Junichiro Tanaka

Prediction of sensitivity of rectal cancer cells in response to preoperative radiotherapy by DNA microarray analysis of gene expression profiles.

Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treatment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radiotherapy. Response to radiotherapy was determined by histopathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expression differed significantly between responders and nonresponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion-related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombospondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in nonresponders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets.

Distal Colorectal Cancers with Microsatellite Instability (MSI) Display Distinct Gene Expression Profiles that Are Different from Proximal MSI Cancers

Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%). These genes were related to phenotypic characteristics of MSI cancers. Next, we identified 24 probe sets that were differentially expressed in proximal and distal MSI cancers. These genes included promoter methylation-mediated genes, whose expression was significantly down-regulated in proximal MSI cancers. Among discriminating genes between MSI and MSS, nine methylation-mediated genes showed down-regulation in MSI cancers. Of these, 7 (77.8%) showed down-regulation in proximal MSI cancers. Furthermore, methylation-specific PCR confirmed that frequency of hMLH1 promoter methylation was significantly higher in proximal MSI cancers (P = 0.0317). These results suggested that there is a difference between proximal and distal MSI cancers in methylation-mediated influence on gene silencing. In conclusion, using DNA microarray, we could distinguish MSI and MSS cancers. We also showed distinct characteristics of proximal and distal MSI cancers. The inactivation form of hMLH, per se, differed between proximal and distal MSI cancers. These results suggested that distal MSI cancers constitute a distinct subgroup of sporadic MSI cancers.

Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation.

BACKGROUND Zoledronic acid (ZOL) is clinically available for the treatment of skeletal complications. In preclinical studies, strong anti-cancer activities against breast cancer, prostate cancer, and leukemia were reported. It also inhibited the proliferation of cultured human endothelial cells, suggestive of an anti-angiogenic activity. Since ZOL has the tendency to accumulate in bone, we investigated the effect of ZOL on endothelial progenitor cells (EPCs), which originate from the bone marrow, and play important roles in angiogenesis. MATERIALS AND METHODS Human peripheral blood mononuclear cells were cultured for 7 d to differentiate into EPCs. Cells were treated without/with ZOL or with geranylgeraniol (GGOH). Their endothelial phenotype was confirmed by the expression of CD144 and vascular endothelial growth factor receptor 2 and the tube-like formation ability on Matrigel (Becton Dickinson, Bedford, MA). Annexin V/propidium iodide staining was used to analyze apoptosis. RESULTS ZOL treatment, even at low doses, from d 2 to 7 of culture resulted in impaired EPC differentiation and could be restored by co-treatment with GGOH. On the other hand, treatment of putative EPCs with ZOL at concentrations higher than 10 mum resulted in induction of apoptosis. CONCLUSION ZOL dose-dependently inhibited the differentiation of EPCs, the effect being observed even at low drug levels. At high concentrations, ZOL also induced the apoptotic death of putative EPCs. Since GGOH restored the inhibitory effect of ZOL on EPCs differentiation, the effect of ZOL appears to be dependent on the inhibition of prenylation of small-G-proteins. From these findings, we conclude that ZOL could be a potential anticancer agent by inhibiting angiogenesis.

Gene Expression Signature and the Prediction of Ulcerative Colitis–Associated Colorectal Cancer by DNA Microarray

Purpose: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients. Experimental Design: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group). Results: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor–related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively. Conclusions: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.

Comparison of human gut microbiota in control subjects and patients with colorectal carcinoma in adenoma: Terminal restriction fragment length polymorphism and next-generation sequencing analyses

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in Japan. The etiology of CRC has been linked to numerous factors including genetic mutation, diet, life style, inflammation, and recently, the gut microbiota. However, CRC-associated gut microbiota is still largely unexamined. This study used terminal restriction fragment length polymorphism (T-RFLP) and next-generation sequencing (NGS) to analyze and compare gut microbiota of Japanese control subjects and Japanese patients with carcinoma in adenoma. Stool samples were collected from 49 control subjects, 50 patients with colon adenoma, and 9 patients with colorectal cancer (3/9 with invasive cancer and 6/9 with carcinoma in adenoma) immediately before colonoscopy; DNA was extracted from each stool sample. Based on T-RFLP analysis, 12 subjects (six control and six carcinoma in adenoma subjects) were selected; their samples were used for NGS and species-level analysis. T-RFLP analysis showed no significant differences in bacterial population between control, adenoma and cancer groups. However, NGS revealed that i), control and carcinoma in adenoma subjects had different gut microbiota compositions, ii), one bacterial genus (Slackia) was significantly associated with the control group and four bacterial genera (Actinomyces, Atopobium, Fusobacterium, and Haemophilus) were significantly associated with the carcinoma-in-adenoma group, and iii), several bacterial species were significantly associated with each type (control: Eubacterium coprostanoligens; carcinoma in adenoma: Actinomyces odontolyticus, Bacteroides fragiles, Clostridium nexile, Fusobacterium varium, Haemophilus parainfluenzae, Prevotella stercorea, Streptococcus gordonii, and Veillonella dispar). Gut microbial properties differ between control subjects and carcinoma-in-adenoma patients in this Japanese population, suggesting that gut microbiota is related to CRC prevention and development.

