Ⅱ型谷氨酰胺转氨酶通过调节相关凋亡因子的表达抑制骨肉瘤MG-63细胞凋亡的机制
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摘要
一、研究背景
骨肉瘤是一种常见于青少年的原发性恶性肿瘤。其发病率约占所有骨肿瘤的20%。骨肉瘤细胞增殖迅速,以浸润性生长破坏周围正常组织,早期即发生远处转移,其中最常见的是肺转移,其预后差。传统治疗方法主要是截肢和放疗,5年生存率不超过20%。近几年辅助化疗、保肢手术的运用使骨肉瘤患者5年生存率和保肢成功率显著提高,但长期疗效及患者的远期存活率并无明显改善。近年来越来越多的学者关注于研究骨肉瘤的发生、发展以及转移的分子机制,希望从基因水平找到治疗骨肉瘤的新方法。随着研究的进展,发现骨肉瘤的发生、发展和转移是一个复杂的过程。除了一系列病理生理反应外,还有许多基因和转录因子参与,涉及到多种调控机制。其中缺氧引起的多种基因的表达以及它们之间的调控机制在肿瘤的增殖过程中发挥重要的作用。
研究证实,在实体恶性肿瘤的生长过程中,由于组织增生过快必然造成局部组织的缺氧和供能与耗能之间的不平衡,这就导致肿瘤细胞发生凋亡。为了适应缺氧环境,肿瘤细胞表型发生一系列变化,如诱导血管形成及增加细胞糖酵解；而对于细胞凋亡的调节也发挥很重要的作用。目前认为与骨肉瘤相关的细胞凋亡途径主要有两条：死亡受体配体途径和线粒体途径。其中线粒体途径是通过部分凋亡刺激因子刺激线粒体释放细胞色素c(Cyt c),释放到细胞浆的细胞色素c在dATP存在的条件下能与凋亡激活因子1结合,使其形成多聚体,并促使caspase-9与其结合形成凋亡小体,caspase-9被激活,被激活的caspase-9能激活其它的caspase如caspase-3等,caspase-3又激活DNA断裂因子,导致静息状态的核酸内切酶激活,最终引起DNA断裂。从线粒体功能障碍到DNA断裂,是细胞凋亡生化途径的共同途径。由于各caspase可相互激活,所以caspase蛋白酶级联反应是导致细胞凋亡结构改变的主要环节。线粒体功能障碍在骨肉瘤细胞凋亡发生机制中起关键作用。
研究证实Bcl-2、凋亡蛋白抑制因子(inhitors of apoptosis proteins, IAPs)、p53、caspases等细胞凋亡因子在调控骨肉瘤细胞凋亡过程中发挥重要的作用。在与凋亡相关的调控基因中,Bcl-2是研究最深入、与凋亡关系最密切的基因之一。主要包括凋亡抑制蛋白Bcl-2类(Bcl-2、Bcl-x1、Bcl2w、Mcl-1、Ced-9等)和促凋亡蛋白Bax类(包括Bax、Bid和仅有BH3结构域的Bid、Bim、PUMA等蛋白)两种。骨肉瘤中对有凋亡抑制作用基因的研究主要以Bcl-2为主,它可编码α和β2种蛋白,存在于线粒体外膜及部分内质网中。目前普遍认为骨肉瘤中Bcl-2发挥凋亡抑制作用的机制是通过阻断线粒体途径,即阻断线粒体内细胞色素c的释放进而抑制下游效应因子的激活,从而发挥凋亡抑制作用；Bcl-2蛋白也可通过与促凋亡蛋白Bax相结合而抑制凋亡。不同肿瘤中Bcl-2的表达及与预后的关系也不尽相同。学者们通过免疫组化法研究人骨肉瘤中的Bcl-2基因,结果显示骨肉瘤组织Bcl-2蛋白染色阳性率和染色程度均较正常骨组织增高,揭示骨肉瘤的发生与凋亡异常有关。体外实验亦证实,骨肉瘤细胞中Bcl-2呈高水平表达,且Bcl-2对骨肉瘤细胞系凋亡有抑制作用。Ferrari等的研究表明,在多数伴有肺转移的骨肉瘤复发病例中,Bcl-2的表达较原发肿瘤强,但Bcl-2蛋白的表达并不随骨肉瘤分期的升高而升高,可见其表达情况并不能作为骨肉瘤恶性程度的指征。促凋亡蛋白Bax主要存在于胞浆,当细胞受到凋亡信号刺激时转入线粒体中,并通过线粒体途径发挥促凋亡作用。Eliseev等证实,骨细胞的特异性转录因子RUNX2以Bax为靶点,通过提高其表达量来增加骨肉瘤细胞Saos-2对凋亡的敏感性。多项研究表明,Bcl-2与Bax比值的高低决定细胞凋亡与否,目前认为其机理是Bax-Bax二聚体可诱导凋亡,但当Bcl-2蛋白量升高时可与Bax形成更稳定的Bcl-Bax二聚体,因此拮抗了Bax的促凋亡作用。Kaseta等的研究还认为,Bcl-2与Bax的比值与骨肉瘤细胞凋亡的抑制呈正相关关系。
谷氨酰胺转氨酶又称转谷氨酰胺酶,是一种催化蛋白质或多肽链中的谷氨酰胺残基里的丫酰胺基和一级氨基之间进行酰胺基转移反应的酶。经研究表明,谷氨酰胺转氨酶广泛分布于血液、细胞外基质及细胞内组分中。参与许多生物化学过程,如凝血、伤口愈合、组织重建、细胞分化和死亡等。到目前为止,已发现的人类谷氨酰胺转氨酶有8型。Ⅱ型谷氨酰胺转氨酶(transglutaminase2,TG2),也叫组织型谷氨酰胺转氨酶(tissue transglutaminase, tTG,E. C.2.3.2.13),它是该家族中研究得较为广泛和深入的一个酶。此酶具有广泛的功能,参与许多病理生理过程,如纤维化、动脉粥样硬化、神经退行性病变、腹泻和肿瘤转移等。近年来,越来越多的研究证实该酶与肿瘤发生、发展有密切的联系。
TG2是以二聚体的形式存在。每个单体由686个氨基酸构成,相对分子质量约为80kD,每个单体含有4个独特的结构域：氨基端的p三明治结构域(1～139)、催化转氨基的核心结构域(147～460)和羧基端的2个p管状结构域(472～583,591～687)。TG2是谷氨酰胺转氨酶家族中的一个独特成员,它不仅具有Ca2+依赖的催化蛋白交联的活性,还有GTP依赖的G蛋白的功能。此外,它还具有蛋白二硫键异构酶活性和蛋白激酶的功能。虽然TG2主要位于胞浆内,但也可以分泌到细胞外,交联细胞外的基质蛋白,起稳定细胞外基质的作用,TG2的这种分泌作用依赖于其活性构象和(或)正确的三级结构。它还可以在输入蛋白(importin)-a3协助下转位进入核内,交联组蛋白、pRb和SP1转录因子,调节细胞周期和凋亡。同样,TG2也可以转位到膜上,和整合素一起,促进细胞表面的整合素和纤连蛋白相互作用。因此TG2可能对肿瘤发生、分化及侵袭行为有重要的影响。
许多研究结果都支持TG2的促凋亡作用,凋亡细胞内通常都有高水平的TG2表达,抑制TG2的表达会显著降低细胞死亡数量。在极端恶劣的环境下(如缺氧、生长因子缺乏、化疗药物处理),细胞内Ca2+稳态受到破坏,这种胞浆内Ca2+的突然改变会使TG2转变成有交联活性的构象,导致细胞内蛋白质的广泛交联、多聚化,形成不溶于酸的结构,稳定了凋亡细胞,防止炎症反应和瘢痕出现。然而TG2表达和凋亡并不是完全一致的。TG2基因敲除的小鼠并未表现出细胞凋亡障碍,这其中也不能排除其他异构酶对TG2缺乏的补偿。此外有几种快速分裂的肿瘤细胞虽表达了高水平的TG2,却没进入凋亡。最近又有直接证据表明,升高的TG2通过阻止凋亡来延长细胞的存活。此外表皮生长因子虽可以抑制多柔比星(阿霉素)诱导的乳腺癌细胞凋亡,但却上调TG2的表达。Antonyak等已经证明了在维甲酸处理某些细胞系中,维甲酸诱导的细胞内TG2水平的增加能够保护细胞免受羟苯维胺脂(N-HPR)诱导的凋亡。这些研究结果提示TG2不仅具有促凋亡作用,而且还有抗凋亡作用,这种似乎矛盾的作用取决于细胞类型、所受刺激类型及TG2在细胞内的定位和酶活性类型。在细胞浆内,TG2通过其转氨基作用来促进凋亡；在核内,TG2通过G蛋白功能或(和)转氨酶活性保护细胞不凋亡；同样地,在细胞膜上,它作为整合素的辅助受体,促进整合素与细胞外基质黏附力和信号转导,保护细胞不凋亡。
最新的研究证实,TG2还能够通过与细胞内蛋白质以非共价形式形成复合物而表现出非酶学的功能。在]HEK293细胞,TG2通过消耗Bax、降低caspase-3和caspase-9、抑制细胞核内的细胞色素c释放进入细胞浆以及在Ca2+增高时线粒体膜的去极化,从而发挥抗凋亡的作用。在缺氧的肿瘤细胞发现同样的机制是通过与caspase-3交叉结合消耗caspase-3,从而发挥抗细胞凋亡的作用。这提示在缺氧环境中,TG2可能通过线粒体途径参与调节骨肉瘤细胞凋亡,但是关于TG2在骨肉瘤细胞凋亡过程中的作用及参与调节的机制还未见报道。本研究在缺氧环境中培养骨肉瘤细胞,通过转染TG2siRNA抑制TG2表达后检测线粒体途径中的细胞因子的表达情况及细胞凋亡率的变化,探讨骨肉瘤MG-63细胞中TG2的抗凋亡作用,以及TG2通过调节Bcl-2、Bax、细胞色素c、caspase-3的表达抑制肿瘤细胞的凋亡的可能机制。
二、目的：
在缺氧条件下体外培养骨肉瘤MG-63细胞,探讨Ⅱ型谷氨酰胺转氨酶在骨肉瘤MG-63细胞中的抗凋亡作用,及其通过调节Bcl-2、Bax、细胞色素c、caspase-3的表达抑制肿瘤细胞的凋亡的机制。
三、方法：
1、设计针对TG2的siRNA,按照Dharmafect3试剂盒步骤进行转染。
2、实验分组：①常氧组：细胞在常氧下培养；②单纯缺氧组：细胞在缺氧培养箱里培养；③对照siRNA缺氧组：转染对照siRNA后在缺氧培养箱里培养：④TG2siRNA缺氧组：转染TG2siRNA后在缺氧培养箱里培养。
3、建立骨肉瘤细胞体外缺氧培养模型,将细胞置缺氧培养箱内进行培养,分不同时间段(6.12、24、48、72h)对MG-63细胞进行相应处理。
4、采用微量滴定板法来测定细胞内TG活性。
5、半定量逆转录聚合酶链反应(RT-PCR)方法检测TG2、Bcl-2及Bax的mRNA水平。
6、免疫组化(SP法)了解蛋白在细胞内的分布,蛋白质印迹法(Western blot)检测TG2、Bcl-2、Bax、细胞色素c及caspase-3蛋白表达变化情况。
7、流式细胞仪检测细胞凋亡率。
四、结果：
1、TG2活性：
在常氧组未检测到原位TG2活性,而单纯缺氧组TG2的活性较常氧组表达增强(P<0.01),且各个时间点之间比较差异有统计学意义；与单纯缺氧组及对照siRNA缺氧组比较,TG2siRNA缺氧组的TG2的活性减弱(P<0.01)。
2、RT-PCR方法检测TG2、Bcl-2及Bax的mRNA水平：
TG2、Bcl-2、Bax mRNA在常氧组仅见低水平的表达；在单纯缺氧组和对照siRNA缺氧组TG2mRNA较常氧组增高,随缺氧时间延长,TG2mRNA水平逐渐提高,并且各个时间段之间比较差异有统计学意义(P<0.01); Bax mRNA活性未见增强(P>0.05)；而Bcl-2mRNA活性明显提高,并且随着缺氧时间延长逐渐增强(P<0.01),各个时间段之间也存在显著性差异(P<0.01)。与单纯缺氧组和对照siRNA缺氧组比较,在TG2siRNA缺氧组,当抑制TG2的表达时,各个时间段TG2、Bcl-2mRNA水平均降低有统计学意义(P<0.01); Bax mRNA水平轻度增强,但与其它三组相比较并无统计学意义(P>0.05)。
3、免疫组织化学结果显示MG-63骨肉瘤细胞的TG2、Bcl-2、Bax蛋白定位于胞浆。常氧组,TG2蛋白表达阴性。单纯缺氧组和对照siRNA缺氧组TG2阳性细胞率较常氧组提高(P<0.01),并且染色强度随缺氧时间延长增强；而与单纯缺氧组和对照siRNA缺氧组比较,TG2siRNA缺氧组的TG2阳性细胞率减少(P<0.01)。
4、Western blot检测TG2、Bcl-2、Bax蛋白表达：
蛋白免疫印迹结果分析表明：常氧组未见明显TG2、Bcl-2蛋白表达,但可见明显Bax蛋白条带。单纯缺氧组和对照siRNA缺氧组TG2、Bcl-2蛋白表达高于常氧组(P<0.01),并且表达强度随缺氧时间延长递增；而Bax蛋白条带逐渐减弱(P<0.01),并且表达强度随缺氧时间延长而递减。而TG2siRNA缺氧组TG2、Bcl-2蛋白表达低于单纯缺氧组和对照siRNA缺氧组P<0.01); Bax蛋白条带却增强,显著高于单纯缺氧组和对照siRNA组(P<0.01)。
5、细胞色素c：
Western-blot结果显示：在常氧组,细胞胞浆内仅有少量的细胞色素c；在单纯缺氧组及对照siRNA缺氧组,细胞胞浆及线粒体内的细胞色素c含量仅受轻微影响。而在TG2siRNA缺氧组,当抑制TG2的表达时,细胞胞浆内的细胞色素c含量增加,而线粒体内的细胞色素c减少(P<0.01)。提示TG2抑制缺氧条件下骨肉瘤MG-63细胞色素c由线粒体向细胞胞浆内释放。
6、caspase-3:
caspase-3活性：与常氧组比较,单纯缺氧组与对照siRNA缺氧组的caspase-3活性未见增强(P>0.05)；而在TG2siRNA缺氧组,caspase-3活性随着缺氧时间延长逐渐增强(P<0.01)。
Western blotting结果显示在单纯缺氧组,可见弱的caspase-3蛋白条带;而在TG2siRNA缺氧组,caspase-3蛋白条带增强(P<0.01),并且表达强度随缺氧时间延长明显递增(P<0.01)。
7、流式细胞仪检测结果显示：
与常氧组比较,单纯缺氧组及对照siRNA缺氧组,细胞凋亡率轻度增加；而在TG2siRNA缺氧组,细胞凋亡率较前三组提高(P<0.01)。
五、结论：
1、在缺氧条件下,骨肉瘤MG63细胞的TG2表达增强,有统计学意义；
2、TG2通过促进Bcl-2的表达和消耗Bax,从而上调Bcl-2/Bax比值,阻止细胞色素c释放至胞浆,降低caspase-3活性；
3、TG2在胞浆内形成的交联聚合体可以与caspase-3形成不溶性复合物,降低caspase-3的表达及活性,从而发挥抗凋亡的作用,抑制肿瘤细胞的凋亡。
4、TG2siRNA转染竞争性抑制TG2的表达后,通过减少Bcl-2的表达和增加Bax的表达,促进细胞色素c释放至细胞胞浆,增强caspase-3的表达及活性,促进肿瘤细胞凋亡。
5、本研究揭示了TG2一种新的抗凋亡途径,提示通过抑制TG2的表达,可以促进骨肉瘤细胞凋亡,为骨肉瘤的治疗提供新的有效的途径。
Research Background:
Osteosarcoma is a primary malignant tumor commonly seen in adolescents. Its incidence rate accounts for20%of all bone tumors. Osteosarcoma cells proliferate rapidly to damage the surrounding normal tissue with its invasive growth. Distant metastasis is found at the early stage, among which, lung metastasis is most common with a poor prognosis. Traditional treatment methods include mainly amputation and radiotherapy, but5-year survival rate is less than20%. In recent years, the use of adjuvant chemotherapy, limb salvage surgery has seen the significant improvement of5-year survival rate and limb salvage success rate, but it still finds no striking progress in terms of the long-term efficacy and long-term survival in patients. More and more scholars these years are focusing on the study of the molecular mechanism of the occurrence, development and metastasis of osteosarcoma in the hope of finding new methods for the treatment of osteosarcoma from the gene level. With the progress of the study, it has been found that the occurrence, development and metastasis of osteosarcoma are a complex process. In addition to a series of pathophysiological reactions, many genes and transcription factors are involved and implicated in a variety of regulatory mechanisms, wherein the expression of multiple genes induced by hypoxia and regulatory mechanisms between them play an important role in the process of tumor proliferation.
