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Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53+/-or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

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.

Cellular senescence and cell competition are important tumor suppression mechanisms that restrain cells with oncogenic mutations at the initial stage of cancer development. However, the link between cellular senescence and cell competition remains unclear. Senescent cells accumulated during the in vivo aging process contribute toward age-related cancers via the development of senescence-associated secretory phenotype (SASP). Here, we report that hepatocyte growth factor (HGF), a SASP factor, inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells in the cell competition assay. Additionally, cellular senescence induced by a high-fat diet promotes the survival of cells with oncogenic mutations, whereas crizotinib, an inhibitor of HGF signaling, provokes the removal of mutated cells from mouse livers and intestines. Our study provides evidence that cellular senescence inhibits cell competition-mediated elimination of oncogenic cells through HGF signaling, suggesting that it may lead to cancer incidence during aging.

Congenital malformations cause life-threatening diseases in pediatrics, yet the molecular mechanism of organogenesis is poorly understood. Here we show that Dyrk2-deficient mice display congenital malformations in multiple organs. Transcriptome analysis reveals molecular pathology of Dyrk2-deficient mice, particularly with respect to Foxf1 reduction. Mutant pups exhibit sudden death soon after birth due to respiratory failure. Detailed analyses of primordial lungs at the early developmental stage demonstrate that Dyrk2 deficiency leads to altered airway branching and insufficient alveolar development. Furthermore, the Foxf1 expression gradient in mutant lung mesenchyme is disrupted, reducing Foxf1 target genes, which are necessary for proper airway and alveolar development. In ex vivo lung culture system, we rescue the expression of Foxf1 and its target genes in Dyrk2-deficient lung by restoring Shh signaling activity. Taken together, we demonstrate that Dyrk2 is essential for embryogenesis and its disruption results in congenital malformation., Yogosawa et al find that mice lacking the Dyrk2 kinase display congenital malformations in multiple organs and die from respiratory failure associated with disrupted lung mesenchyme and reduced expression of Foxf1. Using an ex vivo lung culture system, they rescue the expression of Foxf1 and its target by restoring Shh signalling, altogether providing insights into Dyrk2 function.

Non-thermal atmospheric pressure plasma has been proposed as a new therapeutic tool for cancer treatment. Recently, plasma-activated medium (PAM) has been widely studied in various cancer types. However, there are only few reports demonstrating the anti-tumour effects of PAM in an animal model reflecting pathological conditions and the accompanying mechanism. Here we investigated the inhibitory effect of PAM on the metastasis of ovarian cancer ES2 cells in vitro and in vivo. We demonstrated that ES2 cell migration, invasion and adhesion were suppressed by PAM at a certain PAM dilution ratio, whereas cell viability remained unaffected. In an in vivo mouse model of intraperitoneal metastasis, PAM inhibited peritoneal dissemination of ES2 cells, resulting in prolonged survival. Moreover, we assessed the molecular mechanism and found that MMP-9 was decreased by PAM. On further investigation, we also found that PAM prevented the activation of the MAPK pathway by inhibiting the phosphorylation of JNK1/2 and p38 MAPK. These findings indicate that PAM inhibits the metastasis of ovarian cancer cells through reduction of MMP-9 secretion, which is critical for cancer cell motility. Our findings suggest that PAM intraperitoneal therapy may be a promising treatment option for ovarian cancer.

Plasma medical science is a novel interdisciplinary field that combines studies on plasma science and medical science, with the anticipation that understanding the scientific principles governing plasma medical science will lead to innovations in the field. Non-thermal atmospheric pressure plasma has been used for medical treatments, such as for cancer, blood coagulation, and wound healing. The interactions that occur between plasma and cells/tissues have been analyzed extensively. Direct and indirect treatment of cells with plasma has broadened the applications of non-thermal atmospheric pressure plasma in medicine. Examples of indirect treatment include plasma-assisted immune-therapy and plasma-activated medium. Controlling intracellular redox balance may be key in plasma cancer treatment. Animal studies are required to test the effectiveness and safety of these treatments for future clinical applications.

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2) -tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells.

