【作者】 徐志刚；

【导师】 陈立；

【作者基本信息】 吉林大学 ， 药理学， 2010， 博士

【摘要】 研究背景:肿瘤流行病学资料显示,肝癌和肺癌是威胁人类健康与生命的恶性疾病,其发病率和死亡率极高。目前普遍采用的放化疗以及手术切除治疗,均存在敏感性较差,不能根治的缺点。因此,寻找有效的治疗方法是亟待解决的问题。塞来昔布(celecoxib)属于新一代非甾体类抗炎药物(nonsteroidal anti-inflammatory drugs,NSAIDs),大量的研究结果表明,塞来昔布具有潜在的抑制肿瘤发生、发展以及转移等作用。塞来昔布为环氧合酶-2(cyclooxygenase-2,COX-2)选择性抑制剂,通过抑制COX-2表达、降低前列腺素E2(prostaglandin E2,PGE2)水平促进肿瘤细胞凋亡,而近年来人们还发现了一些COX-2非依赖的靶点,但是其抗肿瘤作用机制目前尚未完全阐明。研究目的:阐述塞来昔布对H22肝癌和Lewis肺癌的治疗作用及其作用机制,为其作为抗肿瘤药物的临床应用提供理论依据。研究内容:建立小鼠实体瘤模型后灌胃给药,计算肿瘤体积和重量,检测在体水平对肿瘤的抑制;MTT方法检测对肿瘤细胞增殖抑制作用;流式细胞仪手段检测肿瘤细胞凋亡和线粒体膜电位变化;Western blot方法检测细胞花生四烯酸(arachidonic acid,AA)代谢通路关键酶磷脂酶A2(cytosolic phospholipase A2,cPLA2)和COX-2,线粒体凋亡通路关键蛋白Bcl-2相关X蛋白(Bcl-2 Associated X protein,Bax)、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)、凋亡诱导因子(apoptosis-inducing factor,AIF)、细胞色素c (cytochrome c,Cyto c)、半胱氨酸天冬氨酸酶-3(cysteine-aspartic proteases,caspase-3)、半胱氨酸天冬氨酸酶-9(cysteine-aspartic proteases,caspase-9),过氧化物酶体增殖物激活受体( peroxisome proliferator activated receptor,PPARγ),核因子-κB( nuclear factor kappa B,NF-κB)的表达;RT-PCR方法检测细胞中cPLA2和COX-2的mRNA转录情况,采用高效液相色谱(high performance liquid chromatography,HPLC)法检测24h细胞培养上清液AA水平的变化,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)方法检测24h细胞培养上清液PGE2水平的变化。研究结果:1、塞来昔布对H22肝癌抑制作用塞来昔布抑制H22肝癌细胞增殖及其移植瘤生长;高剂量塞来昔布抑制cPLA2和COX-2表达,增加AA和降低PEG2水平;低剂量塞来昔布增加cPLA2表达,对COX-2表达无影响,AA含量增高、对PEG2水平无影响;各剂量组塞来昔布均增加AA与PGE2比值;塞来昔布作用于线粒体凋亡途径,破坏线粒体膜,影响凋亡信号蛋白表达,促进肿瘤细胞凋亡;塞来昔布对PPARγ/NF-κB途径无影响。2、塞来昔布对Lewis肺癌抑制作用塞来昔布抑制Lewis肺癌移植瘤生长及细胞增殖;能够促进细胞凋亡、降低线粒体膜电位;塞来昔布抑制COX-2表达,增加cPLA2表达,提高AA含量,降低PEG2水平;各剂量组塞来昔布均增加AA与PGE2比值;塞来昔布作用于PPARγ/NF-κB途径,增加PPARγ表达,降低NF-κB表达。研究结论:塞来昔布对H22肝癌细胞及Lewis肺癌细胞花生四稀酸代谢途径关键酶及其产物调节不同,但均增加AA与PGE2比值;AA与PGE2比值可能反映非甾体抗炎药抗肿瘤作用;塞来昔布可能通过调节COX-2依赖及非依赖途径、激活线粒体凋亡通路,促进细胞凋亡,从而发挥抗H22肝癌作用;塞来昔布抗Lewis肺癌可能通过调节COX-2依赖途径及PPARγ/NF-κB途径,促进细胞凋亡实现。更多还原

【Abstract】 Liver and lung cancer are malignant diseases that threaten health and life of human, and their incidence and mortality keep very high nowadays, according to epidemiology data from cancer. Though many kinds of chemotherapies and surgical resections are used in clinical, there is no sensitive and efficiency method to overcome the problems. Therefore, it is very urgent to find some appropriate ways for the patients.Recent studies have found that, cancer development had close relationship with disorders of cPLA2 and COX-2, the two key enzymes in the AA metabolic pathway. The membrane phospholipids are catalyzed into AA by cPLA2, and then AA is converted into PGE2, which has the activities of inhibiting apoptosis, stimulating division and angiogenesis, promoting invasion and metastasis on tumor cells. It was reported that COX-2 and cPLA2 were over-expressed in cancers, such as colorectal cancer, liver cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer etc. These findings suggested that COX-2 and cPLA2 played a very important part in the development and differentiation of liver cancer and lung cancer.In addition, PPARγ/NF-kB pathway also had very close relationship with tumorigenesis and development. The PPARs are some clasess of nuclear transcription factors, and activated by their ligands. After being activated, the PPARs will bind to the specific sequences of DNA in heterodimer forms and regulate the transcription of the NF-κB. NF-κB activation can inhibit cell apoptosis and promote cell proliferation. The expressions of PPARγand NF-kB in tumor tissue were higher than those in adjacent tissues, depending on tumor histological type, cell differentiation and postoperative TNM staging. The agonists of PPARγpioglitazone and blocking NF-kB could inhibit tumor proliferation and promote apoptosis of tumor cells. Pathw.The mitochondria is an important place for apoptosis. With the stimulations of apoptosis