CN105853400A - AT-101在制备Hedgehog信号通路的选择性抑制剂中的用途 - Google Patents
AT-101在制备Hedgehog信号通路的选择性抑制剂中的用途 Download PDFInfo
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Abstract
本发明属于肿瘤药理学及Hedgehog信号通路研究领域，涉及AT-101在制备Hedgehog信号通路的选择性抑制剂中的用途；具体涉及AT-101在体外NIH3T3细胞中对Hedgehog信号通路的特异性抑制，以及对Hedgehog活性异常升高的实体瘤(如髓母细胞瘤)的生长抑制中的用途。本发明提供了证明AT-101对Hedgehog信号通路特异性抑制的实验方法和证据，和AT-101体内体外抗瘤效应的评价体系，同时证实了AT-101Hedgehog信号通路的作用靶点及其体内的抗瘤效应。本发明可进一步用于制备新的抗肿瘤药。
Description
技术领域
本发明属于肿瘤药理学及Hedgehog信号通路研究领域，涉及AT-101在制备Hedgehog信号通路的选择性抑制剂中的用途；具体涉及AT-101在体外NIH3T3细胞中对Hedgehog信号通路的特异性抑制，以及对Hedgehog活性异常升高的实体瘤(如髓母细胞瘤)的生长抑制中的用途。
背景技术
据报道显示，Hedgehog信号通路最早于1980年在果蝇中发现，它在许多的生理过程中发挥着关键性的作用，如，组织形成，形态的发生，细胞分化，胚胎发育，骨形成等。研究显示，除了在胚胎发育过程中发挥作用，Hedgehog信号通路还在出生后的发育及维持组织/器官的稳态和功能中起着重要作用。有研究证明，Hedgehog信号的异常活化与许多肿瘤的发生密切相关，如髓母细胞瘤、基底细胞癌、急性粒细胞白血病以及前列腺癌、乳腺癌、直肠癌和肺癌等。阻断肿瘤细胞中Hh信号传导通路将为人类肿瘤的治疗提供一个新的有效手段。
现有技术公开了Hedgehog(Hh)信号通路主要由分泌型糖蛋白配体Hedgehog、跨膜蛋白受体Ptched(Ptch)、跨膜蛋白Smoothened(Smo)、核转录因子Gli蛋白及下游靶基因组成。人类和鼠中存在三个Hedgehog的同源基因：Sonic Hedgehog(SHH)、IndianHedgehog(IHH)和Desert Hedgehog(DHH)，分别编码Shh、Ihh和Dhh蛋白；两种patched基因(PTCH1和PTCH2),3种CLi相关基因(GLi 1,GLi2,Gli3)。正常情况下Hedgehog信号通路调控着胚胎期组织细胞的生长分化，胚胎发育成熟后，进入失活状态，此时，Smo蛋白的活性被抑制。Shh、Ihh和Dhh蛋白这三种配体都有相同的受体Ptchl。Ptchl是一种具有12次跨膜结构的膜蛋白，在没有配体激活的情况下，这种膜蛋白抑制它下游的Smo的活性。Smo是一种具有7次跨膜结构的膜蛋白，同时也是G蛋白偶联受体样蛋白。当Hedgehog配体与Ptchl受体结合之后，Ptchl对Smo的抑制作用解除，Smo激活。活化的Smo将信号传递到通路最后的一环，即核转录因子Gli，下游的Gli转录因子被激活，以全长形式转入核内引起靶基因的转录。
已知AT-101是棉酚的左旋异构体，棉酚是从棉花的种子中提取分离出来的一种化合物，具有杀精作用，其最初是用做避孕药。
发明内容
本发明的目的在于提供AT-101在制备Hedgehog信号通路的选择性抑制剂中的新的用途；具体涉及AT-101在体外NIH3T3细胞中对Hedgehog信号通路的特异性抑制，以及对Hedgehog活性异常升高的实体瘤(如髓母细胞瘤)的生长抑制中的用途。
本发明采用棉籽中提取分离的AT-101，经AT-101对Hedgehog信号通路特异性抑制的实验以及AT-101体内体外抗瘤效应的评价体系研究，结果证实，AT-101是Hedgehog信号通路的特异性抑制剂以及它的体内抗瘤作用。
本发明所述的的化合物是棉酚的左旋异构体((-)enantiomer，简称AT-101)具有如下结构：
本发明进行了AT-101对体外NIH3T3细胞中Hedgehog信号通路的特异性抑制作用实验，结果显示，AT-101能显著抑制NIH3T3细胞中Hedgehog信号活性，能显著抑制C3H10T1/2中碱性磷酸酶的活性，也即是对Hh信号通路有抑制作用；
本发明进行了AT-101对Hedgehog信号通路作用靶点的探索实验，结果显示，AT-101能剂量依赖性的抑制转染Smoothened激活的Gli luciferase活性，而对Smoothened突变体SmoM2(W535L)所激活的Gli luciferase活性无抑制作用，表明AT-101是Smoothened的拮抗剂；
