PP6 deficiency in mice with KRAS mutation and Trp53 loss promotes early death by PDAC with cachexia-like features.

To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer.

Associations of cigarette smoking, alcohol drinking and body mass index with survival after colorectal cancer diagnosis by anatomic subsite: a prospective patient cohort study in Japan.

BACKGROUND: Cigarette smoking, alcohol drinking and obesity are known to be risk factors for colorectal cancer. These factors may affect survival after diagnosis, but evidence has been inconsistent. We investigated subsite-specific associations between prediagnosis smoking, alcohol drinking and body mass index and survival in colorectal cancer. METHODS: Subjects were 1300 patients (colon 778; rectum 502; concurrent 20) with histologically confirmed colorectal cancer diagnosed during 1997-2013 at a single institution in Japan. Histories of smoking and alcohol drinking, height and prediagnosis weight were assessed using a self-administered questionnaire. Using Cox proportional hazards model, hazard ratios and 95% confidence intervals of mortality were estimated. RESULTS: During a median follow-up period of 6.7 years, 479 deaths were documented. Ever-smoking was associated with an increased risk of all-cause death among patients with colon cancer (hazard ratio: 1.47; 95% confidence interval: 1.07-2.02 compared with never-smoking). According to colon subsite, this increased risk was clear in patients with proximal colon cancer (hazard ratio: 2.09; 95% confidence interval: 1.28-3.40). There was no association between smoking and rectal cancer survival. Alcohol drinking was not associated with survival for either colon or rectal cancer. Among patients with rectal cancer, higher body mass index was associated with a lower risk of all-cause (Ptrend = 0.0006) and disease-specific death (Ptrend = 0.02). For colon cancer, lower body mass index tended to be associated with a higher risk of all-cause death (Ptrend = 0.05). CONCLUSIONS: The results indicate that lifestyles identified as risk factors for colorectal cancer may impact differently on patient survival according to anatomic subsite.

Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis.

According to TCGA database, mutations in PPP6C (encoding phosphatase PP6) are found in c. 10% of tumors from melanoma patients, in which they coexist with BRAF and NRAS mutations. To assess PP6 function in melanoma carcinogenesis, we generated mice in which we could specifically induce BRAF(V600E) expression and delete Ppp6c in melanocytes. In these mice, melanoma susceptibility following UVB irradiation exhibited the following pattern: Ppp6c semi-deficient (heterozygous) > Ppp6c wild-type > Ppp6c-deficient (homozygous) tumor types. Next-generation sequencing of Ppp6c heterozygous and wild-type melanoma tumors revealed that all harbored Trp53 mutations. However, Ppp6c heterozygous tumors showed a higher Signature 1 (mitotic/mitotic clock) mutation index compared with Ppp6c wild-type tumors, suggesting increased cell division. Analysis of cell lines derived from either Ppp6c heterozygous or wild-type melanoma tissues showed that both formed tumors in nude mice, but Ppp6c heterozygous tumors grew faster compared with those from the wild-type line. Ppp6c knockdown via siRNA in the Ppp6c heterozygous line promoted the accumulation of genomic damage and enhanced apoptosis relative to siRNA controls. We conclude that in the presence of BRAF(V600E) expression and UV-induced Trp53 mutation, Ppp6c haploinsufficiency promotes tumorigenesis.

Associations of Japanese food intake with survival of stomach and colorectal cancer: A prospective patient cohort study.

Dietary factors may affect the prognosis of digestive tract cancer, but evidence has been sparse. We investigated the association between pretreatment intake of 6 Japanese foods (including soy food, miso [soybean paste] soup and seaweed) and the risk of death among patients with histologically confirmed major digestive tract cancers (stomach, 1931; colon, 793; rectum, 510) diagnosed during 1997-2013 at a single institution in Japan. Pretreatment dietary intake was assessed using a food frequency questionnaire, and the patients were followed until December 2016. The Cox proportional hazards model was used to estimate hazard ratios (HR) and 95% confidence intervals (CI). Among the patients with stomach cancer, frequent intake of soy food was inversely associated with the risk of all-cause (Ptrend for four frequency groups = 0.01; HR = 0.72, 95% CI: 0.50-1.04 for highest vs lowest group) and stomach cancer (Ptrend  = 0.03; HR = 0.63, 95% CI: 0.40-0.99) death. A similar inverse association was also found for intake of miso soup. In contrast, frequent seaweed intake was inversely associated with the risk of all-cause death among the patients with colon cancer (Ptrend  = 0.03). Rectal cancer patients who had frequently consumed seaweed tended to have a lower risk of rectal cancer death (Ptrend  = 0.02). These findings indicate that pretreatment intake of Japanese foods such as soybean products and seaweed may have favorable effects on patient survival of stomach and colorectal cancer, although this needs to be confirmed by further research.

