published_or_final_version / Surgery / Master / Master of Surgery

癌症復發往往伴隨著耐藥性和轉移率的增加。目前我們仍未完全瞭解確切的腫瘤逃脫機制。皮下無水酒精注射（PEI）已經被用於治療肝細胞癌（HCC）幾十年，而PEI治療後的癌症復發仍然是該方法的一個主要限制。最近有許多證據表明癌細胞能夠逆轉化學誘導的細胞凋亡過程而得以存活，這有可能是其中一個導致癌細胞復發的原因。這篇論文的重點在於研究肝癌細胞HepG2經歷乙醇誘導凋亡事件後存活下來的後果。 / 這個研究首先證實肝癌細胞 HepG2能從乙醇誘導凋亡事件後存活下來。然後我們對存活下來的肝癌細胞HepG2進行增殖率，耐藥性，運動性以及侵襲性的研究。結果表明，存活下來的HepG2有46%的乙醇耐藥性和84%的高運動性。然後爲了發現存活下來的HepG2是否對其他臨床常用藥物也同樣具有耐藥性，4種臨床常用藥物包括阿黴素，紫杉醇，順鉑，5-氟尿嘧啶（5Fu）均被用於測試。有趣的是，存活下來的HepG2對5-氟尿嘧啶變得更加敏感，平均敏感性下降了58.2%。 / 總的來說，我們的研究結果表明肝癌細胞可從乙醇誘導凋亡事件中恢復過來。此外，存活下來的細胞變得更具有耐藥性和侵入性。這種恢復過程可能是導致癌症復發的原因之一。出乎意料的是，雖然所有存活下來的細胞對乙醇具耐受性，但是它們對於5-氟尿嘧啶均變得更加敏感。這些結果表明，乙醇和5-氟尿嘧啶的聯合治療可能有助於提高PEI治療效果從而預防肝癌癌症復發。 / Cancer relapse, associated with increased drug resistance and higher rate of metastasis, often occurs after chemotherapy. The cancer escape mechanisms are still incompletely understood. Percutaneous ethanol injection (PEI) has been used for treating hepatocellular carcinoma (HCC) for decades, but the recurrence after PEI treatment remains a major limitation. Recently there are mounting evidences showing that cancer cells could survive from chemical-induced apoptosis, suggesting a potential route through which cancer relapse may occur. This thesis focuses on the consequences of the recovery of HepG2 cells from ethanol-induced apoptotic event. / This study verified that HepG2 cells could recover from ethanol-induced apoptosis. Proliferation rate, drug resistance, motility and invasiveness were investigated in recovered HepG2 cells. On average, the recovered HepG2 cell clones were found to be 46% more resistant to ethanol and 84% higher in motility than the parental cell clones. And then four commonly used clinical drugs were assayed to determine whether the recovered cell clones were also resistant to other clinical drugs, including doxorubicin, docetaxel, cisplatin and 5-fluorouracil (5-Fu). Interestingly, the recovered clones became 58.2% more sensitive to 5-fluorouracil on average. / In conclusion, our findings showed that HepG2 cells can recover from ethanol-induced apoptotic event. In addition, some cell clones recovered from apoptosis became more resistant to ethanol and some became more invasive. Such recovery might be one of the reasons causing cancer recurrence. Unexpectedly, although the recovered cell clones were more resistant to ethanol, they became more sensitive to 5-Fu treatment. These results indicated that ethanol-5-Fu combined treatment might be useful in enhancing the PEI treatment and preventing HCC cancer recurrence. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Shanshan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 115-130). / Abstracts also in Chinese.