It has been confirmed that, in the process of the growth of malignant tumors, tissues growing too fast would inevitably causes the local tissue hypoxia and an imbalance between energy consumption and energy supply, thus leads to tumor cell apoptosis. In order to adapt to hypoxic environment, tumor cell phenotype undergoes a series of changes, such as inducing angiogenesis/vascularization and increasing glycolysis in cells, contributing enormously to the regulation of cell apoptosis. It is believed that there are now two major pathways associated with osteosarcoma:death receptor-ligand pathway and mitochondrial pathway. The mitochondrial pathway is stimulating mitochondria to release cytochrome c (Cyt c) with some apoptosis stimulating factors. Cytochrome c (Cyt c) released into the cytoplasm combines with apoptosis activating factor1in the presence of dATP, generating its polymer, and prompts caspase-9to combine with the polymer to generate apoptotic bodies. Thus, caspase-9is activated, and the activated caspase-9activates other caspases such as caspase-3, and caspase-3activates DNA fragmentation factors, resulting in the activation of endonuclease in the resting state, and ultimately causes DNA rupture. It is a common biochemical pathway for apoptosis from the mitochondrial dysfunction to DNA rupture. Since the caspase can be mutually activated, so the caspase protease cascade is a major part leading to changes in the structure of cell apoptosis. Mitochondrial dysfunction plays a key role in the pathogenesis of osteosarcoma apoptosis.
It has been proved that apoptosis-related factors such as Bcl-2, inhibitors of apoptosis proteins (IAPs), p53, caspases, etc., play an important role in the process of regulating osteosarcoma cells apoptosis. Among the regulatory genes related to apoptosis, Bcl-2is the one most closely connected to apoptosis and also has been studied most in-depth, including mainly the anti-apoptosis protein Bcl-2class (Bcl-2, the Bel-x1, Ced, Bcl2w, Mcl-1-9, etc.) and the pro-apoptotic protein Bax class (including Bax, the Bid with only the BH3domain structure, Bim, PUMA, etc). Studies on apoptosis-inhibiting genes in terms of osteosarcoma focus on Bcl-2, which can encode2kinds of proteins, α and β in the mitochondrial outer membrane and part of the endoplasmic reticulum. Now it is generally believed that the pathology that the Bcl-2in osteosarcoma playing its part in inhibiting apoptosis is realized by blocking the mitochondrial pathway, i.e. blocking the release of cytochrome c into mitochondria, whereby, inhibiting the activation of downstream effect factors, and thus exerts apoptosis inhibition; The Bcl-2protein can also inhibit apoptosis through combination with the pro-apoptotic protein Bax. The expression of Bcl-2and its relationship with prognosis vary in different tumor. Some scholars studied the Bcl-2genes in osteosarcoma via immunohistochemical method to, and it was shown that the staining positive rate and the extent of staining of Bcl-2in osteosarcoma tissues were higher than they were in normal bone tissues, which revealed that the occurrence of osteosarcoma was associated with abnormal apoptosis. Vitro experiments also confirmed that osteosarcoma cells showed high expression of Bcl-2, and Bcl-2suggested inhibitory effect on apoptosis of osteosarcoma cell line. Ferrari's research showed that in most cases with pulmonary metastasis of osteosarcoma recurrence, the expression of Bcl-2is stronger than a primary tumor, but the protein expression of Bcl-2does not rise with the rise of osteosarcoma in installment, therefore, its expression doesn't indicate the malignant degree of osteosarcoma. The pro-apoptotic protein Bax are often found in the cytoplasm, transferring into mitochondria when cells are stimulated by apoptosis signals, and they promote apoptosis through the mitochondrial pathway. Eliseev identified that specific transcription factor RUNX2in bone cells, with Bax as targets, enhancing the sensitivity of osteosarcoma cell, Saos-2to apoptosis by increasing its expression. A vast array of studies has stated that the ratio of Bcl-2and Bax determines whether there is apoptosis or not. Now its mechanism has been agreed that, the Bax-Bax dimer induces apoptosis, but once the Bcl-2protein content increases, Bcl-2conbines Bax to form Bcl-Bax dimer more stable than the Bcl-Bax dimer, which compromises the effect on promoting apoptosis of Bax. Kaseta et al also believes that the ratio of Bcl-2and Bax and the inhibition of apoptosis in osteosarcoma were positively correlated.
Glutamine transaminase, also known as transglutaminase, is an enzyme that undergoes transamidating activity between primary amine and acylamino-Y in catalytic protein or glutamine residues in polypeptide chains. It has been discovered that transglutaminase is widely distributed in the blood, extracellular matrix and intracellular subgroups and it is involved in many biochemical processes, such as blood coagulation, wound healing, tissue reconstruction, cell differentiation and death, etc. It is now known to contain eight transglutaminase types in humans. Ⅱ glutamine transaminase (transglutaminase2, TG2), also tissue transglutaminase (tTG, e. c.2.3.2.13), is a member of the TG family of enzymes which is most extensively studied in depth. This enzyme is highly multifunctional, involved in many physiological and pathophysiological processes, including fibrosis, atherosclerosis, neural-degenerative diseases, diarrhea and tumor metastasis. Over the past few years, a growing number of studies have proved that the enzyme is closely related to the occurrence and development of tumor.
TG2exists in the form of dimers with each monomer consisting of686amino acids, relative molecular mass of about80kd, each monomer containing four unique structure domains:an p sandwich structure domain at amino-terminal (1-139), core structure domain for catalyzing transamination (147～460) and2β tubular structure domain at the c-terminal (472～583,591～687). TG2is a unique member of the transglutaminase family, which has both the activity of catalyzing Ca2+-dependent protein crosslinking and the function of GTP-dependent G protein. Moreover, it has an activity of protein-disulfide isomerase and the function of the protein kinase. Although TG2is mainly located in the cytoplasm, it can also be secreted to extracellular and cross-linking extracellular matrix proteins to stablize the extracellular matrix. The secretion of TG2relies on its active conformation and (or) correct tertiary structure. It can also be transferred into the nucleus, cross-linked histones, PRb and SP1transcription factors with the help of the input protein (importin)-a3, to regulate cell cycle and apoptosis. Likewise, TG2is trans-located to the membrane, together with integrin, to promote the interaction between integrin on cell surface and fibronectin. Therefore, TG2may have great impact on the occurrence, differentiation and invasion of tumors.
Many research results have confirmed the role of TG2in promoting apoptosis, since a full expression of TG2is usually found in apoptotic cells, and the inhibited expression of TG2significantly reduces the number of deal cells. Under extreme circumstances (such as hypoxia, the lack of growth factors, the chemotherapy treatment), intracellular Ca2+homeostasis is damaged. The sudden change of Ca2+in cytoplasm transfers TG2into the conformation with crosslinking activity, leading to the extensive crosslinking and multimerization of intracellular proteins, thus forms an acid-insoluble structure, stabilizes the apoptotic cells and prevents inflammation and scarring. However TG2expression and apoptosis are not completely consistent. TG2knockout mice did not show the apoptosis-induced disorders, which didn't rule out the compensation of other isomerase on TG2deficiency. In addition, while several kinds of rapidly dividing cancer cells expressed a high level of TG2, they didn't end up in apoptosis. The latest direct evidences showed that the rising TG2prolonged the survival of the cells by preventing apoptotic cell death. Moreover, though the epidermal growth factor inhibits the doxorubicin-induced apoptotic cell death of breast cancer, but increases TG2expression. Antonyak et al have proved that in retinoic acid's treatment of some cell lines, the increase in the levels of retinoic acid-induced TG2cells protects cells from (N-HPR)-induced apoptosis. These findings suggest that TG2not only promots apoptosis, but also resists apoptosis. The seemingly contradictory role is dictated by the cell type, the stimulation type and the positioning of TG2in the cell and its activity type. TG2promotes apoptosis through transamination in the cytoplasm, while, it protects cells from apoptosis through G protein function or (and) transamidating activity in nucleus; Likewise, on the cell membrane, TG2, as the auxiliary integrin receptor, promotes the adhesion of the integrin and the extracellular matrix and signal transduction, protecting cells from apoptosis.
