Kunishige Onuma

Elevated oxidative stress in erythrocytes due to a SOD1 deficiency causes anaemia and triggers autoantibody production

Reactive oxygen species are involved in the aging process and diseases. Despite the important role of Cu/Zn SOD (superoxide dismutase) encoded by SOD1, SOD1-/- mice appear to grow normally under conventional breeding conditions. In the present paper we report on a novel finding showing a distinct connection between oxidative stress in erythrocytes and the production of autoantibodies against erythrocytes in SOD1-/- mice. Evidence is presented to show that SOD1 is primarily required for maintaining erythrocyte lifespan by suppressing oxidative stress. A SOD1 deficiency led to an increased erythrocyte vulnerability by the oxidative modification of proteins and lipids, resulting in anaemia and compensatory activation of erythropoiesis. The continuous destruction of oxidized erythrocytes appears to induce the formation of autoantibodies against certain erythrocyte components, e.g. carbonic anhydrase II, and the immune complex is deposited in the glomeruli. The administration of an antioxidant, N-acetylcysteine, suppressed erythrocyte oxidation, ameliorated the anaemia, and inhibited the production of autoantibodies. These data imply that a high level of oxidative stress in erythrocytes increases the production of autoantibodies, possibly leading to an autoimmune response, and that the intake of antioxidants would prevent certain autoimmune responses by maintaining an appropriate redox balance in erythrocytes.

Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells.

Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO(2)) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO(2), either uncoated (TiO(2)-1, hydrophilic) or coated with stearic acid (TiO(2)-2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO(2)-1, but not TiO(2)-2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO(2)-1 and TiO(2)-2 treatments. However, TiO(2)-2, but not TiO(2)-1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO(2)-1 and TiO(2)-2 resulted in intracellular ROS formation, TiO(2)-2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO(2)-2, but not TiO(2)-1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO(2) toxicity acquired a tumorigenic phenotype. TiO(2)-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO(2) has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells.

Deterioration of ischemia/reperfusion-induced acute renal failure in SOD1-deficient mice

Reactive oxygen species (ROS) are likely candidates for involvement in ischemia/reperfusion-induced acute renal failure (ARF). In this study, the issue of whether superoxide dismutase (SOD1)-deficiency exacerbates the ischemia/reperfusion-induced ARF was examined. At two weeks after a right nephrectomy of mice, the left renal vessels were clipped to induce renal ischemia and were then released after 45 min. The severe renal damage observed at one day was partially recovered at seven days after the induction of ischemia. SOD1− / − mice suffer from severe ARF compared with SOD1+/ − and SOD1+/+ mice. The damage was more evident in aged animals (24–28 week old) than younger ones (10–12 week old). The expression of major antioxidative and redox enzymes, except for CuZnSOD, were substantially unchanged. Thus, the increased ARF in SOD1− / − mice appears to be mainly attributable to a deficiency in CuZnSOD. These data support the view that ROS are exacerbating factors in ischemia/reperfusion-induced ARF.

Prevention of inflammation-mediated acquisition of metastatic properties of benign mouse fibrosarcoma cells by administration of an orally available superoxide dismutase

Weakly tumorigenic and nonmetastatic QR-32 cells derived from a fibrosarcoma in C57BL6 mouse are converted to malignant cells once they have grown after being coimplanted with a gelatine sponge which induces inflammation. We administered a newly developed peroral superoxide dismutase (SOD), oxykine, and as control vehicle, gliadin and saline, starting 2 days before the coimplantation and continued daily throughout the experiment. In the oxykine group, tumour incidence was lower (41%) than in the gliadin or saline group (83 and 79%, respectively). The inhibitory effect of oxykine was lost when an individual component of oxykine was administered, that is, SOD alone and gliadin alone. The effect was also abolished when administered by intraperitoneal route. When perfused in situ with nitroblue tetrazolium, an indicator of superoxide formation, the tumour masses from gliadin and saline groups displayed intense formazan deposition, whereas, those from oxykine group had less deposition. Enzymatic activity of SOD was also increased in oxykine group. Arising tumour cells in gliadin and saline groups acquired metastatic phenotype, but those in oxykine group showed reduced metastatic ability. These results suggested that the orally active SOD derivative prevented tumour progression promoted by inflammation, which is thought to be through scavenging inflammatory cell-derived superoxide anion.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics‐based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP‐3) that developed from nontumorigenic human colonic adenoma cells (FPCK‐1–1) and were converted to tumorigenic by foreign‐body‐induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK‐1–1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin‐cDNA‐transfected FPCKpP‐3 cells reduced fascin expression and lost their tumor‐forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three‐dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell‐to‐substrate interactions), which is represented by the three‐dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase‐dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase‐associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Fermented Brown Rice and Rice Bran with Aspergillus oryzae (FBRA) Prevents Inflammation-Related Carcinogenesis in Mice, through Inhibition of Inflammatory Cell Infiltration

