【作者】 李萌；

【导师】 李继承；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 第一部分NF-κB/IκB信号通路在TCS抗肝癌HepG2细胞中的作用研究背景：天花粉蛋白(trichosanthin, TCS)是从植物栝楼天花粉块根中分离提取的有效成分,属于Ⅰ型核糖体失活蛋白,具有引产,免疫调节,抗肿瘤及抗病毒等多种生物学功效。近年来TCS的抗肿瘤活性备受关注,多种凋亡机制参与TCS诱导肿瘤细胞凋亡。与宫颈癌、绒毛膜上皮癌等对TCS高度敏感的肿瘤细胞株相比,肝癌HepG2细胞对TCS低度敏感,其机理尚未阐明。核转录因子kappa B (nuclear factor kappa B, NF-κB)广泛存在于真核生物中,参与细胞生长、分化、炎症及免疫反应、凋亡等多种基因表达调控。目前,NF-κB活化是否参与了TCS抗肿瘤过程,以及在该过程中的作用机制,尚未见报道。研究目的：本实验选取对TCS低度敏感的肝癌HepG2细胞作为细胞模型,研究TCS对HepG2细胞NF-κB活性的影响,以及NF-κB活化在TCS诱导HepG2细胞凋亡中的作用,揭示HepG2细胞对TCS低度敏感的机制,为TCS抗肿瘤实验研究,提供理论依据。研究方法：MTT法检测HepG2和MIHA细胞经不同浓度TCS处理48 h后的细胞存活率。提取经TCS处理细胞的胞浆和胞核蛋白,应用Western blot法检测胞浆中IκB-a蛋白、NF-κB p65亚单位、Cox-2蛋白水平,以及胞核中NF-κB p65亚单位水平。采用双荧光素酶报告基因检测系统检测NF-κB转录活性。应用Hoechst33258染色,检测TCS诱导的细胞凋亡,并统计细胞凋亡率。通过生物抑制剂(IκB-DM)和化学抑制剂(PDTC)抑制NF-κB活化,观察IKB-a蛋白、NF-κB p65亚单位的改变,以及细胞存活率和凋亡率改变。研究结果：(1)经200μg/ml TCS处理48 h后,HepG2和MIHA细胞生长抑制率分别约为48%和17%,表明TCS对HepG2具有选择性的抑制作用。(2)TCS能迅速诱导肝癌HepG2 IKB-a蛋白水平显著降低,并伴有胞浆中NF-κB蛋白水平降低及胞核NF-κB蛋白水平增高,即NF-κB核易位。TCS可以明显促进NF-κB转录活性,具有时间依赖性。并且TCS能诱导NF-κB下游Cox-2基因表达增加。(3)通过稳定转染,建立了稳定表达IκB-DM和空白对照(pEGFP-N1) HepG2细胞系。空白质粒对照组细胞胞浆中IκB-a和NF-κB p65蛋白变化趋势与未转染质粒的HepG2细胞相似；IκB-DM组IKB-a蛋白基础水平显著增加,而且TCS处理持续达3h也未见降解。同时IκB-DM组未见NF-κB p65蛋白胞浆胞核蛋白水平变化。经MTT检测,50μg/ml TCS处理48 h后,IκB-DM组的细胞存活率(43%)比空白质粒组(63%)低20%(P＜0.05)。应用PDTC对HepG2细胞进行预处理抑制NF-κB活化,Hoechst 33258染色观察发现,(PDTC+TCS)组的细胞凋亡率较TCS组增加了16%。结论：(1)TCS对肝癌HepG2细胞具有选择性细胞毒性作用,使得TCS具有较好的抗肿瘤应用前景。(2)TCS能引起HepG2细胞胞浆中IKB-a蛋白水平降低,导致NF-κB被释放、发生核转移,并调节下游基因表达。(3)NF-κB活化可抑制TCS诱导的HepG2细胞凋亡,导致HepG2对TCS的低度敏感；而有效抑制NF-κB信号通路能增强HepG2细胞对TCS的敏感性,促进TCS抗肿瘤作用。第二部分地塞米松促进天花粉蛋白诱导的人肝癌HepG2细胞凋亡研究背景：地塞米松(dexamethasone)属于糖皮质类激素(glucocorticoids),具有抗炎及免疫调节等药理作用。地塞米松能通过抑制NF-κB活化诱导多种淋巴瘤细胞凋亡。近年来,学者们发现地塞米松能有效预防化疗药物引起的恶心、呕吐,促进了5-氟尿嘧啶、顺铂等化疗药物的抗肿瘤效果。然而地塞米松对TCS诱导人肝癌细胞凋亡的调节作用尚未见报道。研究目的：本实验研究地塞米松是否能促进TCS诱导的HepG2细胞凋亡,并进一步确认NF-κB活化在TCS诱导HepG2细胞凋亡中的作用,为TCS单独或药物联合抗肿瘤的临床实验研究,提供依据。研究方法：HepG2细胞经地塞米松(1μM,24 h)预处理后,与不同浓度TCS联用处理48 h后,MTT法检测细胞存活率；应用Western blot法检测胞浆中IκB-a蛋白水平；采用Hoechst 33258染色检测细胞凋亡,并统计细胞凋亡率。研究结果：(1)TCS单独处理时,IC50超过200μg/ml, TCS与地塞米松联用后,IC50约为50μg/ml,表明地塞米松促进了TCS诱导的HepG2细胞凋亡。(2)经地塞米松预处理后,IKB-a蛋白明显增加,且联用TCS后,IKB-a蛋白水平未见明显改变。结论：(1)TCS与地塞米松联用使HepG2细胞对TCS的敏感性增加,促进了TCS诱导的HepG2细胞凋亡发生。(2)地塞米松能显著提高IKB-a蛋白的基础水平,提示地塞米松通过促进IκB-a转录水平,使IKB-a蛋白表达增加,从而抑制了胞浆中NFκB的解离,最终抑制NFκB的活化,促进了TCS的抗肿瘤作用。更多还原

【Abstract】 Part I The role of NF-κB/IκB signaling pathway in trichosanthin-induced apoptosis in the HepG2 hepatoma cell lineBackground:Trichosanthin (TCS) is a type I ribosome-inactivating protein, extracted from the Chinese medicinal herb trichosanthes kirilowii, and has various pharmacological activities, including abortifacient, immunoregulatory, anti-tumor, and anti-viral activity. In recent years, TCS has been the subject of much research because of its potential antitumor activities. Many reports have revealed that various mechanisms participated in TCS-induced apoptosis. TCS-induced