The latest researches have shown that TG2displays nonenzymatic functions when it generates compounds with cellular proteins based on noncovalent interactions. In HEK293cells, through the consumption of Bax and the reduction of caspase-3and caspase-9, TG2prevents cytochrome c in the cell nucleus from being released into the cytoplasm as well as the mitochondrial membrane depolarization when Ca2+increases, which in turn contributes to anti-apoptosis. The same mechanism was found in hypoxic tumor cells that caspase-3is consumed through cross binding with caspase-3, and thus plays the role of resistance to apoptosis. It suggested that in hypoxia environment, TG2may get involved in regulating the apoptosis in osteosarcoma cells through the mitochondrial pathway, but it has not been reported about the role of TG2in the process of apoptosis in osteosarcoma and its regulatory mechanism. This study is to develop osteosarcoma cells in hypoxia environment and to measure the expression of cytokines in the mitochondrial pathway as well as the change of apoptosis rate through transfection of TG2siRNA to inhibit TG2expression, with an aim to explore anti-apoptotic effect of TG2in osteosarcoma MG-63cells as well as the possible mechanism of inhibiting apoptosis in tumor cells by TG2through regulating the expression of Bcl-2, Bax, cytochrome c and caspase-3.
Objective:
Under hypoxic conditions, cultivate osteosarcoma MG-63cells in vitro to explore the role of transglutaminase Ⅱ in its anti-apoptosis in osteosarcoma MG-63cells, as well as its mechanism of inhibiting apoptosis in tumor cells through regulating the expression of the Bcl-2, Bax, cytochrome c and caspase-3.
Methods:
1) Design TG2-specific siRNA, and carry out the transfection in accordance with the steps defined in Dharmafect3kit.
2) The experimental groups:(1) normoxic group:cells cultured under normal oxygen;(2) hypoxia group:cells cultured in hypoxia box;(3) control siRNA hypoxia group:si RNA cultured in hypoxia box after transfection and control;(4) TG2siRNA hypoxia group:TG2siRNA cultured in hypoxia box after transfection.
3) Establish culture models of osteosarcoma cells in vitro under hypoxic conditions, place the cells in hypoxia incubators for cultivation, and treat respectively the MG-63cells according to different time periods (6,12,24,48,72h).
4) Determine intracellular TG activity using microtiter plate method.
5) Detect TG2, Bcl-2and Bax mRNA level using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method.
6) Understand the distribution of proteins in cells using immunohistochemical method (SP); detect the expression changes of TG2, Bcl-2, Bax, cytochrome c and caspase-3using West blotting (Western blot).
7) Detect the rate of cell apoptosis with flow cytometry.
Results
1) TG2activity:
The in situ TG2activity was not detected in normoxic group, and the activity of TG2in hypoxia group was found an increased expression compared with that in normoxic group (P<0.01), and it was statistically significant comparing the difference between the various time points;the TG2activity of TG2siRNA hypoxia group decreased significantly, compared with hypoxia group and control siRNA hypoxia group (P<0.01).
2) RT-PCR method for detecting the mRNA level of TG2, Bcl-2and Bax:
TG2, Bcl-2, Bax in normoxic group were restricted to the low level expression of mRNA; TG2mRNA level, in hypoxia group and control siRNA hypoxia group, increased compared with its expression in normoxic group. With the prolonged hypoxia, TG2mRNA level gradually improved, and it was statistically significant comparing the difference between the various time periods (P<0.01); the mRNA activity Bax showed no remarkable enhancement (P>0.05); while the mRNA activity of Bcl-2was signicantly increased, and gradually enhanced with prolonged hypoxia (P<0.01), and there was significant difference between the respective time periods (P<0.01). Compared with hypoxia group and control siRNA hypoxia group, in the TG2siRNA hypoxia group, the mRNA level of both TG2and Bcl-2was significantly reduced in each time period when inhibiting the expression of TG2(P<0.01); the mRNA level of Bax was slightly enhanced, however, not statistically significant when compared with the other three groups(P>0.05).
3) Immunohistochemical results showed that the TG2, Bcl-2, and Bax protein in osteosarcoma MG-63cells were localized in the cytoplasm. In normoxic group, TG2protein expression was negative. In hypoxia group and control siRNA hypoxia group, the rate of TG2positive cells increased significantly(P<0.01), and the staining intensity was enhanced strikingly with prolonged hypoxia; the rate of TG2positive cells TG2siRNA hypoxia group reduced considerably when compared with hypoxia group and control siRNA hypoxia group(P<0.01).
4) Western blot detection of the protein expression of TG2, Bcl-2, Bax:
The result of western blot showed no obvious protein expression of TG2, Bcl-2in normoxic group, but clearly visible Bax protein bands. The protein expression of TG2, Bcl-2in hypoxia group and control siRNA hypoxia group was significantly higher than that that in normoxic group (P<0.01), and the expression intensity increased obviously with prolonged hypoxia; While, Bax protein bands were gradually weakened (P<0.01), and the expression intensity decreased with hypoxia prolonged. In TG2siRNA hypoxia group, the protein expression of TG2and Bcl-2was obviously lower than that in hypoxia group and in control siRNA hypoxia group (P<0.01), whereas Bax protein bands were enhanced obviously, significantly higher than that in hypoxia group and in control siRNA hypoxia group (P<0.01).
5) Cytochrome c:
The results of Western blot showed:In normoxic group, only a small amount of cytochrome c were found in the cytoplasm; In hypoxia group and control siRNA hypoxia group, the cytochrome c content in the cytoplasm and mitochondria was only slightly affected. In TG2siRNA hypoxia group, the cytochrome c content in the cytoplasm increased significantly when inhibiting the expression of TG2, while cytochrome c content in mitochondria decreased remarkably (P<0.01). It suggested the inhibition of cytochrome c in osteosarcoma MG-63from releasing into the cytoplasm by TG2under the hypoxic condition.
6) Caspase-3:
Caspase-3activity:Caspase-3activity in hypoxia group and control siRNA hypoxia group was shown no obvious increase (P>0.05) compared with that in normoxic group; While, in the TG2siRNA hypoxia group, caspase-3activity gradually increased with prolonged hypoxia (P<0.01).
The results of Western blot showed weak caspase-3protein bands in hypoxia group; While, caspase-3protein bands obviously increased in TG2siRNA hypoxia group (P<0.01), and its expression intensity grew significantly with prolonged hypoxia (P<0.01).
7) The results of flow cytometry showed that:
The apoptosis rate in hypoxia group and control siRNA hypoxia group increased slightly compared with that in normoxic group; while, the apoptosis rate in TG2siRNA hypoxia group was improved significantly, compared with that in the first three groups (P<0.01).
Conclusions:
1) Under hypoxic conditions, the expression of TG2in osteosarcoma MG63cells was strikingly impoved;
2) TG2increases the Bel-2/Bax ratio, prevents the cytochrome c from releasing into the cytoplasm, and reduces caspase-3activity by promoting the expression of Bel-2and the depletion of Bax;
3) Crosslinked polymers of TG2generated in the cytoplasm form insoluble compounds with caspase-3, reducing the expression and activity of caspase-3, thus contribute to anti-apoptosis and inhibit the apoptosis of tumor cells.
4) When the TG2siRNA inhibits the expression of TG2, it facilitates the release of cytochrome c into the cytoplasm, enhances the expression and activity of caspase-3, and promotes apoptosis of tumor cells by decreasing the expression of Bcl-2and increasing the expression of Bax.
5) This study reveals a new anti-apoptotic pathway of TG2, suggesting that inhibiting the expression of TG2promotes apoptosis in osteosarcoma cells and providing a new effective way for the treatment of osteosarcoma.
