Hidetsugu Yamagishi

Expression of SDF-1α and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer

Although stromal cell-derived factor (SDF)-1α and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1α and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1α and CXCR4. The relationships between clinicopathological features and SDF-1α or CXCR4 expression were then analysed. Stromal cell-derived factor-1α and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1α and nuclear CXCR4 predicts LN metastasis in CRCs.

Colonic expression of heme oxygenase-1 is associated with a better long-term survival in patients with colorectal cancer

Objective. Heme oxygenase-1 (HO-1) has emerged as a crucial mediator of mucosal defense in the gastrointestinal tract. Its metabolic pathway products, biliverdin/bilirubin and carbon monoxide, can reduce oxidative stress and inflammation, and promote resistance to apoptosis. The role of HO-1 in gastrointestinal malignancies, however, remains to be elucidated. The purpose of this study was to analyze HO-1 expression in human colon adenoma and cancer samples. Material and methods. Fifty-five paraffin-embedded surgical specimens of colorectal cancer and 19 colonic adenoma samples were stained immunhistochemically for HO-1 expression using an anti-HO-1 monoclonal antibody. HO-1 expression was evaluated independently by two different investigators and subsequently correlated to clinical data and patients’ life expectancy. Results. Focal HO-1 expression could be documented in 41.8% (23/55) of patients with colorectal cancer. HO-1 expression in colonic adenoma was detectable in 36.8% (7/19) of cases. The rate of lymphatic tumor invasion was significantly lower in colorectal cancer samples expressing HO-1 (p=0.048). Additionally, fewer lymph node metastases were found in colorectal cancer samples with HO-1 expression, but these differences did not reach statistical significance. Mean observation period was 65.87±3.96 months. Kaplan-Meier analysis showed a significantly better survival for colorectal cancer patients with colonic HO-1 expression (p=0.018). Conclusions. This study demonstrates that colonic HO-1 may be a prognostic marker of colorectal-cancer outcome.

Versatile inhibitory effects of the flavonoid-derived PI3K/Akt inhibitor, LY294002, on ATP-binding cassette transporters that characterize stem cells

Stem cells are undifferentiated cells capable of proliferation, self-renewal, and production of a large number of differentiated progeny. Stem cells exist even in malignancies. They are called cancer stem cells, which may represent the origin of these tumors and may be one of the reasons of chemoresistance. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is important for the maintenance of pluripotency in stem cells. Flow cytometry assay for identifying stem cells defines a side population of cells that displays low fluorescent dye and is highly enriched for stem cells. The dye efflux is attributed to expression of ATP-binding cassette transporters such as P-glycoprotein and breast cancer resistance protein (BCRP)/ABCG2, which also transport a variety of anticancer drugs. The PI3K/Akt pathway can modulate functions of ABC transporters through various mechanisms. Reportedly, inhibition of the PI3K/Akt pathway caused BCRP translocation in hematopoietic stem cells and glioma stem-like cells. On the other hand, a PI3K inhibitor, LY294002, reversed multidrug resistance in cancer cells that overexpress BCRP not by affecting BCRP translocation but putatively as a competitive inhibitor. Other PI3K inhibitors, wortmannin and PI-103, did not reverse BCRP-mediated drug resistance. Since LY294002 is a derivative of quercetin that is a naturally occurring flavonoid, its chemical structure is similar to those of a group of flavonoids but those of wortmannin and PI-103 are quite different. It is known that many flavonoids are inhibitors of BCRP and PI3K. LY294002 has also been reported to exert inhibitory effects on multidrug resistance-associated protein 1 (MRP1) function via dual mechanisms, competitive block of substrate transport and modulation of expression. Furthermore, LY294002 has been found to antagonize transport activity of P-glycoprotein without influencing its expression. Taken together, LY294002 can inhibit all BCRP, P-glycoprotein, and MRP1, which are three major ABC transporters that are highly expressed in stem cells and cause multidrug resistance. Due to its versatile effects, LY294002 could be a lead compound for developing more effective and tolerable reagents for cancer treatment.

Expression profile of REG family proteins REG Iα and REG IV in advanced gastric cancer: Comparison with mucin phenotype and prognostic markers

Regenerating gene family members 1 (REG Iα) and 4 (REG IV) are overexpressed in a subset of gastric cancers. However, comparative characterization of the expression of these family proteins has remained unclear. Therefore, we aimed to elucidate not only the association between REG protein expression and mucin phenotype but also their significance as a prognostic marker for patients with gastric cancer. The expression of REG Iα, REG IV, CDX2, MUC2, and MUC5AC in gastric cancer tissues was examined by immunohistochemistry. The relationship between REG protein expression and clinicopathological parameters or mucin phenotype was then analyzed. REG Iα and REG IV expression was positive in 33 (52%) and 31 (49%) of 63 gastric cancers examined, respectively. REG Iα expression was significantly related to venous invasion and tumor stage, whereas REG IV expression showed no relationship to clinicopathological features. With regard to mucin phenotype, REG IV expression was significantly correlated with MUC2 and CDX2 expression, suggesting an association with the intestinal mucin phenotype of gastric cancer. On the other hand, REG Iα expression had no correlation with MUC2, CDX2, or MUC5AC in gastric cancer tissues. Expression of REG Iα but not REG IV was an independent predictor of poor outcome in patients with gastric cancer. In addition, patients with gastric cancer negative for both REG Iα and REG IV expression had a significantly better outcome than patients positive for either REG Iα or REG IV. Profiling of REG protein expression is useful to for prognostication of patients with gastric cancer.