cytotoxicity of HepG2 cells was not as significant as that of JAR and HeLa cells, the underlying mechanisms remain to be elucidated. Nuclear factor-kappa B (NF-κB) is an important transcription factor that regulates the expression of various genes involved in inflammation, cell proliferation and apoptosis. So far, it is need to elucidate that whether NF-κB activation participated in TCS-induced apoptosis and the potential role of NF-κB in the low sensitivity of hepatoma cells to TCS.Objective:TCS low-sensitive hepatoma cell line HepG2 was employed for investigation. The present study investigated whether NF-κB activation was induced in TCS-induced apoptosis of HepG2 cells and the potential role of NF-κB in the low sensitivity of hepatoma cells to TCS-induced apoptosis.Methods:After treatment with various concentrations of TCS for 48 h, cell viability assays were performed using the MTT method. HepG2 cells were treated with 50μg/ml TCS at different time intervals, and cytoplasmic and nuclear extracts were prepared. The levels of IκB-αand Cox-2 were determined in cytoplasmic extracts, and the level of NF-κB p65 was detected in both cytoplasmic extracts and nuclear extracts by Western blot assay. Luciferase reporter assay detected the NF-κB transcriptional activity induced by TCS. And apoptosis was evaluated with Hoechst 33258 staining. Additionally, we took advantage of dominant-negative IκB (IκB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-κB activation, and analyzed the changes of cell viability and apoptosis induced by TCS.Results:(1) After treatment with 200μg/ml TCS for 48 h, the cell growth inhibition of HepG2 and MIHA cells were about 48% and 17%, respectively. TCS had relatively low toxicity to HepG2 cells, but could still discriminate HepG2 cells and MIHA cells. (2) TCS could rapidly decrease the level of IμB-αprotein, and also decrease the level of cytoplasmic NF-κB p65 subunit and lead to an elevation of the p65 subunit level in the nuclei, indicating nuclear translocation of NF-κB. and decrease of COX-2 expression in HepG2 cells. TCS could induce NF-κB transcriptional activity in a time-dependent manner. In addition, TCS could gradually increase the level of downstream factor, Cox-2 protein. (3) It is established stably-transfected HepG2 cell lines expressing either IκB-DM or a mock control transfected with plasmid pEGFP-N1. After treatment with TCS, the changes of the level of IκB-αprotein and NF-κB p65 subunit in the mock control cells were similar to that observed in untransfected HepG2 cells. In contrast, the basal level of IκB-αprotein increased significantly in IκB-DM-transfected cells. In addition, there was no obvious cytoplasmic down-regulation of IκB-αprotein and p65 subunit in cells induced by TCS-induced for 3 h, and there was also no obvious elevation of the p65 