Efficacy of oral administration of cystine and theanine in colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery: a multi-institutional, randomized, double-blinded, placebo-controlled, phase II trial (JORTC-CAM03).

PURPOSE: Capecitabine-based adjuvant chemotherapy for colorectal cancer patients often causes adverse events (AEs), such as diarrhea, stomatitis, anorexia, and hand-foot syndrome (HFS). Cystine and theanine were reported to attenuate some chemotherapy-associated AEs, and hence are also expected to attenuate capecitabine-induced AEs. Therefore, we aimed to investigate the safety and efficacy of cystine/theanine treatment in colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery. METHODS: A total of 100 colorectal cancer patients treated with capecitabine as an adjuvant chemotherapy after surgery were randomly allocated into the cystine/theanine group (n = 52) or the placebo group (n = 48). The primary endpoint was incidence rate of diarrhea of grade 1 or higher in accordance with the Common Terminology Criteria for AEs (CTCAE) v.4.0, Japanese Clinical Oncology Group (JCOG) version. The secondary endpoints included incidence rates of other AEs (CTCAE v.4.0-JCOG), as well as the incidence rate of HFS according to the HFS grading scale. RESULTS: There were no significant differences in capecitabine-induced AEs between the two groups. However, the incidence rate of diarrhea of grade 1 or higher tended to be lower in the cystine/theanine group than the placebo group (18.4% vs. 28.9%, p = 0.169) as well as the incidence rate of HFS of grade 1 or higher (CTCAE v.4.0-JCOG or HFS grading scale) (67.4% vs. 77.8%, p = 0.185, 67.3% vs. 80.0%, p = 0.124, respectively). CONCLUSION: This trial demonstrated that cystine/theanine treatment of colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery is safe and has the tendency to reduce the incidence rate of diarrhea or HFS. TRIAL REGISTRATION: UMIN000024784.

Associations of cigarette smoking and alcohol drinking with stomach cancer survival: A prospective patient cohort study in Japan.

Cigarette smoking and alcohol drinking may affect the prognosis of stomach cancer, but evidence has been inconsistent. We investigated the associations between pretreatment smoking and alcohol drinking and the risk of all-cause and stomach cancer death among 1,576 patients with histologically confirmed stomach cancer diagnosed during 1997-2010 at a single hospital in Japan. Histories of smoking and alcohol drinking were assessed using a self-administered questionnaire. The patients were followed until December 31, 2013. The Cox proportional hazards model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). During 9,625.1 person-years, 670 all-cause and 419 stomach cancer deaths were documented. Among the patients overall, ever-drinking was significantly associated with an increased risk of all-cause death (HR: 1.25; 95% CI: 1.03-1.51), but not stomach cancer death. Positive linear associations with the frequency of drinking (ptrend = 0.02) and the amount of alcohol consumed per day (ptrend = 0.03) were observed for the risk of all-cause death. Ever-smoking was not related to either the risk of all-cause or stomach cancer death. Conversely, among the patients who underwent curative resection, a significant positive association was found between ever-smoking and the risk of stomach cancer death (HR: 2.44; 95% CI: 1.17-5.08). A positive association was also found for earlier age at start of smoking (ptrend = 0.0046). Pretreatment smoking and alcohol drinking have significant effects on stomach cancer survival. Lifestyle adjustments throughout life may improve survival.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-rasG12D -expressing and Ppp6c-deficient) mice in which K-rasG12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably-treated K-rasG12D -expressing mice did not. H&E-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly-mutant vs K-rasG12D mice revealed that cell proliferation and cell size increased approximately 2-fold relative to K-rasG12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-rasG12D -only mice. Moreover, AKT phosphorylation increased in K-rasG12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-rasG12D -dependent tumor promotion.

Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer-specific metabolism.

Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.

NDRG2, suppressed expression associates with poor prognosis in pancreatic cancer, is hypermethylated in the second promoter in human gastrointestinal cancers.

Although N-myc downstream regulated gene 2 (NDRG2) is frequently downregulated in various cancers and is considered to be a candidate tumor suppressor gene, molecular mechanisms of the expressional suppression that lead to cancers are largely unknown. Recent studies indicated that epigenetic suppression of NDRG2 involved carcinogenesis and progression in several tumor types, and we demonstrated positive association with NDRG2 suppression and poor prognosis in pancreatic cancer. In this study, we analyzed mRNA and protein expressions of NDRG2 in 26 cancer cell lines (20 colorectal and 6 gastric cancers) and found that many cell lines showed variously reduced NDRG2 expressions. Furthermore, NDRG2 expressions were significantly reduced in primary resected cancer tissues compared to corresponding normal tissues immunohistochemically (19 of 20 colorectal and 14 of 17 gastric cancers). Treatment with 5-Aza-2' deoxycytidine predominantly upregulated NDRG2 expressions in NDRG2 low-expressing cell lines. Bisulfite sequencing analyses and methylation specific PCR revealed that methylation status at one of the two promoters (around exon 2) correlated well with the suppressed expression, and this is the major promoter in colorectal and gastric cancer cell lines. Our present results suggest that hypermethylation in promoter around exon 2 is functioning as essential factors of NDRG2 silencing in gastrointestinal cancers.

SERPINI1 regulates epithelial-mesenchymal transition in an orthotopic implantation model of colorectal cancer.

An increasingly accepted concept is that the progression of colorectal cancer is accompanied by epithelial-mesenchymal transition (EMT). In our study, in order to characterize the properties of EMT in 16 colorectal cancer cell lines, the cells were first orthotopically implanted into nude mice, and the tumors in vivo, as well as cells cultured in vitro, were immunostained for EMT markers. The immunostaining revealed that seven of the cells had an epithelial phenotype with a high expression of E-cadherin, whereas other cells showed opposite patterns, such as a high expression of vimentin (CX-1, COLO205, CloneA, HCT116, and SW48). Among the cells expressing vimentin, some expressed vimentin in the orthotopic tumors but not in the cultured cells (SW480, SW620, and COLO320). We evaluated these findings in combination with microarray analyses, and selected five genes: CHST11, SERPINI1, AGR2, FBP1, and FOXA1. Next, we downregulated the expression of SERPINI1 with siRNA in the cells, the results of which showed reverse-EMT changes at the protein level and in the cellular morphology. Along with immunohistochemical analyses, we confirmed the effect of the intracellular and secreted SERPINI1 protein of SW620 cells, which supported the importance of SERPINI1 in EMT. The development of therapeutic strategies targeting EMT is ongoing, including methods targeting the transforming growth factor-ß signaling pathway as well as the Wnt pathway. SERPINI1 is an important regulator of EMT. Our findings help to elucidate the signaling pathways of EMT, hopefully clarifying therapeutic pathways as well.

Gene-expression profiles correlate with the efficacy of anti-EGFR therapy and chemotherapy for colorectal cancer.