本发明进行了AT-101对Hh依赖的髓母细胞瘤的体内抗瘤效应，结果显示，AT-101能显著抑制髓母瘤细胞中Hedgehog信号活性。
本发明经实验证实。所述的AT-101不同于传统的细胞毒性化疗药物，具有对Hedgehog信号通路的特异性抑制作用，即具有分子靶向性，因此对肿瘤细胞的选择性更好，进一步，所述的AT-101可用于制备Hedgehog信号通路的选择性抑制剂，或治疗肿瘤的药物，尤其是治疗Hedgehog信号异常活化的肿瘤(如髓母细胞瘤)的药物。
本发明还提供了一整套Hedgehog信号通路抑制剂体内外抗肿瘤药效评价的体系。
本发明的有益效果是：
1，公开了AT-101是Hedgehog信号通路的特异性抑制剂以及它的体内抗瘤效应。
2，探索获得了AT-101对Hedgehog信号通路的作用靶点，AT-101是Smoothened的拮抗剂。
3、AT-101对Hedgehog信号通路的作用具有特异性，且能在转录水平阻断Hedgehog信号的传递。
5、提供了将AT-101制成靶向Hedgehog信号通路的抗肿瘤药物的理论基础，能明显扩大AT-101的应用领域；
6、可进一步将AT-101制备成新的抗肿瘤药。
以下通过附图并结合具体实施例对本发明作进一步的阐述，但并不限制本发明的保护范围。
附图说明
图1为不同浓度下，AT-101作用于NIH3T3细胞36h后，Gli luciferase活性抑制的量效曲线，结果表明其对SHH刺激的Gli luciferase活性抑制的的IC50为1.84uM，对SAG激活的Gli luciferase活性抑制的IC50为2.72uM。
图2为不同浓度下，AT-101作用于NIH3T3细胞24h后Gli1mRNA表达的柱形图，呈现良好的量效关系。
图3为不同浓度下，AT-101作用于C3H10T1/2细胞72h后碱性磷酸酶活性抑制的柱形图，结果表明其对C3H10T1/2的分化有抑制作用，呈现良好的剂量关系。
图4为转染Smoothened质粒与Smoothened M2(W535L)质粒激活Hedgehog信号后，用不同浓度的AT-101作用NIH3T3细胞36h后，抑制Smoothened激活的Gli luciferase活性；然而对SmoothenedM2(W535L)激活的Gli luciferase活性没有抑制作用，呈现良好量效曲线，说明AT-101是Smoothened的拮抗剂。
图5为AT-101与Bodipy-cyclopamine的相互作用的情况，结果表明AT-101和cyclopamine存在竞争性结合Smoothened的作用，进一步说明了AT-101是Smoothened的拮抗剂。
图6为从裸小鼠身上的髓母细胞瘤组织分离得到的髓母瘤细胞，用AT-101处理24h后Gli 1mRNA表达的柱形图。
图7为AT-101(20mg/kg，或40mg/kg灌胃给药，每天一次)给药21天对裸小鼠接种的髓母细胞瘤的生长抑制效应。
具体实施方式
下列实施例举例说明了本发明的标准实验室实践，用于示范本发明的模式，而不应将本发明理解为限定于这些实施例的范围。
应特别指出的是在下列实施例中，使用的分子生物学技术和动物实验技术是本领域普通技术人员所熟知的，并可在诸如Rudin,C.M.et al.Treatment of medulloblastomawith hedgehog pathway inhibitor GDC-0449.N.Engl.J.Med.361,1173–1178(2009)及Kim J,Tang JY.et al.Itraconazole,a commonly used antifungal that inhibitsHedgehog pathway activity and cancer growth.Cancer Cell.2010Apr13；17(4):388-99等参考文献中获得。
实施例1AT-101对体外NIH3T3细胞中Hedgehog信号通路的特异性抑制作用实验
1.1双荧光素酶报告基因测试
NIH3T3(American Type Culture Collecton)细胞用含10％(v/v)新生牛血清(GIBCO)的Dulbecco’s modified Eagle’s medium(DMEM)(GIBCO)，青霉素/链霉素(Hyclone)，37℃，5％CO2恒温培养，以3×104个/孔种在48孔板中，24h后用转染试剂lipo2000(Invitrogen)转染Gli-dependent firefly luciferase reporter和TK-Renillaluciferase reporter vectors。转染后36h，换成含1％新生牛血清(GIBCO)的培养基，SAG(50nM)或SHHN conditioned medium(SHHN-CM)及配制成不同浓度的AT-101(Selleck)对细胞进行处理，同时设置空白对照组和阳性对照组，37℃，5％CO2恒温孵育36h后，细胞用1XPBS洗2次，按照双荧光素酶报告系统(Promega)的说明书进行Gli 1-luciferase相对活性的检测，本实验每个浓度设三个复孔，至少重复三次；
1.2实时荧光定量PCR(QRT-PCR)
NIH3T3(ATCC)细胞用含10％(v/v)新生牛血清(GIBCO)的Dulbecco’s modifiedEagle’s medium(DMEM)(GIBCO)，青霉素/链霉素，37℃，5％CO2恒温培养，以4×105个/孔种在六孔板中，24h后换成含10％SHHN conditioned medium(SHHN-CM)、1％新生牛血清(GIBCO)的DMEM培养基，细胞以不同浓度梯度的AT-101(selleck)(溶于DMSO(sigma)中)处理，24h后用Trizol(北京鼎国生物)提取总RNA，以内源性的管家基因GUSB作为参照，用逆转录试剂盒(Takara)以及Premix Ex TaqTM(Tli RNaseHPlus)系统(Takara)进行处理后，使用Biorad IQ5.0型实时荧光定量PCR仪，用染料法对Hedgehog的靶基因Gli 1mRNA进行定量测定，本实验每个浓度设三个复孔，至少重复三次，实验结果均为以GUSB标准化后计算出的相对表达水平；
1.3碱性磷酸酶实验
C3H10T1/2(ATCC)细胞用含10％(V/V)胎牛血清(GIBCO)的Dulbecco’s modifiesEagle’s medium(DMEM)(GIBCO),青霉素/链霉素，37℃，5％CO2恒温培养，以4×104个细胞于48孔板中，24h后加入5％SHHN conditioned medium(SHHN-CM)及配制成不同浓度的AT-101(Selleck)对细胞进行处理。37℃，5％CO2恒温孵育72h后，细胞用1XPBS洗2次，用碱性磷酸酶试剂盒测定碱性磷酸酶的活性(碧云天)；
结果显示：AT-101能显著抑制NIH3T3细胞中Hedgehog信号活性，表现为抑制Gl ilucifearse的活性，抑制效果成明显量效关系；其IC50为1.84uM，对SAG激活的Gliluciferase活性抑制的IC50为2.72uM(如图1所示)；AT-101能显著抑制NIH3T3细胞中Hedgehog信号活性，表现为Gli 1mRNA的相对表达降低，抑制效果成明显量效关系(如图2所示)；AT-101能显著抑制C3H10T1/2中碱性磷酸酶的活性，也即是对Hh信号通路有抑制作用，抑制效果成明显剂量关系(如图3所示)。
实施例2AT-101对Hedgehog信号通路作用靶点的实验研究
2.1AT-101对转染Smoothened后激活的Gli luciferase活性的抑制
NIH3T3(ATCC)细胞用含10％(v/v)新生牛血清(GIBCO)的Dulbecco’s modifiedEagle’s medium(DMEM)(GIBCO)，青霉素/链霉素，37℃，5％CO2恒温培养，以3×104个/孔种在48孔板中，24h后用转染试剂lipo2000同时转染hSmo(生博)、Gli-dependentfirefly luciferase reporter和TK-Renilla luciferase reporter vectors。转染后36h，换成含1％新生牛血清(GIBCO)的DMEM培养基，用不同浓度的AT-101(Selleck)对细胞进行处理，37℃，5％CO2恒温孵育36h后，细胞用1XPBS洗2次，用双荧光素酶报告系统测定Gli-luciferase的相对活性(Promega)，本实验每个浓度设三个复孔，至少重复三次；
结果表明AT-101能剂量依赖性的抑制转染Smoothened激活的Gli luciferase活性(如图4A所示)，而对Smoothened突变体SmoM2(W535L)所激活的Gli luciferase活性并无抑制作用(如图4B所示)，说明AT-101是Smoothened的拮抗剂；
2.2AT-101与Bodipy-cyclopamine的相互作用
