Motoko Izumiya

Cytomegalovirus is frequently reactivated and disappears without antiviral agents in ulcerative colitis patients

OBJECTIVE: The clinical significance of cytomegalovirus (CMV) reactivation complicating ulcerative colitis (UC) patients has been uncertain. It has therefore remained undetermined whether or not CMV reactivation should be treated in UC patients under immunosuppression. The aim of the study was to clarify the natural history of CMV reactivation in UC patients.METHODS: Sixty-nine UC patients with moderate to severe activity were enrolled in the study. All of the patients were treated with prednisolone, and/or immunosuppressants such as cyclosporine A. We sequentially monitored CMV reactivation every 2 wk up until 8 wk using the CMV antigenemia (Ag) assay and plasma quantitative real-time polymerase chain reaction (PCR) assay for CMV.RESULTS: Immunoglobulin (Ig) G for CMV was positive in 48 patients (69.6%) and negative in 21 patients (30.4%). CMV was reactivated in 25 patients out of the 48 seropositive patients (52.1%) during the study period. The CMV Ag and PCR values were low and none of the patients showed any evidence of CMV infection on biopsy specimens by hematoxylin and eosin staining. While gancylovir (GCV) was not used except in two patients, clinical outcomes including rates of remission and colectomy were not significantly different among the CMV reactivation-positive, -negative, and CMV IgG negative groups. Furthermore, CMV disappeared without GCV in most of the CMV reactivation-positive patients.CONCLUSIONS: CMV is frequently reactivated in active UC patients; however, it disappears without antiviral agents. Therefore, antiviral therapies should not be necessary for most UC patients with only CMV reactivation as long as CMV Ag values are low.

Side population of pancreatic cancer cells predominates in TGF-β-mediated epithelial to mesenchymal transition and invasion

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up‐regulated E‐cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF‐β, SP cells changed their shape into mesenchymal‐like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E‐cadherin expression level. TGF‐β induced EMT‐associated gene alteration such as reduction of E‐cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)‐2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF‐β treatment, whereas MP cells did not respond to TGF‐β‐mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro‐invasion, and in vivo metastasis, as compared to MP cells. Because micro‐invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells.

BackgroundMyeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer.MethodsSeven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry.ResultsSix out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations.ConclusionThese results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.

Psychological aspects of inflammatory bowel disease

Psychological stress has been described as “a process in which environmental demands tax or exceed the adaptive capacity of an organism, resulting in psychological and biological changes that may place persons at risk for disease.”1 Psychological stress is widely believed to play a major role in functional gastrointestinal disorders, especially irritable bowel syndrome. There is a long history of observations suggesting that psychological stress contributes to the course of infl ammatory bowel disease (IBD). The chronic medical conditions characterizing IBD, chronic diarrhea, bloody stools, abdominal pain, weight loss, malnutrition, and weakness, seem to be exacerbated by physiological and psychological stress. From this viewpoint, we often see an overlap of pathophysiology between IBD and irritable bowel syndrome (IBS). Furthermore, recent studies on IBS have demonstrated that dysregulation of the immune system and its interaction with bacteria/fl ora may contribute to IBS pathophysiology, just as with IBD. Here, we review the role of psychological stress in IBD, including our current preliminary observations of patients with ulcerative colitis (UC), and the possibility of an overlap in pathophysiology between IBD and IBS. The infl uence of psychological stress on gastrointestinal tract homeostasis through corticotrophin-releasing factor, a key player in the brain–gut axis

Granulocyte and Monocyte Adsorption Apheresis Therapy Modulates Monocyte‐Derived Dendritic Cell Function in Patients With Ulcerative Colitis

The aim of this study was to elucidate the molecular mechanisms responsible for the therapeutic effects of granulocyte and monocyte adsorption apheresis (GMA). We investigated the alterations in circulating monocyte subsets and monocyte‐derived dendritic cell (moDC) function after GMA therapy in ulcerative colitis (UC) patients. Eighteen patients with UC were enrolled: 14 patients were responders, and 4 patients were non‐responders. Peripheral venous blood was obtained within 5 min before and 5 min after GMA therapy. Flow cytometric analysis for monocyte markers (CD14/CD16) was then performed. Monocyte‐derived dendritic cells were obtained and alterations in their phenotype were analyzed by flow cytometry. Their function was also analyzed in a mixed lymphocyte reaction assay between allo‐naïve T lymphocytes. Flow cytometric analysis for intracellular interferon (IFN)‐γ (T‐helper 1 cells) and interleukin (IL)‐4 (T‐helper 2 cells) was then performed for the stimulated T lymphocytes. In patients who responded to GMA, the average numbers of monocytes, especially CD16+ monocytes, were significantly decreased after therapy (P < 0.05). In responders, post‐GMA moDCs expressed significantly lower CD80 and B7‐DC, which are one of the stimulation and maturation markers of dendritic cells, compared to pre‐GMA moDCs. CD83, CD86 and human leukocyte antigen‐DR also showed a tendency to decrease. In responders, naïve T lymphocytes stimulated with post‐GMA moDCs produced significantly less IFN‐γ and IL‐4 compared to those stimulated with pre‐GMA moDCs. The results of our study show that some of the immunosuppressive effects of GMA therapy may be associated with the modulation of monocyte subsets and moDC function.

