【作者】 陈逢生；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 原发性肝癌是世界常见的恶性肿瘤之一,每年约有60万人死于此病,其中40%发生在我国。大部分肝癌病人于诊断后1年内死亡,5年生存率不超过5%。早期肝癌手术后5年生存率也仅15%～39%。治疗肝癌的传统方法包括手术、放疗、化疗等,其中手术切除虽然是首选的治疗方法,但手术后复发率高、手术切除率低,加上很多患者确诊肝癌时已是中晚期,失去了手术治疗的机会。近年来,随着肝癌分子生物学的研究深入,分子靶向治疗研究正逐渐成为肝癌治疗的新手段。分子靶向治疗药物酪氨酸激酶抑制剂已经在多种肿瘤的临床试验中取得另人鼓舞的效果。其中多靶点药物索拉非尼（Sorafenib,Nexavar,BAY 43-9006）在晚期肝细胞癌（hepatocellular carcinoma,HCC）的治疗中取得确切的疗效。索拉非尼是一种口服的小分子激酶抑制剂,它对C-RAF、野生型和突变型B- RAF有强效的抑制作用,能抑制C-RAF和B-RAF的丝氨酸/苏氨酸激酶活性;它还能抑制血管内皮生长因子受体-2（vascular endothelial growth factor receptor-2,VEGFR-2）、血管内皮生长因子受体-3（VEGFR-3）以及血小板源生长因子受体-β（platelet-derived growth factor receptor,PDGFR-β）、FLT3、Ret和c-KIT的酪氨酸激酶的活性,具有双重抗肿瘤作用。索拉非尼在多种肿瘤的临床前及临床试验均显示出活性。肝癌方面,一项国际多中心Ⅲ期随机对照治疗HCC的SHARP研究结果显示索拉非尼能延长晚期HCC患者的生存期。该研究结果被认为是首次发现能延长HCC存活期的药物,并认为索拉非尼可视为晚期HCC的一线治疗药物。2007年11月,美国FDA已经批准索拉非尼治疗不能手术切除的HCC。传统化疗药物顺铂（Cisplatin,DDP）的抗肿瘤作用显著,抗肿瘤活性强,抗肿瘤谱广,临床广泛应用于多种实体瘤的治疗。DDP主要靶位点为DNA,可形成DNA链内交联、链间交联、DNA-蛋白质交联,破坏DNA的复制及抑制肿瘤细胞的分裂。肝癌传统的全身化疗效果差,本实验尝试应用DDP与分子靶向药物联用这一新的模式来治疗肿瘤,以期获得更好的治疗效果。生物化疗（Biochemotherapy）,是生物治疗和化学治疗联合应用于肿瘤防治的全新综合治疗模式。有计划地联合应用化疗药物和生物制剂进行治疗,以取得最好的治疗效果,达到最大限度地改善病人生存质量的目的。本研究旨在探讨索拉非尼与DDP联合对HCC的联合抑制作用,并探讨其可能机制,为肝癌的综合治疗、提高疗效提供理论基础。第一部分索拉非尼联合DDP对人肝癌HepG2细胞抑制作用的实验研究一、材料与方法1.HepG2细胞在含10%小牛血清的RPMI-1640培养基中培养,3-5天传一次代,选择对数生长期细胞作为实验用。2.将细胞分成4组,分别为空白对照组、索拉非尼单药组、DDP单药组、索拉非尼联合DDP组。检测细胞增殖抑制率时索拉非尼药物浓度分别为2、4、8、16μmol/L;DDP药物浓度分别为3、6、12、24μmol/L;索拉非尼联合DDP组药物浓度是2+3、4+6、8+12μmol/L。流式细胞仪检测细胞周期、凋亡及Western-blot检测蛋白(ERK、pERK、Cyclin D1及p21^WAF1/CIP1蛋白)时,组中药物浓度索拉非尼均为4μmol/L,DDP均为6μmol/L。3.采取上述分组,用MTT法检测药物对人肝癌HepG2细胞增殖的抑制,根据OD值计算细胞生长抑制率。4.采用协同作用q值判断索拉非尼和DDP联用的性质:q＞1.15为协同作用,0.85≤q≤1.15为相加作用,q＜0.85为拮抗作用。5.采取上述分组,收集细胞用流式细胞仪检测细胞周期及凋亡。6.采取上述分组,收集细胞,提取蛋白行Western-blot检测ERK、pERK、CyclinD1及p21^WAF1/CIP1蛋白的表达。7.统计处理:实验结果以均数士标准差（（?）±s）表示,数据处理采用SPSS 11.5统计软件,各组间细胞抑制率比较采用重复测量方差分析;细胞周期、细胞凋亡率及蛋白表达IOD值采用单向方差分析;多重比较均采用LSD检验。P≤0.05（双侧）表示差异有显著性。二、结果1.索拉非尼、DDP单药对人肝癌HepG2细胞的生长抑制作用经MTT法检测示,不同浓度索拉非尼对人肝癌均有抑制作用,随浓度增高及时间延长抑制率增强（P均＜0.001）。DDP对人肝癌HepG2细胞的抑制作用亦具有时间及剂量依赖效应（P均＜0.001）。2.索拉非尼和DDP不同浓度单独及联合对人肝癌HepG2细胞的抑制作用经MTT法检测示,各浓度单独和联合应用对细胞的抑制率有显著性差异（P＜0.001）;各浓度联合组与相应浓度索拉非尼或DDP单药组相比,抑制率均有显著性差异（P＜0.001）,联合组疗效均优于单药组（P＜0.001）。3.索拉非尼和DDP不同浓度联合作用的q值索拉非尼2μmol/L和DDP3μmol/L联合用药组在24小时未显示出相加作用,48和72小时表现为相加作用。索拉非尼4μmol/L和DDP 8μmol/L联合用药组在各个时间点均显示相加作用（0.85≤q≤1.15）。索拉非尼8μmol/L和DDP12μmol/L联合用药组于24和48小时未出现相加作用,至72小时表现为相加作用。4.索拉非尼、DDP单药及联合用药对人肝癌HepG2细胞周期的影响流式细胞仪检测细胞周期示,与对照组相比,索拉非尼及DDP单药作用均可使细胞周期阻滞于G₀/G₁期（P=0.002,0.032）。联合用药组G₀/G₁期细胞比率高于索拉非尼及DDP单药组（P=0.025,0.002）,S期细胞比率低于单药组（P=0.004,0.022）。5.索拉非尼、DDP单药及联合用药对人肝癌HepG2细胞凋亡的作用索拉非尼或DDP单药作用后经流式细胞仪检测,细胞凋亡率均较对照组升高（P=0.006,0.036）。与索拉非尼（21.18%）和DDP（16.45%）单药组相比,联合用药组凋亡率升高更为明显,达35.28%（P=0.007,0.001）。6.索拉非尼、DDP单药及联合用药对人肝癌HepG2细胞ERK及pERK蛋白表达的影响Western-blot检测结果示,单药及联合用药组对HepG2细胞ERK总蛋白的表达无影响,而pERK蛋白在索拉非尼组及联合用药组的表达均较对照组降低（P＜0.001）,以联合组尤甚;DDP组与对照组条带表达无差异（P=0.173）。7.索拉非尼、DDP单药及联合用药对蛋白的影响经Western-blot检测示,索拉非尼组和联合组的Cyclin D1条带表达低于对照组（P＜0.001,P=0.001）,DDP组和对照组条带无显著性差异（P=0.509）。对照组和索拉非尼组p21^WAF1/CIP1蛋白的表达低;DDP组及联合组条带表达未见差异（P=0.734）,两者均较对照组表达增强（P=0.001）。三、结论1.索拉非尼及DDP单药对人肝癌HepG2细胞都有增殖抑制作用。2.