Retrospective analysis of risk factors for postoperative perineal hernia after endoscopic abdominoperineal excision for rectal cancer.

BACKGROUND: In contrast to open-surgery abdominoperineal excision (APE) for rectal cancer, postoperative perineal hernia (PPH) is reported to increase after extralevator APE and endoscopic surgery. In this study, therefore, we aimed to determine the risk factors for PPH after endoscopic APE. METHODS: A total 73 patients who underwent endoscopic APE for rectal cancer were collected from January 2009 to March 2020, and the risk factors for PPH were analyzed retrospectively. RESULTS: Nineteen patients (26%) developed PPH after endoscopic APE, and the diagnosis of PPH was made at 9-393 days (median: 183 days) after initial surgery. Logistic regression analysis showed that absence of pelvic peritoneal closure alone increased the incidence of PPH significantly (odds ratio; 13.76, 95% confidence interval; 1.48-1884.84, p = 0.004). CONCLUSIONS: This preliminary study showed that pelvic peritoneal closure could prevent PPH after endoscopic APE.

Adverse Effects on the Postoperative Urinary Function After Combined Resection of Inferior Vesical Artery in Laparoscopic Lateral Pelvic Lymph Node Dissection: Retrospective Analysis of Consecutive 95 Series.

BACKGROUND: The combined resection of the vesical artery (VA) in laparoscopic lateral pelvic lymph node dissection (L-LPLD) was reported to facilitate the safe dissection of metastatic lymph nodes. However, whether or not the combined VA resection affects the urinary function remains controversial. PURPOSE: The purpose of the present study was to examine the risk factors for the postoperative urinary dysfunction (PUD) after L-LPLD followed by total mesorectal excision and to clarify the effects of the combined VA resection in L-LPLD on PUD. PATIENTS AND METHODS: L-LPLD was performed in 95 patients with advanced rectal cancer at Saga University Hospital and Kyushu University Hospital from January 2013 to December 2017. The risk factors for PUD after L-LPLD were investigated. RESULTS: The univariate analysis revealed that the combined resection of the inferior vesical artery (IVA) was a risk factor for PUD. To examine by the type of IVA resection, the incidence of PUD significantly increased with the bilateral IVA resection, but the unilateral IVA resection induced PUD on the same level with the preservation of IVA. CONCLUSIONS: Bilateral IVA resection in L-LPLD could increase the incidence of PUD. Thus, if possible, the preservation of the unilateral IVA through L-LPLD should be considered.

Predictive value of the ratio of 8­hydroxydeoxyguanosine levels between cancerous and normal tissues in patients with stage II/III colorectal cancer.

Reactive oxygen species (ROS) accumulation is known to induce carcinogenesis and accelerate cancer progression. 8­Hydroxydeoxyguanosine (8­OHdG) is a specific marker of ROS­mediated DNA damage. Therefore, we analysed 8­OHdG levels in cancerous and normal tissue DNA via enzyme­linked immunosorbent assay (ELISA) using 97 tissue specimens obtained from surgically­treated patients with stage II/III colorectal cancer (CRC). Additionally, 8­OHdG levels in these tissues were also assessed via quantitative immunohistochemistry (qIHC). To eliminate individual background variables, the ratio of 8­OHdG levels between cancerous and normal tissues was calculated using both techniques. A comparative analysis demonstrated that the 8­OHdG ratio in DNA was significantly correlated with both lymph node metastasis and lymphatic invasion. Multivariate analysis revealed that a high 8­OHdG ratio in DNA was independently correlated with poor prognosis. These results suggest that the 8­OHdG ratio in DNA reflects ROS­induced cancer progression. Conversely, a low 8­OHdG ratio as estimated via qIHC was an independent factor for poor prognosis. In Kaplan­Meier analysis, the combination of a high 8­OHdG ratio in DNA (ELISA) and a low 8­OHdG ratio in cytoplasm (qIHC) was associated with markedly worse patient prognosis than other combinations. Combined evaluation of the 8­OHdG ratio using ELISA and qIHC may be pivotal for predicting surgical outcomes for patients with stage II/III CRC.

Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer.