骨肉瘤是一种常见于青少年的原发性恶性肿瘤。其发病率约占所有骨肿瘤的20%。骨肉瘤细胞增殖迅速,以浸润性生长破坏周围正常组织,早期即发生远处转移,其中最常见的是肺转移,其预后差。传统治疗方法主要是截肢和放疗,5年生存率不超过20%。近几年辅助化疗、保肢手术的运用使骨肉瘤患者5年生存率和保肢成功率显著提高,但长期疗效及患者的远期存活率并无明显改善。近年来越来越多的学者关注于研究骨肉瘤的发生、发展以及转移的分子机制,希望从基因水平找到治疗骨肉瘤的新方法。随着研究的进展,发现骨肉瘤的发生、发展和转移是一个复杂的过程。除了一系列病理生理反应外,还有许多基因和转录因子参与,涉及到多种调控机制。其中缺氧引起的多种基因的表达以及它们之间的调控机制在肿瘤的增殖过程中发挥重要的作用。
研究证实,在实体恶性肿瘤的生长过程中,由于组织增生过快必然造成局部组织的缺氧和供能与耗能之间的不平衡,这就导致肿瘤细胞发生凋亡。为了适应缺氧环境,肿瘤细胞表型发生一系列变化,如诱导血管形成及增加细胞糖酵解；而对于细胞凋亡的调节也发挥很重要的作用。目前认为与骨肉瘤相关的细胞凋亡途径主要有两条：死亡受体配体途径和线粒体途径。其中线粒体途径是通过部分凋亡刺激因子刺激线粒体释放细胞色素c(Cyt c),释放到细胞浆的细胞色素c在dATP存在的条件下能与凋亡激活因子1结合,使其形成多聚体,并促使caspase-9与其结合形成凋亡小体,caspase-9被激活,被激活的caspase-9能激活其它的caspase如caspase-3等,caspase-3又激活DNA断裂因子,导致静息状态的核酸内切酶激活,最终引起DNA断裂。从线粒体功能障碍到DNA断裂,是细胞凋亡生化途径的共同途径。由于各caspase可相互激活,所以caspase蛋白酶级联反应是导致细胞凋亡结构改变的主要环节。线粒体功能障碍在骨肉瘤细胞凋亡发生机制中起关键作用。
研究证实Bcl-2、凋亡蛋白抑制因子(inhitors of apoptosis proteins, IAPs)、p53、caspases等细胞凋亡因子在调控骨肉瘤细胞凋亡过程中发挥重要的作用。在与凋亡相关的调控基因中,Bcl-2是研究最深入、与凋亡关系最密切的基因之一。主要包括凋亡抑制蛋白Bcl-2类(Bcl-2、Bcl-x1、Bcl2w、Mcl-1、Ced-9等)和促凋亡蛋白Bax类(包括Bax、Bid和仅有BH3结构域的Bid、Bim、PUMA等蛋白)两种。骨肉瘤中对有凋亡抑制作用基因的研究主要以Bcl-2为主,它可编码α和β2种蛋白,存在于线粒体外膜及部分内质网中。目前普遍认为骨肉瘤中Bcl-2发挥凋亡抑制作用的机制是通过阻断线粒体途径,即阻断线粒体内细胞色素c的释放进而抑制下游效应因子的激活,从而发挥凋亡抑制作用；Bcl-2蛋白也可通过与促凋亡蛋白Bax相结合而抑制凋亡。不同肿瘤中Bcl-2的表达及与预后的关系也不尽相同。学者们通过免疫组化法研究人骨肉瘤中的Bcl-2基因,结果显示骨肉瘤组织Bcl-2蛋白染色阳性率和染色程度均较正常骨组织增高,揭示骨肉瘤的发生与凋亡异常有关。体外实验亦证实,骨肉瘤细胞中Bcl-2呈高水平表达,且Bcl-2对骨肉瘤细胞系凋亡有抑制作用。Ferrari等的研究表明,在多数伴有肺转移的骨肉瘤复发病例中,Bcl-2的表达较原发肿瘤强,但Bcl-2蛋白的表达并不随骨肉瘤分期的升高而升高,可见其表达情况并不能作为骨肉瘤恶性程度的指征。促凋亡蛋白Bax主要存在于胞浆,当细胞受到凋亡信号刺激时转入线粒体中,并通过线粒体途径发挥促凋亡作用。Eliseev等证实,骨细胞的特异性转录因子RUNX2以Bax为靶点,通过提高其表达量来增加骨肉瘤细胞Saos-2对凋亡的敏感性。多项研究表明,Bcl-2与Bax比值的高低决定细胞凋亡与否,目前认为其机理是Bax-Bax二聚体可诱导凋亡,但当Bcl-2蛋白量升高时可与Bax形成更稳定的Bcl-Bax二聚体,因此拮抗了Bax的促凋亡作用。Kaseta等的研究还认为,Bcl-2与Bax的比值与骨肉瘤细胞凋亡的抑制呈正相关关系。
谷氨酰胺转氨酶又称转谷氨酰胺酶,是一种催化蛋白质或多肽链中的谷氨酰胺残基里的丫酰胺基和一级氨基之间进行酰胺基转移反应的酶。经研究表明,谷氨酰胺转氨酶广泛分布于血液、细胞外基质及细胞内组分中。参与许多生物化学过程,如凝血、伤口愈合、组织重建、细胞分化和死亡等。到目前为止,已发现的人类谷氨酰胺转氨酶有8型。Ⅱ型谷氨酰胺转氨酶(transglutaminase2,TG2),也叫组织型谷氨酰胺转氨酶(tissue transglutaminase, tTG,E. C.2.3.2.13),它是该家族中研究得较为广泛和深入的一个酶。此酶具有广泛的功能,参与许多病理生理过程,如纤维化、动脉粥样硬化、神经退行性病变、腹泻和肿瘤转移等。近年来,越来越多的研究证实该酶与肿瘤发生、发展有密切的联系。
TG2是以二聚体的形式存在。每个单体由686个氨基酸构成,相对分子质量约为80kD,每个单体含有4个独特的结构域：氨基端的p三明治结构域(1～139)、催化转氨基的核心结构域(147～460)和羧基端的2个p管状结构域(472～583,591～687)。TG2是谷氨酰胺转氨酶家族中的一个独特成员,它不仅具有Ca2+依赖的催化蛋白交联的活性,还有GTP依赖的G蛋白的功能。此外,它还具有蛋白二硫键异构酶活性和蛋白激酶的功能。虽然TG2主要位于胞浆内,但也可以分泌到细胞外,交联细胞外的基质蛋白,起稳定细胞外基质的作用,TG2的这种分泌作用依赖于其活性构象和(或)正确的三级结构。它还可以在输入蛋白(importin)-a3协助下转位进入核内,交联组蛋白、pRb和SP1转录因子,调节细胞周期和凋亡。同样,TG2也可以转位到膜上,和整合素一起,促进细胞表面的整合素和纤连蛋白相互作用。因此TG2可能对肿瘤发生、分化及侵袭行为有重要的影响。
许多研究结果都支持TG2的促凋亡作用,凋亡细胞内通常都有高水平的TG2表达,抑制TG2的表达会显著降低细胞死亡数量。在极端恶劣的环境下(如缺氧、生长因子缺乏、化疗药物处理),细胞内Ca2+稳态受到破坏,这种胞浆内Ca2+的突然改变会使TG2转变成有交联活性的构象,导致细胞内蛋白质的广泛交联、多聚化,形成不溶于酸的结构,稳定了凋亡细胞,防止炎症反应和瘢痕出现。然而TG2表达和凋亡并不是完全一致的。TG2基因敲除的小鼠并未表现出细胞凋亡障碍,这其中也不能排除其他异构酶对TG2缺乏的补偿。此外有几种快速分裂的肿瘤细胞虽表达了高水平的TG2,却没进入凋亡。最近又有直接证据表明,升高的TG2通过阻止凋亡来延长细胞的存活。此外表皮生长因子虽可以抑制多柔比星(阿霉素)诱导的乳腺癌细胞凋亡,但却上调TG2的表达。Antonyak等已经证明了在维甲酸处理某些细胞系中,维甲酸诱导的细胞内TG2水平的增加能够保护细胞免受羟苯维胺脂(N-HPR)诱导的凋亡。这些研究结果提示TG2不仅具有促凋亡作用,而且还有抗凋亡作用,这种似乎矛盾的作用取决于细胞类型、所受刺激类型及TG2在细胞内的定位和酶活性类型。在细胞浆内,TG2通过其转氨基作用来促进凋亡；在核内,TG2通过G蛋白功能或(和)转氨酶活性保护细胞不凋亡；同样地,在细胞膜上,它作为整合素的辅助受体,促进整合素与细胞外基质黏附力和信号转导,保护细胞不凋亡。
最新的研究证实,TG2还能够通过与细胞内蛋白质以非共价形式形成复合物而表现出非酶学的功能。在]HEK293细胞,TG2通过消耗Bax、降低caspase-3和caspase-9、抑制细胞核内的细胞色素c释放进入细胞浆以及在Ca2+增高时线粒体膜的去极化,从而发挥抗凋亡的作用。在缺氧的肿瘤细胞发现同样的机制是通过与caspase-3交叉结合消耗caspase-3,从而发挥抗细胞凋亡的作用。这提示在缺氧环境中,TG2可能通过线粒体途径参与调节骨肉瘤细胞凋亡,但是关于TG2在骨肉瘤细胞凋亡过程中的作用及参与调节的机制还未见报道。本研究在缺氧环境中培养骨肉瘤细胞,通过转染TG2siRNA抑制TG2表达后检测线粒体途径中的细胞因子的表达情况及细胞凋亡率的变化,探讨骨肉瘤MG-63细胞中TG2的抗凋亡作用,以及TG2通过调节Bcl-2、Bax、细胞色素c、caspase-3的表达抑制肿瘤细胞的凋亡的可能机制。
二、目的：
在缺氧条件下体外培养骨肉瘤MG-63细胞,探讨Ⅱ型谷氨酰胺转氨酶在骨肉瘤MG-63细胞中的抗凋亡作用,及其通过调节Bcl-2、Bax、细胞色素c、caspase-3的表达抑制肿瘤细胞的凋亡的机制。
三、方法：
