Drug resistance in cancer cells is a serious complication that is always continuously evolving. Rather than just one or two factors, drug resistance is a combination of a handful of elusive mechanisms. Many of these mechanisms and factors have been studied in the past, however new methods of analysis and treatment are being developed and tested rigorously. Along with new progress and breakthroughs, the pharmaceutical industry must also recognize the increasing expensive cost factor and its burden on cancer patients of the future. Ultimately, new treatment methods accompanied by cost-efficient analysis will provide patients with the best cancer treatment possible.

<p>  </p> <p>Shikonin is 1,4-naphthoquinones produced exclusively in Boraginaceae species. The compound and its derivatives are predominantly made in roots where they function in mediating plant-plant (allelopathic) and plant-microbe interactions. Moreover, this compound has been a target for drug development due to its strong anti-cancer properties. Our genome assembly and analysis of <em>Lithospermum erythrorhizon</em> uncovered metabolic innovation events that contributed to the evolution of the shikonin biosynthesis. This metabolic innovation also reveals the evolutionary link between shikonin biosynthesis and ubiquinone biosynthesis, one of the central metabolism functions in aerobic cellular respiration. To explore additional links between these two pathways, we used a transcriptome-based network analysis which uncovered a shikonin gene network model that predicts strong associations between primary metabolic pathway genes and known shikonin biosynthesis genes, as well as links with uncharacterized genes. <em>L. erythrorhizon</em> geranyldiphosphate (GPP) synthase (<em>LeGPPS</em>) is one of the candidates predicted by the network analysis, of which encodes a cytoplasmic enzyme shown in vitro to produce GPP. Knocking down of <em>LeGPPS</em> in <em>L. erythrorhizon </em>hairy roots (<em>LeGPPSi </em>lines) results in reduced shikonin content. This result provides functional evidence that cytoplasmic LeGPPS supplies GPP precursor to the shikonin biosynthesis. <em>LeGPPSi </em>lines also increased ubiquinone content, further supporting our hypothesis on the metabolic and evolutionary connection between shikonin and ubiquinone biosynthesis. Further RNA-seq analysis of the <em>LeGPPSi</em> line showed that downregulating <em>LeGPPS</em> significantly reduces the expression of benzenoid/phenylpropanoid genes, indicating the presence of factors that coordinately regulate the pathways providing the 4-hydroxybenzoic acid and GPP precursors to the shikonin pathway. In addition to <em>LeGPPS</em>, we also found<em> ubiquinone biosynthesis protein COQ4-like </em>gene (<em>LeCOQ4-L</em>) which provided another evolutionary link between shikonin and ubiquinone biosynthesis. The enzymatic activity of canonical COQ4 is unknown. In yeast, the protein is essential for ubiquinone biosynthesis and its metabolon formation. With the existing connections between shikonin and ubiquinone biosynthesis, if LeCOQ4 functions in the same manner as yeast COQ4, it is possible that <em>LeCOQ4-L </em>has an analogous function in shikonin biosynthesis as a structural protein for stabilizing biosynthesis metabolon. This leads us to the characterization of<em> COQ4</em> ortholog in Arabidopsis (<em>AtCOQ4</em>) to gain insight into its functional mechanism. Characterization of <em>atcoq4 </em>T-DNA mutant line showed that reduced <em>AtCOQ4</em> expression resulted in reduced ubiquinone. Further subcellular localization study revealed that AtCOQ4 and <em>LeCOQ4-L</em> localize in mitochondria without conventional transit peptide. We also performed pull-down assay to identify AtCOQ4 interactors which might be the missing enzymes that cannot be identified based on homology. 80 potential AtCOQ4 interactors were found including proteins like AtCHLM, GRIM-19, and AtSSLs. However, further study is needed to verify the protein interactions captured by pull-down assay. Taken all together, our study sheds light on the metabolic innovations that give rise to shikonin biosynthesis from ubiquinone biosynthesis and provide insight into the dynamics of the metabolic networks.</p>

Hippo signaling pathway represents organ size control mechanism constrained between all metazoans. Individual components of the Hippo signaling pathway were identified as key tumor-suppressors which phosphorylate and inhibit activity of several oncogenic factors and signaling pathways (such as YAP/TAZ, PI3K and mTOR). MST1 kinase is a part of central protein complex of the Hippo signaling pathway and its activation is involved in anti-cancer activity of several drugs. We have demonstrated activation of the MST1 kinase by natural compounds in leukemic cells followed by inhibition of proliferation and induction of apoptosis. Shikonin represents natural naphthoquinonic compound isolated from Lithospermum erythrorhizon which acts as inhibitor of glycolysis and mitochondrial respiratory chain in human cells. Shikonin induces fast activation of the MST1 protein in leukemic cells however mechanism of this activation remains unknown. Therefore, we tried to characterize posttranslational modifications of the MST1 kinase during shikonin treatment of leukemic cells. Firstly, we isolated MST1 kinase from control and shikonin-treated cells using immunoprecipitation. Then we characterized posttranslational modifications of the MST1 protein employing mass spectrometry. Using this approach we found out...

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24673号 / 農博第2556号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5454(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 矢﨑 一史, 教授 梅澤 俊明, 教授 伊福 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25337号 / 農博第2603号 / 新制||農||1106(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 矢﨑 一史, 教授 山口 信次郎, 教授 飛松 裕基 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Natural bioactive compounds have drawn the interest of many researchers worldwide in their effort to find novel treatments, including prostate cancer (PC) treatment which is estimated to be 13.1% of all new cancer cases in the U.S. in 2021. Many of these bioactive compounds have been identified from treatments in traditional Chinese medicine (TCM), that often have multiple bioactive compounds present. However, in vitro studies frequently focus on the compounds in isolation, or in simple combinations of two compounds. We used mixture design response surface methodology (MDRSM) to assess changes in PC cell viability after 48 hours of treatment to identify the optimal mixture of all 35 three-compound combinations of seven bioactive compounds from TCM. We used Berberine, Wogonin, Shikonin, Curcumin, Triptolide, Emodin, and Silybin to treat PC-3, DU145, and LNCaP human PC cells, and a drug-resistant PC-3 cell line. Berberine and Wogonin most frequently contributed to the optimal combination to reduce cell viability in PC-3 and LNCaP cells; DU145 cells more frequently responded best to a single compound.