BACKGROUND: Imprinted genes are regulated by DNA methylation at imprinting-associated differentially methylated regions (iDMRs). Abnormal expression of imprinted genes is implicated in imprinting disorders and tumors. In colorectal cancer (CRC), methylation and imprinting status of the IGF2/H19 domain have been studied. However, no comprehensive methylation analysis of iDMRs in CRC has been reported. Furthermore, the relationship between iDMR methylation status and other methylation-related issues, such as CpG island methylator phenotype (CIMP) and long interspersed element-1 (LINE-1) methylation, remains unclear. RESULTS: We analyzed the methylation status of 38 iDMRs in 106 CRC patients. We also investigated CIMP, LINE-1 methylation, KRAS and BRAF gene mutations, and loss of imprinting (LOI) of IGF2. We further examined the relationship between these factors and clinicopathological factors. The overall trend in iDMR methylation was towards hypermethylation, and iDMRs could be grouped into three categories: susceptible, resistant, and intermediate-to-aberrant methylation. The susceptible and resistant iDMRs consisted of all types of iDMR (gametic and somatic, maternally and paternally methylated). Hypermethylation of multiple iDMRs (HyMiD)-positive status was statistically associated with CIMP-positive status, but not associated with mutations in the BRAF and KRAS genes. HyMiD-positive status was inversely associated with LINE-1 methylation. Among four iDMRs within the IGF2/H19 domain, IGF2-DMR0 hypomethylation occurred most frequently, but was not associated with IGF2 LOI. Finally, we statistically calculated predictive prognostic scores based on aberrant methylation status of three iDMRs. CONCLUSION: In CRC tissues, some iDMRs were susceptible to hypermethylation independent of the type of iDMR and genomic sequence. Although HyMiD-positive status was associated with CIMP-positive status, this was independent of the BRAF and KRAS pathways, which are responsible for CIMP. Since IGF2-DMR0 hypomethylation and aberrant methylation of other iDMRs within the IGF2/H19 domain were not associated with IGF2 LOI, dysfunction of any of the molecular components related to imprinting regulation may be involved in IGF2 LOI. The prognostic score calculated based on aberrant methylation of three iDMRs has potential clinical applications as a prognostic predictor in patients. Further study is required to understand the biological significance of, and mechanisms behind, aberrant methylation of iDMRs and IGF2 LOI in CRCs.

A survey on selection, use, maintenance, and management of chemical protective gloves at workplaces in Japan.

OBJECTIVES: The aim of this study was to survey the selection, use, and maintenance of chemical protective gloves (CPGs) at real workplaces. METHODS: Subjects comprised 817 workers using CPGs at seven domestic manufacturing plants in Japan. We administered an anonymous questionnaire survey comprising the following aspect related to CPGs: environment of use, conditions of use, method of selection, knowledge/awareness pertaining to their use, method of use, precautionary measures associated with their use, maximum time of use, and maintenance. We compared responses provided by management staff and field workers. Chi square test was used for the analysis. RESULTS: Respondents included 661 (80.9%) manufacturing workers, 121 (14.8%) managers or supervisors, and 35 (4.3%) others. In total, 70.5% organic solvents, 28.9% acid or/and alkali, 18.1% dust, and 10.3% carcinogenic substances were the chemical substances handled using CPGs. The reason for deciding to wear CPGs was "the use of chemical substances" for 46.5%, "notice in safety data sheet (SDS) " for 29.8%, and "management staffs' guidance" for 21.4% respondents. "The grasp of chemical substances" was 70.1% (91.1% excluding "not applicable" ). "Warning of caution on skin and eyes" was indicated by 69.5% (91.0%) and "educational reasons for use of CPGs" was accepted by 68.1% (90.7%) respondents. On the other hand, the rate of responses such as "obtaining permeability test results of target substances" and "mixed substances are selected considering substances with short permeation time" was 25.2% (38.4%) and 29.2% (48.4%), respectively. The rate of "yes" as a response in the item concerning "permeation test" was low. On comparing the response provided by the management staff and field workers, the rate of "the permeation test result of the target substance was obtained" was 27.7% for management staff and 41.2% for field workers (p = 0.022). Regarding the cuffs of CPGs, the rate of "to fold back and to prevent sagging" and "mounted with tape" were 30.5% and 21.8% for management staff and 50.2% and 42.2% for field workers (p = 0.001 and p = 0.001), respectively. DISCUSSION: This survey results suggested that the knowledge of "permeation test" of CPGs was not yet popular at industrial workplaces. It is necessary to disseminate knowledge related to "permeation test" to the users from manufacturers of CPGs. Additionally, the employer should appoint an administrator to ensure that CPGs are worn and increase the understanding of correct knowledge and usage of CPGs among workers.

