Jugoh Itoh

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization.

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both co-culture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.

Methylation of the KEAP1 gene promoter region in human colorectal cancer

BackgroundThe Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.MethodsWe used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.ResultsDNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.ConclusionHypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

Chronic nicotine stimulation modulates the immune response of mucosal T cells to Th1-dominant pattern via nAChR by upregulation of Th1-specific transcriptional factor.

Inflammatory bowel disease (IBD) consists of Crohns disease (CD) and ulcerative colitis (UC). The etiology has not been clarified yet, but immune disorder is thought to be involved in the pathogenic physiology. Recently, general consensus has been reached that CD and UC are distinct, especially in respect of the immune response. Interestingly, smoking has diverse effects on CD, Th1-type enteritis, and on UC, Th2-type. However, the mechanisms remain obscure. Therefore, we hypothesized that nicotine altered the distinct immune responses in each form of IBD to affect their pathophysiology. In this study, we first demonstrated by RT-PCR analysis that human lamina propria T (LPT) cells had nicotinic acetylcholine receptor (nAChR), and express alpha7 nAChR subunit universally. In addition, the expression of T-bet mRNA in human LPT cells was significantly upregulated after the culture with 10(-7)M and 10(-5)M nicotine for 9 days, while chronic nicotine stimulation showed negligible effect on the expression of GATA-3 mRNA by real-time PCR. The effect of nicotine was inhibited by mecamylamine (MEC). These results suggested that nicotine could modulate the immune balance to Th1-dominant via nAChR in the intestine, to improve Th2-type enteritis. This may provide the experimental evidence for the fact that nicotine has a beneficial influence on UC, and exacerbates CD. Furthermore, it is of great interest that nicotine acts oppositely on CD and UC by modulation of the mucosal immune balance via the neurotransmitter receptor.

Intratracheal delivery of CX3CL1-expressing mesenchymal stem cells to multiple lung tumors.

The lung is one of the organs to which cancers from solid tumors frequently metastasize. Multiple tumors in the lung are usually treated by systemic chemotherapy because of the lack of efficient methods of targeting antitumor agents to the lung. Although intratracheal administration is an ideal route for targeting multiple lung tumors, antitumor agents are often harmful to the organ or induce inflammation. Mesenchymal stem cells (MSCs), nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells, have a propensity to migrate to and proliferate in tumor tissues after systemic administration. We intratracheally injected MSCs expressing CX3CL1 (MSC/RGDFKN) into the lung of lung tumor-bearing mice with multiple metastases of C26 or Lewis lung carcinoma (LLC). Antitumor effects were evaluated by counting the number of lung metastases and survival. We demonstrated the tropism of mouse MSCs to lung tumor tissues after intratracheal administration of GFP-positive MSCs. Intratracheal injection of MSC/RGDFKN strongly inhibited growth of lung metastases of C26 or LLC, and thus prolonged survival. Intratracheal injection of MSC/RGDFKN did not induce an inflammatory reaction in the lung. These results suggest that MSCs expressing antitumor agents can be delivered intratracheally into multiple lung tumor tissues without causing inflammation.

High prevalence of diffuse large B-cell lymphoma in occult hepatitis B virus-infected patients in the Tohoku district in Eastern Japan.

Occult hepatitis B virus (HBV) infection is a clinical challenge, but its relationship to clinicopathologic features and the risk of progression to malignant lymphoma (ML) are poorly defined. We estimated the prevalence of HBV infection of 1,358 patients with newly diagnosed ML. HBV infection was more prevalent in ML than in control patients. The occult HBV infection group had a higher median onset age, no liver or spleen involvement, and higher prevalence of diffuse large B‐cell lymphoma than the other groups, indicating that occult HBV infection is a distinct clinicopathologic entity. J. Med. Virol. 88:2206–2210, 2016.

