OBJECTIVES: Recent studies have elucidated that diabetes mellitus (DM) patients exhibit an accelerated tumor progression, but the mechanism of its regulation is not yet fully understood. The following study seeks to examine the role of angiogenic factors in the growth of subcutaneously injected melanoma cancer using a diet-induced type II diabetic mouse model. METHODS: C57BL/6 mice were fed either a regular or high-fat, high-sucrose (HFHS) diet for 2 months (T2DM model; confirmed through a GTT) and subcutaneously injected with B16F0 melanoma cells. After a 1-week or 2-week incubation period, the tumor was extracted to examine its size, weight, vascularity, and gene/protein expression. In vitro studies were performed using endothelial cells to assess the effects of high-glucose on endothelial cell proliferation, migration, and tube formation. GLRX expression was examined in both tumor samples and endothelial cells. RESULTS: The results of the study showed that T2DM induced by a HFHS diet is able to promote tumor growth in both weight (2-week, p = 0.0070) and volume (1-week, p = 0.0351; 2-week, p = 0.0002). Tumors extracted from the HFHS diet group showed reduced expressions of angiogenic markers (ACTA2 (1-week, p = 0.0239; 2-week, p = 0.0123), KDR (1-week, p = 0.0091)) by western blot and a slightly reduced trend of angiogenesis (PECAM1) in histological analyses. GLRX expression was reduced in HFHS tumor samples (1-week, p = 0.0090) and, interestingly, lower amounts of GSH adducts (2-week, p = 0.0317) could be seen in 2-week tumors as well. In vitro studies of endothelial cells showed reduced trends of endothelial cell function (proliferation, migration, and tube formation) in high glucose medium. Also, it has been observed that high glucose may be able to stimulate GLRX expression in endothelial cells. CONCLUSION: The results of the following study have confirmed that B16F0 melanoma growth is, in fact, augmented in diet-induced diabetic mice; however, the vascularity and levels of angiogenic markers from the tumor tissues did not parallel the growth in its size. In vitro studies suggested that high glucose can impair EC function (i.e. proliferation, migration, and tube formation capabilities) as well as promote GLRX expression, which may be related to this discrepancy. Glutaredoxin-1 (GLRX), an enzyme which controls redox signaling, is upregulated in DM. Endothelial cell-specific GLRX overexpression in transgenic mice was found to stimulate subcutaneously injected melanoma (B16F0) growth, despite hindering limb revascularization after hind limb ischemia. The augmented tumor progression in DM may be associated with GLRX upregulation, alongside impaired ischemic limb revascularization and tumor angiogenesis; however, the mechanism of tumor growth in diabetes still lies inconclusive and further studies need to be examined to elucidate this phenomenon.

BACKGROUND: While advances in medicine have led to increased survival rates, cancer is responsible for millions of deaths each year. Melanoma itself accounts for tens of thousands of these deaths, and is capable of progressing rapidly. If left untreated, patient prognosis is bad and survival is unlikely. With distant metastasis, survival rates past five years are under ten percent. The link between angiogenesis and tumor growth has been well established, and therefore anti-angiogenic factors should show a decrease or slowing of cancer growth. Glutaredoxin-1 (Glrx), an enzyme residing in the cytosol, is responsible for removing glutathione (GSH) from GSH-protein adducts. This plays a role in reversible cellular signaling. It has been shown that increased levels of global and endothelial cell (EC) specific Glrx leads to decreased levels of revascularization following hind limb ischemia in mice. Few studies however have compared the overexpression of Glrx to melanoma tumor growth. OBJECTIVE: To examine how Glrx overexpression in endothelial cells (EC) influences B16F0 murine melanoma cell growth in C57BL6 mice. METHODS: Eight Glrx overexpressing double transgenic (DTG) and seventeen control mice (Ve-cadherin or WT) between the ages of 3 and 4 months old each received subcutaneous injection with B16F0 mouse melanoma cells. After 2 weeks, the mice were euthanized to remove tumors. Weight and size of tumor were recorded. Tumor, lung, and tail samples were taken. Real-time polymerase chain reaction (qPCR) of tumor samples were performed. Genotypes were confirmed via western