Gen Sakai

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.

Improvement of gastrointestinal quality of life scores in cases of Helicobacter pylori‐positive functional dyspepsia after successful eradication therapy

Functional dyspepsia (FD) refers to a broad range of chronic upper abdominal symptoms associated with food intake. A definitive treatment for FD has not yet been established, and the effect of Helicobacter pylori (H. pylori) eradication still remains under debate. The Gastrointestinal Symptom Rating Scale (GSRS) is a specific questionnaire for patients with gastrointestinal symptoms. The present study examined the quality of life (QOL) of patients with H. pylori‐positive FD following H. pylori eradication.

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.

Expression levels of CXC chemokine receptors 3 are associated with clinical phenotype of type 1 diabetes.

Abstract:  Type 1 diabetes is recognized as one of T helper 1 cell (Th1)‐mediated diseases. The purpose of this article was to investigate the expression levels of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) on CD4 T cells as Th1 markers in Japanese patients with type 1 diabetes and control subjects. A total of 72 patients with type 1 diabetes and 24 healthy subjects were enrolled. Their peripheral mononuclear cells were obtained and stained with anti‐CXCR3, anti‐CCR5, and anti‐CD4 monoclonal antibodies. Flow‐cytometric analysis was performed and patients were classified according to their onset pattern as fulminant, typical, or slow onset. Statistical analysis was performed using ANOVA. CXCR3 expression on CD4 T cells in patients with a fulminant pattern of onset was significantly lower than that in the other groups, and that in patients with a typical pattern of onset was significantly higher than that in the other groups. CCR5 expression on CD4 T cells was not different among the three clinical phenotypes. CXCR3 expression level is associated with the onset pattern of type 1 diabetes. Further studies are needed to clarify the role of chemokines in type 1 diabetes.

Female Functional Constipation Is Associated with Overactive Bladder Symptoms and Urinary Incontinence

This noninterventional cross-sectional study aims to assess the association between functional constipation (FC) and urinary symptoms in female patients with no treatment for urination and defecation. The Rome III criteria for evaluation of defecation, Overactive Bladder Symptom Score (OABSS) for evaluation of urinary symptoms, and clinical features were investigated in 145 female patients. Latent FC and moderate to severe overactive bladder (OAB) were defined on the basis of positivity for two or more of the Rome III criteria and an OABSS ≥ 6 with OABSS Q3 ≥ 2, respectively. In 60 latent FC patients, the OABSS was higher (5.0 versus 3.2, p = 0.001), and concurrent moderate to severe OAB symptoms and OAB with urinary incontinence were more frequent than those in 85 nonlatent FC patients (33.3 versus 10.6%, p = 0.001, and 31.7 versus 7.1%, p < 0.001). Multivariate analysis demonstrated that moderate to severe OAB symptoms were a significant associated factor of latent FC (odds ratio (OR) = 4.125, p = 0.005), while latent FC was the only associated factor of moderate to severe OAB and OAB with urinary incontinence (OR = 4.227, p = 0.005 and OR = 4.753, p = 0.004). In conclusion, moderate to severe OAB symptoms are correlated with FC. Moreover, FC is related to moderate to severe OAB symptoms and to OAB with urinary incontinence.

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.