索拉非尼与DDP联用具有疗效相加作用。3.索拉非尼和DDP单药或联合能诱导细胞周期阻滞及凋亡,以联合时更显著。4.索拉非尼通过RAF/MEK/ERK途径下调Cyclin D1的表达,而DDP通过p53途径上调的p21^WAF1/CIP1蛋白的表达,导致细胞周期阻滞;与单药相比,两药联用从多个途径引起细胞周期阻滞,促进细胞凋亡,增强了抗肿瘤效果。第二部分索拉非尼联合DDP抑制人肝癌细胞HepG2裸鼠移植瘤的实验研究一、材料与方法1.建立人肝癌HepG2裸鼠移植瘤模型:0.2ml人肝癌HepG2细胞（对数生长期）悬液接种裸鼠,每只裸鼠注射部位为裸鼠右腋下部皮下,注射肿瘤细胞悬液（0.2ml/只）,含活细胞5×10⁶/0.2ml。2.实验分组:将已成瘤小鼠（瘤重约130-150mg）随机分成空白对照组、溶剂对照组、索拉非尼组、DDP组和联合用药组共五组,每组10只。各组裸鼠于治疗起第21天处死。3.给药:索拉非尼组给予索拉非尼30mg/kg,胃管灌入0.1ml,每天一次,共12天。DDP组给予4mg/kg,腹腔注射0.2ml,每4天一次,共3次,联合用药组中索拉非尼给予30mg/kg,DDP给予4mg/kg,两种药物于同一天开始给药,早上给予索拉非尼,下午给DDP,时间间隔为3-5小时。4.裸鼠肿瘤大体形态观察,并测定裸鼠体重、肿瘤大小及计算瘤重:每只裸鼠在给药第1天开始测体重和肿瘤大小,每星期测两次。瘤重根据公式:（1×w²）/2,1指肿瘤的长径;w指肿瘤的短径。5.疗效及毒性评价:疗效以完全缓解（complete remission,CR）、部分缓解（partialremission,PR）和肿瘤生长抑制（tumor growth inhibition,TGI）来评价。毒性评价:以超过20%裸鼠死亡和/或体重减轻（去除瘤重）超过20%称为毒性大。6.肿瘤组织HE染色并置于显微镜观察。7.统计处理:实验结果以均数士标准差（（?）±s）表示,数据处理采用SPSS11.5统计软件,各组肿瘤瘤重比较采用重复测量方差分析,多重比较均采用LSD检验。P≤0.05（双侧）表示差异有显著性。二、结果1.观察裸鼠移植瘤大体形态发现,各治疗组肿瘤生长慢于对照组,以联合组肿瘤生长最慢。治疗后第21天处死的瘤体组织,对照组肿瘤体积最大,联合用药组肿瘤体积最小。切开肿瘤组织,对照组及DDP组肿瘤周边组织血供较丰富,而索拉非尼组及联合用药组小血管较少。2.各治疗组与对照组相比疗效均有显著性差异,P＜0.001;联合用药组疗效优于索拉非尼及DDP单药组（P=0.003,P＜0.001）。3.每组均未有超过20%的死亡率,体重亦未减少超过20%。溶剂对照组、DDP组及联合用药组各死亡1只,索拉非尼死亡2只。各组未出现CR,其中索拉非尼单药组及联合用药组各出现1例PR。索拉非尼及DDP单药的TGI分别为46.43%和38.45%,联合用药组TGI达60.42%。4.肿瘤组织经HE染色:与对照组相比,三种用药处理组瘤体内坏死组织多,肿瘤细胞核固缩、核结构不清、核破裂,部分区域肿瘤细胞全部坏死、消失,其中联合组最显著。三、结论索拉非尼及DDP单药均对人肝癌HepG2裸鼠移植瘤模型有抑制肿瘤生长的作用。联合用药显示出了增强的抗肿瘤效应。两药在该剂量水平联合具有安全性和耐受性。更多还原

【Abstract】 Background and objectionHepatocellular carcinoma（HCC） is the fifth most common cancer in the world and is responsible for＞600,000 deaths annually.The majority of patients with HCC die within 1 year after the diagnosis of their disease.Unfortunately,the disease is often diagnosed at a late stage when potentially curative therapies are least effective. For these patients,medical treatments,including chemotherapy,chemoembolization, ablation,radiotherapy,remain disappointing.Most patients show disease recurrence and their 5-year relative survival rate is less than 5%.The prognosis for HCC patients who have surgically resectable localized tumors is better,but they still have only a 15%to 39%5-year survival rate.Clearly,there is an urgent need for new therapies for this aggressive disease.With the development of the molecular biology of carcinogenesis and tumor progression of HCC,the management of advanced HCC is entering the era of molecular targeting therapy,which is of particular significance for HCC in view of the lack of existing effective systemic therapy for this cancer.Effective agents targeting these molecular abnormalities,such as Raf kinase inhibitors,have been developed and widely used.Several agents have entered clinical trials in HCC patients,and recent data indicated that a multikinase inhibitor targeting Ras kinase and VEGFR-2,sorafenib（Nexavar,BAY 43-9006） is effective in prolonging survival of patients with advanced HCC.Sorafenib is an orally available multikinase inhibitor that targets kinases of wild-type B-Raf,mutant B-Raf and C-Raf thus blocking tumor growth.Furthermore, sorafenib shows