CN108653291A - Thz1在治疗卵巢癌中的用途 - Google Patents
Thz1在治疗卵巢癌中的用途 Download PDFInfo
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- CN108653291A CN108653291A CN201710214021.7A CN201710214021A CN108653291A CN 108653291 A CN108653291 A CN 108653291A CN 201710214021 A CN201710214021 A CN 201710214021A CN 108653291 A CN108653291 A CN 108653291A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及THZ1在治疗卵巢癌中的用途;还涉及THZ1在制备用于治疗卵巢癌的药物中的用途。
Description
技术领域
本发明涉及THZ1在治疗卵巢癌中的用途;还涉及THZ1在制备用于治疗卵巢癌的药物中的用途。
背景技术
卵巢癌是在卵巢中形成的癌症[1]。其导致异常细胞具有侵入或扩散到身体其他部分的能力[2]。当这一过程开始时,没有或仅有一些不明显的症状[3][4]。症状随癌症的进展变得越来越明显。这些症状可包括腹胀、盆腔疼痛、腹部肿胀、食欲不振等[3]。卵巢癌通常可扩散的区域包括腹部、肠和膀胱的衬里、淋巴结、肺和肝[5][6]。2012年,全世界一共新增约239000名卵巢癌患者,导致约152000例死亡[7]。
卵巢癌的发病风险在排卵更多的女性中更高。这包括未生育的女性、在更年轻的时候就开始排卵的女性或者在更大的年龄才绝经的女性[7]。其他风险因素包括绝经后的激素治疗、生育医疗和肥胖[1][8]。降低风险的因素包括激素节育、输卵管结扎术和母乳喂养[8]。浆液型卵巢癌是最常见的亚型,较不常见的类型包括生殖细胞肿瘤和性索间质肿瘤[7]。卵巢癌的诊断通过组织活检确认,并一般通过手术切除[3]。
对卵巢癌的常规化疗和药物治疗效果不佳,虽然近年来发现BET(bromodomainand extra terminal domain,溴结构域)抑制剂,例如JQ1等,对多种卵巢癌有不错的疗效,但在用药一段时间后会产生抗性,并且一些亚型对BET抑制剂天然具有抗性,治疗效果不理想。因此有需要寻找新的药物靶点和用于治疗卵巢癌的有效药物。
发明内容
本发明人发现了CDK7和E2F家族在卵巢癌中的重要作用,并出人意料的发现THZ1对绝大多数卵巢癌具有显著效果,特别是对JQ1抵抗的卵巢癌细胞系具有显著效果。
本发明的一些方面涉及THZ1在制备用于治疗卵巢癌的药物中的用途;其中所述卵巢癌可以为对BET抑制剂具有抗性的卵巢癌,进一步地,所述对BET抑制剂具有抗性的卵巢癌为对BET抑制剂天然具有抗性的卵巢癌或者在经BET抑制剂治疗后对BET抑制剂具有抗性的卵巢癌。
本发明的一些方面涉及THZ1在制备通过抑制CDK家族和E2F家族来治疗患有卵巢癌之对象的药物中的用途;其中所述卵巢癌可以为对BET抑制剂具有抗性的卵巢癌,进一步地,所述对BET抑制剂具有抗性的卵巢癌为对BET抑制剂天然具有抗性的卵巢癌或者在经BET抑制剂治疗后对BET抑制剂具有抗性的卵巢癌。
本发明的另一些方面涉及用于治疗卵巢癌的药物组合物,其包含治疗有效量的THZ1。所述药物组合物还包含可药用赋形剂和/或THZ1以外的可用于治疗卵巢癌的一种或更多种成分。
本发明的另一些方面涉及药盒,其包含治疗有效量的含有THZ1药物组合物。上述药盒还可包含THZ1和所述药物组合物以外的可用于治疗卵巢癌的一种或更多种药物,其中所述含有THZ1的药物组合物和另外的可用于治疗卵巢癌的一种或更多种药物可以同时施用或者分开施用。
本发明的另一些方面涉及治疗卵巢癌的方法,其包括向有此需要的对象施用治疗有效量的THZ1或包含其的药物组合物。