blot of lung and PCR of tail DNA. Excised tumor tissue was in part fixed to examine for angiogenesis and macrophage markers. VEGF ELISA was performed on tumor samples. Immunohistochemistry (IHC) testing for Isolectin B4 and CD68 was performed on frozen sections. RESULTS: Slightly higher average tumor weight was found in DTG mice (m=1229.45 mg ± 202.89 mg, n=8) when compared to the control (m=827.32 mg ±172.62 mg, n=17). Lower gene expression of VCAM, VEGFA, IL-6, IL-1B, Kdr, and HIF1 was found in the DTG mice when compared to the control. The expression of CD68 was significantly lower (P<0.05). VEGF ELISA showed higher concentrations of VEGF per total protein in the DTG mice versus the control mice. IHC showed DTG had higher endothelial cell signaling by Isolectin B4, and higher macrophage signaling by CD68. CONCLUSIONS: The growth of B16F0 murine melanoma is not suppressed by the overexpression of Glrx in EC of C57BL6 mice. / 2019-07-11T00:00:00Z

Parkinson’s disease (PD) is a debilitating movement disorder. The cardinal symptoms of PD are bradykinesia, resting tremors and rigidity. PD is characterized by degeneration of dopaminergic neurons of A9 region, substantia nigra pars compacta (SNpc) and loss of dopaminergic terminals in striatum while the dopaminergic neurons of A10 region, ventral tegmental area (VTA) are relatively protected. Putative mechanisms, such as mitochondrial dysfunction, dysregulation of the ubiquitin proteasome system and increased oxidative stress have been hypothesized to mediate PD pathology. However, precise mechanisms that underlie selective vulnerability of SNpc dopaminergic neurons to degeneration are unknown. The aim of this thesis was to evaluate the pathological mechanisms that may contribute to degeneration of SNpc dopaminergic neurons in PD. Dopaminergic neurons of SNpc are pacemakers and constant calcium entry through L-type calcium channel, Cav1.3 has been reported in these neurons during pacemaking. In addition, these neurons have poor calcium buffering capacity. Together, this leads to dysregulation of calcium homeostasis in the SNpc dopaminergic neurons leading to increased oxidative stress. Gene expression of the full length channel and the variant was investigated in the mouse midbrain and further their presence was verified in mouse SNpc and VTA and also in SNpc and VTA in the MPTP mouse model of PD. Gene expression of Cav1.3 -42 and its variant was also studied in SNpc from autopsy tissue from PD patients and age matched controls. Having studied differential expression of the calcium channels, global changes in gene expression in SNpc from the MPTP mouse model of PD and PD autopsy tissues were next examined. This is the first report of transcriptome profile alterations from SNpc in mouse model and PD tissue performed using RNA-seq. Gene expression profiles were examined from SNpc 1 day post single exposure to MPTP, in which case there is no neuronal death and 14 days after daily MPTP treatment where SNpc has undergone ~50% cell death. Further, RNA- seq was performed to study gene expression alterations in SNpc from human PD patients and age- matched controls. The RNA-seq data was taken through extensive analyses; analysed for differential gene expression, gene-set enrichment analysis, pathway analysis and network analysis. Glutaredoxin 1 (Grx1) is a thiol disulfide oxidoreductase that catalyses the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis. Down-regulation of Grx1 has been established to exacerbate neurodegeneration through impairment of cell survival signalling. Previous work from our laboratory has demonstrated that perturbation of protein thiol redox homeostasis through diamide injection into SNpc leads to development of PD pathology and motor deficits. It was therefore investigated if Grx1 down-regulation in vivo, leading to increased glutathionylation and protein thiol oxidation, could result in PD pathology. This work is thus the first study of RNA-seq based transcriptomic profile alterations in SNpc from human PD patients. This work also highlights several differences between mouse model and human PD tissue indicating that the underlying mechanisms of PD pathogenesis differ from mouse to humans in addition to developing a novel model for PD.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23043号 / 理博第4720号 / 新制||理||1676(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 曽田 貞滋, 教授 沼田 英治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM