞ishikawa细胞。
正常糖敲低-阴性对照:sh-NC组细胞在正常葡萄糖下培养,条件同实施例1;
正常糖敲低-PDK1:sh-PDK1组细胞在高浓度葡萄糖下培养,条件同实施例1;
高糖敲低-阴性对照:sh-NC组细胞在正常葡萄糖下培养,条件同实施例1;
高糖敲低-PDK1:sh-PDK1组细胞在高浓度葡萄糖下培养,条件同实施例1;
结果如图4E-4F所示,E图为,正常糖和高糖培养条件下,敲低-阴性对照(sh-NC)和敲低PDK1(sh-PDK1)对子宫内膜癌ishikawa细胞外酸化率的影响情况,F图为E图的统计结果分析图,可以看出,结果表明sh-PDK1组细胞的糖酵解能力明显小于sh-NC组(ECAR平均值:26mPH/min vs 46mPH/min),提示长期高糖培养促进子宫内膜癌细胞糖酵解,而抑制PDK1的表达能够抑制高糖对糖酵解的促进作用。
3、干预PDK1的表达抑制子宫内膜癌肿瘤大小
为了在活体水平进一步验证假设,随后构建了BALB/c裸鼠子宫内膜癌合并糖尿病的模型。
(一)糖尿病小鼠模型构建
本研究采用高脂饲料联合低剂量链脲佐菌素(streptozotocin,STZ)法诱导II型糖尿病小鼠模型。STZ是一种含有亚硝基的化合物,进入体内后可特异性的破坏胰岛细胞,导致胰岛细胞变性,坏死,最终诱发糖尿病。
(1)5周龄的BALb/c裸鼠由北京大学人民医院动物实验室(北京,中国),SPF实验动物中心提供,实验开展前已通过我院伦理委员会的批准,并在实验操作过程中严格遵守实验动物的伦理要求和动物福利法的要求。按照实验设计将裸鼠随机分组,每组6只。糖尿病组裸鼠连续五天腹腔注射STZ 50mg/kg,并给予高脂饲料饮食;正常对照组裸鼠连续五天腹腔注射与STZ等体积的柠檬酸缓冲液,并给予正常饲料饮食。
(2)STZ注射液配制:首先配柠檬酸缓冲液A液和B液。
A液:柠檬酸(MW:210.14)2.1g加入蒸馏水100ml,混匀;
B液:柠檬酸(MW:294.10)2.94g加入蒸馏水100ml,混匀;
再将A液和B液按照1:1混合,调整PH值至4.2-4.5。
(3)STZ称量后用锡纸包好和柠檬酸缓冲液一起置于冰浴中,一起带到动物房备用。
(4)小鼠禁食12h后称重,用柠檬酸缓冲液配1%(质量体积比g:ml)STZ注射液,按照50mg/kg的剂量对裸鼠进行腹腔注射。(注:STZ容易失活,STZ称取后应注意干燥避光,并在30分钟内快速完成注射。)
(5)检测血糖:末次注射一周后,禁食6h后,尾静脉采血,血糖仪测定血糖值,当血糖浓度大于11.1mmol/L时判为建模成功。
(二)裸鼠皮下成瘤实验
(1)取处于对数生长期的各组细胞,细胞达80-90%左右密度为宜。
(2)胰酶消化各组细胞后用预冷的PBS洗两遍,用无血清培养基重悬细胞,吹打细胞沉淀至单个细胞悬液,计数细胞,调整细胞密度至3×107/ml,皮下接种量为3×106个细胞/只,接种体积0.1ml。
(3)将细胞悬液放置于冰上带进动物房,应尽快在半个小时内完成裸鼠皮下接种,接种于裸鼠血供丰富的区域腋窝中后部。
(4)接种前用枪将细胞悬液充分的吹散,接种时,针头在皮下进针约1cm深,避免细胞悬液随着针拔出的过程溢出。
(5)定期观察裸鼠皮下成瘤情况,当开始观察到成瘤后每周游标卡尺检测肿瘤的体积大小,4周后处死裸鼠,取下皮下瘤拍照记录,并将组织进行固定石蜡包埋和﹣80度冰箱保存备用(肿瘤体积计算公式:V=A×B2×0.52,A代表肿瘤长径,B代表肿瘤短径)。
上述各组细胞为:敲低-阴性对照(sh-NC组细胞)和敲低-PDK1(sh-PDK1组细胞)
结果如图4G和4H所示,可以看出,与敲低-阴性对照相比,敲低PDK1的表达具有明显的抗肿瘤作用。