Abstract 102: Hypermethylation of Keap1 promoter and expression of the downstream genes in human colorectal cancer cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Nrf2-Keap1 system is a master regulator of phase 2 detoxifying and antioxidant genes including NAD(P)H-quinon oxidoreductase 1[NQO-1] and aldo-keto reductase C1 (AKRC1). Somatic mutation of Keap1 gene and aberrant hypermethylation of Keap1 gene promoter region was found in human lung cancer. Loss of keap1 function activates Nrf2 activity and enhanced the lung cancer cell growth in vitro and in vivo. However, there has not been studied Keap1 gene status in colorectal cancer. In this study, we investigated status of keap1 gene in 10 human colorectal cancer cell lines. No somatic mutation of keap1 gene was found in all 10 cell lines. Hypermethylation of Keap1 promoter region was found in 8 cell lines. Keap1 mRNA and protein expression tended to be lower in these hypermethylated cell lines. The keap1 mRNA in a methylated cell line (HT29) was re-expressed by treatment with the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-azadC) and Trichostatin A (TSA). mRNA of the downstream genes, such as NQO-1 and AKRC1, were over expressed in the methylated cell line (HT29) compared to a nonmethylated cell line (Colo320DM) in Real time PCR analysis. In addition, we examined the methylation status of keap1 promoter region in primary tumors of 30 patients with colorectal cancer using methylation-specific PCR. An aberrant methylation in keap1 promoter was detected in 16 of 30 (53%). These findings suggest that hypermethylation of Keap1 promoter region in human colorectal cancer upregulates the expression of the downstream genes of keap1/nrf2 system which can be associated with the advantage for cancer progression and resistance to the anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 102. doi:10.1158/1538-7445.AM2011-102

Clinicopathological characteristics of malignant lymphoma in patients with hepatitis C virus infection in the Tohoku district in Eastern Japan

Katsushi Tajima, Naoto Takahashi, Kenichi Ishizawa, Kazunori Murai, Tomoaki Akagi, Hideyoshi Noji, Osamu Sasaki, Masaharu Wano, Jugoh Itoh, Yuichi Kato, Tsutomu Shichishima, Hideo Harigae and Yoji Ishida: for the Tohoku Hematology Forum Department of Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba, Japan; Department of Neurology, Hematology, Metabolism, Endocrinology, and Diabetology, Yamagata University School of Medicine, Yamagata, Japan; Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine, Akita, Japan; Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan; Department of Hematology/ Oncology, Internal Medicine, Iwate Medical University, Morioka, Japan; Department of Hematology, Aomori Prefectural Central Hospital, Aomori, Japan; Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan; Division of Hematology, Department of Internal Medicine, Miyagi Cancer Center, Natori, Japan; Department of Hematology, Iwate Prefectural Hospital, Morioka, Japan; Department of Medical Oncology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

Abstract 536: Mesenchymal stromal cells promote tumor growth both in vitro and in vivo

[Background] Mesenchymal stromal cells (MSCs), also referred as mesenchymal stem cells are pluripotent progenitor cells that differentiate into chondrocytes, adipocytes, osteoblasts and other types of cell. Although MSCs are resident mainly in bone marrow, MSCs are found in adipose tissue, lung, and many other organs where MSCs maintain and regenerate the connective tissues of organs. Recent report proposed that MSCs also migrate and function as stromal cells in the tumor tissues. Effects of MSCs on tumor growth in vitro and in vivo are still unclear. In this study, we verified tumor promotion by MSCs and analyzed mechanisms. [Method] B16-LacZ, a melanoma cell line expressing β-galactosidase protein, was tested for their growth rate by X-gal colorizing assay in the presence or absence of mouse bone marrow MSCs in vitro. Growth of B16-LacZ was also examined in the detached condition using cell disk or conditioned medium from MSCs. Next, B16-LacZ and Lewis Lung Carcinoma (LLC) cells were mixed with MSCs at 1/1 and 1/5 ratios and subcutaneously inoculated into mice. Tumor sizes were measured and immunostaining of CD31 was performed to evaluate angiogenesis. We also determine the secretion of soluble factors associated with angiogenesis in cultured medium from B16-LacZ alone, MSCs alone, or B16-LacZ and MSCs ELISA. [Result] Proliferation of B16-LacZ co-cultured with MSCs exhibited 5.8 times greater than that of B16-LacZ alone in vitro (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 536.