Hedgehog信号通路与肿瘤发生机制的研究进展

恶性肿瘤是危害人类健康的主要疾病之一,确切的发病机制尚不清楚,但可以肯定的是,其形成具有多因素、多步骤的特点,涉及一系列原癌基因异常的激活以及抑癌基因突变导致的失活,从而引起一些信号转导通路的异常.近年来,通过对Hedgehog(Hh)信号通路的研究显示,Hh基因编码一系列分泌型信号蛋白,对胚胎发育过程中的细胞分化和增...     

Increased expression of multidrug resistance-associated genes after chemotherapy in pediatric solid malignancies

Background/Purpose

The overexpression of multidrug resistance (MDR)-associated genes in primary untreated tumors has been proven to be associated with worse prognosis in various pediatric malignancies. This study compared the expression of 3 MDR-associated genes, MDR1, MDR-associated protein 1 (MRP1), and lung resistance-related protein (LRP), in pediatric tumors before and after chemotherapy to elucidate the mechanism of MDR during chemotherapy.

Methods

Surgical specimens of both primary and chemotherapy-treated tumors were obtained from 24 patients with pediatric malignancies (neuroblastoma [NB] 8; hepatoblastoma [HB] 8; Wilms tumor [WT] 4; rhabdomyosarcoma [RB] 4). The expression of MDR1, MRP1, and LRP was evaluated using the immunohistochemical staining.

Results

In primary tumors, MDR1 expression was observed in 6 NBs, 8 HBs, 3 WTs, and 3 RBs. MRP1 expression was observed in 3 NBs and 1 HB. LRP expression was not detected in any of the primary tumors. After chemotherapy, MDR1 expression was observed to increase in 5 NBs, 4 HBs, 2 WTs, and 3 RBs. MRP1 expression was newly observed or increased in 7 NBs, 4 HBs, and 3 RBs. LRP expression was newly observed in 3 HBs and 2 WTs.

Conclusions

These results indicate that these 3 MDR-associated genes were upregulated after chemotherapy in various pediatric malignancies. These findings may be useful to understand the mechanism of drug resistance in pediatric malignancies.     

Hedgehog信号通路对胰腺癌细胞增殖的影响

目的 研究特异性阻断hedgehog信号通路对人胰腺癌Mia PaCa-2细胞增殖的影响.方法 用Western blot方法检测7种胰腺癌细胞系中hedgehog信号蛋白表达情况,筛选出hedgehog通路活化明显的Mia PaCa-2细胞系进行研究;应用特异性hedgehog通路阻断剂KAAD-cyclopamine阻断Mia PaCa-2细胞的hedgehog信号通路,Western blot方法检测Gli1蛋白表达变化;MTT法检测阻断hedgehog信号通路对胰腺癌细胞增殖的影响;RT-PCR方法检测胰岛素样生长因子2(IGF2)mRNA表达的变化.结果 KAAD-cyclopamine处理后hedgehog信号通路中关键效应蛋白Gli1表达水平明显下调(P=0.032);KAAD-cyclopamine明显抑制胰腺癌Mia PaCa-2细胞增殖,其作用呈剂量及时间依赖性;阻断hedgehog信号通路后,胰腺癌细胞中胰岛素样生长因子2(IGF2)mRNA表达水平下调(P=0.037).结论 Hedghog信号通路异常活化参与促进胰腺癌细胞增殖;特异性阻断hedgehog信号通路后胰腺癌细胞增殖明显受抑制,其部分机制可能通过下调IGF2表达水平实现.     

Hedgehog Signaling in Mammary Gland Development and Breast Cancer

The Hedgehog pathway is critical for many developmental processes, including the formation of several epidermal appendages. In the mammary gland strict regulation of the Hedgehog pathway is required for normal development. Alterations in Hedgehog signaling result in defects in both the embryonic and postnatal mammary gland. Activation of Hedgehog signaling either by mutation or misexpression of pathway members can lead to the development and/or progression of cancers in multiple organs. This review addresses the current understanding and controversies of Hedgehog signaling in mammary gland development and its potential role in promoting breast carcinogenesis and cancer progression.     