本发明的另一些方面涉及治疗卵巢癌的方法,其包括向有此需要的对象施用包含治疗有效量THZ1的药物组合物,所述药物组合物还可包含THZ1以外的可用于治疗卵巢癌的一种或更多种成分。
附图说明
图1A示出了根据本发明所建立的3种具有JQ1抗性的卵巢癌细胞系(抗性)及其对照(敏感)的显微照片。图1B示出了不同浓度的JQ1和BET151对具有或不具有JQ1抗性之细胞系的细胞生存力的影响。
图2A示出了高通量小分子药物筛选的流程图。图2B示出了JQ1敏感和耐药细胞(COV413B、OVCA420和SKOV3)用不同浓度THZ1处理后的细胞生存力曲线;其中后缀.S表示敏感,后缀.R表示抗性。图2C示出了蛋白质印迹分析敏感和耐药细胞经THZ1处理后的RNA聚合酶II(RNAPII)羧基末端磷酸化情况。
图3A示出18种卵巢癌细胞系用不同浓度THZ1处理后的细胞生存力曲线及其用0.1μM THZ1或DMSO处理10天后的结晶紫染色照片。图3B示出了五种细胞系用不同浓度JQ1或THZ1处理10天后的细胞结晶紫染色图,与JQ1相比,THZ1在抑制肿瘤细胞生长方面通常更加有效。图3C示出将A2780、COV362和ES-2细胞系用JQ1或THZ1处理10天后的细胞结晶紫染色图。图3D示出A2780、COV413B和OVCA420细胞系用THZ1处理,蛋白质印迹分析经切割聚(ADP-核糖)聚合酶(poly ADP-ribose polymerase,PARP)和RNAPII羧基末端磷酸化情况。图3E示出THZ1对A2780和HEY两种细胞异种移植肿瘤生长的影响,每组10只小鼠(BALB/c裸鼠)。每条线代表一只小鼠的肿瘤生长曲线,带方块的线/带圆点的线分别表示对照组和实验组平均肿瘤体积。THZ1处理组的肿瘤体积统计学上显著小于对照组(*P<0.05,不成对t检验)。图3F示出了肿瘤异种移植切片中ki-67和经切割胱天蛋白酶3的免疫组织化学染色。
图4.卵巢癌细胞对CDK7的广泛依赖性。图4A示出蛋白质印迹分析18种卵巢癌细胞系RNAPII羧基末端磷酸化情况及CDK分子的表达,CDK7在所有细胞系中均表达。图4B.应用CRISPR-Cas9系统敲除多种细胞(A2780、COV413B、ES-2、HEY、OVCA420、SKOV3、DOV13、IGROV1、OVCA433、OVCAR8和TOV-112D)的CDK7蛋白,蛋白质印迹分析证明了CDK7水平下调。图4C.卵巢癌细胞在CDK7敲除后生存力减弱。左侧,中间及右侧的部分分别表示明视野图,结晶紫染色图,细胞活性定量(*P<0.05,单因素方差分析及Tukey’s实验后检验)图4D.HEY和ES-2细胞敲除CDK7后,异种移植肿瘤的照片及其平均重量(*P<0.05,单因素方差分析及Tukey’s实验后检验),转染含sgEGFP的慢病毒作为对照。
图5A在4种卵巢癌系(A2780、OVCA420、HEY和ES-2)中用CRISPR-Cas9技术敲除各CDK基因后的结晶紫染色。图5B示出了图5A中4种细胞系之生存力的量化。
图6A示出了在多个卵巢癌数据集中CDK7的表达作为总生存之单变量预测的森林图。图6B.免疫组织化学分析CDK7在卵巢癌组织中的表达。顶部为典型的CDK7阴性或阳性样本照片,底部为CDK7阴性和阳性卵巢癌患者Kaplan-Meier生存曲线比较。
图7A.100nM THZ1处理多种细胞系6小时,mRNA的总体水平出现急剧下降。图7B.基因集富集分析(Gene Set Enrichment Analysis,GSEA)图示出,与DMSO处理的卵巢癌细胞相比,THZ1处理的卵巢癌细胞中E2F靶基因下调。图7C.THZ1处理后,细胞系中E2F家族基因下调,仅示出一部分结果。图7D.THZ1处理后,E2F家族基因的下游基因下调,仅示出部分示例性基因。(*P<0.05)
具体实施方式
定义:
本文所述的“THZ1”是指具有如下结构的化合物:
本文所用的术语“CDK7”是指周期素依赖性激酶7(cyclin dependent kinase 7),其是由人CDK7基因编码的蛋白质,属于丝氨酸/苏氨酸激酶家族。该蛋白与周期素H和MAT1形成3聚体,作为Cdk激活激酶。其是转录因子TFIIH的重要组分,TFIIH参与转录起始和DNA修复。