subunit in the nuclei. MTT assays demonstrated that the viability of the IKB-DM-transfected cell group treated with 50μg/ml TCS for 48 h was 43%, compared with 63% in the mock-transfected cell group (P＜0.05). In addition, HepG2 cells were pretreated with inhibitor of NF-κB, PDTC, and stained with Hoechst 33258. There were 16% more apoptotic cells in the PDTC pretreatment group compared with the TCS alone group (P<0.05).Conclusion:(1) TCS can discriminate tumor cells (HepG2 cells) and normal cells (MIHA cells), which make TCS possessed a good application prospect of anti-tumor. (2) TCS can cause rapid down-regulation of IKB-a protein in the cytoplasm of HepG2 cells, liberation of NF-κB dimmers, entrance to the nucleus, and activation of NF-κB. (3) NF-κB activation played an anti-apoptotic role in TCS-treated cells. At least in part, by inhibiting the NF-κB signaling pathway can enhance trichosanthin-induced apoptosis in the HepG2, and thus strengthening the antitumor effects of TCS.PartⅡDexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell lineBackground:Dexamethasone, a synthetic glucocorticoid, is widely used in clinical settings for its anti-inflammatory and immunomodulatory effects. Dexamethasone can treat cancers, such as lymphocytic leukemia, Hodgkin’s disease and non-Hodgkin’s myeloma, through effective inhibition of NF-κB activity and induction of apoptosis. In recent years, researchers have tested the effect of dexamethasone co-treated with antitumor drugs, such as 5-fluorouracil, cisplatin and so on, on solid cancers. The modulatory effects of dexamethasone on TCS mediated apoptosis in human hepatoma cells have not yet been reported.Objective:The present study investigated the modulatory effects of dexamethasone on TCS induced apoptotic death in the human HepG2 hepatoma cell line, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.Methods:After pretreated with or without 1μM dexamethasone for 24 h, and then treated with different concentrations of TCS, cell viability assays were performed using the MTT method. The levels of IκB-a in cytoplasmic extracts was detected by Western blot assay. And apoptosis was evaluated with Hoechst 33258 staining.Results:(1) Our results demonstrated that dexamethasone could enhance TCS induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200μg/ml to 50 ug/ml. (2) Dexamethasone increased the level of IκB-αprotein and effectively inhibited TCS-induced down-regulation of IκB-α.Conclusion:(1) Dexamethasone was able to enhance the sensitivity of hepatoma HepG2 cells to TCS, and promote trichosanthin-induced apoptosis in the HepG2. (2) Dexamethasone might promote the transcription of the IκB-αgene to increase the expression of IκB-αprotein, and efficiently inhibited TCS induced down-regulation of IκB-αprotein, and enhance the antitumor effect of TCS itself.更多还原