BACKGROUND: Comprehensive gene-expression analysis is very useful for classifying specific cancers into subgroups on the basis of their biological characteristics; it is used both prognostically and predictively. The purpose of this study was to classify unresectable advanced or recurrent colorectal cancer (CRC) by gene-expression profiling of formalin-fixed paraffin-embedded tissues and to correlate CRC subgroups with clinicopathological and molecular features and clinical outcomes. METHODS: One hundred patients with advanced or recurrent CRC were enrolled. RNA extracted from FFPE tissues was subjected to gene-expression microarray analysis. RESULTS: The patients were stratified into four subgroups (subtypes A1, A2, B1, and B2) by unsupervised hierarchical clustering. By use of principle-components analysis (PCA), the patients were divided into subtypes A and B on the basis of component 1 and into subtypes 1 and 2 on the basis of component 2. Subtype A was significantly enriched among patients without the KRAS mutation and with an earlier clinical stage at diagnosis. With regard to anti-EGFR therapy, progression-free survival (PFS) was better for patients in subtype A without the KRAS mutation than for those with the KRAS mutation (P = 0.047). PFS for patients without the KRAS mutation in subtype B was comparable with that for patients with the KRAS mutation (P = 0.55). Similar results were observed in a validation set. CONCLUSION: We found that gene-expression profiles enabled stratification of CRC patients into four subgroups. The efficacy of anti-EGFR therapy was correlated with component 1 from PCA. This comprehensive study may explain the heterogeneity of unresectable advanced or recurrent CRC and could be useful for identifying novel biomarkers for CRC treatment.

Gastrointestinal toxicities of 5-fluorouracil increase the proportion of regulatory T cells in intestinal tract: advantages of alternate-day S-1 administration.

BACKGROUND: 5-Fluorouracil (5-FU), a core anticancer agent for malignancies, induces gastrointestinal (GI) toxicities. Despite recent advances in tumor immunology, it still remains unknown how GI toxicities affect antitumor immunity. S-1 is a tegafur-based oral 5-FU prodrug which has been widely introduced in Japan and other countries. The alternate-day S-1 administration has been proposed to minimize its GI and other toxicities without reducing its anticancer efficacy. METHODS: In this study, two S-1 administration regimens were compared in mice to evaluate their impact of GI toxicities on immunity. In the daily group as a standard administration model, S-1 was administered for 14 days on and 14 days off, and in the alternate-day group as a non-GI toxicity model, S-1 was administered every other day for 28 days. As well as physical findings, regulatory T cells, Th1 cells and other cells in murine lymphoid tissues were analyzed with flow cytometry. RESULTS: Only the daily group exhibited body weight loss and GI toxicities. In the daily group, a proportion of regulatory T cells in the intestinal lymphoid tissue were demonstrated to be six-fold higher than in the control without S-1, and the proportion of Th1 cells showed a decreasing trend. However, the alternate-day group exhibited almost no change in T-cell subsets. CONCLUSION: GI toxicities of 5-FU may have a negative influence on antitumor immunity due to increased proportions of regulatory T cells and decreased proportions of Th1 cells. The alternate-day S-1 administration may be a useful regimen with its minimal influence on T-cell subsets.

Usefulness of alternate-day administration of S-1 and leucovorin in a xenograft mouse model of colorectal cancer: a shorter drug-free interval leads to more efficient antitumor effects.

BACKGROUND: A clinical trial of S-1 with leucovorin (S-1/LV) in metastatic colorectal cancer (CRC) patients demonstrated promising efficacy; however, the gastrointestinal toxicities were so severe that it has not been applied in the clinical setting. On the other hand, alternate-day administration of S-1 has been proposed to attenuate the adverse events without reducing its anticancer activity. Our present study was conducted to confirm the feasibility of alternate-day administration of S-1/LV in in vivo xenograft tumor models. METHODS: Mice were treated with S-1/LV in a daily group (2 weeks of administration followed by 2 weeks of withdrawal) or an alternate-day group (administration on alternate days for 4 weeks), then the mice were killed and the xenograft tumors were resected. We compared body weight changes, condition of feces, mucosal injury and myelosuppression and assessed adverse reactions, tumor volume, tumor growth inhibition (TGI) and expression of Ki67, TUNEL, cIAP2 and XIAP to evaluate the antitumor activity and tumor apoptosis. RESULTS: Severe weight loss, diarrhea, mucosal injury and myelosuppression were observed only in the daily group; however, some myelosuppression was also observed in the alternate-day group. The TGI in the alternate-day group was better than in the daily group, possibly resulting from apoptosis due to the suppression of cIAP2 but not XIAP. CONCLUSION: Our findings suggest that alternate-day administration of S-1/LV for CRC treatment can achieve high antitumor activity without severe adverse reactions, and we propose that clinical trials with this regimen should be conducted in CRC patients.

Effects of duodeno-jejunal bypass on glucose metabolism in obese rats with type 2 diabetes.