本文所用的术语“BET”是指溴结构域(bromodomain and extra terminaldomain),其是识别单乙酰化残基(例如组蛋白的N末端尾上的单乙酰化残基)的约110个氨基酸的蛋白质结构域。其亲和力在相邻存在多个乙酰化位点的区域更高。这种识别通常是蛋白质-组蛋白相互作用和染色体重塑的先决条件。该结构域本身采用全α蛋白质折叠,由长度可变的环区域隔开的四个α螺旋形成识别乙酰化赖氨酸的疏水性口袋。
本文所用的术语“BET抑制剂”是指能够抑制含有BET之蛋白的功能的任何物质,包括但不限于JQ1和BET151等。
本文所用的术语“IC50”是指被测量的拮抗剂的半抑制浓度,其能指示某一药物或者物质(抑制剂)在抑制某些生物程序(或者是包含在此程序中的某些物质,比如酶,细胞受体或是微生物)的半量。更具体地,可以理解为诱导肿瘤细胞凋亡50%的抑制剂浓度,抑制剂诱导凋亡的能力越强,该数值越低。
本文所用的术语“E2F家族”是高等真核生物中的转录因子家族,包括E2F1、E2F2、E2F3a、E2F3b和E2F4-8等。它们均参与哺乳动物细胞中的细胞周期调控和DNA合成。
实施例
以下将结合附图,对本发明的具体实施方式进行详细说明,所述实施方式是示例性的,而非对本发明做出进一步的限制。
实验材料
本文中所使用的以下肿瘤细胞系获自美国典型培养物保藏中心(ATCC):SKOV3(ATCC编号:HTB-77)、TOV-112D(ATCC编号:CRL-1173)、OV90(ATCC编号:HTB-77)、HEY(ATCC编号:CRL-3252)、ES-2(ATCC编号:CRL-1978)。本发明所使用的其他细胞系SW982、A2780、DOV13、OVTOKO、COV318、OVCA420、MCAS、IGROV1、PA1、OVCAR5、OVCA433、OVCAR8、和COV413B由美国国家癌症研究所和美国德克萨斯州立大学安德森癌症研究中心获得。所有细胞用含10%胎牛血清的RPMI1640培养基(Sigma)培养。对于CRISPR-Cas9系统的基因敲除实验,细胞用编码单链向导RNA(sgRNA)的慢病毒感染。本发明所使用的sgRNA的编号和对应名称请参见下表1,具体序列还参见序列表。JQ1购自Millipore。THZ1购自MedChem Express。I-BET151和小分子抑制剂库购自Selleck Chemicals。所有的抑制剂溶于DMSO(Sigma-Aldrich)。
表1:所使用的单链向导RNA(sgRNA)的编号和序列
实验方法
高通量抑制剂筛选
各种细胞以最佳密度(约4000个细胞/孔)接种到96孔板的RPMI1640培养基中,用等量浓度的CDR7抑制剂处理。每三天换一次新鲜培养基和药物。处理后,使用ArrayScanInfinity(Thermo Scientific)拍照,并测量生存力。
THZ1 IC50值测量
为了确定THZ1作用于卵巢癌细胞系的IC50值,选择了7个药物浓度,三倍地连续稀释。细胞活性根据厂商说明书用CellTiter-Glo试剂(Promega)测量。根据GraphPad Prism6(GraphPad Software,Inc.)作图得到的7条剂量-反应曲线,估计IC50值。
细胞周期和凋亡分析
细胞经THZ1处理24小时后进行细胞周期分析。除去培养基并用生理盐水清洗两次,接着将细胞以冰冷乙醇固定,之后用碘化丙啶(Propidium Iodide,PI)/RNA酶染色溶液(Cell Signaling Technology)孵育15分钟。细胞凋亡按厂商(Life Technologies)说明书用含Annexin V-FITC和PI的死细胞凋亡试剂盒(Dead Cell Apoptosis Kit)分析。用FACSAriaII cytometer(BD Biosciences)进行流式细胞分析,数据用FlowJo软件处理。
蛋白质印迹实验
细胞用含蛋白酶抑制剂(Roche)和磷酸酶抑制剂(Roche)的RIPA缓冲液(Tris pH7.4 50mM,NaCl 150mM,NP-40 1%,SDS 0.1%,EDTA 2μM)裂解,进行SDS-PAGE电泳和蛋白质印迹实验。使用针对以下蛋白质的抗体:BRD2、BRD3、BRD4(购自Abcam);FoxM1、CDK4、CDK6、CDK7、CDK9、经切割PARP、H3、肌动蛋白(Actin)、微管蛋白(Tubulin)(购自CellSignaling Technology);POL II S-2、POL II S-5、POL II S-7(购自Millipore)。
染色质免疫沉淀(CHIP)
染色质免疫沉淀是本领域技术人员公知的实验技术。简言之,细胞用11%新鲜配制的甲醛交联并以甘氨酸终止反应。裂解细胞团块,以Vibra cell Sonics VCX 500超声,得到清亮的超声裂解产物,用结合了IgG的磁珠孵育过夜。用H3K27ac(Abcam)抗体富集目的蛋白相关的DNA片段。洗涤沉淀的免疫复合物,过夜解交联。以RNA酶A(Roche)和蛋白酶K(Roche)分别消化RNA和蛋白。用Qiaquick PCR纯化试剂盒(Qiagen)纯化DNA。
肿瘤异种移植物模型
为检验THZ1的效力,把肿瘤细胞(约1×106个)和Matrigel(BD Biosciences)混合,植入BALB/c裸鼠背侧皮下。当肿瘤达约150mm3大小时,把鼠随机分为2组,每组10只。一组作为对照(含10%DMSO的D5W(5%葡萄糖水溶液),另一组以10mg/kg的THZ1处理,一天两次。为了检验CDK7的作用,将对照或已敲除CDK7的肿瘤细胞植入BALB/c裸鼠皮下。用数字卡尺测量肿瘤体积(每组10只),用公式V=1/2×L×W2计算体积(V代表肿瘤体积,L代表肿瘤长度,W代表肿瘤宽度。所有的动物实验经过仁济医院动物保护和使用协会许可。
组织学和免疫组织化学
用福尔马林固定、石蜡包埋肿瘤组织,制成5μm厚的组织切片,进行组织学和免疫组织化学实验。以二甲苯脱蜡,在浓度渐减的乙醇中复水,在加有10mM的柠檬酸盐缓冲液(pH 6.0)的高压蒸汽锅中进行抗原修复。预处理组织用苏木精-伊红(H&E)染色,或用Peroxidase Block(Dako)淬灭内源性的过氧化物酶活性,用Protein Block(Dako)封闭,随后用Ki-67或经切割胱天蛋白酶3的抗体(Cell Signaling Technology)孵育。用50mMTris-HCl(pH 7.4)清洗切片,用辣根过氧化物酶标记的二抗孵育。免疫过氧化物酶染色用3,3’-二氨基联苯胺(DAB,diaminobenzidine)(SIGMA)显色,切片用苏木精复染,不同浓度渐增的乙醇和二甲苯脱水,封闭液封片。
数据分析
基因集富集分析用GSEA软件进行。在所有的实验中,用双侧t检验进行两组比较,单因素方差分析(ANOVA)用于分析多组间差别,P值小于0.05认为有统计学意义。
实验结果
建立卵巢癌耐药细胞系
我们利用JQ1对COV413B,OVCA420和SKOV3进行长期处理,即将细胞以约4000个细胞/孔的密度接种到96孔板的RPMI1640培养基中,先用低浓度JQ1 0.3μM(μmol)/mL处理2周,再用0.6μM/mL处理2周,最后1μM/mL处理2月,每三天换一次新鲜培养基和药物,最终建立了3种卵巢癌耐药细胞系COV413B.R、OVCA420.R和SKOV3.R,它们与敏感细胞系对照COV413B.S、OVCA420.S和SKOV3.S的显微照片见图1A。
鉴定THZ1为卵巢癌的潜在抑制剂
我们利用含有181种FDA批准的或其他临床相关的小分子化合物库对JQ1敏感和耐药细胞系进行高通量功能筛选,化合物库中包括的小分子化合物参见表2。出人意料地发现,THZ1在6种细胞系中都具有理想的IC50(图2B),通过进一步的western印迹实验证明,无论在JQ1敏感细胞系和JQ1耐药细胞系,THZ1都能导致RNA聚合酶II CTD S2,S5,S7磷酸化水平的下降,并呈现出剂量依赖性(图2C)。