We have established an inflammation-related carcinogenesis model in mouse, in which regressive QR-32 cells subcutaneously co-implanted with a foreign body—gelatin sponge—convert themselves into lethal tumors due to massive infiltration of inflammatory cells into the sponge. Animals were fed with a diet containing 5% or 10% fermented brown rice and rice bran with Aspergillus oryzae (FBRA). In 5% and 10% FBRA diet groups, tumor incidences were lower (35% and 20%, respectively) than in the non-treated group (70%). We found that FBRA reduced the number of inflammatory cells infiltrating into the sponge. FBRA administration did not cause myelosuppression, which indicated that the anti-inflammatory effects of FBRA took place at the inflammatory lesion. FBRA did not have antitumor effects on the implanted QRsP-11 tumor cells, which is a tumorigenic cell line established from a tumor arisen after co-implantation of QR-32 cells with sponge. FBRA did not reduce formation of 8-hydroxy-2′-deoxyguanine adducts, a marker of oxidative DNA damage in the inflammatory lesion; however, it reduced expression of inflammation-related genes such as TNF-α, Mac-1, CCL3 and CXCL2. These results suggest that FBRA will be an effective chemopreventive agent against inflammation-related carcinogenesis that acts by inhibiting inflammatory cell infiltration into inflammatory lesions.

Amigo2 -upregulation in Tumour Cells Facilitates Their Attachment to Liver Endothelial Cells Resulting in Liver Metastases

Since liver metastasis is the main cause of death in cancer patients, we attempted to identify the driver gene involved. QRsP-11 fibrosarcoma cells were injected into the spleens of syngeneic mice to isolate tumour sub-populations that colonize the liver. Cells from liver metastatic nodules were established and subsequently injected intrasplenically for selection. After 12 cycles, the cell subline LV12 was obtained. Intravenous injection of LV12 cells produced more liver metastases than QRsP-11 cells, whereas the incidence of lung metastases was similar to that of QRsP-11 cells. LV12 cells adhered to liver-derived but not to lung-derived endothelial cells. DNA chip analysis showed that amphoterin-induced gene and open reading frame 2 (Amigo2) was overexpressed in LV12 cells. siRNA-mediated knockdown of Amigo2 expression in LV12 cells attenuated liver endothelial cell adhesion. Ex vivo imaging showed that suppression of Amigo2 in luciferase-expressing LV12 cells reduced attachment/metastasis to liver to the same level as that observed with QRsP-11 cells. Forced expression of Amigo2 in QRsP-11 cells increased liver endothelial cell adhesion and liver metastasis. Additionally, Amigo2 expression in human cancers was higher in liver metastatic lesions than in primary lesions. Thus, Amigo2 regulated tumour cell adhesion to liver endothelial cells and formation of liver metastases.

Fascin protein stabilization by miR-146a implicated in the process of a chronic inflammation-related colon carcinogenesis model

ObjectiveIn sporadic colon tumors, multistep process of well-known genetic alterations accelerates carcinogenesis; however, this does not appear to be the case in inflammation-related ones. We previously established a model of inflammation-related colon carcinogenesis using human colonic adenoma cells, and identified fascin as a driver gene of this process. We analyzed the microRNAs involved in the stable fascin expression in colon adenocarcinoma cells.Materials and methodsmiRNA microarray analysis was performed using FPCK-1-1 adenoma cells and its-derived FPCKpP1-4 adenocarcinoma cells through chronic inflammation. To assess the involvement of miRNA in the inflammation-related carcinogenesis, sphere-forming ability, expression of colon cancer stemness markers, and stability of fascin protein via the proteasome using tough decoy RNA technique.ResultsWe found that 17 miRNAs including miR-146a were upregulated and 16 miRNAs were downregulated in FPCKpP1-4 adenocarcinoma cells. We revealed that miR-146a in the adenocarcinoma cells brought about acquisition of sphere formation, cancer stemness, and inhibition of proteasomal degradation of the fascin protein.ConclusionsWe found that stable fascin expression is brought about via the inhibition of proteasome degradation by miR-146a in the process of a chronic inflammation-related colon carcinogenesis.

MP60-20 IDENTIFICATION OF MICRORNA REGULATING SUNITINIB RESISTANCE IN RENAL CELL CARCINOMA CELLS

patients have distal metastasis at the time of diagnosis. Despite great improvement in therapeutic treatment including molecular targeted therapy, the prognosis of patients with distant metastases remains unfavorable. HOX transcript antisense RNA (HOTAIR) is one of the long non-coding RNAs (lncRNAs). Accumulating evidence demonstrates that HOTAIR plays essential roles in cancer development and metastasis in several types of cancer such as lung cancer and gastric cancer. However, the precise mechanism by which HOTAIR enhances cancer malignancy is still unknown, especially in RCC. The object of this study is to elucidate the function of HOTAIR in RCC. METHODS: The tumor and adjacent normal renal tissues were obtained with informed consent from 64 patients who underwent radical or partial nephrectomy in Miyagi Cancer Center. All tumor tissues were diagnosed pathologically as clear cell renal cell carcinoma. We evaluated the clinical correlates of HOTAIR expression determined by realtime PCR using RNA extracted from the tumor and normal tissues. The functional role of HOTAIR was examined using HOTAIR-overexpressing or knockdown human renal cell carcinoma cell lines (ACHN, A498 and Caki-1). RESULTS: The expression of HOTAIR was significantly correlated with tumor nuclear grade, lymph node metastasis, lung metastasis, and AJCC stage. Migration capacity was enhanced in a HOTAIR-depending manner in vitro. Overexpression of HOTAIR in human renal cell carcinoma accelerated tumorigenicity in immunodeficient mice. Microarray analysis revealed that Insulin Growth Factor Binding Protein 2 (IGFBP2) gene was up-regulated in HOTAIR-overexpressing cells, which was validated by real-time PCR and Western blotting. Co-expression of IGFBP2 and HOTAIR was observed in clinical samples (P 1⁄4 0.04, Fisher0s exact test). Enhanced migration activity in HOTAIR-expressing cells was attenuated by IGFBP2knockdown. CONCLUSIONS: We newly identified IGFBP2 as a downstream molecule of HOTAIR, which is involved in migration capacity. Our findings suggest that a HOTAIR-IGFBP2 axis plays critical roles in RCC progression, and serves as a novel therapeutic target for advanced RCC treatment.

Lysosome‑associated membrane protein 2 (LAMP‑2) expression induced by miR‑194‑5p downregulation contributes to sunitinib resistance in human renal cell carcinoma cells

Sunitinib is a tyrosine kinase inhibitor that is used as the primary treatment in metastatic renal cell carcinoma (RCC). The main difficulty associated with its use is the development of drug resistance. In the present study, ACHN cells, a human renal cell carcinoma cell line, were used to establish sunitinib-resistant (SR) cells. Microarray analysis and reverse transcription-quantitative polymerase chain reaction revealed that miR-194-5p expression was significantly decreased in SR-ACHN cells when compared with that observed in ACHN cells (P<0.05). Transfection of miR-194-5p, though not with negative control miR, in SR-ACHN cells could significantly inhibit cell proliferation following sunitinib treatment (2.5–40 µM; P<0.05). Western blotting demonstrated that the expression of lysosome-associated membrane protein-2 (LAMP-2), which attenuates the anti-proliferative effect of sunitinib, was significantly higher in SR-ACHN than in ACHN cells (P<0.01). In addition, LAMP-2 expression was suppressed by miR-194-5p transfection in SR-ACHN cells. These data suggested that miR-194-5p downregulation may be associated with sunitinib resistance via the induction of LAMP-2 expression in human RCC.