Chromosome 18q deletion and Smad4 protein inactivation correlate with liver metastasis: a study matched for T- and N- classification

Smad4 protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 protein, we focused on the specific impact of Smad4 protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P=0.024) in the level of Smad4 protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.

Laparoscopic surgery for ulcerative colitis: a review of the literature

Despite the development of new therapies, including anti-TNF alpha antibodies and immunosuppressants, a substantial proportion of patients with ulcerative colitis (UC) still require surgery. Restorative proctocolectomy with ileal-pouch anal anastomosis is the standard surgical treatment of choice for UC. With the advent of laparoscopic techniques for colorectal surgery, ileal-pouch anal anastomosis has also been performed laparoscopically. This paper reviews the history and current trends in laparoscopic surgery for UC. The accumulation of experience and improvement of laparoscopic devices have shifted the paradigm of UC surgery towards laparoscopic surgery over the past decade. Although laparoscopic surgery requires a longer operation, it provides significantly better short and long-term outcomes. The short-term benefits of laparoscopic surgery over open surgery include shorter hospital stays and fasting times, as well as better cosmesis. The long-term benefits of laparoscopy include better fecundity in young females. Some surgeons favor laparoscopic surgery even for severe acute colitis. More efforts are being made to develop newer laparoscopic methods, such as reduced port surgery, including single incision laparoscopic surgery and robotic surgery.

Microsatellite instability in poorly differentiated adenocarcinomas of the colon and rectum: relationship to clinicopathological features

Background: Poorly differentiated adenocarcinomas of the colon and rectum (Por) feature the worst prognosis among the various types of colorectal carcinomas. Por is highly associated with microsatellite instability (MSI), although MSI is associated with an improved prognosis in colorectal cancers. Aim: To investigate the influence of MSI on clinicopathological features and survival of patients affected by Por. Methods: 53 patients affected by Por were investigated. DNA extracted from tumour sections and the corresponding normal tissue was analysed by PCR at five microsatellite loci: BAT25, BAT26, D2S123, D5S346 and D17S250. Tumours with alterations at two or more loci were classified as MSI-Por. The others were classified as microsatellite stability (MSS)-Por. The clinicopathological features and survival of patients with MSI-Por and MSS-Por were investigated. Results: Of the 53 patients who were examined, 12 (22.6%) were MSI-Por, whereas 41 (77.4%) were MSS-Por. Significant differences were found between MSI-Por and MSS-Por regarding the following clinicopathological features: age, gender, lymph-node metastasis (MSI-Por: 4/12; MSS-Por: 33/41), TNM stage (MSI-Por: T1/T2/T3/T4 = 2/6/2/2; MSS-Por: 3/3/19/16) and lymphatic invasion (MSI-Por: 4/10; MSS-Por: 27/35). Kaplan–Meier survival curves and log-rank analysis showed that MSI-Por was associated with better prognosis than MSS-Por, although no significant difference was found. Conclusions: Compared with MSS-Por, MSI-Por is significantly associated with a low incidence of lymph-node metastases and a low stage. This indicates that MSI-Por is a less aggressive subtype.

Predictive factors for distant recurrence of HCV‐related hepatocellular carcinoma after radiofrequency ablation combined with chemoembolization

Background  Radiofrequency ablation (RFA) therapy for hepatocellular carcinoma has enabled good local control to be possible. However, after successful local control, distant recurrences frequently occur in the remnant liver.

The expression and function of Toll-like receptors 3 and 9 in human colon carcinoma

Toll-like receptors (TLRs) are pattern-recognition receptors that are important in immune signaling. TLR recognition of various viral components including double-stranded RNA (TLR3) and unmethylated CpG-DNA (TLR9) plays a crucial role in cell survival. However, TLR expression and function in colon carcinoma cells are not well clarified. We investigated the expression of TLR3 and TLR9 in colon carcinoma cells using immunohistochemical methods. The function of TLR3 and TLR9 signaling in carcinoma cell lines was studied by direct cell stimulation with, or by cell transfection of, polyinosinic-polycytidylic acid (Poly I:C), a synthetic form of dsRNA, and by cell stimulation with CpG-oligodeoxynucleotides (ODNs), respectively. Positive TLR3 and TLR9 immunohistochemical staining was observed in 91 and 86% of human hepatocellular carcinoma (HCC) tissues, respectively. Cell surface stimulation of TLR3 with Poly I:C did not affect cell viability but it did activate NF-κB activity. By contrast, stimulation of intracellular TLRs with transfected Poly I:C significantly induced apoptosis. Cell surface stimulation of TLR9 with CpG-ODNs promoted cell proliferation, and, furthermore, these CpG-ODN TLR9 agonists reduced the cytotoxicity of the anticancer drug adriamycin. Cell surface expression of TLR3 and TLR9 in colon carcinoma cells plays an important role in cell survival. In addition, the proapoptotic activity of intracellularly expressed TLR3 may provide the possibility of using TLR3 agonists as novel clinical cytotoxic agents against colon carcinoma cells.