The PI3K/Akt inhibitor LY294002 reverses BCRP-mediated drug resistance without affecting BCRP translocation

Cellular responses toward cytotoxic drugs are influenced by crosstalk between oncogenic signals and resistance mechanisms. Inhibition of the PI3K/Akt pathway is effective in sensitizing cancer cells of various organs, although the mechanisms largely remain to be elucidated. Breast cancer resistance protein (BCRP)/ABCG2, a drug efflux pump, confers resistance to multiple anticancer agents such as SN-38 and topotecan. Previous studies reported that inhibition of the PI3K/Akt pathway, by gene knockout or PI3K inhibitors, modulated BCRP-mediated drug transport via BCRP translocation in hematopoietic stem cells, renal polarized cells and glioma stem-like cells of mammals. In this study, we assessed the effects of PI3K inhibitors, LY294002 and wortmannin, on BCRP-mediated anticancer drug resistance of human cancer MCF-7 and A431 cells. LY294002, but not wortmannin, reversed the BCRP-mediated SN-38 and topotecan resistance. LY294002 treatment did not affect total or cell surface BCRP levels as determined by western blotting and flow cytometry but blocked BCRP-mediated topotecan efflux in a dose-dependent manner. Immunohistochemical analyses also demonstrated unchanged cellular BCRP distribution. BCRP overexpression in MCF-7 and A431 cells did not confer LY294002 resistance, suggesting that LY294002 is not a transported substrate of BCRP. LY294002 is a derivative of quercetin, a member of flavonoids. Taken together, these results suggest that LY294002 inhibits BCRP-mediated drug transport not by BCRP translocation through the PI3K/Akt signal but putatively as a competitive inhibitor in a major subset of cancer cells. Due to its dual effects, LY294002 could be a lead compound for developing more effective and tolerable reagents for cancer treatment.

Differential mucin phenotypes and their significance in a variation of colorectal carcinoma

AIM To investigate mucin expression profiles in colorectal carcinoma (CRC) histological subtypes with regard to clinicopathologic variables and prognosis. METHODS Mucin (MUC)2 and MUC5AC expressions were assessed by immunohistochemistry for a total of 250 CRC cases that underwent surgical resection. CRCs included 63 well-to-moderately differentiated adenocarcinomas (WMDAs), 91 poorly differentiated adenocarcinomas (PDAs), 81 mucinous adenocarcinoma (MUAs), and 15 signet-ring cell carcinomas (SRCCs). MUC2 and MUC5AC were scored as positive when ≥ 25% and ≥ 1% of cancer cells were stained positive, respectively. The human mutL homolog 1 and human mutS homolog 2 expressions were assessed by immunohistochemistry in PDAs to investigate mismatch-repair (MMR) status. Tumors that did not express either of these two were considered MMR-deficient. Results were analyzed for associations with clinicopathologic variables and the prognosis in individual histological CRC subtypes. RESULTS MUC2-positive and MUC5AC-positive WMDA percentages were 49.2% and 30.2%, respectively. In contrast, MUC2-positive and MUC5AC-positive PDA percentages were 9.5% and 51.6%, respectively. MUC2 levels tended to decrease and MUC5AC levels tended to increase from WMDA to PDA. In 21 tumors comprising both adenoma and adenocarcinoma components in a single tumor (4 WMDAs, 7 PDAs, and 10 MUAs), MUC2 was significantly downregulated in PDA and MUC5AC was downregulated in PDA and MUA in the adenoma-carcinoma sequence. These results suggested that MUC2 levels might be associated with malignant potential and that MUC5AC expression was an early event in tumorigenesis. Despite worse prognoses than WMDA, high MUC2 expression levels were maintained in MUA (95.1%) and SRCC (71.5%), which suggested a pathogenesis for these subtypes distinct from that of WMDA. No significant associations were found between MUC2 expression and any clinicopathologic variables in any histological subtype. MUC5AC expression in PDA was closely associated with right-sided location (P = 0.017), absence of nodal metastasis (P = 0.010), low tumor node metastasis stage (P = 0.010), and MMR deficiency (P = 0.003). MUC2 expression in WMDA was a marginal prognostic factor for recurrence/metastasis-free survival (RFS) by univariate Cox analysis (P = 0.077) but not by multivariate Cox analysis (P = 0.161). MUC5AC expression in PDA was a significant prognostic factor for RFS by univariate Cox analysis (P = 0.007) but not by multivariate Cox analysis (P = 0.104). Kaplan-Meier curves and log-rank tests revealed that MUC2 expression was marginally associated with a better WMDA prognosis [P = 0.064 for RFS and P = 0.172 for overall survival (OS)] but not for PDA. In contrast, MUC5AC expression was significantly and marginally associated with a better PDA prognosis in terms of RFS and OS, respectively (P = 0.004 for RFS and P = 0.100 for OS), but not for WMDA and MUA. CONCLUSION Mucin core protein expression profiles and clinical significance differ according to histological CRC subtypes. This may reflect different pathogeneses for these tumors.

Role of Necl-5 in the pathophysiology of colorectal lesions induced by dimethylhydrazine and/or dextran sodium sulphate.

Necl‐5 is an immunoglobulin‐like molecule that was originally identified as a poliovirus receptor. Although Necl‐5 expression is often up‐regulated in cancer cells, its pathophysiological significance in the development of cancer remains unclear. We investigated the roles of Necl‐5 in the development of colitis‐associated neoplasia. Necl‐5‐deficient mice were generated and treated with dimethylhydrazine (DMH) and/or dextran sodium sulphate (DSS) to induce colitis and its associated neoplasias. Colon tissues were examined for histology, Ki‐67 expression by immunohistochemistry and K‐ras gene mutation. Colon tumours occurred significantly less frequently in heterozygous (Necl‐5+/−) or homozygous Necl‐5‐deficient (Necl‐5−/−) mice than in wild‐type (WT) mice with DMH/DSS treatment. Total ulcer index and inflammatory cell infiltration were significantly lower in Necl‐5−/− mice than in WT mice with DSS alone or DMH/DSS treatment. Colon tumours in both WT and Necl‐5−/− mice showed high cell proliferation ability but lacked K‐ras mutation. The total Ki‐67 labelling index in non‐neoplastic colon epithelium was significantly higher in WT (45.9 ± 0.94) than in Necl‐5+/− (34.3 ± 1.40) or Necl‐5−/− (27.7 ± 1.15) mice with DMH/DSS treatment (p < 0.001). Necl‐5 plays a role in the development of colitis‐associated cancer by up‐regulating colonic mucosal cell proliferation. Copyright

Molecular pathogenesis of sporadic colorectal cancers

Colorectal cancer (CRC) results from the progressive accumulation of genetic and epigenetic alterations that lead to the transformation of normal colonic mucosa to adenocarcinoma. Approximately 75% of CRCs are sporadic and occur in people without genetic predisposition or family history of CRC. During the past two decades, sporadic CRCs were classified into three major groups according to frequently altered/mutated genes. These genes have been identified by linkage analyses of cancer-prone families and by individual mutation analyses of candidate genes selected on the basis of functional data. In the first half of this review, we describe the genetic pathways of sporadic CRCs and their clinicopathologic features. Recently, large-scale genome analyses have detected many infrequently mutated genes as well as a small number of frequently mutated genes. These infrequently mutated genes are likely described in a limited number of pathways. Gene-oriented models of CRC progression are being replaced by pathway-oriented models. In the second half of this review, we summarize the present knowledge of this research field and discuss its prospects.

Involvement of REG Iα protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi‐gene family in humans. REG Iα protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Iα expression in the MSG of patients with primary Sjögrens syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Iα protein, Ki67 and single‐strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Iα protein, Ki67 and ssDNA in the MSG were then analysed. REG Iα protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Iα‐positive than in negative SS patients. REG Iα protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.

Evaluation of a novel high-resolution magnifying videoendoscope that is capable of photodynamic diagnosis and therapy for gastric cancer

OBJECTIVE To evaluate the usefulness of a novel high-resolution magnifying videoendoscope called the XG-0001 (Fujifilm, Tokyo, Japan) that is capable of PDD and PDT in experimental and clinical situations. MATERIALS AND METHODS The fluorescences of three photosensitizers (i.e., porfimer sodium (Photofrin), protoporphyrin IX and talaporfin sodium (Laserphyrin)) were studied experimentally via excitation with a purple diode laser (VDL, wavelength 405nm). Five consecutive patients with superficial early gastric cancer not indicated for surgery or other curative endoscopic treatment due to complicated serious diseases were enrolled in this study. After close endoscopic examinations, 2mg/kg of Photofrin were intravenously injected into the patients for PDT, and 5-aminolevulinic acid (ALA; 15-20mg/kg) was orally taken for PDD. PDD using VDL and PDT using an excimer-dye laser (630nm, 4mJ, 60Hz) were performed with the XG-0001. RESULTS Photofrin and Laserphyrin had experimentally the lowest and highest fluorescence intensities, respectively. The five patients comprised four men and one woman with a mean age 75.2 year and an age range of 56-83 years. Two additional cancerous lesions were newly detected by magnifying pharmacoendoscopy. In each patient, PDD was successfully performed. PDT could also safely performed and CR was obtained in 71.4% (5/7) of the cancerous lesions in five patients, and no serious complications were encountered. CONCLUSION The XG-0001, which is based on a simultaneous videoendoscopy method that uses an RGB color chip CCD, proved extremely useful in routine use and also in PDD and PDT for gastric cancer.