Abstract 3146: Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background] Rac1 is one of the key regulators in a variety of physiological processes, including membrane trafficking, cell motility, and cancer invasiveness. Although evidence describing the function of the Rho family small GTPases in colorectal cancer has been recently accumulated, its mechanism in invasion and metastasis remains poorly explored. Here, we directly explore the interaction between Rac1 and β-catenin in colon cancer morphogenesis and metastasis. [Methods] Expressions of cell-cell junctional molecules, small GTPases and some tyrosine kinases such as IGF-1R and c-MET were evaluated by immunohistochemistry in the surgical specimens with human colon cancers. Ten colon cancer cell lines were assessed for Rac1 activation by pull-down assay. Invasion assay was performed to evaluate cells’ behavior. Cell adhesion associated proteins such as E-cadherin and β-catenin and actin cytoskeleton were evaluated with several kinase inhibitors by immunocytochemistry. [Results] First, we found that active Rac1 expression at the invasion front and distant liver metastatic site correlates with loss of E-cadherin and nuclear β-catenin accumulation in colon cancer patients. Next, rho-GTPase activation status was evaluated in various colon cancer cell lines with GST-fused Cdc42 and Rac interactive binding (CRIB) domain of PAK1 (PAK-CRIB). Expression level of active Rac1 correlates with cell invasiveness in colon cancer cell lines. The cell invasiveness was enhanced or inhibited by over expression of either constitutive active mutant of Rac (V12Rac) or dominant negative mutant of Rac (N17Rac) with Matrigel assay. The downstream signaling of Rac1-dependent pathway was inhibited by either dominant negative WAVE (WAVE-MT) or CA region of N-WASP (CA region). The finding that endogenous β-catenin and Rac1 directly were bound in nuclear fractions of cells was detected by co-immunoprecipitation assay. Rac1 inhibition by not only NSC23766, but also IGF-1R inhibitor restores the adhesion of colon cancer cells through inhibiting translocation of β-catenin to the nucleus. We confirmed that Rac1 inhibition induces cell-cell adhesion through restoring E-cadherin localization to the cell membrane and redistribution of β-catenin to the cytoplasm. [Conclusions] Taken together, our results raise the possibility that nuclear translocation of Rac1 and β-catenin at the invasive front could play a role in the invasive activity of colorectal cancer cells. Thus present study provides a concept for a new therapeutic strategy that targeting Rac1 molecul would be effective to suppress colon cancer invasive cell. Citation Format: Shinsuke Funakoshi, Hiromasa Takaishi, Ayano Niibe-Kabashima, Tomohiro Suzuki, Gen Sakai, Motoko Izumiya, Masayuki Adachi, Yasuo Hamamoto, Hajime Higuchi, Akinori Hashiguchi, Nobuhiro Tsukada, Takanori Kanai. Rac1 accumulates in the nucleus at the invasive front of colorectal cancer through promoting cell motility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3146. doi:10.1158/1538-7445.AM2014-3146

Abstract 1017: Wnt signalings contributes to the maintenance of cancer stem cell-like populations in Huh7 hepatocellular carcinoma cell line: Role of hepatic stellate cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Cancer stem cells play crucial roles in cancer development and progression. The function and survival of stem cells appears to be regulated by local microenvironment or niche. In many tumors, tumor cells actively recruit a variety type of cells including myofibroblasts and create a tumor niche that participates in the process of cancer progression, chemo-resistance, and metastasis. During liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblasts and the resulting fibrosis is associated with the development of hepatocellular carcinoma (HCC). However, the possible contribution of HSCs to tumor-stroma interactions is totally unknown. Therefore, our AIM is to investigate precise role of HSCs on interactions with tumor cells, especially with cancer stem cells. METHODS: Hepatoma cell line Huh7 was used in this study. FACS analysis was used to evaluate stem cell markers such as side population (SP) and CD133. Each subpopulation was isolated and stem cell-like properties were evaluated by in vivo tumorigenesity and in vitro sphere-forming activity. To evaluate the role of HSCs on tumor-stromal interactions, Huh7 cells (at lower dish) and immortalized HSC line hTERT HSCs (at upper dish) were co-cultured in Transwell culture dishes. In some experiments, Huh7 and hTERT HSCs were mix-cultured in the upper dish and Huh7 cells in the lower dish was analyzed by FACS. RESULTS: We identified a small percentage (approximately 0.6%) of Huh7 cells as a side population (SP) relative to the main population (MP) cells by Hoechst 33342-based FACS profiles. In SP subpopulation, CD133-positive cells were enriched as compared with MP subpopulation (60.3% in total cells, 81.3% in SP, and 58.0% in MP). Only CD133-positive SP cells, but not CD133-negative SP cells nor CD133-positive MP cells, displayed tumor forming capacity and sphere forming activity, suggesting that distinct CD133-positive SP subpopulation have stem cell-like properties in Huh7 cells. The SP subpopulation of Huh7 is increased after the co-culture with hTERT HSCs for 4 days (7.6 hold), suggesting that HSCs maintains self-renewal of cancer stem cells in Huh7. CD133-positive cells were not increased after the co-culture. An increased number of SP subpopulation was attenuated by the treatment with Wnt antagonist DKK-1. The SP subpopulation in the lower dish was further increased when cancer cells and HSCs were mix-cultured in the upper dish (13.5 hold v.s. single culture). CONCLUSION: CD133-positive SP cells are a distinct cancer stem cell population in Huh7 cells. HSCs play an important role in supporting cancer stem cells in the tumor niche via Wnt signaling. 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 1017.

Abstract 3325: Cancer stem cell like properties of 5-fluorouracil resistant Panc-1 cell

Cancer stem cells (CSCs), a small fraction of tumor-initiating cells, are known to be resistant to chemotherapy-induced apoptosis. CSCs are considered to be responsible for self-renewal/differentiation, invasion, and metastasis of malignant tumor. We have previously reported that pancreatic cancer derived side population (SP) cells, a cancer stem cell enriched fraction, were more resistant to chemotherapeutic agents and more likely to cause epithelial-to-mesenchymal transition (EMT) compare to major population (MP) cells. Therefore, we formulated the HYPOTHESIS that 5-fuluorouracil (5-FU) resistant population cells are enriched with CSCs. The AIM of this study is to evaluate whether apoptosis-resistant cancer cells have CSC-like properties, i.e., self-renewal, local invasion and EMT. METHODS: Seventy to eighty percent confluent pancreatic cancer cells, PANC-1, were incubated in the presence of 5-FU (30mg/ml) for 24 hours, and further incubated for one to five weeks without 5-FU. To assess the capacity of self-renewing, cells were planted using a method for anchorage-independent culturing to form spheroids. EMT was induced with TGF-beta (7.5 ng/ml, for 3 days), then evaluated mRNA expression by real time PCR for E-cadherin, Snail, and vimentin. E-cadherin protein level was also examined for immunoblot analysis. Activity of local invasion was analyzed by Matrigel invasion assay. To evaluate tumorigenicity, 5-FU resistant cells were injected into NOD/SCID mice. RESULTS: 5-FU induced significant apoptosis in PANC-1 cells, reducing the number of surviving cells to less than one percent. The survived cells showed a colony-forming activity as they started to re-proliferate at four weeks after 5-FU removal. There was a group of cells among the 5-FU resistant surviving cells, that gradually regains sensitivity to 5-FU by re-plating. The frequency of cells that were capable of initiating spheres was higher in 5-FU resistant cells and they over expressed stem cell marker genes, Oct 4 and Nanog. Incubation with TGF-beta induced EMT-associated gene alterations. These alterations were greater in 5-FU resistant surviving cells as compared to untreated cells. Matrigel invasion activity of apoptosis-resistant PANC-1 cells was greater by seven fold than controlled cells. In addition, in vivo tumor experiments showed 5-FU resistant cells to be more tumorigenic than conventional Panc-1 cells. In CONCLUSION, apoptosis-resistant cancer cells have CSC-like properties, i.e., initiating sphere formation, expressing stem cell genes, and respond to EMT stimulation. 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 3325.