La chimio-embolisation hépatique (CHE) est recommandée pour le carcinome hépatocellulaire (CHC) non curable. L'utilisation de plateformes de release au cours des CHE permet la libération lente de la chimiothérapie dans les artères tumorales et l'exposition prolongée des cellules tumorales tout en minimisant les effets systémiques. L’émulsion lipiodolée est l'une des plateformes les plus couramment utilisées pour la CHE du CHC. Cependant, la libération de chimiothérapie se produit trop rapidement car les émulsions lipiodolées sont très instables Tout d'abord, nous avons démontré qu’une meilleure stabilité (utilisation d’un tensioactif synthétique comme émulsifiant) améliore les propriétés thérapeutiques de l'émulsion lipiodolée dans un modèle de lapin porteur de tumeurs VX2. Une revue de la littérature indique également que l'émulsion lipiodolée doit être de type eau dans l'huile (w/o) pour une meilleure sélectivité tumorale. Ensuite, nous avons analysé les paramètres techniques qui formulent des émulsions w/o avec la meilleure stabilité et sans émulsifiant supplémentaire. Nous avons constaté que l'incorporation progressive de la chimiothérapie dans le Lipiodol aboutit à un type d’émulsion w/o beaucoup plus prévisible et à une plus grande stabilité. Nous avons également constaté qu’un ratio volumique élevé Lipiodol / chimiothérapie est un paramètre important pour la stabilité de l’émulsion. Ensuite, nous avons développé un nouveau concept d'émulsion pour CHE qui utilise des nanoparticules d'acide polylactique-co-glycolique (PLGA) (Nps) pour stabiliser le Lipiodol et la chimiothérapie (Emulsion de Pickering). L'analyse in-vitro a démontré que l'émulsion de Pickering est la seule plateforme de release (versus émulsion lipiodolée ou billes chargées) qui permet une libération lente et complète de diverses chimiothérapies (doxorubicine, irinotécan, oxaliplatine) mais aussi d'anticorps (anti-CTLA4), ce qui fait de l'émulsion Pickering une plateforme de release universelle, non seulement pour la CHE mais aussi pour l'immunothérapie intra-artérielle. Enfin, la CHE avec de l'oxaliplatine a été réalisé dans un modèle de lapin porteurs de tumeurs VX2 pour comparer l'émulsion de Pickering et l'émulsion lipiodolée. Grâce à son release lent, l'émulsion de Pickering a considérablement diminué l'exposition systémique, mais également augmenté de manière significative le ratio tumeur / foie de l'exposition à l’oxaliplatine. / Liver Trans-Arterial Chemo-Embolization (TACE) is recommended for non-resectable, intermediate Hepatocellular carcinoma (HCC). The use of drug delivery platforms during TACE allows slow release of chemotherapy into the tumors’ arterial supply and prolonged exposure of the tumor cells while minimizing systemic exposures. Lipiodol-emulsion is one of the most widely used drug delivery platforms for TACE of HCC. However, the release of chemotherapy occurs very rapidly because Lipiodol-emulsions have poor stability. First, we have demonstrated that high stability (using synthetic surfactant as emulsifier) improves the therapeutic properties of Lipiodol-emulsion in a VX2-tumor rabbit model. A literature review also indicates that Lipiodol-emulsion must be water-in-oil (w/o) for better tumor’ uptakes. Next, we analyzed technical parameters that formulate in-vitro w/o emulsions with better stability without any additional emulsifier. We found that progressive incorporation of chemotherapy in the Lipiodol results in much more predictable w/o type and higher stability and that higher ratio of Lipiodol/chemotherapy is also a critical parameter for stability. Then, we develop a new concept of emulsion for TACE that uses Polylactic-co-glycolic acid (PLGA) nanoparticles (Nps) to stabilize Lipiodol and chemotherapy (Pickering-emulsion). In-vitro analysis demonstrated that Pickering-emulsion is the only drug delivery system (versus Lipiodol-emulsion and drug eluding beads) that allows slow and complete release of various chemotherapies (doxorubicin, irinotecan, oxaliplatin) but also of antibodies (anti-CTLA4), making Pickering-emulsion an universal carrier, not only for TACE but also for trans-arterial immunotherapy. Finally, TACE with oxaliplatin were performed in a VX2-tumor rabbit model to compare in-vivo Pickering-emulsion and Lipiodol-emulsion. Thanks to its slow drug release, Pickering-emulsion significantly decreased the systemic exposure but significantly increased the tumor/liver ratio oxaliplatin exposure.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20607号 / 医博第4256号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 松田 道行, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Simple Summary Gene therapy is a novel approach to treat diseases by introducing corrective genetic information into target cells. Adeno-associated virus vectors are the most frequently applied gene delivery tools for in vivo gene therapy and are also studied as part of innovative anticancer strategies. Here, we report on the natural preference of AAV2 vectors for hepatocellular carcinoma (HCC) compared to nonmalignant liver cells in mice and human tissue. This preference in transduction is due to the improved intracellular processing of AAV2 vectors in HCC, resulting in significantly more vector genomes serving as templates for transcription in the cell nucleus. Based on this natural tropism for HCC, novel therapeutic strategies can be designed or existing therapeutic approaches can be strengthened as they currently result in only a minor improvement of the poor prognosis for most liver cancer patients. Abstract Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

