宿主防御肽，如抗菌肽和防御素，是固有性免疫的重要组成部分。LL-37是由37个残基组成的阳离子宿主防御肽，是目前唯一被发现的人源宿主防御肽。它在不同的生物过程中都起着关键作用。新证据表明，LL-37与肿瘤进展也有关系。在许多类型的人类恶性肿瘤中它有不同表达，但在结肠癌中的表达和作用，仍未知。在此，我们将对LL-37及其17至32残基片断（ 简称FK-16）对结肠癌的影响进行研究。 / 免疫组化染色结果表明，LL-37在人类结肠癌组织中的表达比正常组织有显著减少。并且，LL-37的表达与TUNEL阳性细胞数量呈正比。合成的LL-37能够诱导不同的结肠癌细胞发生不依赖半胱天冬酶激活的凋亡细胞死亡。并且，LL-37通过激活p53下调Bcl2及上调Bax与Bak来诱导凋亡。LL-37也促使肿瘤坏死因子和核酸内切酶G 向核内转移，以其为目标的siRNA沉默能使细胞对LL-37诱导的凋亡呈现出耐受现象。更重要的是，LL-37的促凋亡作用被发现可以通过对百日咳敏感的Gαi蛋白偶联受体来介导。同时，宿主防御肽敲除的小鼠肠黏膜中，p53、Bax和Bak表达减少而Bcl2表达增加，凋亡的基础水平量也减少。由此说明，LL-37可通过激活GPCR-p53-Bax / Bak / Bcl-2的新信号级联反应来激活AIF / EndoG调控的结肠癌细胞凋亡。 / 与LL-37类似，其片断FK-16也促使不同结肠癌细胞株死亡。但其死亡诱导机制与LL-37不尽相同。FK-16引发了一种独特的死亡方式，即初始诱导不依赖半胱天冬酶激活的凋亡之后紧随引发自噬性细胞死亡。而LL-37没有明显引起这种自噬性死亡。孵育FK-16 24小时后，结肠癌细胞被证明发生凋亡。延长孵育至48小时，细胞的生化和形态学体征符合自噬，包括增加LC3阳性自噬体，积累酸性自噬泡与自溶酶体，和提高 LC3-II水平。敲除两个自噬有关基因 ATG5 和ATG7, 能够部分逆转由FK-16所引起的细胞死亡。并且，细胞凋亡和细胞自噬机制相关信号通路之间存在的交叉调控，在此研究中也被深入提及。 / Host defense peptides, such as cathelicidins (LL-37) and defensins, are important components in the innate immunity. LL-37, a human cationic host defense peptide composed of 37 residues, is the only cathelicidin described so far in humans. It plays a key role in diverse biological processes, including natural immunity, inflammation and tissue repair. Emerging evidence suggests that LL-37 is implicated in cancer development. In this regard, the expression of LL-37 is found to be dysregulated in many types of human malignancy, including lung, breast, ovarian, and gastric cancers. The expression and function of LL-37 in colon cancer, however, are still unknown. In this thesis, the roles of LL-37 and its 17-32 fragment (hereafter referred to as FK-16) in colon cancer development were investigated. / By immunohistochemical staining, it is demonstrated that the expression of LL-37 was significantly reduced in human colon cancer tissues as compared with the cancer adjacent normal tissues. Moreover, LL-37 expression was positively correlated with the number of TUNEL-positive cells. Furthermore, synthetic LL-37 induced caspase-independent apoptotic cell death in different cultured colon cancer cells. In this connection, LL-37 induced apoptosis via downregulation of Bcl-2 and upregulation of Bak and Bax in a p53-dependent manner. It also induced the upregulation and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG), whose targetings by siRNAs rendered the cells resistant to LL-37-induced apoptosis. Above all, the pro-apoptotic effect of LL-37 was found to be mediated through a pertussis toxin-sensitive Gαi protein-coupled receptor. Concordantly, colonic mucosa of cathelicidin-knockout mice exhibited reduced expression of p53, Bax and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Taken together, we demonstrated that LL-37 activates a novel signaling cascade involving the GPCR-p53-Bax/Bak/Bcl-2 axis to activate AIF/EndoG-mediated apoptosis in colon cancer cells. / Similar to the effect of LL-37 peptide, the fragment FK-16 also induced cell death in colon cancer cell lines. However, the action is different. Results demonstrated that FK-16 triggered a unique pattern of cell death characterized by initial caspase-independent apoptosis followed by autophagic cell death, the latter of which was not observed obviously in cells treated with LL-37. Treating colon cancer cells with FK-16 for 24 h induced apoptosis as evidenced by phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Prolonged treatment