[A survey of the otoacoustic emissions (OAEs) of workers exposed to noise in manufacturing factories].

OBJECTIVES: Otoacoustic emission (OAE) testing is not widespread in Japanese industrial health. This study examined the association between hearing thresholds (HTs) and OAEs among workers exposed to noise in actual workplaces. METHODS: In two metal products manufacturing factories, 34 workers with noise exposure in the workplace (exposure group, mean age 40.6 ± 9.4 years), and 9 workers with no noise exposure (control group, 49.0 ± 14.3 years) were surveyed. The time-weighted average (LAeq) and maximum (LAMAX) of environmental noise monitoring (ENM), and the time-weighted average (LTWA) and sound exposure level (LAE) of personal noise monitoring (PNM) were measured for each subject at the same time. As hearing effect indicators of noise exposure levels, HTs (0.5, 1, 2, 4 and 8 kHz) and distortion product OAEs (DPOAEs) (2, 3 and 4 kHz) were performed before and after 5 days of work. The results of the ENM, PNM, HTs and OAEs were compared between the groups using Student's t test, and their correlations were investigated using Pearson correlation coefficients. RESULTS: Noise exposure levels of the exposure group were significantly higher than those of the control group. In the exposure group, LAeq, LAMAX, LTWA and LAE were respectively 84.5 ± 4.1 dB(A),89.5 ± 6.3 dB(A), 83.4 ± 4.7 dB(A) and 153.1 ± 15.7 dB(A), and in the control group, they were 53.2 ± 2.6 dB(A), 56.4 ± 2.4 dB(A), 67.8 ± 5.6 dB(A) and 119.5 ± 5.6 dB(A). There was no difference between hearing effect indicators (HTs and OAEs) before and after work in either group. There was no correlation between the noise exposure level (LAeq, LTWA, LTWA and LAE) and HTs or OAEs, but there was a significant correlation between the HTs and OAEs at most of frequencies. The HTs and OAEs of subjects with noise-induced hearing loss (NIHL) were significantly lower than those of subjects without NIHL. CONCLUSIONS: This study revealed there is a significant correlation of the HTs and OAEs before and after 5 days of work. In the future, OAE is expected to be used as a screening test of hearing management of noise-exposed workers in Japan.

Usefulness of a portable internet-enabled ECG recording system for monitoring heart health among Japanese workers residing abroad.

OBJECTIVE: The aim of this study was to examine the usefulness of a portable Internet-enabled ECG recording system (iECG) in monitoring the heart health of Japanese individuals working abroad. METHODS: This study included 24 male Japanese individuals working abroad, who had been found to have risks of cardiovascular diseases (CVDs) at periodic health checkups. The subjects were instructed to send ECG recordings obtained by the iECG via a personal computer from their countries of residence. Interpretations of the ECG results were shared with the participant and the participant's occupational physician via e-mail. Further, a questionnaire survey was conducted among the subjects to determine the usefulness of the iECG. RESULTS: Only 21 subjects (53.7 ± 5.2 years) could send their ECG recordings from their countries of residence. During the monitoring period (average, 0.7 ± 0.3 years), 504 ECG recordings were obtained using the iECG. No new CVD events were observed. The iECG monitoring recorded ST-T abnormality in 4 subjects (19%), ST-T abnormality plus left bundle branch block in 1 subject (5%), bradycardia in 1 subject (5%), atrial fibrillation in 1 subject (5%), short PR interval in 1 subject (5%) and normal findings in 13 subjects (61%). The questionnaire results showed a 68% rating for satisfaction, 58% rating for comfort, 68% rating for device utility and 37% rating for lifestyle improvements brought about by device use. CONCLUSIONS: The iECG could serve as a useful support tool for monitoring heart health in Japanese workers residing abroad with risk factors of CVDs.

Assessment of aggressiveness of rectal cancer using 3-T MRI: correlation between the apparent diffusion coefficient as a potential imaging biomarker and histologic prognostic factors.