Insensitivity to chemotherapy is an ongoing issue in cancer treatment, one that appears to be highly dependent on patient-specific variations. It has been shown clinically that while a subset of patients will successfully respond to a particular chemotherapeutic regimen, there exists another subset of patients who when exposed to the same course of therapy will remain resistant to treatment or exhibit signs of relapse after treatment has been administered. This discrepancy raises interesting questions regarding the role that patient-specific variations play in controlling the efficacy of chemotherapy treatment regimens. Doxorubicin (Dox) is a common chemotherapeutic agent used in the treatment of a variety of solid tumors and leukemias and resistance to Dox treatment is a major issue in cancer chemotherapy, oftentimes leading to patient relapse. To gain a deeper understanding of the processes that influence Dox resistance, we must first understand the mechanisms that underlie and contribute to Dox's toxicity. To this end, the metabolic reactions that activate Dox have been implicated as major determinants of Dox cytoxicity and as possible factors that control Dox resistance in cancer cells. There are several lines of evidence that redox-dependent metabolism plays a large role in Dox toxicity. The Dox bioactivation network is comprised of a system of reduction/oxidation (redox) reactions that lead to the formation of toxic Dox metabolites and reactive oxygen species (ROS). Moreover, multi-drug resistant acute lymphoblastic leukemia cells derived from relapsed patients have elevated levels of the antioxidant glutathione and show insensitivity to Dox treatment. The redox dependence of Dox bioactivation, the understanding that Dox treatment generates ROS, and the evidence that Dox resistant cells exhibit increased antioxidant capacity, suggest the possibility that redox pathways modulate the efficacy of Dox treatment in cancer cells. The overall objectives of the proposed dissertation, therefore, were to investigate how the redox properties of the Dox bioactivation network influence Dox toxicity in acute lymphoblastic leukemia cells, and to provide evidence that cell-specific variations in the intracellular levels of these redox components influences the degree to which Dox treatment will induce cancer cell death. The significant findings of this study are that the redox reactions involved in Dox metabolism are dual-natured, containing a toxicity-generating module characterized by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Dox reductive conversion, as well as an ROS signal-generating module characterized by NADPH- and oxygen-dependent Dox redox cycling. The balance between the coupled redox reactions that comprise the toxicity- and ROS signal-generating modules of Dox bioactivation determines the sensitivity-phenotype of leukemia cells and phenotypic changes in the Dox-sensitivity of leukemia cells can be induced by the successful modulation of the Dox bioactivation network through the pharmacological inhibition of NADPH in a concentration- and cell type-dependent manner. This study highlights the importance of the intracellular redox network in controlling chemotherapy-induced ROS. The unequal distribution in antioxidant burden across the various intracellular antioxidant enzymes suggests a significant role for NADPH supply, as controlled by the enzyme glucose-6-phosphate dehydrogenase (G6PD), to the intracellular ROS buffering capacity of cells during instances of oxidative stress. Changes in G6PD activity were shown to promote protein-S-glutathionylation during oxidative stress conditions, thereby implicating G6PD in the modulation of redox-sensitive signal transduction pathways. The intracellular glutathione redox balance, a measure of the intracellular redox environment, can effectively regulate Dox-induced NF-κB signal transduction in leukemia cells. The systematic modulation of intracellular glutathione redox balance in leukemia cells by N-acetylcysteine (NAC) revealed an important role for protein S-glutathionylation mechanisms in the control of NF-κB signal transduction induced by Dox treatment. These findings identify the glutathione redox network as a potential therapeutic target for the systematic modulation of Dox sensitivity in cancer cells and elucidate the complex role that antioxidants such as NAC can play in modulating the effectiveness of Dox chemotherapy treatment regimens. Lastly, this study highlights the need for and the capacity of computational models to accurately describe the complex redox-reactions that contribute to Dox metabolism in leukemia cells. This study is groundbreaking in its use of computational modeling to analyze reversible electron transfer events between proteins using mass-action kinetics. The models developed in this study can accurately explain cytosolic doxorubicin bioactivation, intracellular hydrogen peroxide clearance, and kinase-specific S-glutathionylation, thereby showing that the use of comprehensive and/or relatively simple computational models can provide semi-quantitative predictions about the behavior of redox systems in mammalian cells as they relate to Dox-induced toxicity and Dox-induced cell signaling.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

Esse trabalho contribui com o desenvolvimento de um novo modelo para dados de sobrevivência com sobreviventes de longo termo visando uma formulação e interpretação mais realista do que a apresentada pelos modelos com fração de curados usuais. Motivados pelo estudo do tempo de sobrevivência residual para pacientes oncológicos, o modelo usa o processo de nascimento e morte para permitir a variação do número de fatores de risco latentes durante um período precedente ao acompanhamento médico, considerando, então, um cenário de riscos competitivos para obtenção da função da sobrevivência (imprópria) dos pacientes. Simulações a aplicações à dados do Instituto do Câncer do Estado de São Paulo mostraram vantagens sobre o modelo de tempos de promoção. / This work contributes with a new cure rate survival model developed aiming more realistic formulation and interpretations than the usual long-term survival models. Motivated by studying residual survival times in oncological patients, the model uses birth and death process to allow free variation on the number of latent risk factors during a pre-follow up period, then considers competing risks scenario for accessing the patients survival. Simulations and application to Instituto do Câncer do Estado de São Paulo data showed improvement over the promotion time model.

Esse trabalho contribui com o desenvolvimento de um novo modelo para dados de sobrevivência com sobreviventes de longo termo visando uma formulação e interpretação mais realista do que a apresentada pelos modelos com fração de curados usuais. Motivados pelo estudo do tempo de sobrevivência residual para pacientes oncológicos, o modelo usa o processo de nascimento e morte para permitir a variação do número de fatores de risco latentes durante um período precedente ao acompanhamento médico, considerando, então, um cenário de riscos competitivos para obtenção da função da sobrevivência (imprópria) dos pacientes. Simulações a aplicações à dados do Instituto do Câncer do Estado de São Paulo mostraram vantagens sobre o modelo de tempos de promoção. / This work contributes with a new cure rate survival model developed aiming more realistic formulation and interpretations than the usual long-term survival models. Motivated by studying residual survival times in oncological patients, the model uses birth and death process to allow free variation on the number of latent risk factors during a pre-follow up period, then considers competing risks scenario for accessing the patients survival. Simulations and application to Instituto do Câncer do Estado de São Paulo data showed improvement over the promotion time model.

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras. / Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.