The functional characterization of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone in hepatocellular carcinoma: targeting heat shock protein 27 to mediate mitochondrial apoptosis.

研究背景：肝癌是全球常見的惡性腫瘤之一，世界上每年大約有50萬死亡病例，並且呈逐年上升之勢， 是全球第3位的腫瘤死亡原因。慢性乙型和丙型肝炎病毒感染是肝癌的主要成因。肝癌惡性程度高、預後差，並且目前的治療手段非常有限，術後易復發和轉移，迄今尚無正式獲准有效治療藥物。現階段，治療肝癌的主要方法是手術切除，但是隨之引起的併發症以及較高的復發機率嚴重影響了治療的療效，大大降低肝癌病人的存活期。研究目的：分析TDP對肝癌細胞和肝腫瘤旁細胞生長的影響；分析TDP抑癌的分子靶標蛋白及其分子機理；驗證TDP對肝癌動物模型的抑制效果。開發一種新型有效的肝癌治療藥物。研究方法：首先用MTT法從102種來源於嶺南山竹子的純複合物中分離出了TDP,它是一種甾醇類化合物。採用MTT法檢測TDP對腫瘤細胞生長的影響；流式細胞實驗驗證TDP能否引起腫瘤細胞的凋亡；採用蛋白組學和質譜分析找出TDP抑癌的分子靶標；進一步的蛋白功能增加和缺失實驗證明Hsp27的功能和作用；生物資訊學驗證HSP27和TDP的作用結果；最後利用動物模型驗證TDP對肝腫瘤的治療效果。結果：TDP不但能效率極高的抑制肝癌細胞的生長而且可以大量誘發肝癌細胞的凋亡，而對正常的肝癌旁細胞沒有影響。二維電泳以及質譜分析TDP處理的肝癌細胞對比DMSO處理的肝癌細胞發現了具有不同表達水準的18種蛋白，Hsp27是其中一個在TDP誘導下調變化倍數較大並且與細胞凋亡有密切關係的蛋白，Hsp27的過表達以及Knock-down都充分驗證了TDP通過調節Hsp27的表達參與了依賴於caspase的線粒體凋亡途徑，在Western Blotting以及RT-PCR中得到了充分的驗證。生物資訊學預測TDP可以與Hsp27結合，實驗結果表明TDP可以誘導Hsp27的聚集並導致功能喪失。動物實驗腫瘤生長結果以及免疫組化結果證明，TDP可以在很大程度上對肝癌有抑製作用。結論：本研究首次表明，TDP如果不是完全的,最起碼也是部分通過誘導依賴於caspase的線粒體凋亡的途徑來抑制肝癌細胞的增值和分化, 具有明顯的抗腫瘤的功效，特別是對Hsp27高表達的腫瘤細胞有比較明顯的作用，是一種值得繼續深入研究的有較高潛在價值的藥物。Background：Hepatocellular carcinoma (HCC), the most common primary hepatic malignancy, is a global public health problem that accounts for approximately 500,000 deaths annually. Chronic hepatitis B and hepatitis C infections are the major risk factors for the development of HCC. Due to the high rate of these infections, the incidence of HCC remains alarmingly high globally. Although great advances have been made in HCC treatment, poor prognosis and high risk of recurrence have been major challenges to patients. Currently, surgical resection is the main treatment option for HCC patients; however, complications arising from surgery can threaten its therapeutic effect and patients’ survival.Objectives：To characterize the functions of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b]Xanthone (TDP) in cell proliferation of HepG2 cells; to discover the molecular target genes and elucidate the underlying molecular mechanism of TDP; to examine the in vivo function of TDP in a nude mouse tumor model of HCC. Finally, to investigate TDP’s potential as an anti-HCC drug candidate.Methods and Results：In this study, we discovered that TDP, isolated from the Chinese medicinal herb, Garcinia oblongifolia, strongly inhibited cell growth and induced caspase-dependent mitochondrial apoptosis in HCC, as evidenced from MTT assay and flow cytometry analysis. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to find the molecular targets of TDP in HCC cells, and eighteen proteins were identified with altered expression, with Hsp27 protein being one of the proteins most significantly down-regulated by TDP. Further Hsp27-siRNA knockdown and Lenti-Hsp27 overexpression studies found that Hsp27 was involved in TDP induced mitochondrial apoptosis, with bioinformatics predictions and biological results revealing that TDP might cause Hsp27 protein form dimer and consequent degradation via the ubiquitin-proteasome