1、设计针对TG2的siRNA,按照Dharmafect3试剂盒步骤进行转染。
2、实验分组：①常氧组：细胞在常氧下培养；②单纯缺氧组：细胞在缺氧培养箱里培养；③对照siRNA缺氧组：转染对照siRNA后在缺氧培养箱里培养：④TG2siRNA缺氧组：转染TG2siRNA后在缺氧培养箱里培养。
3、建立骨肉瘤细胞体外缺氧培养模型,将细胞置缺氧培养箱内进行培养,分不同时间段(6.12、24、48、72h)对MG-63细胞进行相应处理。
4、采用微量滴定板法来测定细胞内TG活性。
5、半定量逆转录聚合酶链反应(RT-PCR)方法检测TG2、Bcl-2及Bax的mRNA水平。
6、免疫组化(SP法)了解蛋白在细胞内的分布,蛋白质印迹法(Western blot)检测TG2、Bcl-2、Bax、细胞色素c及caspase-3蛋白表达变化情况。
7、流式细胞仪检测细胞凋亡率。
四、结果：
1、TG2活性：
在常氧组未检测到原位TG2活性,而单纯缺氧组TG2的活性较常氧组表达增强(P<0.01),且各个时间点之间比较差异有统计学意义；与单纯缺氧组及对照siRNA缺氧组比较,TG2siRNA缺氧组的TG2的活性减弱(P<0.01)。
2、RT-PCR方法检测TG2、Bcl-2及Bax的mRNA水平：
TG2、Bcl-2、Bax mRNA在常氧组仅见低水平的表达；在单纯缺氧组和对照siRNA缺氧组TG2mRNA较常氧组增高,随缺氧时间延长,TG2mRNA水平逐渐提高,并且各个时间段之间比较差异有统计学意义(P<0.01); Bax mRNA活性未见增强(P>0.05)；而Bcl-2mRNA活性明显提高,并且随着缺氧时间延长逐渐增强(P<0.01),各个时间段之间也存在显著性差异(P<0.01)。与单纯缺氧组和对照siRNA缺氧组比较,在TG2siRNA缺氧组,当抑制TG2的表达时,各个时间段TG2、Bcl-2mRNA水平均降低有统计学意义(P<0.01); Bax mRNA水平轻度增强,但与其它三组相比较并无统计学意义(P>0.05)。
3、免疫组织化学结果显示MG-63骨肉瘤细胞的TG2、Bcl-2、Bax蛋白定位于胞浆。常氧组,TG2蛋白表达阴性。单纯缺氧组和对照siRNA缺氧组TG2阳性细胞率较常氧组提高(P<0.01),并且染色强度随缺氧时间延长增强；而与单纯缺氧组和对照siRNA缺氧组比较,TG2siRNA缺氧组的TG2阳性细胞率减少(P<0.01)。
4、Western blot检测TG2、Bcl-2、Bax蛋白表达：
蛋白免疫印迹结果分析表明：常氧组未见明显TG2、Bcl-2蛋白表达,但可见明显Bax蛋白条带。单纯缺氧组和对照siRNA缺氧组TG2、Bcl-2蛋白表达高于常氧组(P<0.01),并且表达强度随缺氧时间延长递增；而Bax蛋白条带逐渐减弱(P<0.01),并且表达强度随缺氧时间延长而递减。而TG2siRNA缺氧组TG2、Bcl-2蛋白表达低于单纯缺氧组和对照siRNA缺氧组P<0.01); Bax蛋白条带却增强,显著高于单纯缺氧组和对照siRNA组(P<0.01)。
5、细胞色素c：
Western-blot结果显示：在常氧组,细胞胞浆内仅有少量的细胞色素c；在单纯缺氧组及对照siRNA缺氧组,细胞胞浆及线粒体内的细胞色素c含量仅受轻微影响。而在TG2siRNA缺氧组,当抑制TG2的表达时,细胞胞浆内的细胞色素c含量增加,而线粒体内的细胞色素c减少(P<0.01)。提示TG2抑制缺氧条件下骨肉瘤MG-63细胞色素c由线粒体向细胞胞浆内释放。
6、caspase-3:
caspase-3活性：与常氧组比较,单纯缺氧组与对照siRNA缺氧组的caspase-3活性未见增强(P>0.05)；而在TG2siRNA缺氧组,caspase-3活性随着缺氧时间延长逐渐增强(P<0.01)。
Western blotting结果显示在单纯缺氧组,可见弱的caspase-3蛋白条带;而在TG2siRNA缺氧组,caspase-3蛋白条带增强(P<0.01),并且表达强度随缺氧时间延长明显递增(P<0.01)。
7、流式细胞仪检测结果显示：
与常氧组比较,单纯缺氧组及对照siRNA缺氧组,细胞凋亡率轻度增加；而在TG2siRNA缺氧组,细胞凋亡率较前三组提高(P<0.01)。
五、结论：
1、在缺氧条件下,骨肉瘤MG63细胞的TG2表达增强,有统计学意义；
2、TG2通过促进Bcl-2的表达和消耗Bax,从而上调Bcl-2/Bax比值,阻止细胞色素c释放至胞浆,降低caspase-3活性；
3、TG2在胞浆内形成的交联聚合体可以与caspase-3形成不溶性复合物,降低caspase-3的表达及活性,从而发挥抗凋亡的作用,抑制肿瘤细胞的凋亡。
4、TG2siRNA转染竞争性抑制TG2的表达后,通过减少Bcl-2的表达和增加Bax的表达,促进细胞色素c释放至细胞胞浆,增强caspase-3的表达及活性,促进肿瘤细胞凋亡。
5、本研究揭示了TG2一种新的抗凋亡途径,提示通过抑制TG2的表达,可以促进骨肉瘤细胞凋亡,为骨肉瘤的治疗提供新的有效的途径。
Research Background:
Osteosarcoma is a primary malignant tumor commonly seen in adolescents. Its incidence rate accounts for20%of all bone tumors. Osteosarcoma cells proliferate rapidly to damage the surrounding normal tissue with its invasive growth. Distant metastasis is found at the early stage, among which, lung metastasis is most common with a poor prognosis. Traditional treatment methods include mainly amputation and radiotherapy, but5-year survival rate is less than20%. In recent years, the use of adjuvant chemotherapy, limb salvage surgery has seen the significant improvement of5-year survival rate and limb salvage success rate, but it still finds no striking progress in terms of the long-term efficacy and long-term survival in patients. More and more scholars these years are focusing on the study of the molecular mechanism of the occurrence, development and metastasis of osteosarcoma in the hope of finding new methods for the treatment of osteosarcoma from the gene level. With the progress of the study, it has been found that the occurrence, development and metastasis of osteosarcoma are a complex process. In addition to a series of pathophysiological reactions, many genes and transcription factors are involved and implicated in a variety of regulatory mechanisms, wherein the expression of multiple genes induced by hypoxia and regulatory mechanisms between them play an important role in the process of tumor proliferation.
It has been confirmed that, in the process of the growth of malignant tumors, tissues growing too fast would inevitably causes the local tissue hypoxia and an imbalance between energy consumption and energy supply, thus leads to tumor cell apoptosis. In order to adapt to hypoxic environment, tumor cell phenotype undergoes a series of changes, such as inducing angiogenesis/vascularization and increasing glycolysis in cells, contributing enormously to the regulation of cell apoptosis. It is believed that there are now two major pathways associated with osteosarcoma:death receptor-ligand pathway and mitochondrial pathway. The mitochondrial pathway is stimulating mitochondria to release cytochrome c (Cyt c) with some apoptosis stimulating factors. Cytochrome c (Cyt c) released into the cytoplasm combines with apoptosis activating factor1in the presence of dATP, generating its polymer, and prompts caspase-9to combine with the polymer to generate apoptotic bodies. Thus, caspase-9is activated, and the activated caspase-9activates other caspases such as caspase-3, and caspase-3activates DNA fragmentation factors, resulting in the activation of endonuclease in the resting state, and ultimately causes DNA rupture. It is a common biochemical pathway for apoptosis from the mitochondrial dysfunction to DNA rupture. Since the caspase can be mutually activated, so the caspase protease cascade is a major part leading to changes in the structure of cell apoptosis. Mitochondrial dysfunction plays a key role in the pathogenesis of osteosarcoma apoptosis.
It has been proved that apoptosis-related factors such as Bcl-2, inhibitors of apoptosis proteins (IAPs), p53, caspases, etc., play an important role in the process of regulating osteosarcoma cells apoptosis. Among the regulatory genes related to apoptosis, Bcl-2is the one most closely connected to apoptosis and also has been studied most in-depth, including mainly the anti-apoptosis protein Bcl-2class (Bcl-2, the Bel-x1, Ced, Bcl2w, Mcl-1-9, etc.) and the pro-apoptotic protein Bax class (including Bax, the Bid with only the BH3domain structure, Bim, PUMA, etc). Studies on apoptosis-inhibiting genes in terms of osteosarcoma focus on Bcl-2, which can encode2kinds of proteins, α and β in the mitochondrial outer membrane and part of the endoplasmic reticulum. Now it is generally believed that the pathology that the Bcl-2in osteosarcoma playing its part in inhibiting apoptosis is realized by blocking the mitochondrial pathway, i.e. blocking the release of cytochrome c into mitochondria, whereby, inhibiting the activation of downstream effect factors, and thus exerts apoptosis inhibition; The Bcl-2protein can also inhibit apoptosis through combination with the pro-apoptotic protein Bax. The expression of Bcl-2and its relationship with prognosis vary in different tumor. Some scholars studied the Bcl-2genes in osteosarcoma via immunohistochemical method to, and it was shown that the staining positive rate and the extent of staining of Bcl-2in osteosarcoma tissues were higher than they were in normal bone tissues, which revealed that the occurrence of osteosarcoma was associated with abnormal apoptosis. Vitro experiments also confirmed that osteosarcoma cells showed high expression of Bcl-2, and Bcl-2suggested inhibitory effect on apoptosis of osteosarcoma cell line. Ferrari's research showed that in most cases with pulmonary metastasis of osteosarcoma recurrence, the expression of Bcl-2is stronger than a primary tumor, but the protein expression of Bcl-2does not rise with the rise of osteosarcoma in installment, therefore, its expression doesn't indicate the malignant degree of osteosarcoma. The pro-apoptotic protein Bax are often found in the cytoplasm, transferring into mitochondria when cells are stimulated by apoptosis signals, and they promote apoptosis through the mitochondrial pathway. Eliseev identified that specific transcription factor RUNX2in bone cells, with Bax as targets, enhancing the sensitivity of osteosarcoma cell, Saos-2to apoptosis by increasing its expression. A vast array of studies has stated that the ratio of Bcl-2and Bax determines whether there is apoptosis or not. Now its mechanism has been agreed that, the Bax-Bax dimer induces apoptosis, but once the Bcl-2protein content increases, Bcl-2conbines Bax to form Bcl-Bax dimer more stable than the Bcl-Bax dimer, which compromises the effect on promoting apoptosis of Bax. Kaseta et al also believes that the ratio of Bcl-2and Bax and the inhibition of apoptosis in osteosarcoma were positively correlated.
Glutamine transaminase, also known as transglutaminase, is an enzyme that undergoes transamidating activity between primary amine and acylamino-Y in catalytic protein or glutamine residues in polypeptide chains. It has been discovered that transglutaminase is widely distributed in the blood, extracellular matrix and intracellular subgroups and it is involved in many biochemical processes, such as blood coagulation, wound healing, tissue reconstruction, cell differentiation and death, etc. It is now known to contain eight transglutaminase types in humans. Ⅱ glutamine transaminase (transglutaminase2, TG2), also tissue transglutaminase (tTG, e. c.2.3.2.13), is a member of the TG family of enzymes which is most extensively studied in depth. This enzyme is highly multifunctional, involved in many physiological and pathophysiological processes, including fibrosis, atherosclerosis, neural-degenerative diseases, diarrhea and tumor metastasis. Over the past few years, a growing number of studies have proved that the enzyme is closely related to the occurrence and development of tumor.
TG2exists in the form of dimers with each monomer consisting of686amino acids, relative molecular mass of about80kd, each monomer containing four unique structure domains:an p sandwich structure domain at amino-terminal (1-139), core structure domain for catalyzing transamination (147～460) and2β tubular structure domain at the c-terminal (472～583,591～687). TG2is a unique member of the transglutaminase family, which has both the activity of catalyzing Ca2+-dependent protein crosslinking and the function of GTP-dependent G protein. Moreover, it has an activity of protein-disulfide isomerase and the function of the protein kinase. Although TG2is mainly located in the cytoplasm, it can also be secreted to extracellular and cross-linking extracellular matrix proteins to stablize the extracellular matrix. The secretion of TG2relies on its active conformation and (or) correct tertiary structure. It can also be transferred into the nucleus, cross-linked histones, PRb and SP1transcription factors with the help of the input protein (importin)-a3, to regulate cell cycle and apoptosis. Likewise, TG2is trans-located to the membrane, together with integrin, to promote the interaction between integrin on cell surface and fibronectin. Therefore, TG2may have great impact on the occurrence, differentiation and invasion of tumors.
Many research results have confirmed the role of TG2in promoting apoptosis, since a full expression of TG2is usually found in apoptotic cells, and the inhibited expression of TG2significantly reduces the number of deal cells. Under extreme circumstances (such as hypoxia, the lack of growth factors, the chemotherapy treatment), intracellular Ca2+homeostasis is damaged. The sudden change of Ca2+in cytoplasm transfers TG2into the conformation with crosslinking activity, leading to the extensive crosslinking and multimerization of intracellular proteins, thus forms an acid-insoluble structure, stabilizes the apoptotic cells and prevents inflammation and scarring. However TG2expression and apoptosis are not completely consistent. TG2knockout mice did not show the apoptosis-induced disorders, which didn't rule out the compensation of other isomerase on TG2deficiency. In addition, while several kinds of rapidly dividing cancer cells expressed a high level of TG2, they didn't end up in apoptosis. The latest direct evidences showed that the rising TG2prolonged the survival of the cells by preventing apoptotic cell death. Moreover, though the epidermal growth factor inhibits the doxorubicin-induced apoptotic cell death of breast cancer, but increases TG2expression. Antonyak et al have proved that in retinoic acid's treatment of some cell lines, the increase in the levels of retinoic acid-induced TG2cells protects cells from (N-HPR)-induced apoptosis. These findings suggest that TG2not only promots apoptosis, but also resists apoptosis. The seemingly contradictory role is dictated by the cell type, the stimulation type and the positioning of TG2in the cell and its activity type. TG2promotes apoptosis through transamination in the cytoplasm, while, it protects cells from apoptosis through G protein function or (and) transamidating activity in nucleus; Likewise, on the cell membrane, TG2, as the auxiliary integrin receptor, promotes the adhesion of the integrin and the extracellular matrix and signal transduction, protecting cells from apoptosis.