signal external or internal in tumor cells, the mitochondrial membrane permeability was changed, and then cytochrome c, AIF and other apoptotic factors were released into the cytoplasm. The cytochrome c would lead to cell apoptosis after forming apoptosome with the Apaf-1 and caspase-9, activating caspase-3, caspase-6 or caspase-7 in the downstream. The Bcl-2 family proteins, including anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax, are some important factors that regulated the changes of the mitochondrial membrane permeability, and play a very important role in the mitochondrial apoptosis pathway.Celecoxib belongs to the new generation of non-steroidal anti-inflammatory drugs, and a large number of studies declare the potential inhibition effects of celecoxib on occurrence, development and transfer of tumor. Celecoxib is a kind of selective inhibitors of COX-2. It promoted tumor cell apoptosis by inhibiting the expression of COX-2 and decreasing the level of PGE2 in traditional theory. But, in recent years, some COX-2 independent pathways were also found a number of studies. Those showed that its anti-tumor mechanisms were not yet fully understood. In the present study, we attempted to elucidate the inhibition effects of celecoxib on the H22 hepatoma and Lewis lung cancer and their mechanisms by COX-2 dependent and -independent Pathway in vivo and in vitro. Moreover, we tried to explain the preventive and therapeutic effect of celecoxib on cancer through its regulations on the AA metabolic pathway, the PPARγ/NF-kB pathway and the mitochondrial apoptosis pathway. These findings will provide some theoretical basis to its clinical application as antitumor drugs in the future. 1 Inhibition Effects of Celecoxib on H22 Hepatoma and its MechanismForty BALB/c mice receiving tumor implantation were divided randomly into control group and celecoxib groups at low, middle, high dosage (100、200、400mg/kg). All the groups were gavaged continuously with normal saline or celecoxib on the third day after implantation. The mice were killed on the 15th day, and the volume and weight of the tumor tissues were calculated. The protein expressions of cPLA2 and COX-2 were examined in the tumor tissues by Western blot analysis. The proliferation of H22 Hepatoma cells, treated with different doses (0, 25, 50, 100, 200μM) of celecoxib for 12, 24, 48 and 72 h, was measured by MTT assay. After being treated with celecoxib of different doses (0, 50, 100, 200 and 400μM) for 24h, the H22 cells were measured as followed: The apoptosis ratio and the mitochondrial membrane potential were detected by flow cytometry; The protein expressions of cPLA2, COX-2, Bax, Bcl-2, AIF, cytochrome c, caspase-3 and caspase-9 were examined by Western blot analysis, and the mRNA levels of cPLA2 and COX-2; The concentrations of arachidonic acid and prostaglandin E2 in the culture supernatants for 24h were measured by the methods of RP-HPLC and PGE2-specific ELISA respectively.The results in vivo showed that: The solid tumor volume and weight of treatment groups were lower than those of control group, and there was significant difference among the groups (P<0.05). The tumor inhibitory ratios of treatment groups were more than 30%, especially 75% in high dose group. The protein expressions of cPLA2 and COX-2 in treatment groups were lower than control group in a dose-dependent manner.The results in vitro showed that: Celecoxib exposure significantly reduced the viability of H22 cells in a dose- and time-dependent manner. IC50 values of celecoxib were 134.7±0.5μM, 112.4±0.3μM, 78.0±0.6μM and 56.9±0.8μM at 12, 24, 48, and 72h; Compared with controlgroup, the apoptosis ratio of tumor cell in treated groups notably raised and