【作者】 潘跃银；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 博士

【摘要】 肝细胞性肝癌（hepatocellular carcinoma, HCC）是我国常见的恶性肿瘤之一，近二十年来，我国HCC的死亡率增加了41.7％，约占全世界HCC死亡人数的42％。目前，手术切除仍是治疗HCC的最佳手段，但本病起病隐匿，病情发展迅速，大多数患者就诊时已发展到中晚期，失去了手术治疗的机会，患者5年生存率低于10％。而多柔比星（doxorubicin, DOX）等临床一线化疗药物疗效不理想，有效率不超过20％，且大剂量时可引起严重的不良反应，从而限制了临床应用及疗效的进一步提高。近年来的研究发现环氧化酶2（cyclooxygenase-2, COX-2）在肝癌细胞中有高的表达，并且与癌细胞的分化程度、有无转移及愈后密切相关。COX-2的过度表达可能通过其改变某些与增殖及凋亡有关基因的表达而促进肿瘤生成，也可能通过调节某些细胞因子如血管内皮生长因子（vascular endothelial growth factor,VEGF）的分泌而促进肿瘤的侵袭及转移。因而，选择性阻断或抑制COX-2的活性，可能有利于HCC的预防和治疗。尼美舒利（nimesulide, NIM）是一新型的非甾体抗炎药（non-steroidal anti-inflammatory drugs, NSAIDs），对COX-2有选择性的抑制作用。有研究表明NIM在体外对多种癌细胞株有抑制作用。到目前为止化疗药物包括DOX在内治疗HCC疗效仍然十分有限且毒性很大，因此探讨选择性NSAIDs联合化疗的抗肝癌作用及其作用机制有重要的理论及临床意义。本研究考察了NIM的体外抑制原发性肝癌的作用，首次探讨了NIM与DOX联合使用时的增效作用，并初步研究了其作用机制，并在临床研究中观察了其对HCC患者血清VEGF水平的影响及生活质量的改善情况。1 NIM对人肝癌细胞株HepG₂和SMMC-7721增殖的影响选用人肝癌细胞株HepG₂和SMMC-7721，采用MTT法检测了NIM体外抑制人肝癌细胞株增殖的量效关系。结果显示：加入不同浓度(25、50、100、200、400μmol·L^-1)的NIM，48h后测定细胞增殖抑制率。结果发现，NIM在上述浓度范围内对HepG₂和SMMC-7721细胞的增殖均有抑制作用，药物浓度越高，抑制作用越强，呈现明显的浓度依赖效应关系。以浓度的对数与抑制率进行直线相关分析：HepG₂，r=0.9807，P＜0.01，其IC₅₀值：285.41μmol·L^-1；SMMC-7721，r=0.9715，P＜0.01，其IC₅₀值：358.99μmol·L^-1。2 NIM与DOX联合应用对人肝癌细胞增殖的影响将25、50、100μmol／L三种浓度的NIM与阿霉素0.156、0.32、0.63、1.25 mg／L联用，显示NIM和DOX联合使用对HepG₂和SMMC-7721细胞的抑制作用明显增强。以金氏公式计算结果显示具有协同作用。结果表明在合适的浓度下NIM与DOX对HepG₂和SMMC-7721细胞的增殖有明显协同抑制作用。3 NIM增效作用的可能机制探讨3．1 NIM与DOX联合应用诱导人肝癌细胞HepG₂凋亡的形态学改变采用吖啶橙（Acridine orange, AO）荧光染色，观察细胞的形态学改变。在荧光显微镜下可见未处理的HepG₂肿瘤细胞增殖旺盛，细胞质和核仁内有大量RNA，被染为桔黄或桔红色荧光，细胞核中有大量DNA，表现为黄绿色荧光深浅不一的结构样特征；药物处理组作用24h后，细胞密度减少，并可见典型的凋亡细胞形态特征：核固缩、体积缩小和表面起泡，细胞核或细胞质内可见致密浓染的黄绿色荧光。3．2 NIM与DOX联合应用诱导肿瘤细胞凋亡的流式细胞仪分析应用流式细胞仪（Flow Cytometry, FCM）检测细胞凋亡率。NIM单独应用可诱导人肝癌HepG₂细胞发生凋亡，在DNA直方图上G₀／G₁期前可见亚G₁峰即凋亡峰。NIM与DOX联合应用24h后凋亡率明显高于单独用药组，提示两药合用对诱导人肝癌细胞凋亡有协同作用。3．3 NIM与DOX联合应用HepG2细胞VEGF产生的影响不同浓度（25、50μmol／L）的NIM作用于HepG₂细胞后，NIM可导致细胞上清液中VEGF的水平显著下降；当NIM与DOX联合使用后，VEGF的水平进一步下降，提示两药对HepG₂细胞VEGF的产生的抑制有协同作用。进一步的临床研究显示原发性肝癌血清中VEGF水平显著高于正常对照组。NIM对晚期原发性肝癌血清中VEGF水平有明显影响，NIM单药治疗后VEGF明显下降。患者VEGF治疗前为230.17±147.87μg·L^-1，VEGF治疗后为124.33±253.67μg·L^-1，差异有显著性（P＜0.05）。当与DOX联合使用时血清VEGF的水平下降也有明显下降，但未见统计学差异，推测可能与联合治疗组病例过少有关。结合实验与临床研究的结果说明，NIM与DOX的协同抗原发性肝癌的作用可能部分是通过下调肿瘤的VEGF水平来实现的。3．4 NIM对HepG2细胞COX-2蛋白表达及PPGE2产生的影响本研究中发现不同浓度的（25、50、100μmol／L）NIM作用于人肝癌细胞株HepG₂后，其COX-2的表达水平并无明显的变化，与其浓度依赖性的细胞毒性作用并不一致。而PGE₂的水平与对照组相比，25、50、100μM的NIM作用48h后水平分别下降约80％、65％和40％，则呈现浓度依赖性的下降。说明NIM对人肝癌细胞株的增值抑制作用可能是通过抑制COX-2的活性导致PGE₂的合成减少来实现的。4 NIM改善原发性肝癌患者的生活质量NIM单药对原发性肝癌患者EORTC QLQ-C30（V3.0）生活质量15项领域有效改善率为50％，躯体、角色、情绪等5项功能领域改善率为30％～44.4％，乏力、恶心呕吐、疼痛等8项症状领域改善率为20％～62.5％，整体生活质量领域改善50％，生活质量15项领域中位有效改善率为44.4％。以上结果显示，NIM单药显著提高原发性肝癌患者的生活质量，包括躯体、角色、情绪、症状等多个方面。NIM与DOX为主的化疗联合使用时生活质量的改善较NIM单药治疗组未体现出优势。可能与问卷调查时间过早及联合治疗组病例数过少有关。总之，本研究实验研究的结果表明NIM有明显的抗肝癌作用，并首次观察了NIM与DOX的协同作用。其作用机制可能与降低PGE₂水平、诱导细胞凋亡、下调VEGF的水平有关。将NIM应用于肝癌患者发现其显著改善病人的生活质量。可考虑扩大病例数进一步研究。更多还原

【Abstract】 Hepatocellular carcinoma （HCC） is one of the most common malignancies in China. Although the clinical diagnosis and management of early-stage HCC has significantly improved, HCC prognosis remains poor. Only 10-20% of patients qualify for curative surgery. The median survival of patients who have unresectable tumors is only 4 months. Chemotherapy is one of the commonly used strategies in HCC treatment, especially for unresectable patients. But antineoplastic agents often achieve limited antineoplastic activity at the expense of close to unacceptable toxicity, which have been demonstrated a consistent response rate of less than 