system. Finally, subcutaneously injecting cancer cells with Hsp27 expression vector into the dorsal flank of nude mice tumor model also demonstrated the suppressive effect of TDP on HCC.Conclusions：In summary, our study discovered that TDP, a natural xanthone, was a potent inhibitor of Hsp27 in HCC. TDP inhibited cell growth and induced apoptosis by inducing Hsp27 degradation, which stimulated mitochondrial cytochrome C release which resultantly activated caspase-3 and caspase-9. These data combined with the results of the animal model strongly supported TDP’s potential as a novel anti-cancer drug candidate, especially for cancers with an abnormally high expression of Hsp27.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Fu, Weiming.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 111-151).Abstract also in Chinese.Abstract (English) --- p.iAbstract (Chinese) --- p.iivAcknowledgment --- p.viPublications --- p.viiiList of Contents --- p.ixList of Tables --- p.xiiList of Figures --- p.xiiiList of Abbreviations --- p.xvChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- General Introduction --- p.1Chapter 1.1.1 --- Overview of HCC --- p.1Chapter 1.1.2 --- Epidemiology of HCC in China and Hong Kong --- p.3Chapter 1.2 --- Etiology of HCC --- p.7Chapter 1.2.1 --- Cirrhosis --- p.8Chapter 1.2.2 --- HBV infection --- p.9Chapter 1.2.3 --- HCV infection --- p.10Chapter 1.2.4 --- Viral Co-Infection --- p.11Chapter 1.2.5 --- Fatty Liver Disease and Cryptogenic Cirrhosis --- p.12Chapter 1.2.6 --- Alcohol --- p.13Chapter 1.2.7 --- Iron --- p.13Chapter 1.2.8 --- Aflatoxin --- p.14Chapter 1.2.9 --- Others --- p.14Chapter 1.3 --- Diagnosis of HCC --- p.14Chapter 1.4 --- Prognosis of HCC --- p.17Chapter 1.5 --- Treatment of HCC --- p.19Chapter 1.5.1. --- Early stage --- p.19Chapter 1.5.2. --- Intermediate and advanced stage --- p.24Chapter 1.5.3. --- Terminal stage --- p.28Chapter 1.6 --- Signaling pathways in HCC --- p.28Chapter 1.6.1 --- Proliferation signaling pathways --- p.29Chapter 1.6.2 --- Signaling pathways frequently dysregulated in HCC --- p.30Chapter 1.6.3 --- Pathways involved in liver development and cell differentiation --- p.34Chapter 1.6.4 --- Inflammation pathways involved in hepatocarcinogenesis --- p.35Chapter 1.6.5 --- Pathways involved in neoangiogenesis --- p.37Chapter 1.6.6 --- The P53 tumor suppressor --- p.38Chapter 1.6.7 --- Heat shock proteins in HCC --- p.39Chapter 1.7 --- The roles of microRNAs in liver cancer progression --- p.42Chapter 1.8 --- TCM in the treatment of HCC --- p.45Chapter 1.8.1 --- Introduction --- p.45Chapter 1.8.2 --- Garcinia --- p.49Chapter 1.9 --- Objectives of the study --- p.51Chapter Chapter 2 --- Materials and Methods --- p.52Chapter 2.1 --- Preparation of the pure compounds --- p.52Chapter 2.2 --- Liver cell lines and tissue culture --- p.52Chapter 2.3 --- Human tissue samples --- p.52Chapter 2.4 --- Cell viability assessment with MTT assay --- p.53Chapter 2.5 --- Apoptosis analysis --- p.53Chapter 2.6 --- Two-dimensional