上述结果表明了高糖对子宫内膜癌细胞的促增长作用,以及下调PDK1能够抑制高糖对子宫内膜癌促生长作用。
实施例4、靶向PDK1小分子抑制剂JX06联合二甲双胍的抗肿瘤作用
(一)细胞水平-靶向PDK1小分子抑制剂JX06联合二甲双胍的抗肿瘤作用
一、靶向PDK1小分子抑制剂JX06抑制子宫内膜癌
JX06(MCE公司,货号:HY-19564,化学式如下)是一种有效的,选择性的,共价的PDK抑制剂,具有显著抗肿瘤作用,其作用机制为JX06以不可逆的方式与半胱氨酸残基共价结合来抑制PDK1活性。
JX06化学式
为证实JX06对子宫内膜癌是否具有抗癌活性,进行如下实验:
1)CCK-8实验
将Ishikawa细胞接种于96孔板,每孔接种100μl包含3000个细胞。培养过夜后,换包含不同浓度的JX06的新鲜培养基,继续培养24-48h。然后在每孔中加入10μl的CCK-8试剂,37℃培养箱孵育2h,随后用酶标仪检测450nm处的吸光度值。
2)流式检测细胞凋亡
将ishikawa细胞接种于六孔板中,并在高糖培养条件下培养,依据实验分组分别给予细胞PBS和0.5μM JX06处理48h。然后,收获细胞并用预冷的PBS清洗两遍,1000转离心3min,1X Binding Buffer重悬细胞浓度为1x 10^6cells/ml,吸取100ul(含1x 10^5cells)于1mLEP管中。随后,加5μl的FITC-annexin V和5μl碘化丙啶(PI)于EP管中,混匀。室温(25℃)避光孵育15min。加400μl of 1X Binding Buffer到每个EP管中,最后用流式细胞仪定量检测细胞凋亡情况。
3)Western blot实验
将ishikawa细胞接种于六孔板中,并在高糖培养条件下培养,依据实验分组分别给予细胞PBS和0.5μM JX06处理48h。
通过Western blot实验分析检测PDK1在蛋白水平的表达。在培养皿中加入细胞裂解液(RIPA:蛋白酶抑制剂:磷酸化蛋白酶抑制剂=100:2:1)提取细胞总蛋白。采用考马斯亮蓝蛋白定量法进行蛋白定量。根据分子量大小选择合适的分离胶。每个泳道加入40μg蛋白样品,采用SDS-PAGE凝胶电泳分离蛋白样品,并将蛋白转到NC膜上,5%牛奶或5%BSA封闭1小时,用抗体稀释液将PDK1一抗(CST,货号:3820T)按照1000:1稀释,4℃孵育过夜。第二天洗涤NC膜后孵育荧光二抗,并在Odyssey红外荧光扫描成像系统进行扫描定量分析。
CCK-8实验结果如图5A所示,可以看出,在高糖培养条件下,随着JX06浓度的增加,子宫内膜癌细胞的增殖活性减弱,JX06对子宫内膜癌细胞在24小时的半数抑制浓度IC50约为0.65μM,48小时为0.35μM(图5A)。
进一步通过流式凋亡实验,结果表明,与PBS组相比,JX06组细胞凋亡率明显增加(图5B)。
Westernblot结果提示在高糖培养条件下JX06能够明显抑制PDK1的表达(图5C和图5D)。
上述结果表明,JX06作为糖酵解关键酶PDK1的抑制剂能够有效抑制高糖对子宫内膜癌细胞促增长作用。
二、JX06和二甲双胍联合抑制高糖对子宫内膜癌细胞促增长作用
高糖可能是导致肿瘤细胞对二甲双胍产生抗性的重要原因。也发现,在正常糖条件下,5mM,10mM和20mM二甲双胍均能够显著抑制子宫内膜的增殖,而在高糖培养条件下这种抑制作用不如正常糖培养显著,如5mM二甲双胍在正常糖条件下对子宫内膜癌细胞的抑制率是35%,而在高糖条件下对子宫内膜癌细胞的抑制率是30%。这证实了高糖培养导致子宫内膜癌对二甲双胍的抵抗性增加,而这种抵抗性可能与高糖通过促进PDK1的表达,从而促进子宫内膜癌细胞糖酵解增加相关。因此,认为PDK1抑制剂(JX06)联合二甲双胍可能具有协同抗肿瘤效应。