potent inhibition of receptor tyrosine kinases（RTKs） involved in angiogenesis,including human vascular endothelial growth factor receptors-2 and -3 （VEGFR-2/-3）,the platelet derived growth factor receptor-b（PDGFR-b）,FLT3,Ret, and c-KIT.Sorafenib has demonstrated preclinical and clinical activity against several tumor types.At the recent Annual Meeting of the American Society of Clinical Oncology,results of a phaseⅢ,randomized,placebo-controlled trial were presented in which sorafenib demonstrated improved survival in patients with advanced HCC.This landmark study represents the first agent that has demonstrated an improved overall survival benefit in this disease and sets the new standard for first-line treatment of advanced HCC.FDA has approved sorafenib for patients with inoperable liver cancer in 2007.Cisplatin（DDP） is a DNA-damaging agent that is widely used in cancer chemotherapy.DDP cross-links to DNA,forming intra- and inter-strand adducts, which bend and unwind the duplex and attract high-mobility-group domain and other proteins.Systematic chemotherapies are least effective in HCC,so we try to combine molecular targeting therapy with cytotoxic agents,DDP,to improve efficacy. Biochemotherapy,a new synthesis therapy mode,is the use of biotherapy in conjunction with chemotherapy.Rationally combining biotherapy with cytotoxic agents may be more effective at extending overall survival and quality of life for patients.Our study aimed at further improving efficacy of sorafenib by combination with DDP in HCC and providing preclinical evidence for biochemotherapy of HCC.PartⅠ:Coadministration of Sorafenib with DDP inhibits cell proliferation in hepatocellular carcinoma cells HepG2Methods1.HepG2（p53 wild type） human HCC tumor cells were cultured in RPMI 1640 containing 10%fetal calf serum and subcultured every 3 to 5 days.Exponentially growing cells were chosen for experiment. 2.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group, DDP-treated group and combination treatment group.MTT assay included cells treated with Sorafenib（2,4,8,16μmol/L）,DDP（3,6,12,24μmol/L） alone and combination（2 + 3,4 + 6,8 + 12μmol/L） for 24,48 and 72 hours respectively.Cell cycle,apoptosis and Western-blot analysis included cells treated with Sorafenib （4μmol/L） and DDP（6μmol/L） alone and combination.3.Group as the above mentioned,the inhibitory of HepG2 cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value.4.Interaction between Sorafenib and DDP was assessed using the q value,where q＞1.15,0.85≤q≤1.15,and q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.5.Group as the above mentioned,collected cells and analyzed cell cycle and apoptosis by flow cytometry.6.Group as the above mentioned,expression of ERK,pERK,Cyclin D1 and p21^WAF1/CXP1 were detected by Western-blot.7.Statistical analysis:Unless otherwise stated,data were expressed as（x±s）.The inhibitory rate was analyzed by Repeated Measure followed by LSD multiple comparison test.Data of cell cycle and apoptosis were analyzed by One-way