electrophoresis (2-DE), protein visualization and image analysis --- p.54Chapter 2.6.1 --- Materials --- p.54Chapter 2.6.2 --- Protein extraction --- p.54Chapter 2.6.3. --- 2-DE protein profiling --- p.55Chapter 2.6.4. --- Gel staining and image analysis --- p.55Chapter 2.6.5. --- In-gel protein digestion with trypsin --- p.56Chapter 2.6.6. --- MALDI-TOF mass spectrometric analysis --- p.56Chapter 2.6.7. --- Database search --- p.57Chapter 2.7.1 --- Sample preparation --- p.58Chapter 2.7.2 --- SDS-PAGE --- p.58Chapter 2.7.3 --- Protein transfer --- p.58Chapter 2.7.4 --- Blocking --- p.59Chapter 2.7.5 --- Incubation with primary and secondary antibodies --- p.59Chapter 2.7.6 --- Proteins Visualization --- p.59Chapter 2.8 --- Real-time PCR --- p.60Chapter 2.9 --- Vector construction and lentivirus production --- p.61Chapter 2.9.1 --- Lenti-vector construction for Hsp27 expression --- p.61Chapter 2.9.2 --- Lentivirus production --- p.62Chapter 2.9.3 --- Lentivirus infection --- p.63Chapter 2.10 --- SiRNAs transfection. --- p.63Chapter 2.11 --- Identification of potential protein targets for TDP --- p.64Chapter 2.12 --- In Vivo Tumorigenesis --- p.64Chapter 2.13 --- Assay of chaperone activity of Hsp27 using lysozyme as substrate --- p.65Chapter 2.14 --- Mitochondria and cytosolic proteins preparation --- p.66Chapter 2.15 --- Immunohistochemistry (IHC) --- p.67Chapter 2.15.1 --- Preparation of paraffin tissue sections --- p.67Chapter 2.15.2 --- Immunostaining --- p.67Chapter 2.16 --- Methodology of this study --- p.68Chapter 2.17 --- Statistical analysis --- p.68Chapter CHAPTER 3 --- Results --- p.69Chapter 3.1 --- Introduction --- p.69Chapter 3.2 --- TDP significantly suppressed cell growth and induced apoptosis in HCC cells. --- p.69Chapter 3.2.1 --- TDP was identified from 102 pure compounds by using MTT assay --- p.69Chapter 3.2.2 --- TDP significantly suppressed HCC cell growth --- p.73Chapter 3.2.3 --- TDP induced the apoptosis of HCC cells --- p.74Chapter 3.3 --- Study of the molecular mechanism of TDP on HCC --- p.76Chapter 3.3.1 --- The comparative proteomic profiling --- p.76Chapter 3.3.2 --- Hsp27 was one of the molecular targets of TDP in HepG2 cells. --- p.80Chapter 3.3.3 --- TDP induced apoptosis through the caspase-dependent mitochondrial pathway. --- p.82Chapter 3.3.4 --- Hsp27 involved in the mitochondrial apoptosis induced by TDP --- p.84Chapter 3.3.5 --- Enforced Hsp27 overexpression rescued the mitochondrial apoptosis induced by TDP in HepG2 cells --- p.87Chapter 3.3.6 --- The possible regulatory signaling by TDP --- p.91Chapter 3.4 --- TDP directly targeted Hsp27 and destroyed its chaperone action --- p.92Chapter 3.5 --- Degradation of Hsp27 aggregation stimulated by TDP was mediated by ubiquitin-proteasome system (UPS) pathway --- p.96Chapter 3.6 --- Nude mice model demonstrated the suppressive effect of TDP on HCC --- p.97Chapter Chapter 4 --- Discussion and Conclusions --- p.100Chapter 4.1 --- Discussion --- p.100Chapter 4.2 --- Conclusion --- p.110Reference --- p.11