1、流式检测凋亡实验
为了初步验证该猜想,进行流式检测凋亡实验,具体方法如下:
将ishikawa细胞接种于六孔板中,依据实验分组分别给予细胞PBS,二甲双胍(培养体系加入二甲双胍,使其浓度为5mM),JX06(培养体系加入JX06,使其浓度为0.5μM),二甲双胍+JX06(培养体系加入二甲双胍和JX06,使二甲双胍的浓度为5mM,JX06的浓度为0.5μM),各组均培养24h。然后收获细胞并用预冷的PBS清洗两遍,1000转离心3min,1X BindingBuffer重悬细胞浓度为1x10^6 cells/ml,吸取100ul(含1x10^5 cells)于1mLEP管中。随后,加5μl的FITC-annexin V和5μl碘化丙啶(PI)于EP管中,混匀。室温(25℃)避光孵育15min。加400μl of 1X Binding Buffer到每个EP管中,最后用流式细胞仪定量检测细胞凋亡情况。
结果如图6A和6B所示,JX06(0.5μM)与二甲双胍(5mM)联合应用对子宫内膜癌细胞的诱导凋亡作用较JX06和二甲双胍单药明显增强,这再一次表明了对子宫内膜癌合并糖尿病的患者,联合应用二甲双胍和JX06可能具有协同抗肿瘤效应。
2、PDC细胞模型
为了更加真实的模拟体内情况,利用患者来源的原代子宫内膜癌细胞(PDC细胞)模型验证了药物对糖尿病患者来源子宫内膜癌细胞的抑制作用。具体方法如下:
获取新鲜的子宫内膜癌患者切除标本1例,患者诊断为IAG2,患者病理免疫组化结果(图6C)提示:孕激素受体(ER)(60%++),Ki67(40%+),孕激素受体(PR)(80%+++),PTEN(-)。采用II胶原酶消化法,进行细胞分离原代培养。将原代子宫内膜癌细胞接种于96孔板,每孔接种100μl包含3000个细胞。培养过夜后,根据实验分组分别给予予细胞PBS,二甲双胍(培养体系加入二甲双胍,使其浓度为5mM),JX06(培养体系加入JX06,使其浓度为0.5μM),二甲双胍+JX06(培养体系加入二甲双胍和JX06,使二甲双胍的浓度为5mM,JX06的浓度为0.5μM),各组均继续培养1周。在每孔中加入10μl的CCK-8试剂,37℃培养箱孵育2h,随后用酶标仪检测450nm处的吸光度值。
结果发现,单用5mM二甲双胍或0.5/μM的JX06均表现出对子宫内膜癌病人来源的肿瘤细胞具有明显抑制效果,其中5mM二甲双胍的抑制率为65%,0.5μMJX06的抑制率为34%。此外,JX06联合二甲双胍的抗肿瘤效应明显强于单药(图6D)。
上述结果表明了在糖尿病患者中JX06联合二甲双胍有可能产生协同抗肿瘤作用。
(二)体内水平-靶向PDK1的JX06小分子抑制剂联合二甲双胍协同抗肿瘤效应
构建了裸鼠皮下荷瘤模型来从活体水平验证PDK1抑制剂JX06联合二甲双胍对子宫内膜癌合并糖尿病的治疗作用。
1、构建糖尿病裸鼠皮下荷瘤模型
选用5周龄的BALB/c裸鼠,构建糖尿病小鼠模型(方法同实施例3的二3),并设计正常对照。当小鼠空腹6小时,血糖浓度大于11.1mmol/L时判为建模成功。按照如下步骤构建皮下荷瘤模型。
(1)取处于对数生长期的ishikawa细胞,细胞达80-90%左右密度为宜。
(2)胰酶消化各组细胞后用预冷的PBS洗两遍,用无血清培养基重悬细胞,吹打细胞沉淀至单个细胞悬液,计数细胞,调整细胞密度至3×107/ml,皮下接种量为3×106个细胞/只,接种体积0.1ml。