PURPOSE: To evaluate the foregut and hindgut hypotheses for metabolic surgery in obese rats with diabetes. METHODS: Otsuka Long-Evans Tokushima fatty rats were divided into a sham operation group, a partial duodeno-jejunal bypass (P-DJB) group, and a complete DJB (C-DJB) group. P-DJB is a model to test foregut hypothesis, whereas C-DJB is a model to test both hypotheses. We performed oral glucose tolerance tests (OGTT) on all groups at baseline, and then 4 and 8 weeks postoperatively. The rats were killed thereafter and the plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were measured. A separate sub-group of C-DJB rats underwent OGTT after treatment with the GLP-1 antagonist, the PYY antagonist, or saline. RESULTS: Marked improvement of the blood glucose control during the OGTT was noted 8 weeks after C-DJB, but not 8 weeks after P-DJB or the sham operation. The serum GLP-1 and PYY levels were higher in the C-DJB group than in the other two groups. Pretreatment with the GLP-1 antagonist increased the blood glucose levels 30 min after the OGTT in the C-DJB rats. CONCLUSIONS: Improvement in glucose metabolism after DJB was associated with the inflow of bile and pancreatic juice into the ileum, supporting validity of the hindgut hypothesis. GLP-1 appears to play a role in this improvement.

Role of the intraluminal contents and the continuity of intrinsic neurons in intracolonic capsaicin-induced contraction and defecation in dogs.

PURPOSE: We, herein, examined the role of the intraluminal contents and continuity of colonic intrinsic neurons in intracolonic capsaicin-induced enhancement of colonic motility and defecation. METHODS: Five beagle dogs were equipped with three strain gauge force transducers throughout the colon. The colonic contractile activity in response to intracolonic capsaicin was studied in intact dogs, dogs after colonic cleansing and dogs with transection/re-anastomosis (T/R) between the proximal and middle colon. The effects of intravenous yohimbine, a α2 adrenergic antagonist, on the colonic motility and defecation were also studied in the same models. RESULTS: In intact dogs, capsaicin (10 mg) and yohimbine (2 mg/kg) immediately induced contractions throughout the colon, with defecation occurring in all experiments. In dogs after colonic cleansing and T/R, the capsaicin (10 mg)-induced enhancement of colonic motility was decreased in the middle and distal colon, and capsaicin-induced defecation was observed in 0-20 % of experiments (p < 0.05 compared to intact dogs). The effect of yohimbine (2 mg/kg) in inducing colonic contractions was unaltered after colonic cleansing and T/R; in contrast, yohimbine-induced defecation was not observed after colonic cleansing, but was unchanged after T/R. CONCLUSIONS: The continuity of the colonic intrinsic nerves as well as the intraluminal contents appear to play an important role in the colonic motor response to intracolonic capsaicin.

Association between being breastfed in infancy and adult colorectal cancer risk among Japanese men and women.

It has been postulated that being breastfed in infancy affects not only health status in childhood but also disease risk in adulthood. To investigate the association of being breastfed with the risks of adult colorectal cancer and benign tumor, we conducted a case-control study including 1190 colorectal cancer and 1585 benign tumor cases and 5301 controls, admitted to a single hospital in Miyagi Prefecture, Japan, between 1997 and 2013. History of having been breastfed was assessed using a self-administered questionnaire, and odds ratios (ORs) were estimated using unconditional logistic regression. There was no association between being breastfed and colorectal cancer risk (breastfed versus formula-only fed, OR = 1.21; 95% CI 0.87-1.67). There was also no association with the risk of benign tumor (OR = 1.04). On the other hand, analyses stratified by sex and birth year found heterogeneous associations. Women born after 1950 who had been breastfed tended to have increased risks of colorectal cancer (OR = 1.58) and benign tumor (OR = 1.51) relative to those who had been formula-only fed, although not statistically significant. In men born after 1950, being breastfed was associated with a significantly decreased risk of benign tumor (OR = 0.57; 95% CI 0.33-0.98).

Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer.

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P=0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2' deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.

Results of modified uncut Roux-en-Y reconstruction after distal gastrectomy for gastric cancer.