ANOVA followed by LSD multiple comparison test.P-Values were considered to be significant at≤0.05.Results1.Sorafenib and DDP inhibit proliferation of HepG2 cells aloneSorafenib and DDP alone inhibited cell proliferation time- and dose-dependently （P＜0.001）.2.Combination of Sorafenib and DDP enhanced inhibition of proliferation in HepG2 cellsInhibitary rate was different among Sorafenib and DDP alone and combination at three concentration level respectively（P＜0.001）.Combination group showed better inhibitory effect than single agent group（P＜0.001）.3.q value of Sorafenib combined with DDP Coadministration of Sorafenib with DDP showed additive effect of all concentration levels at 72h（0.85≤q≤1.15）.Only Sorafenib（4μmol/L） together with DDP（8μmol/L） showed additive effect at 24,48,and 72h（0.85≤q≤1.15）.4.Sorafenib and DDP alone and together affected cell cycle of HepG2 cellsSorafenib and DDP alone can induced accumulation of cells in G₁ phase compared with untreated control（P=0.002,0.032）.Accordingly,the number of cells in the S-phase decreased（P=0.004,0.022）.Combinaton group showed greater increase of G₁（P=0.025,0.002） and decrease of S-phase than single agent group（P=0.036, 0.006）.5.Sorafenib and DDP alone and together induced HepG2 cells apoptosisCells apoptosis was detected after treated with Sorafenib and DDP alone（P=0.006, 0.036）.Coadministration of Sorafenib with DDP increased cells apoptosis compared with each agent individually（P=0.007,0.001）.6.Sorafenib and DDP alone and together affected the expression of ERK and pERKThe expression of ERK showed no difference in all groups.The decrease of pERK expression were seen after treated with sorafenib alone and in combination with DDP for 24h（P＜0.001）,and the latter was more obvious.7.Sorafenib and DDP alone and together affected the expression of Cyclin D1 p21^WAF1/CIP1The expression of Cyclin D1 in Sorafenib-treated group and combination group were lower than that in DDP-treated group and control group（P≤0.001）.The increase of p21^WAF1/CIP1 were seen in DDP-treated group and combination group（P=0.001）, the Sorafenib-treated group and control group showed lower expression of p21^WAF1/CIP1Conclusion1.Sorafenib and DDP alone inhibited cell proliferation time- and dose-dependently.2.Coadministration of Sorafenib with DDP showed additive inhibitory effect.3.Sorafenib and DDP alone or combination induced cell cycle arrest and potentiated apoptosis.4.Sorafenib and DDP induced cell cycle arrest by down regulating the expression of cyclin D1 through RAF/MEK/ERK signal pathway and up regulating the expression of p21^WAF1/CIP1 through p53 pathway respectively,Sorafenib and DDP showed no interaction of cyclin D1 and p21^WAF1/CIP1 expression in combination group, promoted cell cycle arrested and contributed to more cells apoptosis and enhanced antitumor effect.PartⅡ:Efficacy of Sorafenib combined with DDP against HepG2 hepatocellular carcinoma tumor xenografts.Methods1.Establish HepG2 HCC xenograft models:HepG2 tumors were generated by harvesting