No description available.

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). The virally encoded X antigen, HBx, contributes importantly to the development of HCC through its trans-activating role in various signal transduction pathways. Pathways implicated in stem cell self-renewal also contribute to carcinogenesis. Thus, experiments were designed to test if HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HBx expressing (HepG2X) and control (HepG2CAT) human cell lines were assayed for phenotypic and molecular characteristics of "stemness." Western blotting of protein extracts from HepG2X and HepG2CAT cells as well as immunohistochemical staining of HCC and adjacent liver tissue sections from HBV infected patients showed up-regulation of "stemness"-associated (EpCAM and beta-catenin) and "stemness" (Oct-4, Nanog, Klf-4) markers by HBx. Moreover, HBx stimulated cell migration and spheroid formation. HBx expression was also associated with depressed levels of E-cadherin and subsequent activation of beta-catenin and EpCAM. Results from ChIP-chip data performed previously in this lab suggest an associative link between HBx and the expression of epigenetic co-repressor, mSin3A, which is known to repress E-cadherin when complexed with histone deacetylases. Thus, experiments were also designed to test if HBx represses the E-cadherin gene (CDH1) through histone deacetylation by the mSin3A/HDAC complex. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was showed by immunofluorescent staining. Chromatin immunoprecipitation revealed that HBx mediated the recruitment of the mSin3A/HDAC complex to the CDH1 promoter. HDAC inhibition by Trichostatin A treatment restored E-cadherin expression. Thus, HBx-associated epigenetic repression of E-cadherin and up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristic of CSCs. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. / Biology

Chronic infection with hepatitis B virus (HBV) is a primary risk factor for the development of hepatocellular carcinoma (HCC). HCC is the fifth most common cancer type worldwide with few treatment options. The hepatitis B encoded x antigen (HBx) plays a crucial role in the pathogenesis of HCC through several mechanisms. HBx alters signaling pathways shown to promote carcinogenesis and mediates epigenetic changes that silence tumor suppressor genes and activate host oncogenes. Short chain fatty acids (SCFAs) are made by selected gut bacteria with largely anti-inflammatory properties. They alter gene expression by functioning as histone deacetylase inhibitors (HDACi) and can bind to G protein coupled receptors (GCPR) to stimulate signaling pathways. Due to the documented anti-cancer properties of SCFAs, experiments were designed to test the hypothesis that SCFAs delay the development of HCC in HBx transgenic (HBxTg) mice. A diet of SCFAs was fed to HBxTg for three months prior to the expected appearance of dysplastic nodules and HCC. The results showed a statistically significant reduction in the number of dysplastic nodules as well as the presence and frequency of HCC. The effect of SCFAs on tumor growth was also evaluated in nude mice subcutaneously injected with human HCC cells. Tumor size in SCFA-treated mice was statistically smaller compared to the controls. The effect of SCFAs on cell viability of cancer and primary human hepatocytes was evaluated. SCFAs were shown to reduce cell viability in cancer cells only, with no effect on primary hepatocytes. Proteomics was performed on SCFA-treated compared to control livers from HBxTg to investigate changes on the molecular level that are associated with reduced preneoplastic and neoplastic nodule formation. Pathway analysis showed a decrease in important cancer-promoting pathways altered by HBx in HCC, including inflammation, oxidative stress, PI3K, VEGF, EGF, and Ras. These pathways are involved in biological processes central to carcinogenesis such as cell proliferation, survival, and angiogenesis. The ability of SCFAs to decrease these pathways has never been demonstrated. Further investigation confirmed that Ras activity was decreased in 12-month old livers treated with SCFAs. Taken together, these results show that SCFAs are capable of delaying the rate of tumor growth and tumor frequency in two mouse models of HBV-associated HCC, as well as reduce cell viability in cancer cells specifically. This data suggests that SCFAs may be a novel treatment option for HBV-associated HCC. / Biology