with FK-16 induced biochemical and morphological features consistent with autophagy, including increased formation of LC3+ autophagosomes, the accumulation of acidic vesicular organelles and autolysosomes, and increased levels of LC3-I/II, Atg5 and Atg7. Knockdown of Atg5 or Atg7 partially reversed the cytotoxic effect of FK-16, suggesting that FK-16-induced autophagy was pro-death in nature. Furthermore, the novel cross-talks between apoptotic and autophagic signalings were also noted. / Collectively, the present study not only contributes to understanding the role of host defense peptide cathelicidin in tumorigenesis, but also provides pre-clinical evidence to propel the development and application of these peptides as novel therapeutic agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / 综上所述，目前的研究不仅有助于理解宿主防御肽在肿瘤发生, 同时也提供了临床前研究证据，推动了宿主防御肽的开发和应用, 这些肽片段为治疗结肠癌提供了新的治疗手段 。 / Ren, Shunxiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 176-208). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Declaration --- p.vi / Acknowledgements --- p.vii / Publications --- p.ix / Table of contents --- p.xiii / List of illustrations --- p.xviii / Abbreviations --- p.xxiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology of colorectal cancer --- p.1 / Chapter 1.1.2 --- Etiology of colorectal cancer --- p.3 / Chapter 1.1.3 --- Pathogenesis of CRC --- p.8 / Chapter 1.1.4 --- Chemotherapy of colorectal cancer --- p.9 / Chapter 1.2 --- Programmed cell death (PCD) --- p.10 / Chapter 1.2.1 --- Cell death --- p.10 / Chapter 1.2.2 --- Apoptosis --- p.11 / Chapter 1.2.2.1 --- Mechanisms of apoptosis --- p.12 / Chapter 1.2.2.1.1 --- Caspase-dependent apoptosis --- p.13 / Chapter 1.2.2.1.2 --- Caspase-independent apoptosis --- p.15 / Chapter 1.2.2.1.3 --- Tumor suppressor p53 --- p.17 / Chapter 1.2.2.2 --- G-coupled protein receptors in apoptosis --- p.18 / Chapter 1.2.3.1 --- Types of Autophagy --- p.20 / Chapter 1.2.3.2 --- Biological process --- p.22 / Chapter 1.2.3.3 --- Biological functions --- p.24 / Chapter 1.2.3.4 --- Autophagic machinery --- p.27 / Chapter 1.2.3.5 --- Autophagy in cancer --- p.30 / Chapter 1.2.3.6 --- Autophagy and apoptosis --- p.32 / Chapter 1.3 --- Biological functions of cathelicidin --- p.33 / Chapter 1.3.1 --- Antimicrobial activity --- p.34 / Chapter 1.3.2 --- Immunological functions --- p.35 / Chapter 1.3.3 --- Wound healing, angiogenesis and mitogenesis --- p.36 / Chapter 1.3.4 --- Programmed cell death --- p.38 / Chapter 1.4 --- Aim of the present study --- p.39 / Chapter Chapter 2 --- Methods / Chapter 2.1 --- General --- p.41 / Chapter 2.1.1 --- Chemicals and reagents --- p.41 / Chapter 2.1.2 --- Antibodies --- p.44 / Chapter 2.1.3 --- Commercial kits --- p.45 / Chapter 2.1.4 --- Peptide synthesis --- p.46 / Chapter 2.1.5 --- Experimental Animals --- p.46 / Chapter 2.1.6 --- Cell Culture --- p.47 / Chapter 2.2 --- DNA Methylation Analysis --- p.48 / Chapter 2.2.1 --- 5-aza-2’-deoxycytidine (5’Aza-dC) Treatment --- p.48 / Chapter 2.2.2 --- Bisulfite Genomic Sequencing and Methylated-DNA capture (MethylCap)-qPCR --- p.48 / Chapter 2.3 --- Effects of LL-37 and its analogue FK-16 in colon cancer cells in vitro --- p.48 / Chapter 2.3.1 --- Cell viability Assay --- p.49 / Chapter 2.3.2 --- Lactic dehydrogenase (LDH) activity --- p.49 / Chapter 2.3.3 --- Cell cycle analysis --- p.49 / Chapter 2.3.4 --- Measurement of apoptosis in vitro --- p.50 / Chapter 2.3.4.1 --- Quantitation of DNA fragmentation --- p.50 / Chapter 2.3.4.2 --- Quantitation of phosphatidylserine externalization --- p.50 / Chapter 2.3.5 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.3.6 --- Nuclear protein extraction --- p.52 / Chapter 2.3.7 --- Western Blot --- p.53 / Chapter 2.3.8 --- Immunofluorescence --- p.54 / Chapter 2.3.9 --- Bcl-2 overexpression --- p.54 / Chapter 2.3.9.1 --- Transforming competent cells --- p.55 / Chapter 2.3.9.2 --- Plasmid DNA purification --- p.55 / Chapter 2.3.10 --- RNA interference --- p.56 / Chapter 2.3.11 --- Detection of acidic vesicular organelles (AVOs) with acridine orange --- p.57 / Chapter 2.3.12 --- Labeling of autophagic vacuoles with monodansylcadaverine (MDC) --- p.58 / Chapter 2.3.13 --- Transmission electron microscopy --- p.59 / Chapter 2.4 --- Cathelicidin-knockout (Cnlp/) mice model --- p.60 / Chapter 2.4.1 --- Normal mouse colon sample collection --- p.60 / Chapter 2.4.2 --- Tissue Processing --- p.61 / Chapter 2.4.3 --- Measurement of basal apoptosis in normal colon tissues --- p.61 / Chapter 2.5 --- Clinical samples --- p.62 / Chapter 2.5.1 --- Immunohistochemistry of clinical samples --- p.62 / Chapter 2.5.2 --- Measurement of colonocyte apoptosis of clinical samples --- p.62 / Chapter 2.5.3 --- Evaluation of colonocyte proliferation of clinical samples --- p.63 / Chapter 2.6 --- Statistical analysis --- p.63 / Chapter Chapter 3 --- Results and Discussion / Chapter 3.1 --- LL-37 was downregulated in colon cancer tissues --- p.64 / Chapter 3.2 --- Effects of LL-37 on human colon cancer cells --- p.72 / Chapter 3.2.1 --- LL-37 induced DNA fragmentation and phosphatidylserine externalization without caspase activation in colon cancer cells --- p.72 / Chapter 3.2.2 --- LL-37 induced AIF- and EndoG-dependent apoptosis --- p.79 / Chapter 3.2.3 --- Altered expression of Bcl-2 family members was required for AIF- and EndoG-mediated apoptosis induced by LL-37 --- p.84 / Chapter 3.2.4 --- p53 activation was required for LL-37-induced apoptosis --- p.90 / Chapter 3.2.5 --- The apoptogenic action of LL-37 was mediated by G protein-coupled receptor (GPCR) --- p.94 / Chapter 3.2.6 --- Reduced basal apoptotic rate in colonic mucosa of cathelicidin-knockout mice --- p.95 / Chapter 3.2.7 --- Preliminary Discussion --- p.103 / Chapter 3.3 --- Effects of FK16 on human cancer cells --- p.108 / Chapter 3.3.1 --- FK-16 induced AIF- and EndoG-dependent apoptosis in colon cancer cells --- p.108 / Chapter 3.3.2 --- FK-16 induced autophagic cell death in colon cancer cells --- p.116 / Chapter 3.3.3 --- Activation of p53 was required for FK-16-indcued apoptosis and autophagy cell death --- p.123 / Chapter 3.3.4 --- Altered expression of Bcl-2 and Bax was required for FK-16-indcued apoptosis and autophagic cell death --- p.129 / Chapter 3.3.5 --- FK-16-induced apoptosis and autophagic cell death were reciprocally regulated --- p.134 / Chapter 3.3.6 --- Preliminary Discussion --- p.139 / Chapter Chapter 4 --- Summary and Finial Conslusion --- p.142 / References --- p.144
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328798 |
| Date | January 2012 |
| Contributors | Ren, Shunxiang., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, xxvii, xxvi, 208 leaves : ill. (chiefly col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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