cells from mid-log phase cultures.A volume of 0.2 ml（5×10⁶ cells） of the cell suspension was injected subcutaneously into the right flank of each mouse.2.HepG2 HCC xenograft mice（tumors averaging from 130 to 150 mg） were divided into 5 groups:untreated control group,vehicle control group, Sorafenib-treated group,DDP-treated group and combination treatment group,with 10 mice per group.Xenograft mice were killed in day 21 after the treatment of first day.3.Administration:Sorafenib was administered po on a qd×14 schedule at 30mg/kg in the morning of each day.DDP was administered ip on a q4d×3 dose of 4 mg/kg alone or in combination with sorafenib on the same schedule of single agent.DDP was administered in the afternoon approximately 3-5 h after sorafenib.4.Tumor morphous were observed.Tumor dimensions and body weights were recorded twice weekly starting with the first day of treatment.Tumor weights were calculated using the equation（1×w²）/2,where 1 and w represent the largest and smallest dimensions collected at each measurement.5.Evaluated the efficacy and toxicity:Anti-tumor efficacy was evaluated by the incidence of durable complete regressions（CR）,partial regressions（PR）,and tumor growth inhibition（TGI）.Treatments resulting in greater than 20%lethality and/or 20%net body weight loss were considered ’toxic’. 6.Tumor tissues were HE dyed and were viewed under microscope.7.Statistical analysis:Unless otherwise stated,data were expressed as（x±s）.The tumor weights were analyzed by Repeated Measure followed by LSD multiple comparison test.P-Values were considered to be significant at≤0.05.Results1.Tumor morphous showed the growth of tumor in combination group was slower than that in other groups.At 21st day after treatment,tumor tissues showed the smallest in the combination group.Discissioed the tumor tissues,and less vessels were seen in Sorafenib-treated group and combination group.2.Sorafenib and DDP alone were efficacious against HepG2 hepatocellular carcinoma tumor xenografts.The efficacy of combination was significantly better than Sorafenib or DDP administered alone（P=0.003,P＜0.001）.3.All treatments resulted in less than 20%lethality and/or 20%net body weight loss.1/10 mice died during the treatment period in the vehicle group,DDP-treated group and combination treatment group respectively.2/10 mice died in the Sorafenib-treated group.CR was not observed.1/10 mice achieved PR in Sorafenib-treated group and combination treatment group respectively.TGI of Sorafenib-treated group and DDP-treated group were 46.43%and 38.45% respectively,and combination group（60.42%） was higher than single treatment group.4.HE dyeing showed more necrotic tissue,pyknosis,unclear structure of nuclear, caryoclasis in tumor tissues of combination group than that of other groups.ConclusionSorafenib and DDP alone were efficacious and tolerated in HepG2 hepatocellular carcinoma tumor xenografts.Coadministration of Sorafenib with DDP showed better efficacy than single treatment and well tolerated.更多还原