The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 .- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

LYL1, a member of the class II basic helix-loop-helix transcription factors, is aberrantly expressed in a fraction of human T-cell acute lymphoblastic leukemia. Here, we generated transgenic mice ubiquitously overexpressing LYL1 using a construct expressing full-length cDNA driven by a human elongation factor 1alpha promoter. Four independent lines exhibiting high LYL1 expression were established. Of these transgenic mice, 96% displayed loss of hair with a short kinked tail. Furthermore, 30% of them developed malignant lymphoma, with an average latent period of 352 days. In these mice, histological examination revealed tumor cell infiltration in multiple organs and immunohistochemical analysis showed that the infiltrated tumor cells were either CD3 or CD45R/B220-positive; fluorescence-activated cell sorter analysis indicated that each tumor consisted either of mainly CD4, CD8 double-positive T cells or mature B cells; the clonality of LYL1-induced lymphoma was confirmed by T-cell receptor rearrangement and immunoglobulin heavy-chain gene rearrangement analyses. Mammalian two-hybrid analysis and luciferase assay suggested that excess LYL1 blocked the dimerization of E2A and thus inhibited the regulatory activity of E2A on the CD4 promoter. Reverse transcription-polymerase chain reaction results showed that the expression of certain E2A/HEB target genes was downregulated. Taken together, our results provide direct evidence that aberrant expression of LYL1 plays a role in lymphomagenesis.

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous W(v) mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (W(v)/W(v):Fas(-/-)) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged W(v)/W(v) mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of W(v)/W(v) mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl4) administration in rats — one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl4 intragastrically. Liver damage was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl4 administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P

Ferric nitrilotriacetate induces oxidative damage in renal proximal tubules, a consequence of Fenton-like reaction, that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. In order to find common genetic alterations in this oxystress-induced carcinogenesis model, RCCs were produced in F1 hybrid rats between Wistar and Long-Evans strains and genomes were screened for loss of heterozygosity (LOH) with microsatellite polymorphic markers by PCR. Five consecutive markers on chromosome 5 (D5Mgh5, D5Mit9, D5Mgh6, D5Mit11 and D5Mit6) showed LOH in ≥40% of the RCCs. As possible candidate tumor suppressor genes on chromosome 5, p15INK4B and p16INK4A were investigated for genetic alteration and aberrant methylation by Southern blot, PCR/SSCP/sequencing and methylation-specific PCR. Genetic alteration (homozygous or hemizygous deletion with or without point mutation) or aberrant methylation were found in 30.7 and 53.8% of the RCC cases, respectively, which was proportionally associated with the histological nuclear grade and metastatic activity. Our data suggest that inactivation of p15 and p16 genes could be one of the major pathways responsible for oxystress-induced carcinogenesis.

The paraoxonase enzyme (PON) gene polymorphism causes a change of methionine (M-allele) to leucine (L-allele). PON may reduce low density lipoprotein oxidation and prevent atherosclerosis. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive index of oxidative DNA damage. We have studied the association between the PON genotypes and the urinary excretion of 8-OHdG. The study population consisted of 93 Finnish type 2 diabetes patients and 106 non-diabetic control subjects. The 24-h excretion of 8-OHdG was significantly higher in diabetic patients than in control subjects (P < 0.001). In control subjects, the ratio of the 8-OHdG/glomerular filtration rate increased in order of genotype from MM to ML to LL (P < 0.0412). These results suggest that lipid peroxidation may have an effect on DNA oxidation.

Iron appears to play a major role in catalysing free radical production, leading to lipid peroxidation and DNA damage. We, therefore, investigated the effect of colloidal iron deposited in the peritoneum. Wistar male rats were given either ferric saccharate, ferric saccharate and nitrilotriacetic acid (NTA), NTA or saline. NTA was shown previously to 'free' iron to promote lipid peroxidation and an iron chelate of NTA is known to be carcinogenic to the kidney. Iron at a dose of 5 mg kg-1 day-1, and saline at a dose of 0.5 ml day-1 were injected i.p. for 3 months. NTA at a dose of 83.5 mg kg-1 day-1 was give i.p. for 5 months. All the rats were killed about a year later for histological examination. In nine of the 19 rats treated with ferric saccharate, mesothelial tumors were induced in the serosa of the tunica vaginalis or the length of the spermatic cord. Among rats treated with ferric saccharate and NTA, seven had localised mesotheliomas in the above locations and six had wide-spread peritoneal mesotheliomas. No mesothelial tumors developed in either NTA treated or saline treated rats. No pleural mesotheliomas were found in any group. These findings add to the evidence that iron is involved in some carcinogenic processes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6