Chronic Hepatitis B Virus (HBV) infection is a global health problem because of its connection to acute and chronic liver diseases as well as hepatocellular carcinoma (HCC). There is increasing evidence showing that HBV contributes to HCC due to persistently high levels of trans-activating protein---hepatitis B encoded x antigen (HBxAg). Studies have shown that the HBxAg affects and alters the activity of many different transcription factors and plays an essential role in several cytoplasmic signaling transduction pathways, such as Wnt signaling pathways. One of the upregulated genes, designated URG11, was found transactivated by HBxAg. URG11 could stimulate the ß-catenin promoter and hepatocellular growth and survival which suggest that URG11 may be a regulatory element in the ß-catenin signaling pathways. microRNA148a (miR148a) was identified from two miRNA microarrays as one of the up-regulated miRNAs in cells stably expressing HBxAg or over-expressing URG11. Moreover, the expression of miR148a was also elevated in HBV-mediated HCC patient tissue samples. To study the function of miR148a, HepG2 (hepatoblastoma) and Hep3B (hepatoma) cells stably expressing HBxAg or over-expressing URG11 were transduced by recombinant lentiviruses encoding anti-miR148a. anti-miR148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Further, introduction of anti-miR148a increased PTEN protein and mRNA expression, suggesting that PTEN was suppressed by miR148a. In addition, anti-miR148a blocked the stimulation of Akt signaling, resulting in decreased expression of ß-catenin. Thus, miR148a may play a central role in HBxAg/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. / Biology

Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus. / Ph. D. / The circadian clock is the system allowing the body to stay in synchrony with its environment. Clocks are found in organisms ranging from bacteria to humans, and use environmental cues such as light and temperature to coordinate important processes inside the cell. Many of these processes require enzymes which contain iron in order to function. Iron is obtained almost exclusively through feeding, and high iron levels are toxic to the cell. In this work, we looked at how the circadian clock helps maintain the amount of iron within the cell at healthy levels. We showed that the genes which are involved in managing iron are expressed in different amounts depending on the time of day, and that this causes the amount of iron within the cell to vary over time. We also examined how the amount of iron in the cells goes on to alter the circadian clock. The way the circadian rhythm oscillates is altered when either too much or too little iron is available to the cells. These findings have health impacts, especially in the context of the liver where poor management of the circadian clock or iron metabolism have been linked to the development of various forms of liver cancer.

Objective: The primary aim of this study was to assess patients’ preferences between radiofrequency ablation (RFA) versus hepatic resection (HR) for the treatment of small hepatocellular carcinomas (HCC). Methods: Decision analysis was performed by using probability trade-off (PTO) technique to elicit patients’ preferences and the strength of their decisions. Results: The vast majority of the study population preferred RFA over HR (70% vs. 30%, p=0.001). Their initial choice changed if 5-year survival benefit after surgery was at least 14% superior to RFA and if the 3-year disease-free survival advantage was at least 13% better than ablation. Conclusions: The results of this study suggest that fully informed cirrhotic patients would prefer RFA if diagnosed with early stage HCC even if able to undergo surgery.