表2:高通量功能筛选所使用的181种小分子化合物
THZ1在多种卵巢癌细胞系中的抗肿瘤作用
为了进一步确认THZ1作为候选药物的潜力,我们选择了18种卵巢癌细胞系(COV362、A2780、TOV-112D、COV 413B、DOV 13、OV90、SW982、OVCA433、PA1、OVCAR5、HEY、IGROV1、SKOV3、OVCA420、OVTOKO、OVCAR8、COV 318、ES-2),代表了上皮性卵巢癌的主要几种组织亚型(7中浆液型,5种非浆液型,6种未确定),进行了药理学功能筛选,以确定它们对THZ1的反应。IC50和结晶紫染色的实验结果如图3A所示,这些研究证实绝大多数细胞系对于THZ1诱导的细胞毒性呈高度敏感性,具体的IC50在表3中列出。值得注意的是,结晶紫实验可以看出,与JQ1相比,THZ1在抑制肿瘤细胞生长方面通常更加有效(图3B)。而且,我们发现那些对JQ1天然耐药的细胞,包括A2780,COV362和ES-2,都被THZ1有效地抑制了(图3C)。因此,THZ1是可以克服天然的以及后天获得的JQ1耐药性。因此THZ1可以用作治疗天然对JQ1具有耐药性或在一段时间的JQ1施用后对JQ1产生耐药性的癌症的特别有用的药物。A2780、COV413B和OVCA420细胞系用THZ1处理后,蛋白质印迹分析经切割PARP和RNAPII羧基末端磷酸化,THZ1都能导致RNA聚合酶II CTD S2,S5,S7磷酸化水平的下降,同时增加了THZ1的,并都呈现出剂量依赖性(图3D)。
表3:THZ1在18种卵巢癌细胞系中的IC50
细胞系 | IC50(μM) | 细胞系 | IC50(μM) | 细胞系 | IC50(μM) |
COV362 | 0.005 | HEY | 0.045 | SK-OV-3 | 0.074 |
A2780 | 0.007 | OV90 | 0.048 | OVCA420 | 0.105 |
TOV-112D | 0.015 | SW982 | 0.048 | OVTOKO | 0.057 |
COV413B | 0.013 | OVCA433 | 0.053 | OVCAR8 | 0.112 |
DOV-13 | 0.032 | PA1 | 0.055 | COV318 | 0.129 |
OVCAR5 | 0.032 | IGROV-1 | 0.057 | ES-2 | 0.178 |
如前所述,我们利用A2780和HEY这2个细胞系建立独立了异种异位移植瘤模型,以评价THZ1体内的疗效。当肿瘤长到约150mm3时,将动物随机分成2组(每组10只),进行给药。很显然地,THZ1给药的老鼠荷瘤体积显著减少(图3E)。组织学分析显示THZ1处理后的肿瘤组织普遍坏死,ki-67指示细胞增殖生存力下降,凋亡标志物经切割的胱天蛋白酶3被激活,然而,对照组肿瘤组织的上皮细胞依然保有生存力(图3F)。由此我们得出结论,在卵巢癌不同的病理学亚型中,THZ1通过阻碍细胞周期和细胞存活来抑制肿瘤的增殖。
卵巢癌细胞对CDK7的依赖性
THZ1是CDK7的特异性抑制剂,而CDK7激酶活性涉及到基因转录和细胞周期的调控,因此发明人考虑进一步研究THZ1是否是通过靶向CDK7抑制卵巢癌的,即CDK7的抑制剂均可起到相同的作用。CDK7以及在S2、S5和S7磷酸化的RNA聚合酶II CTD广泛表达于几乎所有细胞系(图4A)。接下来,我们应用CRISPR-Cas9系统将CDK7在11株卵巢癌细胞系进行基因敲除。3条独立的靶向CDK7的单链向导RNA序列(sgCDK7-1、sgCDK7-2、sgCDK7-3)都能导致不同肿瘤细胞中CDK7蛋白水平的显著下降(图4B)。很明显,在体外所有用到的细胞模型中,CDK7的下调极大地抑制了细胞的生存力(图4C)。相应地,体内试验中,CDK7的消除显著破坏了HEY和ES-2细胞的异种移植物的形成(图4D)。这些实验结果证明了卵巢癌中CDK7是THZ1发挥功能的靶点之一。