肝细胞肝癌中Hedgehog信号通路效应蛋白的功能分析

目的 探讨Gli1、Gli3蛋白在肝细胞癌组织中的表达及临床意义.方法 免疫组化法检测36例肝细胞癌组织及其癌旁肝组织中Gli1、Gli3蛋白的表达,并与临床因素进行相关性分析.结果 在肝细胞癌组织及癌旁肝组织中Gli1的阳性表达率分别为75％和36.1％,Gli3的阳性表达率分别为58.3％和30.6％.Gli1和Gli3在肝细胞癌组织中的阳性表达率显著高于癌旁肝组织(P＜0.05).Gli1和Gli3两种蛋白的表达呈正相关(r=0.423,P＜0.05).Gli3的表达与临床指标(年龄、肿瘤大小、分化和淋巴结转移)无关.Gli1的表达与年龄、肿瘤大小无关,但与肿瘤分化程度和淋巴结转移相关(P＜0.05).结论 Gli1、Gli3蛋白在肝细胞癌组织中有较高的阳性表达率且具有相关性,且Gli1的表达率与肿瘤分化程度和淋巴结转移相关.检测Gli1、Gli3蛋白表达有助于肝细胞癌的诊断,可能作为判断肝细胞癌恶性程度及预后的指标.     

FOXC2介导Hedgehog/Gli信号通路对乳腺癌侵袭及迁移的影响

目的探讨FOXC2介导Hedgehog/Gli信号通路通过调控上皮间质转化（EMT）途径参与乳腺癌侵袭及迁移的机制。 方法应用不同浓度环靶明（Cyclopamine）处理乳腺癌MDA-MB-231细胞,MTT法检测细胞增殖抑制率及计算药物半数抑制浓度（IC₅₀）;采用Cyclopamine或沉默FOXC2基因表达以阻断Hedgehog/Gli信号通路活化;通过Transwell小室体外侵袭实验及划痕实验分别检测阻断Hedgehog/Gli信号通路对MDA-MB-231细胞侵袭及迁移影响;Western blotting检测阻断前后Hedgehog/Gli信号通路分子Smoothened（Smo）、Gli1和EMT相关标志物FOXC2,E-cadherin及Vimentin蛋白表达变化。 结果与空白对照组比较,Cyclopamine可显著地抑制MDA-MB-231细胞增殖并呈现时效-量效关系,48 h的IC₅₀为25 μmol/L。Transwell小室体外侵袭实验及划痕实验均显示,与未转染组及Control-siRNA组相比,FOXC2-siRNA组和Cyclopamine组细胞穿膜数及迁移率均显著降低,差异有统计学意义（P<0.05）。Western blotting显示,与未转染组及Control-siRNA组相比较,FOXC2-siRNA组及Cyclopamine组Smo、Gli1及FOXC2蛋白表达显著降低,差异有统计学意义（P<0.05）。而沉默FOXC2表达可显著降低Vimentin蛋白表达及增加E-cadherin蛋白表达,差异有统计学意义（P<0.05）。 结论FOXC2通过介导Hedgehog/Gli信号通路从而调控EMT促进乳腺癌侵袭及迁移,提示阻断该信号通路有望成为乳腺癌靶向治疗新线索。     

Hedgehog signaling pathway in neuroblastoma differentiation

Purpose

The hedgehog (Hh) signaling pathway is activated in some adult cancers. On the other hand, the Hh signaling pathway plays an important role in the development of the neural crest in embryos. The aim of this study is to show the activation of Hh signaling pathway in neuroblastoma (NB), a pediatric malignancy arising from neural crest cells, and to reveal the meaning of the Hh signaling pathway in NB development.

Methods

This study analyzed the expression of Sonic hedgehog (Shh), GLI1, and Patched 1 (Ptch1), transactivators of Hh signaling pathway, by immunohistochemistry in 82 NB and 10 ganglioneuroblastoma cases. All 92 cases were evaluated for the status of MYCN amplification.

Results

Of the 92 cases, 67 (73%) were positive for Shh, 62 cases (67%) were positive for GLI1, and 73 cases (79%) were positive for Ptch1. Only 2 (10%) of the 20 cases with MYCN amplification were positive for Shh and GLI1, and 4 cases (20%) were positive for Ptch1 (MYCN amplification vs no MYCN amplification, P ≦ .01). The percentage of GLI1-positive cells in the cases with INSS stage 1 without MYCN amplification was significantly higher than that with INSS stage 4. Of 72 cases without MYCN amplification, 60 were GLI1-positive. Twelve cases were GLI1-negative, and the prognosis of the GLI1-positive cases was significantly better than that of the GLI1-negative cases (P = .015).