20% when used as single-agent in inoperable HCC patients. Thus it is imperative to develop more effective and low-toxic chemopreventive and chemotherapy agents for HCC.The cyclooxygenase-2 （COX-2） is highly expressed in HCC. The expression pattern of COX-2 protein in HCC is well correlated with the differentiation grade, suggesting that abnormal COX-2 expression plays an important role in hepatocarcinogenesis. Overexpression of COX-2 may increase the resistance of apoptosis in cancer cells and secretion of cytokine such as vascular endothelial growth factor （VEGF） which, one of the most potent angiogenic factors, has been shown to play a pivotal role in tumor angiogenesis, including HCC. Thus, the reduction of the COX-2 enzyme activity or protein expression may inhibit cell growth in cancer cells. Non-steroidal anti-inflammatory drugs （NSAIDs） have been shown to inhibit COX enzymes and may be employed for the chemoprevention of cancer. Some NSAIDs such as nimesulide exhibit high selectivity for COX-2 enzyme. Inhibition of COX-2 by several inhibitors including nimesulide induces growth inhibition and marked apoptosis in cultured HCC cells. There is no convincing evidence from randomized trails that the combination chemotherapy prolongs the survival of patients with unresectable HCC better than single agents. Whether COX-2 inhibitors are beneficial in the treatment of HCC if given in combination with these effective agents such as doxorubicin with complementary mechanisms remains unknown.In this study, we will firstly investigate the growth-inhibitory effect of NIM with DOX in HCC cell lines HepG₂ and SMMC-7721 and the possible mechanism. In clinical trial, we will detect whether NIM can improves the quality of life （QOL） of the patients with hepatocellular carcinoma.1. Effect of NIM on the proliferation of hepatocellular carcinoma cell lines HepG₂ and SMMC-7721The antiproliferative effect of NIM on HepG₂ and SMMC-7721 cell lines in vitro was measured by MTT assay. The results suggested that NIM at concentrations of 25、50、100、200、400μmol·L^-1 , had inhibitory effects on the proliferation of both HepG₂ and SMMC-7721 cells. The higher the concentration of NIM was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NIM. The r values of dose-effect curves for single-agent NIM on HepG₂ and SMMC-7721 cell lines were 0.9807 and 0.9715 respectively. The IC₅₀ values were 285.41μmol·L^-1 （HepG₂） and 358.99μmol·L^-1（SMMC-7721）. 2. Effect of NIM combined with DOX on the proliferation of human和hepatocellular carcinoma cell lines HepG₂ and SMMC-7721To investigate the synergistically inhibitory effects of NIM and DOX on the proliferation of HepG₂ and SMMC-7721 cell lines, three doses of NIM (25、50、100μmol·L^-1) were used to combine with DOX. The Jin’s formula was used to analyse the synergistically inhibitory effect of drug combination. NIM combining with different concentrations of DOX showed different extent synergistically inhibitory effect on the proliferation of the two cell lines in appropriate concentrations.3. Possible mechanisms of the synergistic antiproliferation effect of NIM in combination with DOX on human hepatocellular carcinoma cell lines HepG₂3.1 Morphological evidence of apoptosis induced by NIM combined with DOXMorphological evidence of apoptosis was demonstrated by using acridine orange （AO） fluorescence staining. The results showed that AO permeated through cell membrane and made the nuclei appear