CDK7属于约20个丝氨酸/苏氨酸激酶的细胞周期素依赖性激酶(CDK)家族,这个家族可以粗略分成细胞周期相关CDK和转录相关CDK。CDK7是CDK激酶的主要组成成员,不仅能够磷酸化RNA聚合酶II CTD,而且还参与调节所有的CDK。我们利用CRISPR-Cas9技术将每个CDK基因都敲除,观察哪些CDK在卵巢肿瘤发生中是必不可少的。有趣的是,我们发现有几个细胞周期相关CDK(CDK1、CDK2和CDK6)及转录相关CDK(CDK7、CDK9和CDK12)对于卵巢肿瘤的生长是必需的(图5A)。因此,与三阴性乳腺癌不同,卵巢癌则广泛依赖于多种CDK,而且CDK7与MAT1和CyclinH形成的复合物细胞周期活化激酶(Cyclin-activating kinase,CAK)可以磷酸化多种CDK分子,所以进一步确认了CDK7作为最主要的细胞周期蛋白激酶的中枢作用,并且THZ1直接和/或者通过CDK7间接影响CDK家族的多个成员,从而对卵巢癌细胞系的增殖产生抑制。
为了评价CDK7在卵巢癌病例中的临床相关性,我们利用curated Ovarian Data数据库进行了meta分析。森林图显示CDK7和病人不良预后相关(图6A;CDK7总危险比为1.07,95%可信度1.01-1.15)。我们也做了50例上皮性卵巢癌临床标本的免疫组化分析,发现超过40%样品呈CDK7阳性(表4)。根据CDK7的表达水平将病人分成2组,CDK7阳性组总的生存率明显差于阴性组(图6B)。所有实验通过上海市仁济医院的伦理委员会审查,病人或者病人家属知情且同意实验。
表4:50例上皮性卵巢癌临床标本的免疫组化分析
THZ1抑制卵巢癌基因的转录
我们利用RNA测序来分析THZ1对卵巢癌整体基因组表达的影响。100nM THZ1处理6种细胞系COV413B敏感、COV413B抗性、OVCA420敏感、OVCA420抗性、SKOV3敏感、SKOV3敏感)6小时,仅添加DMSO作为对照,按照“诺禾致源”提供的方案提取总RNA并送诺禾致源进行转录组测序(使用illumina测序仪),实验结果参见图7A,与仅使用DMSO处理的对照相比,使用THZ1处理的实验组中,mRNA的总体水平出现急剧下降,其中41%-87%转录本下降1.5倍以上,推定THZ1可能不仅通过对CDK家族的影响起作用,还可能通过其他信号通路对细胞产生影响,经过数据分析,发明人发现在对THZ1敏感的细胞系中,E2F家族部分基因在THZ1处理的细胞中表达显著下降,部分结果在图7C中示出,相对于未经THZ1处理的对照进行归一化。而且,不管在敏感细胞还是耐药细胞,根据诺禾致源提供的基因集富集分析结果,我们发现E2F家族以及包含E2F结合基序的基因簇在THZ1的作用下,发生了显著下调(图7B);在对THZ1敏感的细胞系中,将E2F家族下游(包含E2F结合基序)的几个示例性基因(MYC、FOSL1(FOS-like antigen 1)、FOSL2(FOS-like antigen 2)、ELK3(ETS domain-containingprotein 3))的表达在图7D中示出,相对于未经THZ1处理的对照进行归一化,在THZ1处理的三种细胞细中均出现表达下调。而且已知在JQ1耐药细胞中,E2F是过度激活的,结合此结果,可以认为THZ1通过影响E2F家族的表达,对于卵巢癌细胞的增殖产生了重要影响。因此综上所述,不仅证明THZ1对细胞的总体mRNA表达产生了显著影响,还说明了THZ1通过影响CDK家族和E2F家族及E2F家族下游包含E2F结合基序的基因簇对于抑制卵巢癌细胞增殖发挥了重要作用。
本领域技术人员将意识到或者能够仅使用常规实验确定本文所述本发明的具体实施方案的许多等同方案。这样的等同方案旨在由所附权利要求涵盖。
参考文献
1."Ovarian Cancer Prevention".NCI.December 6,2013.Retrieved 1 July2014.
2."Defining Cancer".National Cancer Institute.Retrieved 10 June 2014.
3."Ovarian Epithelial Cancer Treatment".NCI.2014-05-12.Retrieved 1July 2014.
4.Ebell,MH;Culp,MB;Radke,TJ(March 2016)."A Systematic Review ofSymptoms for the Diagnosis of Ovarian Cancer.".American journal of preventivemedicine.50(3):384–94.doi:10.1016/j.amepre.2015.09.023.
5."Treating advanced ovarian cancer".www.cancerresearchuk.org.Retrieved2015-05-16.
6.Ruddon,Raymond W.(2007).Cancer biology(4th ed.).Oxford:OxfordUniversity Press.p.223.
7.World Cancer Report 2014.World Health Organization.2014.pp.Chapter5.12.ISBN 9283204298.
8."Ovarian Cancer Prevention".NCI.2014-06-20.Retrieved 1 July 2014.
9.Piek JM,van Diest PJ,Verheijen RH(2008)."Ovarian carcinogenesis:analternative hypothesis".Adv.Exp.Med.Biol.Advances in Experimental Medicineand Biology.622:79–87.doi:10.1007/978-0-387-68969-2_7.ISBN 978-0-387-68966-1.PMID 18546620.
序列表
<110> 上海市肿瘤研究所
<120> THZ1在治疗卵巢癌中的用途
<160> 14
<170> PatentIn version 3.3
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<211> 20
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<213> 人工序列
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<213> 人工序列
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<223> CDK4-sgRNA
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<210> 4
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<212> DNA
<213> 人工序列
<220>
<223> CDK6-sgRNA
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<213> 人工序列
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<223> CDK7-sgRNA-1
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<223> CDK10-sgRNA
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<223> CDK19-sgRNA
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Claims (10)
1.THZ1在制备用于治疗患有卵巢癌之对象的药物中的用途。
2.THZ1在制备通过抑制CDK家族和E2F家族来治疗患有卵巢癌之对象的药物中的用途。
3.权利要求1或2所述的用途,其中所述卵巢癌为对BET抑制剂具有抗性的卵巢癌。
4.权利要求3所述的用途,其中所述卵巢癌为对BET抑制剂天然具有抗性的卵巢癌。
5.权利要求3所述的用途,其中所述卵巢癌为在经BET抑制剂处理后对BET抑制剂具有抗性的卵巢癌。
6.权利要求3至5中任一项所述的BET抑制剂优选为JQ1或BET151。
7.用于治疗卵巢癌的药物组合物,其包含治疗有效量的THZ1。
8.权利要求7所述的药物组合物,其还包含可药用赋形剂和/或THZ1以外的可用于治疗卵巢癌的一种或更多种成分。
9.药盒,其包含治疗有效量的权利要求8所述的药物组合物。
10.权利要求9所述的药盒,其还包含另外的可用于治疗卵巢癌的一种或更多种药物,其中所述药物组合物和另外的可用于治疗卵巢癌的一种或更多种药物可以同时施用,或者其中所述药物组合物和另外的可用于治疗卵巢癌的一种或更多种药物可以分开施用。
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CN111321172A (zh) * | 2019-12-27 | 2020-06-23 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | 一种利用CRISPR-Cas9技术建立CDK13基因敲除动物模型的方法 |
CN112716957A (zh) * | 2020-12-28 | 2021-04-30 | 复旦大学附属肿瘤医院 | Cdk7靶向抑制剂在制备治疗car-t治疗引起的细胞因子释放综合征药物中的用途 |
CN112716957B (zh) * | 2020-12-28 | 2022-10-14 | 复旦大学附属肿瘤医院 | Cdk7靶向抑制剂在制备治疗car-t治疗引起的细胞因子释放综合征药物中的用途 |
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