Conclusions

Most of NBs without MYCN amplification were positive for Shh, GLI1, and Ptch1. In the cases without MYCN amplification, the high expression of GLI1 was significantly associated with early clinical stage and a good prognosis of the patients. In contrast to adult cancers, the activation of the Hh signaling pathway in NB may be associated with the differentiation of the NB.     

Hedgehog信号通路在人肝癌细胞株中的表达及意义

目的 探讨Hedgehog信号通路在人肝细胞癌细胞系中的表达及意义。 方法 采用半定量 RT-PCR 法检测PLC/PRF/5、HepG2及 SMMC-7721肝癌细胞株中Shh、Ptch1、Smo、Gli1、Gli2 mRNA 的表达;Western blot法检测PLC/PRF/5、HepG2及 SMMC-7721肝癌细胞株中Gli2蛋白的表达。 结果 除Shh外, Ptch1、Smo、Gli1、Gli2 mRNA在PLC/PRF/5、HepG2及 SMMC-7721细胞中均有不同程度表达。其中,Ptch1、Smo、Gli1、Gli2 mRNA在PLC/PRF/5及SMMC-7721细胞中的表达强度显著高于成人正常肝细胞,而Gli1 mRNA在HepG2细胞中的表达量与正常肝细胞无明显差异;Gli2蛋白在成人正常肝细胞中几乎没有表达,在HepG2细胞中的表达也与正常肝细胞无明显差别,而在PLC/PRF/5及 SMMC-7721细胞中,尤其是SMMC-7721细胞,Gli2的表达量异常增高。 结论 Hedgehog 信号通路的异常激活参与了肝细胞癌的发生发展过程,Gli2可能成为肝细胞癌重要生物学标志和生物治疗靶点。     

Shh/Gli1信号通路在异氟醚后处理减轻大鼠脑缺血-再灌注损伤中的作用

目的评价Shh/Gli1信号通路在异氟醚后处理减轻大鼠脑缺血-再灌注损伤中的作用。方法清洁级健康雄性SD大鼠44只,6~8周龄,体重220~280 g,采用随机数字表法将其分为四组:假手术组(S组)、缺血-再灌注组(IR组)、缺血-再灌注+异氟醚后处理组(ISO组)和环巴胺+缺血-再灌注+异氟醚后处理组(CYC组),每组11只。采用线栓法栓塞大脑中动脉90 min、再灌注24 h制备脑缺血-再灌注损伤模型。ISO组大鼠在再灌注即刻吸入1.5%异氟醚。CYC组大鼠在缺血前30 min腹腔注射Shh/Gli1信号通路特异性抑制剂环巴胺10 mg/kg。再灌注24 h后,所有大鼠进行神经行为学评分,采用TTC法测定脑梗死体积,HE染色和尼氏染色观察病理学改变,TUNEL染色观察海马CA1区细胞凋亡,免疫荧光和Western blot法测定Shh和Gli1蛋白含量。结果与S组比较,IR组、ISO组和CYC组大鼠神经行为学评分明显升高,脑梗死体积明显增大,坏死和凋亡细胞明显增多,组织病理学损伤严重,Shh和Gli1蛋白含量明显增加(P<0.05)。与IR组比较,ISO组神经行为学评分和脑梗死体积明显降低,坏死和凋亡明显减少,组织病理学损伤明显减轻,Shh和Gli1蛋白含量明显增加(P<0.05)。与ISO组比较,CYC组神经行为学评分明显升高,脑梗死体积明显增大,坏死和凋亡细胞明显增多,组织病理学损伤明显加重,Shh和Gli1蛋白含量明显减少(P<0.05)。结论 Shh/Gli1信号通路激活参与了异氟醚后处理减轻大鼠脑缺血-再灌注损伤的过程。     

