【作者】 刘兵；

【导师】 陶惠民；

【作者基本信息】 浙江大学 ， 外科学， 2008， 硕士

【摘要】 骨肉瘤(osteosarcoma)是儿童和青少年最常见的起源于间充质细胞的恶性骨肿瘤。随着骨肉瘤治疗手段的进展,如扩大切除术、放疗和新辅助化疗,其5年生存率已经增加到50～60%。以铂类为基础的药物,例如顺铂,是骨肉瘤化疗常用的有效的药物。环氧化酶(cyclooxygenase,COX)是催化花生四烯酸合成前列腺素(PG)的限速酶。研究表明,COX至少有两种同工酶,结构型COX即COX-1基因定位于染色体9q32～33.3,在各种组织细胞中稳定表达;诱导型COX即COX-2基因定位于染色体1q25.2～25.3,是一种诱导性即刻反应基因,在正常组织中一般没有明显表达;COX-3是COX-1的一种剪接变体,在哺乳动物的大脑皮层有丰富的表达。研究发现COX-2.高表达存在潜在的致瘤性,并且与肿瘤的发生发展及其生物学行为密切相关。非甾体类抗炎药(NSAIDs)是COX的抑制剂,能够诱导细胞凋亡和促进化疗药物的细胞毒作用。选择性COX-2抑制剂Celecoxib是近年来开发的新型非甾体类抗炎药,其特点是选择性作用于COX-2,对COX-1的活性几乎没有抑制作用。尽管Celecoxib精确的机制尚不明确,然而它的抑制细胞增殖和诱导凋亡是众所周知的,这在结直肠肿瘤和家族性腺瘤性息肉病中已经得到了证实。在本实验中,我们研究Celecoxib和/或Cisplatin诱导骨肉瘤MG-63细胞的凋亡现象,并探讨其机制。我们研究发现,MG-63细胞经过Celecoxib(50,100μmol/L)和/或Cisplatin(10μg/ml)处理48h后,细胞增殖抑制实验(MTT)证实Celecoxib和Cisplatin均能够抑制MG-63细胞的增殖,并且Celecoxib呈现剂量依赖效应。Celecoxib联合Cisplatin对MG-63细胞的生长具有协同抑制作用。倒置显微镜观察药物干预后的MG-63细胞,发现细胞体积变小、皱缩、变圆、脱落。电镜检测发现药物作用后细胞变圆收缩,染色质凝集成半月形附于核膜,核裂解并可见凋亡小体。在Celecoxib(100μmol/L)和Cisplatin(10μg/ml)联合作用于MG-63 48h后可见到特征性的梯形DNA ladder条带。利用流式细胞仪分析药物作用后的MG-63细胞,检测到G1期阻滞和凋亡,并且Celecoxib(100μmol/L)和Cisplatin(10μg/ml)联合的凋亡率明显的高于其他的实验组,这与DNA ladder的结果吻合。Celecoxib为选择性的COX-2抑制剂,其诱导凋亡可分为COX-2和非COX-2途径。在我们的实验中,我们通过RT-PCR和Western blot证实Celecoxib联合Cisplatin作用于骨肉瘤细胞MG-63并没有发现COX-2表达的下调。研究发现,Celecoxib联合Cisplatin明显的降低了PI3K/Akt、survivin、bcl-2的表达,更为重要的是检测到了procaspase-9,procaspase-3和PARP的裂解片段。因此,Celecoxib通过非COX-2途径诱导MG-63的凋亡,可能与PI3K/Akt、survivin、bcl-2相关。为了证实PI3K/Akt途径在西乐葆抗肿瘤中的作用,我们使用特异性的PI3K抑制剂wortmannin(1μmol/L)作用于MG-63细胞48h,检测到pAkt(Thr308),bcl-2,survivin表达的下调,这证实西乐葆诱导肿瘤凋亡作用是通过PI3K/Akt途径。因此,PI3K/Akt途径在survivin、bcl-2表达调控中发挥重要作用,可能是Celecoxib药物干预的中心环节。更多还原

【Abstract】 Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. With current therapies of OS, such as extensive surgical excision, radiotherapy and neoadjuvant chemotherapy, five-year survival rate has increased up to 50%-60%. Platinum-based drugs, such as cisplatin, are an important class of the most active chemotherapeutic agents that are widely used to OS. Cisplatin cytotoxicity involves the formation of intra and interstrand DNA adducts, and the resulting DNA damage triggers apoptosis.The cyclooxygenase (COX) isoenzymes known as a prostaglandins (PGs) ratelimiting synthase catalyze the metabolism of arachidonic acid to PGs. Three isoforms of COX isoenzymes have been identified: COX-1, COX-2, and COX-3. COX-1 is considered a "housekeeping enzyme", constitutively expressed in human cells. COX-3, an alternate splice variant of COX-1, is most abundant in the canine cerebral cortex. COX-2 is an inducible enzyme and is associated with inflammatory diseases and carcinogenesis. The activity of COX-2 is suspected to promote angiogenesis and tissue invasion of tumors and COX-2 over-expression has been mentioned in connection with resistance to apoptosis. Over-expression of COX-2 has been found in many human malignancies, such as colorectal, liver, pancreatic, breast, lung tumors and osteosarcoma. Thus selective pharmacologic inhibition of COX-2 represents a viable therapeutic option for the treatment of malignancies.Nonsteroidal anti-inflammatory drugs (NSAIDs) are COX inhibitors that have been shown to induce apoptosis as well as potentialize the effects of chemotherapeutic agents including cisplatin in vitro and in vivo experimental studies. Consistent with the anti-apoptotic effect of COX-2, induction of cancer cell apoptosis is the principle mechanism by which NSAIDs inhibit cancer cell growth. Celecoxib is a new generation of NSAIDs that specifically inhibits COX-2 activity .Although the precise mechanisms of celecoxib are not yet known, the inhibition of cell proliferation and the induction of apoptosis have been well-known, which have been confirmed in colorectal tumors and familial adenomatous polyposis (FAP). Celecoxib induces apoptosis through COX-2-dependent and -independent pathways. So we exert celecoxib and cisplatin to osteosarcoma cell in vivo and detect the effects of the combination or each agent alone.Cyclooxygenase-2 (COX-2), involved in the inhibition of cell apoptosis and the potentiation of cell growth, is frequently overexpressed in human malignancies including osteosarcoma (OS).We have attempted to identify the anti-proliferation of celecoxib, a selective COX-2 inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the potential molecular mechanisms involved.MG-63 cells were treated with the combination of celecoxib and cisplatin or either agent alone for 48 h in serum-supplemented medium. Celecoxib caused G1 phase arrest and significantly inhibited cell growth, as well as potentiating cisplatin-induced apoptosis. The effect was dose-dependent, and apoptotic changes such as DNA fragments and apoptotic bodies were observed.However, downregulation of COX-2 did not occur in cells treated with celecoxib. Phosphoinositide-3-kinase(PI3K)/Akt, survivin, bcl-2 were significantly downregulated in cells treated with the combination of celecoxib and cisplatin, and decreased survivin and bcl-2 levels were found in cells with wortmannin, a specific PI3K inhibitor. Moreover, the decreased expressions of procaspase-9, procaspase-3 and cleaved PARP-1 were detected by Western blot analysis. Therefore, celecoxib exerts its anti-tumor activities through COX-2-independent mechanisms, which may be PI3K/Akt-dependent, and survivin and bcl-2-related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of survivin and Bcl-2.更多还原