mitochondrial membrane potential decreased; The mRNA and protein expression of COX-2 were not altered in H22 cells treated with celecoxib at 50 and 100μM, however the COX-2 mRNA and protein levels were significantly decreased in H22 cells at 200 and 400μM dosage of celecoxib when compared to untreated H22 cells. Exposure to celecoxib caused an enhancement of the mRNA and protein levels of cPLA2 at dosages of 50 and 100μM, but resulted in an inhibition on the expression of cPLA2 at dosages of 200 and 400μM; The concentration of AA was elevated in the H22 cells after incubation with celecoxib at a dose of 50-400μM for 24h, and AA concentration was the highest at 100μM celecoxib, and the PGE2 production in H22 cells was not significantly influenced by treatment with 50 and 100μM celecoxib, but he levels of PGE2 production were significantly decreased when the cells were treated with 200 and 400μM celecoxib; The ratio of AA to PGE2 concentration was increased by celecoxib treatment in a dose-dependent manner in H22 cells; Compared with control group, Bax、caspase-3、caspase-9 were up-regulated and Bcl-2 was down-regulated; The protein levels of cytochrome c and AIF were up-regulated in the cytoplasmic, and down-regulated in the mitochondrias of the cells.2 Inhibition Effects of Celecoxib on Lewis lung cancer and its MechanismTwenty C57BL/6 mice receiving tumor implantation were divided randomly into control group and treatment (200 mg/kg) groups. All the groups were gavaged continuously with normal saline or celecoxib on the third day after implantation. The mice were killed on the 15th day, and the volume and weight of the tumor tissues were calculated. The proliferation of Lewis lung carcinoma cells, treated with different doses (50, 100, 200μM) of celecoxib for 12, 24, 48 and 72 h, was measured by MTT assay. The apoptosis and mitochondrial membrane potential were detected by flow cytometry. The protein expression of cPLA2, COX-2, PPARγand NF-κB was examined using Western blot analysis. The concentrations of AA and PGE2 in culture supernatants were measured by the methods of RP-HPLC and PGE2-specific ELISA respectively. The results in vivo showed that: The solid tumor volume and weight of treatment groups were lower than those of control group, and there was significant difference among the groups (P<0.05). The tumor inhibitory ratio of treatment groups was 50%.The results in vitro showed that: The proliferation of Lewis lung carcinoma cells was inhibited by celecoxib in a dose and time-dependent manner. The IC50 value of 72 h was 134.06±2.97μM; the apoptosis ratio of tumor cell in treated groups notably raised; The expressions of cPLA2 and PPARγwere up-regulated, but COX-2 and NF-κB were down-regulated in the Lewis cells exposed to celecoxib. The amount of AA was increased and PGE2 was decreased in the culture supernatant respectively. The ratio of AA to PGE2 was increased in a dose-dependent manner.In summary, our findings suggest that celecoxib could adjust the productions of AA and PGE2 by regulating on cPLA2 and COX-2, the two key enzymes in AA metabolic pathway. Although the regulations were different in H22 hepatoma and Lewis lung cancer cells, the ratio of AA and PGE2 was increased in both cell lines treated by celecoxib. The ratio of AA and PGE2 maybe was the a key factor in tumor development and reflecting the anti-tumor effect of celecoxib and other NSAIDs. Celecoxib could significantly inhibit and Lewis lung cancer in vivo and in vitro. Celecoxib may inhibited the H22 hepatoma by COX-2 dependent, non-dependent pathway, and activating mitochondrial apoptosis pathway. Celecoxib may inhibited the Lewis lung cancer by COX-2 dependent pathway, and PPARγ/ NF-κB channels.更多还原