green and chromatin appear yellow. The HepG₂ cell line treated with NIM and DOX showed the typically apoptotic changes, such as chromatin condensation, membrane blebing, deformed and fragmented nuclei, and so on.3.2 Apoptosis caused by NIM combined with DOXA Flow Cytometry （FCM） assay was performed to analyze apoptotic rate of HepG₂ cells treated with NIM or DOX or a combination of the two agents for 24 h. The sub-G₁ peak appeared before G₁ phase, which represents apoptotic cell population, was observed. The apoptotic rate of combination group was higher than drugs used alone, which demonstrated that NIM in combination with DOX had synergistic apoptosis-induction effect on the HepG₂ cell line.3.3 Co-treatment with doxorubicin and nimesulide reduced production of VEGF in hepatocellular carcinoma cells.It is not known whether the inhibition of COX-2 reduces VEGF production in hepatocellular carcinoma cells. To test this possibility, we treated HepG₂ cells with 25μM or 50μM of nimesulide and measured VEGF levels in the culture supernatants by an ELISA. Ttreatment with nimesulide alone led to significant reduction in VEGF levels. VEGF production was also significantly reduced if the cells were co-treated with doxorubicin and a lower concentration （25μM） of nimesulide, suggesting the synergistic inhibition of VEGF production in HepG₂ cells by two agents.3.4 Nimesulide inhibits the COX-2 activity in HepG₂ hepatocellular carcinoma cells.We performed the Western blot analysis to examine whether COX-2 protein is expressed in HepG₂ cells. COX-2 protein was highly expressed in HepG2 cells. As treatment with 25-100 Mm nimesulide for 24 h did not significantly alter expression levels of COX-2 protein in HepG₂ cells. Next we tested whether treatment with NIM inhibits the COX-2 enzyme activity by measuring PGE₂ levels of culture supernatant. Compared with the control, the PGE₂ production was reduced by about 80%, 65% and 40% 24 h after the treatment with 25μM, 50μM or 100μM of nimesulide, respectively, indicating the dose-dependent inhibition.4. NIM improves the quality of life of the patients with hepatocellular carcinoma After the patient were treated with NIM, the response rates for function of body, role, emotion, cognition and social in 15 scales of EOREC QLQ-30 were 30 %-44.4 %. The response rates of inertia, nausea/vomiting, pain dyspnea, sleeplessness, anorexia, constipation, diarrhea, financial straits was 20%-62.5%. The response rate of total QOLwas 50%.The mean response rate for 15 scales of EOREC QLQ-30 was 44.4%. These results display that NIM can improve QOL involving body, role, emotion, symptoms and so on.When the patients were treated with NIM and DOX, the improvement of QOL was not superior to that in the patients treated with NIM alone. The reasons for this satuation might be too earlier time for questionnaire and limited cases in combined group.To sum up, this study for the first time presents evidence that a combination of COX-2 inhibitor nimesulide and doxorubicin additively inhibits growth of the human hepatocellular carcinoma cells. This effect was mainly observed owing to increased apoptosis and reduced VEGF production. In clinical trial, we found that NIM had improved the quality of life of the patients with hepatocellular carcinoma. The study provides a new strategy that the combination of COX-2 inhibitors and doxorubicin may be effective in the treatment of human hepatocellular carcinoma.更多还原