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Mitsuru Yashiro,1 Masaki Ohya,1 Toru Mima,1 Yumi Ueda,2 Yuri Nakashima,1 Kazuki Kawakami,1 Yohei Ishizawa,2 Shuto Yamamoto,1 Sou Kobayashi,1 Takurou Yano,1 Yusuke Tanaka,1 Kouji Okuda,1 Tomohiro Sonou,1 Tomohiro Shoshihara,1 Yuko Iwashita,1 Yu Iwashita,1 Kouichi Tatsuta,1 Ryo Matoba,2 Shigeo Negi,1 Takashi Shigematsu1 1Department of Nephrology, Wakayama Medical University, Wakayama, Japan; 2DNA Chip Research Inc., Minato, Japan Background: FGF23 plays an important role in calcium–phosphorus metabolism. Other roles of FGF23 have recently been reported, such as commitment to myocardium enlargement and immunological roles in the spleen. In this study, we aimed to identify the roles of FGF23 in the kidneys other than calcium–phosphorus metabolism. Methods: DNA microarrays and bioinformatics tools were used to analyze gene expression in mIMCD3 mouse renal tubule cells following treatment with FGF23, erythropoietin and/or an inhibitor of ERK. Results: Three protein-coding genes were upregulated and 12 were downregulated in response to FGF23. Following bioinformatics analysis of these genes, PPARγ and STAT3 were identified as candidate transcript factors for mediating their upregulation, and STAT1 as a candidate for mediating their downregulation. Because STAT1 and STAT3 also mediate erythropoietin signaling, we investigated whether FGF23 and erythropoietin might show interactive effects in these cells. Of the 15 genes regulated by FGF23, 11 were upregulated by erythropoietin; 10 of these were downregulated following cotreatment with FGF23. Inhibition of ERK, an intracellular mediator of FGF23, reversed the effects of FGF23. However, FGF23 did not influence STAT1 phosphorylation, suggesting that it impinges on erythropoietin signaling through other mechanisms. Conclusion: Our results suggest cross talk between erythropoietin and FGF23 signaling in the regulation of renal epithelial cells. Keywords: FGF23, STAT1, PPARγ, DNA microarray, bioinformatics analysis, nonprotein-coding gene

Mitsunori Harada,1 Caname Iwata,2 Hiroyuki Saito,1 Kenta Ishii,1 Tatsuyuki Hayashi,1 Masakazu Yashiro,3 Kosei Hirakawa,3 Kohei Miyazono,2 Yasuki Kato,1 Mitsunobu R Kano21Research Division, NanoCarrier Co, Ltd, Chiba, Japan; 2Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 3Department of Surgical Oncology, Osaka City University, Osaka, JapanAbstract: Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors' own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity.Keywords: drug release, breast cancer, gastric cancer

Byline: H. Yanagawa (1), Y. Nakamura (1), K. Sakata (1), M. Yashiro (1) Keywords: Key words: Gamma-globulin treatment -- Kawasaki disease -- Cardiac sequelae Abstract: The administration of intravenous I3-globulin (IVGG) for Kawasaki disease was investigated throughout Japan in 1993 by obtaining information from pediatric departments in 2652 hospitals that had more than 100 beds. Of 11,221 reported patients, 8958 patients (79.8%) received IVGG treatment. Of all the patients to whom IVGG was administered, the most common total dose was 1000 mg/kg (36.3%) followed by 2000 mg/kg (16.9%) and 1200 mg/kg (16.8%). The treatment was started in 53.8% by day 5 of the illness and in 83.7% by day 7. The proportion of those with cardiac sequelae was higher among patients administered &gt 2000 mg/kg or in those started on IVGG on day 9 of their illness or later. The possible reasons are (1) those who were more severely affected were treated with high-dose IVGG earlier or (2) IVGG does not effectively prevent cardiac sequelae. We concluded that there is a risk of unfavorable effects with IVGG regarding cardiac sequelae when the IVGG dose is &gt 2000 mg/kg or if IVGG is started on day 9 or later. We believe that only a randomized controlled trial, undertaken prospectively, can adequately address the question of the optimal use of IVGG. Author Affiliation: (1) Department of Public Health, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi-ken 329-04, Japan, JP

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Abstract Cancer is the primary cause of human mortality in Japan since 1981. Although numerous novel therapies have been developed and applied in clinics, the number of deaths from cancer is still increasing worldwide. It is time to consider the strategy of cancer prevention more seriously. Here we propose a hypothesis that cancer can be side effects of long time-use of iron and oxygen and that carcinogenesis is an evolution-like cellular events to obtain “iron addiction with ferroptosis-resistance” where genes and environment interact each other. Among the recognized genetic risk factors for carcinogenesis, we here focus on BRCA1 tumor suppressor gene and how environmental factors, including daily life exposure and diets, may impact toward carcinogenesis under BRCA1 haploinsufficiency. Although mice models of BRCA1 mutants have not been successful for decades in generating phenotype mimicking the human counterparts, a rat model of BRCA1 mutant was recently established that reasonably mimics the human phenotype. Two distinct categories of oxidative stress, one by radiation and one by iron-catalyzed Fenton reaction, promoted carcinogenesis in Brca1 rat mutants. Furthermore, mitochondrial damage followed by alteration of iron metabolism finally resulted in ferroptosis-resistance of target cells in carcinogenesis. These suggest a possibility that cancer prevention by active pharmacological intervention may be possible for BRCA1 mutants to increase the quality of their life rather than preventive mastectomy and/or oophorectomy.

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics.