Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

13301乙第2085号博士（医学）金沢大学博士論文要旨Abstract 以下に掲載：Oncotarget 7(43) pp.70779-70793 2016. Impact Journals. 共著者：Eri Adachi, Katsuya Sakai, Takumi Nishiuchi, Ryu Imamura, Hiroki Sato, Kunio Matsumot

Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

13301乙第2085号博士（医学）金沢大学博士論文本文Full 以下に掲載：Oncotarget 7(43) pp.70779-70793 2016. Impact Journals. 共著者：Eri Adachi, Katsuya Sakai, Takumi Nishiuchi, Ryu Imamura, Hiroki Sato, Kunio Matsumot

Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

金沢大学がん研究所附属分子標的がん医療研究開発センターHepatocyte growth factor (HGF) is biosynthesized as a biologically inactive, single-chain form (pro-HGF). Its activation is associated with cleavage at Arg494-Val495 into a two-chain mature form composed of disulfide-linked α- and β-chains. Because serum is a major source of HGF activator (the predominant serine protease responsible for the processing of pro-HGF), serum-free production of recombinant, two-chain HGF had not been established. In this study, to enable serum-free production of two-chain HGF, we generated engineered human pro-HGFs that can be specifically cleaved and activated by Genenase I. Since Genenase I specifically cleaves the C-terminus of the His-Tyr sequence, which does not exist in human HGF, Arg494 (the C-terminus of the HGF α-chain) was replaced by His-Tyr, Ala-Ala-His-Tyr, Pro-Gly-His-Tyr, or Pro-Gly-Ala-Ala-His-Tyr. Genenase I cleaved engineered pro-HGFs specifically at the replaced amino acid sequences, forming a disulfide-linked two-chain form. The cleavage was most efficient in the case of the Pro-Gly-Ala-Ala-His-Tyr sequence, and cleaved HGFs displayed biological activities identical to those of wild-type HGF. Considering a potential medical application of HGF, the present technique is valuable because it enables the production of recombinant, two-chain HGF entirely without serum and extends the choice of host cells and organisms for recombinant production. © 2007 Elsevier B.V. All rights reserved

Cross Talk Mechanism among EMT, ROS, and Histone Acetylation in Phorbol Ester-Treated Human Breast Cancer MCF-7 Cells

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the progression of cancer, and some transcription factors including Slug and Snail are known to be involved in EMT processes. It has been well established that the excess production of reactive oxygen species (ROS) and epigenetics such as DNA methylation and histone modifications participate in carcinogenesis; however, the cross talk mechanism among EMT, ROS, and epigenetics remains unclear. In the present study, we demonstrated that the treatment of human breast cancer MCF-7 cells with phorbol ester (TPA), a protein kinase C activator, significantly induced cell proliferation and migration, and these were accompanied by the significant induction of Slug expression. Moreover, the TPA-elicited induction of Slug expression was regulated by histone H3 acetylation and NADPH oxidase (NOX) 2-derived ROS signaling, indicating that ROS and histone acetylation are involved in TPA-elicited EMT processes. We herein determined the cross talk mechanism among EMT, ROS, and histone acetylation, and our results provide an insight into the progression of cancer metastasis

Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma. Nevertheless, mechanisms for a phenotypic change have remained to be addressed. Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics. In metastatic melanoma, the cells are composed of Met-low and Met-high populations. Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation. Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs. Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Met-high cells facilitates lung metastasis by Met-low cells. Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations. Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high. Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells. These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression

Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma. Nevertheless, mechanisms for a phenotypic change have remained to be addressed. Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics. In metastatic melanoma, the cells are composed of Met-low and Met-high populations. Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation. Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs. Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Methigh cells facilitates lung metastasis by Met-low cells. Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations. Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high. Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells. These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression

α-Lipoic acid-induced inhibition of proliferation and met phosphorylation in human non-small cell lung cancer cells

α-Lipoic acid (α-LA), a naturally occurring anti-oxidant and co-factor for metabolic enzymes, suppresses the growth of different types of tumor cells. The mechanisms that are responsible for these results, however, remain to be elucidated. In the present study, we investigated the effects of α-LA on the proliferation and activation status of definitive receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and Met/hepatocyte growth factor (HGF) receptor, in gefitinib-sensitive human non-small cell lung cancer cells harboring EGFRs with an activating mutation. The enantiomers R-α-LA and S-α-LA suppressed cell proliferation and increased the level of reactive oxygen species in HCC-827 and PC-9 human non-small cell lung cancer cells in an indistinguishable dose-dependent fashion. A phospho-receptor tyrosine kinase array and cell cycle analysis indicated that α-LA decreased tyrosine phosphorylation levels of EGFR, ErbB2, and Met, and this was associated with an inhibition in the cell-cycle transition from the G1 phase to the S phase without inducing apoptosis. Gefitinib, an inhibitor for EGFR tyrosine kinase, inhibited EGFR tyrosine phosphorylation/activation and proliferation of the cells. Instead, the addition of HGF induced Met tyrosine phosphorylation, and this was associated with a resistance to gefitinib-induced growth inhibition, which meant a gain in proliferative ability. In the presence of gefitinib and HGF, the addition of α-LA suppressed Met tyrosine phosphorylation, and this was associated with an inhibition in cell growth. These results suggest that the suppression of tyrosine phosphorylation/activation of growth factor receptors that is critical for the proliferation of human non-small cell lung cancer cells is a mechanism by which α-LA exerts growth inhibition for cancer cells. © 2013 Elsevier Ireland Ltd. All rights reserved

Comprehensive behavioral analysis of mice repeatedly treated with propofol

Propofol, known as “milk of anesthesia”, is used for the induction and maintenance of anesthesia. Recently, propofol has attracted increasing concerns about its safety and abuse potential because of its psychostimulant effects such as euphoria and sexual hallucinations. Previous reports focused on the effects of postoperative and neonatal exposure to propofol. However, the lasting effects of repetitive propofol administration during adulthood have not been well investigated. It is conceivable that prolonged use of propofol affects brain function and the behavioral characteristics of the abused patient. Thus, we performed a comprehensive behavioral analysis of mice exposed to propofol. Adult male C57BL/6J mice were repeatedly administered with propofol (20 or 80 mg/kg/day i.p.), intralipos (vehicle control), or saline only once a day for seven days. We then performed a behavioral test battery to evaluate various behaviors. Afterwards, we resumed the propofol treatment for three days and subsequently conducted contextual and cued fear conditioning tests. In the three‐chamber social approach test, propofol treatment attenuated social novelty preference in mice. In the fear conditioning test, high dose-treated mice exhibited impaired long-term cued-dependent memory retention. In the rotarod test, propofol- and intralipos-treated mice tended to have decreased motor coordination than the saline-treated mice. Our results demonstrated that repetitive propofol treatment has the potential to induce some behavioral changes in mice. Additionally, the solvent itself might have effects different from that of propofol. Our findings provide basic data on the safe use and risk of propofol abuse. Highlights Propofol, known as “milk of anesthesia”, has attracted increasing concerns about its safety and abuse potential. The lasting effects of repetitive propofol administration during adulthood have not been well investigated. To clarify the effects of repetitive propofol use on brain function and behavioral characteristics, we performed a comprehensive behavioral analysis of mice exposed to propofol. In this study, propofol treatment attenuated the social novelty preference and the performance of the cued long-term memory task in mice. Additionally, treatment with propofol and intralipos tended to induce decreased motor coordination. Our results demonstrated that repetitive propofol treatment has the potential to induce some behavioral changes in mice. Furthermore, the solvent itself might have effects different from that of propofol. Our findings provide basic data for the safe use and risk of propofol abuse

LDL-C/HDL-C Ratio Predicts Carotid Intima-Media Thickness Progression Better Than HDL-C or LDL-C Alone

High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) are strong predictors of atherosclerosis. Statin-induced changes in the ratio of LDL-C to HDL-C (LDL-C/HDL-C) predicted atherosclerosis progression better than LDL-C or HDL-C alone. However, the best predictor of subclinical atherosclerosis remains unknown. Our objective was to investigate this issue by measuring changes in carotid intima-media thickness (IMT). A total of 1,920 subjects received health examinations in 1999, and were followed up in 2007. Changes in IMT (follow-up IMT/baseline IMT × 100) were measured by ultrasonography. Our results showed that changes in IMT after eight years were significantly related to HDL-C (inversely, P < 0.05) and to LDL-C/HDL-C ratio (P < 0.05). When the LDL-C/HDL-C ratios were divided into quartiles, analysis of covariance showed that increases in the ratio were related to IMT progression (P < 0.05). This prospective study demonstrated the LDL-C/HDL-C ratio is a better predictor of IMT progression than HDL-C or LDL-C alone