293T(ATCC)细胞用含10％(V/V)胎牛血清(GIBCO)的Dulbecco’s modifies Eagle’smedium(DMEM)(GIBCO),青霉素/链霉素，37℃，5％CO2恒温培养，以1×105个细胞接种于24孔板中，24h后用转染试剂lipo2000(Invitrogen)转染hsmo(Origene)500ng，转染后24h，换成含1％新生牛血清(GIBCO)的DMEM培养基，加入Bodipy-cyclopamine(Biovision)(1uM)以及不同浓度的AT-101(Selleck)对细胞进行处理，37℃，5％CO2恒温孵育10h后，细胞用1XPBS洗2次,4％多聚甲醛固定10mins，1×PBS洗2次，0.1％Triton冰上孵育10mins，0.5ug/ml DAPI(鼎国昌盛)染色5mins,1×PBS洗2次,封片；
结果表明AT-101与Bodipy-cyclopamine存在竞争性结合(如图5所示)，进一步说明AT-101是Smoothened的拮抗剂。
实施例3AT-101对Hh依赖的髓母细胞瘤的体内抗瘤效应
3.1AT-101对髓母细胞瘤细胞中Gli 1mRNA表达的影响
从生长旺盛的髓母细胞瘤组织分离得到瘤细胞，Neurobasal-A Medium(Gibco)，B-27添加剂(Gibco)，丙酮酸钠(Gibco)，L-谷氨酰胺(Gibco)，青霉素/链霉素(Hyclone)，37℃，5％CO2恒温培养，以5×106个/孔种在六孔板中，24h后细胞以不同浓度的AT-101(selleck)(溶于DMSO(sigma)中)处理，24h后用Trizol(北京鼎国生物)提取总RNA，以内源性的管家基因GUSB作为参照，用逆转录试剂盒(Takara)以及PremixEx TaqTM(Tli RNaseH Plus)系统(Takara)进行处理后，使用Biorad IQ5.0型实时荧光定量PCR仪，用染料法对Hedgehog的靶基因Gli 1mRNA进行定量测定，本实验每个浓度设三个复孔，至少重复三次，实验结果均为以GUSB标准化后计算出的相对表达水平；
结果显示：AT-101能显著抑制髓母瘤细胞中Hedgehog信号活性，表现为Gli1mRNA的相对表达降低，抑制效果成明显量效关系(如图6所示)；
3.2AT-101对Hh依赖的髓母细胞瘤的生长抑制效应
取生长旺盛的Ptch+/-P53-/-C57B/L小鼠自发长出的髓母细胞瘤组织剪切成1.5mm3左右，在无菌条件下，接种于4-6周龄的远交系无胸腺雌性裸鼠(Slac)的左侧腋窝皮下，待移植瘤平均体积长至～110mm3，剔除移植瘤过大、过小以及未见生长的裸小鼠，随机分组给药，每2-3天用游标卡尺测量一次肿瘤体积和裸鼠体重，实验组按照10mg/kg灌胃给药，每天一次；GDC-0449阳性对照组按照12.5mg/kg灌胃给药，每天2次，阴性对照组同时给等量的溶媒0.5％CMC。肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b2，其中a、b分别表示长和宽。根据测量的结果计算出相对瘤体积(relative tumorvolume，RTV)，计算公式为：RTV＝Vt/V0其中V0为分笼给药时测量所得的肿瘤体积，Vt为每一次测量时的肿瘤体积，抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：
T/C(％)＝TRTV/CRTV×100其中TRTV表示治疗组RTV，CRTV表示阴性对照组RTV；
结果显示：AT-101对同种异体移植的髓母细胞瘤具有较为明显的抗瘤效应(如图7所示)。
Claims (6)
1.如下结构的化合物AT-101在制备Hedgehog信号通路的选择性抑制剂中的用途
2.按权利要求1所述的用途，其特征在于，所述的AT-101抑制NIH3T3细胞中Hedgehog信号活性，和抑制C3H10T1/2中碱性磷酸酶的活性。
3.按权利要求1所述的用途，其特征在于，所述的AT-101是Smoothened的拮抗剂，其中，AT-101抑制转染Smoothened激活的Gli luciferase活性，对Smoothened突变体SmoM2(W535L)激活的Gli luciferase活性无抑制作用。
4.按权利要求1所述的用途，其特征在于，所述的AT-101用于制备靶向治疗肿瘤的药物。
5.按权利要求4所述的用途，其特征在于，所述的肿瘤是髓母细胞瘤。
6.按权利要求4或5所述的用途，其特征在于，所述的AT-101抑制髓母瘤细胞中Hedgehog信号活性。
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