【作者】 金娟；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2012， 博士

【摘要】 肝细胞癌（hepatocellular carcinoma, HCC）在常见的恶性肿瘤中居于第五位，是第三大肿瘤死亡原因。我国每年HCC的发病率约占世界55％，HCC已成为我国第二位恶性肿瘤致死原因。由于HCC发病机制尚不清楚，目前抗肿瘤药物不良反应大，临床上迫切需要寻找高效、低毒的有效物质来抑制HCC细胞侵袭和转移，从而给予更好的干预。在培养的HCC细胞和人HCC组织中COX-2的表达与Akt磷酸化正相关。体内外实验表明，COX-2参与了抑制caspases3和caspases9的活性、降低p53和Bax的表达、增强Akt的活性，可能有抑制肝细胞的调亡的作用。这些现象提示COX-2的表达与肿瘤的发生、发展有着密切关系。而PGE₂作为COX-2的催化产物，在HCC中的水平随着COX-2水平的升高而升高。已知PGE₂通过激活细胞表面的PGE₂受体（prostaglandin E receptor, EP），与不同的G蛋白偶联，启动下游信号通路而发挥其对细胞功能的调节作用。研究发现，在HCC中EP₁的表达最为丰富。表没食子儿茶素没食子酸酯（（-）-Epigallocatechin-3-gallate, EGCG）是绿茶或一些天然药物中的活性成分，具有抑制肿瘤细胞增殖的活性。EGCG具有较强的抗氧化活性，能够保护细胞和DNA免受损害，并有抗辐射和紫外线等生物学功能。近年来，大量实验证明EGCG能干扰癌细胞生存所需的信号传递，如EGCG能与Bcl-2、Bcl-xL的疏水域紧密地结合，有效抑制抗凋亡Bcl-2家族成员，促进肿瘤细胞凋亡。EGCG降低某些致癌物质的活力、提高癌细胞对化疗的敏感性等作用。结合以上背景资料，探讨PGE₂/EP₁信号通路在HCC发展中的作用和EGCG对其的影响，寻求HCC治疗新靶点以及为EGCG用于HCC的治疗提供实验依据。目的：观察HCC患者血清中PGE₂水平及EP₁在HCC细胞和人肝细胞中的表达，分析PGE₂及EP₁激动剂ONO-DI-004对HCC增殖和移行的相关性，研究EGCG对PGE₂或ONO-DI-004刺激的HCC的抑制作用及对HCC细胞周期、凋亡、PGE₂、EP₁和Bcl-2表达的影响，初步探讨EGCG抗HCC的作用靶点和机制。方法：HepG2细胞设置组别为：对照组、PGE₂（0,4,40,400,4000,40000nM）组、EGCG （12.5,25,50,100μg/mL）组、PGE₂（4,40,400,4000）+EGCG（100μg/mL）组、ONO-DI-004（4,40,400,4000nM）组、ONO-DI-004+EGCG（100μg/mL）组、ONO-8711（210nM,1,5,10μM）组。MTT法检测HepG2的增殖；细胞划痕实验和Transwell侵袭实验检测HepG2的移行；Western blot assay和免疫荧光实验检测HepG2中EP₁、Gq和Bcl-2蛋白的表达；酶联免疫分析法检测HCC患者血清PGE₂及HepG2产生PGE₂和VEGF的水平；流式细胞技术检测HepG2细胞周期和凋亡。结果：1. HCC患者的血清中PGE₂的水平呈高表达与正常志愿者血清中的PGE₂水平比较，HCC患者的血浆中PGE₂的水平呈高表达。2.降低PGE₂和VEGF水平、下调EP₁和Bcl-2的表达是EGCG抑制HCC的重要作用与对照组相比，EGCG （12.5,25,50,100μg/mL）显著降低HepG2中PGE₂和VEGF水平。EP₁在肝癌细胞株MHCC-97L和HepG2中的表达明显高于在人肝细胞株L02中的表达。EGCG （100μg/ml）能显著抑制EP₁受体以及ONO-DI-004诱导的Bcl-2的表达（**P<0.01）。3. EGCG对外源性PGE₂或ONO-DI-004刺激HCC增殖和移行具有显著的抑制作用观察EGCG （12.5,25,50,100μg/mL）作用于HepG2后对细胞的增殖作用，100μg/mL EGCG对HepG2作用24h后可显著抑制HepG2的生长（**P<0.01）,细胞的平均抑制率为41%。24h的药物浓度与抑制率具有相关性（r=0.8，P=0.02）。EGCG对HepG2具有显著抑制作用。细胞划痕实验和Transwell侵袭实验观察PGE₂（4μM）和ONO-DI-004（400nM）刺激后，HepG2移行较对照组明显增加，EGCG和ONO-8711均能显著抑制HepG2移行，且给予EGCG （100μg/mL）后可显著降低PGE₂和ONO-DI-004刺激的HepG2的移行（**P<0.01）。4. EGCG能促进HepG2凋亡流式细胞术检测细胞凋亡率，HepG2经ONO-DI-004（400nM）、PGE₂（4μM）、EGCG （100μg/mL）、EGCG （100μg/mL）+ONO-DI-004（400nM）以及EGCG （100μg/mL）+PGE₂（4μM）作用24h，对照组的细胞凋亡率为（2.36±2.51）%, EGCG （100μg/mL）处理组的凋亡率为（16.8±1.73）%，EGCG （100μg/mL）处理组与对照组的细胞凋亡率相比差异性显著（**P<0.01）。ONO-DI-004（400nM）处理组的凋亡率为（0.6±1.86）%, EGCG （100μg/mL）+ONO-DI-004（400nM）处理组的凋亡率为（12.25±2.64）%，两组间的差异具有显著性（**P<0.01）。EGCG （100μg/mL）可显著的诱导HepG2细胞的凋亡，对PGE₂、ONO-DI-004刺激的HepG2细胞也有明显的促凋亡作用。结论1. HCC患者血清中PGE₂表达异常升高，提示PGE₂在HCC患者中发挥重要作用；2. HCC细胞株中EP1表达较正常肝细胞异常升高，PGE₂可能通过EP1发挥促HCC作用；3. EGCG呈浓度依赖性抑制由PGE₂和ONO-DI-004刺激引起的HepG2的增殖和转移，抑制HCC异常增殖、移行、细胞周期，诱导凋亡是EGCG的重要作用；4. EGCG抑制HepG2产生VEGF和PGE₂，降低EP1及ONO-DI-004刺激的Bcl-2的表达，但对Gq蛋白的表达没有影响，降低PGE₂和VEGF水平、下调EP1和Bcl-2的表达是EGCG抑制HCC增殖和移行、诱导凋亡的重要机制之一。更多还原