Gli1蛋白在乳腺癌组织中的表达及意义

目的探讨Gli1在乳腺癌组织中的表达及其意义。方法采用免疫组化S-P方法测定64例乳腺癌组织以及52例癌旁正常乳腺组织、42例乳腺纤维腺瘤组织中Gli1蛋白的表达情况。结果 Gli1蛋白在癌旁乳腺组织及纤维腺瘤组织中不表达或弱表达,在乳腺癌组织中的明显表达,差异有统计学意义（P〈0.05）;Gli1蛋白在乳腺癌患者腋窝淋巴结有无转移中的表达差异有统计学意义（P〈0.05）,在Ⅲ期乳腺癌中的阳性表达率高于Ⅰ~Ⅱ期乳腺癌（P〈0.05）。结论 Gli1蛋白与乳腺癌组织的发生发展有一定的关系。     

大豆苷元干预Hedgehog信号通路调控去卵巢骨质大鼠基质金属蛋白酶代谢

目的 探究大豆苷元对骨质疏松（osteoporosis,OP）模型大鼠Hedgehog信号通路的调控作用,分析大豆苷元对OP大鼠基质金属蛋白酶代谢的影响。方法 随机将60只雌性SPF级Wistar大鼠分为假手术组、模型组、大豆苷元低、中、高剂量治疗组5组,每组12只。除假手术组,其他4组均采用去卵巢法建立OP模型。造模成功后开始干预治疗,假手术组、模型组等剂量生理盐水灌胃,其他给予大豆苷元低、中、高剂量治疗,连续干预12周。酶联免疫吸附法测定各组大鼠血清雌激素（E2）、骨钙素（BGP）、血清碱性磷酸酶（ALP）含量水平;免疫组化法检测各组大鼠股骨组织中基质金属蛋白酶组织抑制剂-1（TIMP-1）和基质金属蛋白酶-7（MMP-7）免疫蛋白的表达情况;TUNEL染色法检测各组骨细胞凋亡情况;RT-PCR法和WB法检测各组骨组织中Shh、PTC1、Gli1 mRNA及蛋白表达水平。结果 大豆苷元能显著提高OP大鼠大鼠E2、TIMP-1水平,降低细胞凋亡及 BGP、ALP、MMP-7水平;显著改善股骨组织中Shh、PTC1、Gli1 mRNA及蛋白表达水平（P＜0.05）,且大豆苷元不同组具有剂量依赖性（P＜0.01）。结论 大豆苷元可有效调控OP模型大鼠基质金属蛋白酶代谢水平,这可能与大豆苷元能够有效调节Hedgehog信号通路表达相关。     

Hedgehog信号通路相关蛋白在人胰腺癌SW1990耐药株中的表达及意义

目的:探讨Hedgehog信号通路蛋白在人胰腺癌吉西他滨耐药细胞株SW1990中的表达,为克服胰腺癌获得性耐药提供实验基础。方法:采用浓度梯度递增法建立人胰腺癌SW1990耐药株,采用噻唑蓝法测定SW1990亲代与耐药细胞IC50。实时荧光定量PCR检测亲代与耐药细胞mRNA中hedgehog信号通路成员Shh、SMO、Gli-1的表达差异。Western印迹法检测亲代与耐药细胞中上述蛋白质的表达。结果:人胰腺癌耐药株SW1990的IC50从亲代的(3.1±0.2)μmol/L提高到(232.2±12.3)μmol/L。荧光定量PCR结果显示耐药株中Shh、Gli-1的表达提高了(12.07±1.71)倍和(4.15±0.42)倍。亲代SW1990中未检测到SMO表达,而耐药细胞中却可以检测到SMO的表达。Western印迹结果同样显示,人胰腺癌SW1990耐药细胞株中高表达上述蛋白质。结论:人胰腺癌耐药株中高表达部分hedgehog信号通路蛋白。针对hedgehog信号通路的靶向治疗可能为克服胰腺癌耐药提供新的理论基础。     

Expression and regulation of hedgehog signaling pathway in pancreatic cancer

Introduction  

Pancreatic cancer is an aggressive malignancy with a poor prognosis. The overall 5-year survival rate of pancreatic cancer is less than 5%, and has not improved significantly for years. Further understanding of the molecular carcinogenesis of pancreatic cancer is critical for designing effective ways to treat this type of malignancy.