BACKGROUND/AIMS: The frequent occurrence of bile gastritis and esophagitis associated with dehiscence of 'uncut' jejunal portion was of concern for "uncut" Roux-en-Y reconstruction after distal gastrectomy. Our aim was to study if our technique of a modified, uncut Roux-en-Y procedure would decrease this dehiscence. METHODOLOGY: Ten patients with gastric cancer underwent distal gastrectomy with a modified, uncut Roux-en-Y reconstruction. Transmural silk stitches were added around the staples at the "uncut" portion in attempt to prevent dehiscence of the staple line. Dehiscence of the jejunum at the enterically closed site was investigated endoscopically or fluoroscopically. RESULTS: Mean operative time and intraoperative blood loss were 246 minutes and 381 mL, respectively. Morbidity occurred in three patients. No dehiscence was observed in any of the patients examined. CONCLUSIONS: These results suggest the possibility that our technique of a modified, uncut Roux-en-Y reconstruction after distal gastrectomy decreases dehiscence of enterically closed portion.

Infiltration of CD40-positive tumor-associated macrophages indicates a favorable prognosis in colorectal cancer patients.

BACKGROUND/AIMS: Recently the role of tumor-associated macrophages (TAMs) on immunity has been variously discussed. We studied a series of cell surface antigens in TAMs in colorectal cancer tissues and their corresponding normal tissues using flow cytometry to find out prognostic indicators of these patients. METHODOLOGY: We assessed the numbers of CD14+ macrophages positive for each of the cell surface antigens (CD80, CD86, HLA-DR, CD1a, CD40 and CD83) in cancer tissues and corresponding normal tissues among 31 patients with colorectal cancer, and performed the univariate and multivariate analysis to find out prognostic indicators for overall survival among the patients. RESULTS: The numbers of CD80+, CD86+ and HLA-DR+ TAMs in the cancer tissues were higher than those in corresponding normal tissues. Inversely CD40+ and CD83+ macrophages in cancer tissues were less than those in normal tissues. With the multivariate analysis, the number of CD40+ TAMs, as well as lymph node metastasis and distant metastasis, was shown to be an independent prognostic factor of colorectal cancer patients. CONCLUSIONS: The dense infiltration of CD40+ TAM in colorectal cancer tissues indicates a favorable prognosis, which suggests that CD40 plays an important role in the tumor immunity of colorectal cancer.

Intragastric Dai-Kenchu-To, a Japanese herbal medicine, stimulates colonic motility via transient receptor potential cation channel subfamily V member 1 in dogs.

Japanese herbal medicine, also known as Kampo, is used for various diseases in Japan. One of those medicines, Dai-Kenchu-To (DKT), is considered clinically effective for adhesive bowel obstruction and chronic constipation. Although scientific evidence of DKT to improve adhesive bowel obstruction was shown in several previous reports, mechanism of DKT to improve constipation remains unknown. Our aim was to study the effect of intragastric DKT on colonic motility and defecation, and the involvement of various receptors in DKT-induced colonic contractions. Five beagle dogs were instructed with serosal strain-gauge force transducers to measure circular muscle activity at the proximal, middle, and distal colon. Dogs are suitable for a present study to administer the drugs repeatedly to the same individual and look at its effect on colonic motility. We studied the effects of DKT (2.5 or 5 g) administered into the stomach on colonic motility. Muscarinic receptor antagonist atropine, nicotinic receptor antagonist hexamthonium, or 5-hydroxytryptamine-3 receptor antagonist ondansetron was injected intravenously 10 min before DKT administration. Capsazepine, an antagonist to transient receptor potential cation channel subfamily V member 1 (TRPV1), was administered into the stomach 5 min before DKT administration. Intragastric DKT (2.5 or 5 g) induced colonic contractions within 10 min after administration but did not induce defecation. Pretreatment with atropine, hexamthonium, ondansetron, or capsazepine inhibited DKT-induced colonic contractions. These results indicate that orally administered DKT stimulates colonic motility via TRPV1, muscarinic, nicotinic, and 5-hydroxytryptamine-3 receptors, thereby providing scientific support for the efficacy of oral DKT in chronic constipation.