The latest researches have shown that TG2displays nonenzymatic functions when it generates compounds with cellular proteins based on noncovalent interactions. In HEK293cells, through the consumption of Bax and the reduction of caspase-3and caspase-9, TG2prevents cytochrome c in the cell nucleus from being released into the cytoplasm as well as the mitochondrial membrane depolarization when Ca2+increases, which in turn contributes to anti-apoptosis. The same mechanism was found in hypoxic tumor cells that caspase-3is consumed through cross binding with caspase-3, and thus plays the role of resistance to apoptosis. It suggested that in hypoxia environment, TG2may get involved in regulating the apoptosis in osteosarcoma cells through the mitochondrial pathway, but it has not been reported about the role of TG2in the process of apoptosis in osteosarcoma and its regulatory mechanism. This study is to develop osteosarcoma cells in hypoxia environment and to measure the expression of cytokines in the mitochondrial pathway as well as the change of apoptosis rate through transfection of TG2siRNA to inhibit TG2expression, with an aim to explore anti-apoptotic effect of TG2in osteosarcoma MG-63cells as well as the possible mechanism of inhibiting apoptosis in tumor cells by TG2through regulating the expression of Bcl-2, Bax, cytochrome c and caspase-3.
Objective:
Under hypoxic conditions, cultivate osteosarcoma MG-63cells in vitro to explore the role of transglutaminase Ⅱ in its anti-apoptosis in osteosarcoma MG-63cells, as well as its mechanism of inhibiting apoptosis in tumor cells through regulating the expression of the Bcl-2, Bax, cytochrome c and caspase-3.
Methods:
1) Design TG2-specific siRNA, and carry out the transfection in accordance with the steps defined in Dharmafect3kit.
2) The experimental groups:(1) normoxic group:cells cultured under normal oxygen;(2) hypoxia group:cells cultured in hypoxia box;(3) control siRNA hypoxia group:si RNA cultured in hypoxia box after transfection and control;(4) TG2siRNA hypoxia group:TG2siRNA cultured in hypoxia box after transfection.
3) Establish culture models of osteosarcoma cells in vitro under hypoxic conditions, place the cells in hypoxia incubators for cultivation, and treat respectively the MG-63cells according to different time periods (6,12,24,48,72h).
4) Determine intracellular TG activity using microtiter plate method.
5) Detect TG2, Bcl-2and Bax mRNA level using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method.
6) Understand the distribution of proteins in cells using immunohistochemical method (SP); detect the expression changes of TG2, Bcl-2, Bax, cytochrome c and caspase-3using West blotting (Western blot).
7) Detect the rate of cell apoptosis with flow cytometry.
Results
1) TG2activity:
The in situ TG2activity was not detected in normoxic group, and the activity of TG2in hypoxia group was found an increased expression compared with that in normoxic group (P<0.01), and it was statistically significant comparing the difference between the various time points;the TG2activity of TG2siRNA hypoxia group decreased significantly, compared with hypoxia group and control siRNA hypoxia group (P<0.01).
2) RT-PCR method for detecting the mRNA level of TG2, Bcl-2and Bax:
TG2, Bcl-2, Bax in normoxic group were restricted to the low level expression of mRNA; TG2mRNA level, in hypoxia group and control siRNA hypoxia group, increased compared with its expression in normoxic group. With the prolonged hypoxia, TG2mRNA level gradually improved, and it was statistically significant comparing the difference between the various time periods (P<0.01); the mRNA activity Bax showed no remarkable enhancement (P>0.05); while the mRNA activity of Bcl-2was signicantly increased, and gradually enhanced with prolonged hypoxia (P<0.01), and there was significant difference between the respective time periods (P<0.01). Compared with hypoxia group and control siRNA hypoxia group, in the TG2siRNA hypoxia group, the mRNA level of both TG2and Bcl-2was significantly reduced in each time period when inhibiting the expression of TG2(P<0.01); the mRNA level of Bax was slightly enhanced, however, not statistically significant when compared with the other three groups(P>0.05).
3) Immunohistochemical results showed that the TG2, Bcl-2, and Bax protein in osteosarcoma MG-63cells were localized in the cytoplasm. In normoxic group, TG2protein expression was negative. In hypoxia group and control siRNA hypoxia group, the rate of TG2positive cells increased significantly(P<0.01), and the staining intensity was enhanced strikingly with prolonged hypoxia; the rate of TG2positive cells TG2siRNA hypoxia group reduced considerably when compared with hypoxia group and control siRNA hypoxia group(P<0.01).
4) Western blot detection of the protein expression of TG2, Bcl-2, Bax:
The result of western blot showed no obvious protein expression of TG2, Bcl-2in normoxic group, but clearly visible Bax protein bands. The protein expression of TG2, Bcl-2in hypoxia group and control siRNA hypoxia group was significantly higher than that that in normoxic group (P<0.01), and the expression intensity increased obviously with prolonged hypoxia; While, Bax protein bands were gradually weakened (P<0.01), and the expression intensity decreased with hypoxia prolonged. In TG2siRNA hypoxia group, the protein expression of TG2and Bcl-2was obviously lower than that in hypoxia group and in control siRNA hypoxia group (P<0.01), whereas Bax protein bands were enhanced obviously, significantly higher than that in hypoxia group and in control siRNA hypoxia group (P<0.01).
5) Cytochrome c:
The results of Western blot showed:In normoxic group, only a small amount of cytochrome c were found in the cytoplasm; In hypoxia group and control siRNA hypoxia group, the cytochrome c content in the cytoplasm and mitochondria was only slightly affected. In TG2siRNA hypoxia group, the cytochrome c content in the cytoplasm increased significantly when inhibiting the expression of TG2, while cytochrome c content in mitochondria decreased remarkably (P<0.01). It suggested the inhibition of cytochrome c in osteosarcoma MG-63from releasing into the cytoplasm by TG2under the hypoxic condition.
6) Caspase-3:
Caspase-3activity:Caspase-3activity in hypoxia group and control siRNA hypoxia group was shown no obvious increase (P>0.05) compared with that in normoxic group; While, in the TG2siRNA hypoxia group, caspase-3activity gradually increased with prolonged hypoxia (P<0.01).
The results of Western blot showed weak caspase-3protein bands in hypoxia group; While, caspase-3protein bands obviously increased in TG2siRNA hypoxia group (P<0.01), and its expression intensity grew significantly with prolonged hypoxia (P<0.01).
7) The results of flow cytometry showed that:
The apoptosis rate in hypoxia group and control siRNA hypoxia group increased slightly compared with that in normoxic group; while, the apoptosis rate in TG2siRNA hypoxia group was improved significantly, compared with that in the first three groups (P<0.01).
Conclusions:
1) Under hypoxic conditions, the expression of TG2in osteosarcoma MG63cells was strikingly impoved;
2) TG2increases the Bel-2/Bax ratio, prevents the cytochrome c from releasing into the cytoplasm, and reduces caspase-3activity by promoting the expression of Bel-2and the depletion of Bax;
3) Crosslinked polymers of TG2generated in the cytoplasm form insoluble compounds with caspase-3, reducing the expression and activity of caspase-3, thus contribute to anti-apoptosis and inhibit the apoptosis of tumor cells.
4) When the TG2siRNA inhibits the expression of TG2, it facilitates the release of cytochrome c into the cytoplasm, enhances the expression and activity of caspase-3, and promotes apoptosis of tumor cells by decreasing the expression of Bcl-2and increasing the expression of Bax.
5) This study reveals a new anti-apoptotic pathway of TG2, suggesting that inhibiting the expression of TG2promotes apoptosis in osteosarcoma cells and providing a new effective way for the treatment of osteosarcoma.
引文
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