(3)将细胞悬液放置于冰上带进动物房,应尽快在半个小时内完成裸鼠皮下接种,接种于裸鼠血供丰富的区域腋窝中后部。
(4)接种前用枪将细胞悬液充分的吹散,接种时,针头在皮下进针约1cm深,避免细胞悬液随着针拔出的过程溢出。
(5)定期观察裸鼠皮下成瘤情况,当开始观察到成瘤后每周游标卡尺检测肿瘤的体积大小,4周后处死裸鼠,取下皮下瘤拍照记录,并将组织进行固定石蜡包埋和﹣80度冰箱保存备用(肿瘤体积计算公式:V=A×B2×0.52,A代表肿瘤长径,B代表肿瘤短径)。
2、PDK1抑制剂JX06联合二甲双胍对子宫内膜癌合并糖尿病的治疗
将以上小鼠根据是否伴有糖尿病被分为两个大组:正常对照组和糖尿病。其中正常对照组分为:生理盐水组,二甲双胍组;糖尿病组分为:生理盐水组,二甲双胍组,JX06组,JX06+二甲双胍组。当小鼠皮下瘤体积大约为500mm2时,根据实验分组分别给予裸鼠如下处理:
正常对照-生理盐水组:给予生理盐水治疗。
正常对照-二甲双胍组:二甲双胍(500mg/L溶解于水中)通过口服给药。
糖尿病-生理盐水组:给予生理盐水治疗。
糖尿病-二甲双胍组:二甲双胍(500mg/L溶解于水中)通过口服给药。
糖尿病-JX06组:JX06(1.5mg/kg)通过尾静脉给药。
糖尿病-JX06+二甲双胍组:JX06(1.5mg/kg)通过尾静脉给药,二甲双胍(500mg/L溶解于水中)口服给药。
3天给药一次,在治疗两周后,杀死小鼠并将肿瘤组织取出并称重。
结果如下:
称重肿瘤组织肿瘤,结果如图7A所示,可以看出,未经治疗的糖尿病小鼠(糖尿病生理盐水)肿瘤组织重量是正常对照组(正常对照生理盐水)小鼠的1.3倍,这表明了高糖的确促进了肿瘤的进展。
肿瘤组织重量(肿瘤体重)结果如图7B所示,单纯二甲双胍治疗在正常小鼠和糖尿病小鼠模型中均表现出对肿瘤的抑制效果,其中正常对照-生理盐水组肿瘤组织重量是正常对照-二甲双胍组的1.6倍,糖尿病-生理盐水组肿瘤组织体积是糖尿病二甲双胍组的1.4倍。JX06+二甲双胍组肿瘤体积和重量明显小于糖尿病-生理盐水组和JX06组,提示JX06能够明显抑制子宫内膜癌的增长,且其抑制效果优于单纯JX06小分子。
以上结果提示,PDK1的小分子抑制剂JX06联合二甲双胍具有良好的协同抗肿瘤效果。
SEQUENCE LISTING
<110>北京大学人民医院
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Claims (5)
1.丙酮酸脱氢酶激酶1(PDK1)作为靶点在开发、筛选和/或制备治疗或辅助治疗I 型子宫内膜癌合并糖尿病的试剂中的应用。
2.抑制或者干扰丙酮酸脱氢酶激酶1(PDK1)表达的物质在制备具有如下至少一种功能的产品中的应用:
1)治疗或辅助治疗I 型子宫内膜癌合并糖尿病;
2)增强二甲双胍治疗I 型子宫内膜癌合并糖尿病疗效;
3)联合二甲双胍治疗I 型子宫内膜癌合并糖尿病。
3.抑制或者干扰丙酮酸脱氢酶激酶1(PDK1)表达的物质和二甲双胍在制备治疗或辅助治疗I 型子宫内膜癌合并糖尿病的产品中的应用。
4.根据权利要求3所述的应用,其特征在于:
所述抑制或者干扰丙酮酸脱氢酶激酶1(PDK1)表达的物质为干扰丙酮酸脱氢酶激酶1(PDK1)表达的shRNA或PDK1抑制剂。
5.一种具有治疗或辅助治疗I 型子宫内膜癌合并糖尿病功能的产品,其为二甲双胍和抑制或者干扰丙酮酸脱氢酶激酶1(PDK1)表达的物质;
所述抑制或者干扰丙酮酸脱氢酶激酶1(PDK1)表达的物质为JX06。
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