根據文獻研究記載，化療藥物可誘導癌細胞的凋亡，這是公認的化療療法的主要治療效果。作為一種程式性細胞死亡，積累的實驗證據表明，誘導所致的細胞凋亡是可逆轉的。這就引出了對於細胞凋亡恢復及其調節機制的相關問題。 / 在這項研究中，我們證明了在質膜不對稱的散失和半胱天冬酶（caspase）啟動後，HeLa細胞的凋亡的啟動可逆轉。我們發現，除了被廣泛研究的抗增殖作用外，金雀異黃素(genistein)可抑制細胞凋亡的復蘇。即時定量PCR發現抗凋亡基因MDM2和XIAP在凋亡逆轉過程中表達水準上調，金雀異黃素可抑制其表達水準的上調。金雀異黃素，MDM2蛋白抑制劑和XIAP抑制劑的利用，造成復原細胞內持續的半胱天冬酶活性和增強的細胞死亡效果。然而，半胱天冬酶抑制劑並不能挽救金雀異黃素的抑制作用。流式細胞儀的研究表明，金雀異黃素可以導致凋亡逆轉細胞持久磷脂醯絲氨酸（PS）外化和逆轉細胞的細胞壞死。抑制半胱天冬酶活性將金雀異黃素的主要作用轉移到壞死效果。這些結果揭示了金雀異黃素抑制細胞凋亡逆轉的兩個可能的機制。 / 金雀異黃素能維持現有的細胞凋亡信號從而增強細胞凋亡。它也可以破壞凋亡恢復過程，導致繼發性壞死。金雀異黃素對於細胞凋亡逆轉的抑制可與常規化療相結合，以提高治療結果. / It is well documented that chemotherapeutical agents could induce apoptosis of cancer cells, which is recognized as a major treatment effect of chemotherapy. Accumulating evidence indicates that chemopreventive agents like soybean isoflavone genistein could potentiate the antitumor effect of chemotherapeutic drugs both in vivo and in vitro. The mechanistic basis of this augmentation effect by genistein remains to be fully elucidated. / In this study, we demonstrated while low-concentration ethanol stressed cancer cells could recover, the presence of genistein promoted the cell death of stressed cancer cells that displayed apoptotic features. In HeLa cells, quantitative real-time PCR revealed the up-regulation of anti-apoptotic genes MDM2 and XIAP during the recovery process, and genistein suppressed their expression. The application of genistein, MDM2 inhibitor and XIAP inhibitor to the recovering HeLa cells caused persistent caspase activity and enhanced cell death. However, the death-promoting effect of genistein was not rescued by caspase inhibitor. Flow cytometry study indicated that genistein treatment could lead to persistent phosphatidylserine (PS) externalization and necrotic events in the recovering HeLa cells. Caspase activity inhibition shifted the major effect of genistein to secondary necrosis. / These results suggested two possible mechanisms through which genistein promoted cell death in stressed HeLa cells. Genistein could maintain the existing apoptotic signal to enhance apoptotic cell death. It could also disrupt the recovering process in caspase-independent manner, which lead to secondary necrosis. These effects may account for the enhanced antitumor effect of chemotherapeutic drugs when they were combined with genistein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Xin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 79-90). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in also in Chinese. / Cover Page / Statement --- p.i / Thesis Committee members --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Table of contents --- p.vi / List of abbreviations --- p.ix / List of figures and tables --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction to general cancer biology --- p.1 / Chapter 1.1.1 --- Overview of cancer --- p.1 / Chapter 1.1.1.1 --- Classification of cancer --- p.1 / Chapter 1.1.1.2 --- Risk factors of carcinogenesis --- p.2 / Chapter 1.1.1.3 --- Cancer prevention and therapies --- p.4 / Chapter 1.1.2 --- Models of cancer development --- p.6 / Chapter 1.1.2.1 --- Multistage model of carcinogenesis --- p.6 / Chapter 1.1.2.2 --- Colorectal cancer as an example of multistep / multigene carcinogenesis --- p.7 / Chapter 1.1.2.3 --- Driving force for cancer development --- p.9 / Chapter 1.1.3 --- Properties of cancer cells --- p.11 / Chapter 1.2 --- Apoptosis and its roles in cancer development and treatment --- p.14 / Chapter 1.2.1 --- Overview of apoptosis --- p.14 / Chapter 1.2.2 --- Molecular mechanism of apoptosis --- p.15 / Chapter 1.2.3 --- Positive and negative regulation of apoptosis --- p.18 / Chapter 1.2.4 --- Apoptotic defects in cancer development --- p.20 / Chapter 1.2.5 --- Apoptosis in cancer treatment --- p.23 / Chapter 1.3 --- The reversibility of apoptotic events --- p.25 / Chapter 1.4 --- Genistein and its relevance to cancer therapy --- p.27 / Chapter 1.5 --- Objectives of the study --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Cancer cell lines --- p.30 / Chapter 2.1.2 --- Cell culture media and additives --- p.30 / Chapter 2.1.3 --- Biochemical kits --- p.30 / Chapter 2.1.4 --- Chemicals and reagents --- p.30 / Chapter 2.1.5 --- Antibodies --- p.31 / Chapter 2.1.6 --- Primers used for quantitative real-time PCR --- p.32 / Chapter 2.1.7 --- Buffers and solutions --- p.32 / Chapter 2.2 --- Methods and procedures --- p.33 / Chapter 2.2.1 --- Cell culture establishment and cryopreservation --- p.33 / Chapter 2.2.2 --- Living cell staining and imaging --- p.34 / Chapter 2.2.3 --- MTT cell viability assay --- p.34 / Chapter 2.2.4 --- BrdU cell proliferation assay --- p.35 / Chapter 2.2.5 --- LDH cytotoxicity assay --- p.35 / Chapter 2.2.6 --- Quantitative real-time PCR --- p.36 / Chapter 2.2.7 --- Western blotting --- p.37 / Chapter 2.2.8 --- Annexin V/ Propidium Iodide Assay --- p.38 / Chapter 2.2.9 --- Trypan Blue Dye Exclusion Assay --- p.39 / Chapter 2.2.10 --- Cleaved-Caspase 3 Immunostaining --- p.39 / Chapter 2.2.11 --- Statistical Analysis --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Low concentration ethanol stressed cancer cells displayed apoptotic features and the stressed cell could recover after stress removal --- p.40 / Chapter 3.1.1 --- Morphological changes and apoptotic marker activation in low concentration ethanol stress --- p.40 / Chapter 3.1.2 --- In situ study of morphological changes and caspase 3 activation in HeLa --- p.44 / Chapter 3.2 --- Genistein promoted the cell death of stressed cancer cells at non-cytotoxic concentration towards unstressed cells --- p.46 / Chapter 3.2.1 --- Dose-dependent response of genistein on stressed and unstressed cells --- p.46 / Chapter 3.2.2 --- In HeLa cells, genistein suppressed the recovery from stress treatment at non-cytotoxic concentration --- p.48 / Chapter 3.2.3 --- Genistein promoted both apoptosis and necrosis in stressed cells. . --- p.49 / Chapter 3.3 --- Genes involved in the recovery from stress treatment were influenced by genistein --- p.53 / Chapter 3.3.1 --- Stressed HeLa cells were more sensitive to the inhibition of de novo synthesis --- p.53 / Chapter 3.3.2 --- Expression profiles of genes involved in recovery and the influence of genistein --- p.55 / Chapter 3.4 --- Like genistein, MDM2 and XIAP inhibitor potentiated the cell death and caused persistent caspase-3 activity in stressed cells --- p.58 / Chapter 3.4.1. --- Stressed HeLa cells were much more sensitive to the inhibition of XIAP and MDM2 --- p.58 / Chapter 3.4.2 --- The presence of inhibitor at non-cytotoxic concentration to unstressed cells suppressed the recovery of the stressed cells --- p.60 / Chapter 3.4.3 --- Genistein, MDM2 inhibitor and XIAP inhibitor caused persistent apoptotic signals in recovering cells. --- p.61 / Chapter 3.5 --- The death-promoting effect by genistein could be caspase-independent --- p.64 / Chapter 3.6 --- Caspase activity abrogation shifted genistein’s action profile --- p.66 / Chapter Chapter 4 --- Discussion and prospect --- p.70 / Chapter 4.1 --- The apoptotic features were induced by low concentration ethanol stress --- p.70 / Chapter 4.2 --- The apoptotic features caused by ethanol stress were reversible --- p.71 / Chapter 4.3 --- Genistein showed death-promoting effects on the recovering cells --- p.72 / Chapter 4.4 --- The genes (XIAP and MDM2) that were involved in the recovery process may function to terminate apoptotic signal --- p.73 / Chapter 4.5 --- Genistein suppressed the upregulation of anti-apoptotic genes and promoted the expression of pro-apoptotic genes --- p.74 / Chapter 4.6 --- The XIAP and MDM2 activity were essential for the recovery from stress --- p.75 / Chapter 4.7 --- Caspase inhibition increased the secondary necrosis in recovering cells with genistein treatment --- p.76 / Chapter 4.8 --- Hypothetic mechanism of genistein’s inhibitory effect on the recovery of stressed cells --- p.77 / Chapter 4.9 --- Summary and prospects --- p.78 / Reference list --- p.79
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328003 |
| Date | January 2012 |
| Contributors | Xie, Xin., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xi, 90 leaves) : ill. (some 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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