【Abstract】 (Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory ofAnti-inflammatory and Immunopharmacology, Anhui Medical University, Ministryof Education, Anhui Key Laboratory of Research and Development of ChineseMedicine, Engineering Technology Research Center of Anti-inflammatory andImmunodrugs in Anhui Province, Scientific and Technological Team ofAnti-inflammatory and Immunopharmacology of Chinese Medicine in AnhuiProvince, the115industial innovation team of basic and applied research intoanti-inflammation and immunodrugs in anhui province, Hefei,230032)Hepatocellular carcinoma （HCC） is the fifth most common cancer and the thirdmost common cause of death from cancer worldwide. In China HCC accounts for55%of all cases worldwide, and is the second leading cause of cancer death. So farthe tumorigenic mechanism of HCC remains undefined. And the antineoplastic agentsoften achieve low antineoplastic activity at the expense of close to unacceptabletoxicity. Therefore， there is a tremendous interest and urgency to search forrecurrence/metastasis related biomarkers and new agents with high effectiveness andlow toxicity for inhibiting HCC invasion and metastasis, which would better providenovel measures for intervention. The study found that the well-differentiated HCCexpressed COX-2more frequently and strongly than less-differentiated HCC,indicating that COX-2may participate in the early onset of HCC process. The level ofCOX-2expression and Akt phosphorylation was correlated positively in culturedHCC cells and human liver cancer tissues. In vitro and in vivo experiments showedthat COX-2inhibited the activity of caspases3, and caspases9, reduced theexpression of p53and Bax, enhanced Akt activity. COX-2expression was closely related to HCC occurrence and development. More and more evidences revealed thatCOX-2-derived prostaglandin E2（PGE₂） plays pivotal roles in tumor invasion. InHCC the level of PGE₂was highest compared with other PG productions. PGE₂exertsits biological actions primarily via their respective G protein-coupled receptors（GPCR） superfamily of prostaglandin E receptor （EP）. Reports showed that EP₁expression was most abundant in HCC.（-）-epigallocatechin-3-gallate （EGCG） is apolyphenolic compound from tea and natural plants that has been shown to haveanti-tumor activities. However, the delicate mechanisms and signaling pathwaysunderlying the potential anticancer effects of EGCG in HCC cells remain unclear.This study is designed to investigate the possible mechanism of PGE₂/EP₁signaling pathway in HCC and the effects of EGCG on this signaling. Thus, thesefindings might provide the novel target for HCC treatment and the rationale forEGCG served as a novel therapeutic agent to treat HCC patients, and future study onits clinical application might be worthwhile.AIMS:To observe EP1expression in HCC cells and human hepatocyte L02and theserum PGE₂levels of patients with HCC, to analysis of the effects of PGE₂and theEP1agonist ONO-DI-004on proliferation and migration of HCC, to investigate theeffects of EGCG on cell cycle, apoptosis, PGE₂, EP1and Bcl-2expression of HCC, todiscuss the anti-HCC mechanism of EGCG.METHODS:HepG2cells were designed into several groups: control group, PGE₂（0,4,40,400,4000,40000nM） group, EGCG （12.5,25,50,100μg/mL） group, PGE₂（4,40,400,4000）+EGCG（100μg/mL） group, ONO-DI-004（4,40,400,4000nM） group,ONO-DI-004+EGCG （100μg/mL） group, ONO-8711（210nM,1,5,10μM） group.MTT assay was performed to determine the proliferation of HepG2cells; Would healing assay and Transwell filter cell migration assay were used to determine themigration of HepG2cells; Western blot and Immunofluorescence microscopy wereused to determine the expression of EP1, Gq and Bcl-2protein in HepG2cells; Elisaassay was performed to determine the PGE₂production in serum from patients withHCC or in HepG2cells; Flow cytomertry was performed to determine the cell cycleand apoptosis of HepG2cells.Results1. Serum PGE₂level was higher in HCC patientsCompared with the serum PGE₂level from normal people, serum PGE₂level washigher in HCC patients.2. That EGCG reduced PGE₂and VEGF levels, and lowered the expression ofEP1and Bcl-2is the important role of EGCG on HCC inhibitionCompared with control group, EGCG （12.5,25,50,100μg/mL） reduced PGE₂and VEGF levels in HepG2in dose-dependent way （r=0.758, r=0.958）. EP₁expression was higher in the MHCC-97L and HepG2cells compared with humanhepatocytes L02cells. EGCG significantly inhibited EP1expression andONO-DI-004-induced Bcl-2expression in HCC cells.3. EGCG significantly inhibited PGE₂and ONO-DI-004-induced proliferationand migration of HepG2cells.Elisa assay was used to detected the effects of EGCG （12.5,25,50,100μg/mL）on proliferation of HepG2.100μg/mL EGCG significantly inhibited HepG2growthafter24h incubation, the mean inhibition ratio was41%（r=0.8）. EGCG significantlyinhibited HepG2growth. Wound healing assay and Transwell assay were used to testEGCG and ONO-8711suppressed migration of HepG2and EGCG inhibited PGE₂orONO-DI-004-induced migration of HepG2（**P<0.01）. 3.3EGCG induced the apoptosis of HepG2cells.HepG2cells were incubated with ONO-DI-004（400nM）、PGE₂（4μM）、EGCG（100μg/mL）、 EGCG （100μg/mL）+ONO-DI-004（400nM） or EGCG （100μg/mL）+PGE₂（4μM） after24h. The apoptosis ratio in control group was（2.36±2.51）%, that was （16.8±1.73）%in EGCG （100μg/mL） group. EGCGsignificantly induced HepG2cell apoptosis （**P<0.01）. The apoptosis ratio inONO-DI-004（400nM） was （0.6±1.86）%, that was （12.25±2.64）%in EGCG （100μg/mL）+ONO-DI-004（400nM） group.Conclusion1. High serum PGE₂levels in HCC patients indicated the important role of PGE₂inHCC patients;2. EP1expression in HCC cell lines compared with normal liver cells was abnormalelevation of PGE₂, which indicated that PGE₂might promote HCC by binding to EP₁receptors.3. EGCG inhibited PGE₂or ONO-DI-004-induced proliferation and migration ofHepG2, and induced cell cycle arrest and apoptosis. These finding suggested thatinducing cell apoptosis and inhibiting proliferation, migration and cell cycle were theimportant role of EGCG on anti-HCC.4. EGCG reduced VEGF and PGE₂levels in HepG2cells, and inhibited EP₁expression and ONO-DI-004-induced Bcl-2expression but almost no effects on Gqprotein. That reducing PGE₂, VEGF and EP1and Bcl-2expression was one ofmechanisms of EGCG on proliferation and migration of HepG2cells.更多还原