BACKGROUND: Diffusion-weighted magnetic resonance imaging (DW-MRI) permits non-invasive assessment of tumor characteristics. PURPOSE: To assess the value of DW-MRI as a potential non-invasive marker of tumor aggressiveness in rectal cancer by analyzing the relationship between tumoral apparent diffusion coefficient (ADC) values of MRI and histopathologic prognostic parameters that are not affected by preoperative chemoradiation therapy. MATERIAL AND METHODS: Forty patients with rectal cancer were assessed with primary staging 3-T MRI, including DWI, before undergoing surgical therapy. In all patients, surgery was performed without neoadjuvant therapy. Mean tumor ADC was measured and compared between subgroups based on pretreatment carcinoembryonic antigen (CEA) levels, MRI parameters (e.g. postoperative local recurrence), and histopathologic parameters, including A (invasive distance: A1, T-stage; A2, mesorectal fascia [MRF] status), B (differentiation grade: B1, poorly differentiated; B2, moderately differentiated; B3, well differentiated), C (others: C1, N-stage; C2, lymphangiovascular invasion). RESULTS: Mean tumor ADCs were different when comparing groups stratified by histologic differentiation grades (P=0.0192). There was no significant difference in mean ADCs when stratifying patients according to CEA levels, T-stage, N-stage, MRF status, presence of lymphangiovascular invasion, or the presence of local recurrence. CONCLUSION: Significant correlations were found between mean ADC values and differentiation grade. ADC may be useful as an imaging biomarker of tumor aggressiveness, but it cannot serve as an independent biomarker of advanced rectal cancer.

Loss of trefoil factor 1 is regulated by DNA methylation and is an independent predictive factor for poor survival in advanced gastric cancer.

Trefoil factor 1 (TFF1) is considered to be a tumor suppressor gene in gastric cancer. However, the role of TFF1 expression and its regulation in gastric cancer patients remain unclear. The aims of this study were to clarify the clinical significance of TFF1 and to determine its regulatory mechanisms. We assessed the immunohistochemical expression of TFF1 in 182 gastric cancer patients and examined whether or not TFF1 is associated with the clinicopathological factors and patient survival. In vitro study using TFF1 knockdown gastric cancer cells evaluated the role of TFF1 in cancer invasion. Bisulfite sequencing was performed to assess DNA methylation of TFF1 in cells and resected tissues. Patients with low expression of TFF1 showed a significantly deeper invasion of the tumor than those with high expression (p=0.037). Low expression of TFF1 was also associated with a poor survival (p=0.029) in 108 patients who were treated by surgery alone. Both TFF1 expression and lymph node metastasis are independent predictive factors for disease-specific survival in a multivariate analysis. In an in vitro study, invasive power of the cells was significantly increased in the TFF1­deficient cells compared with the control cells. Bisulfate sequencing showed that TFF1 expression is strongly dependent on DNA methylation in both gastric cancer cells and tissues. Interestingly, methylation status of two specific CpG sites, which are located close to a TATA box and hypoxia response element (HRE), determined the TFF1 expression in the resected tissues. TFF1 expression is silenced by DNA methylation and is associated with tumor invasion and a poor survival in gastric cancer patients. The expression and̸or methylation status of TFF1 may, therefore, serve as a useful biomarker for predicting survival in patients with advanced gastric cancer.

Primary squamous cell carcinoma of the liver concomitant with primary colon cancer: report of a case.

A 55-year-old Japanese female was admitted to our hospital to treat colon cancer. Computed tomography revealed a 2.6 × 2.0 cm liver mass considered to be liver metastasis. She synchronously underwent right colectomy with D3 lymph node dissection and subsegmentectomy 8 under the diagnosis of advanced colon cancer with liver metastasis. The pathology examination revealed the liver nodule was pure squamous cell carcinoma (SCC), whereas histology of colon cancer was a well differentiated tubular adenocarcinoma containing no squamous component. The patient underwent intensive checkup by imaging for a primary site of SCC. However, no lesion considered as possible primary site of SCC was found. Therefore, the liver nodule was finally diagnosed as a primary hepatic SCC. Primary SCC of the liver is a rare and high-grade malignant tumor. Recurrent multiple liver nodule was found at 13 months after surgery and the patient died of cancer 17 months after surgery.

Aberrant gene methylation is a biomarker for the detection of cancer cells in peritoneal wash samples from advanced gastric cancer patients.

PURPOSE: To assess whether gene methylation in peritoneal fluid (PF) is clinically feasible for determining micrometastasis to the peritoneum in gastric cancer. METHODS: The gene methylation of BNIP3, CHFR, CYP1B1, MINT25, SFRP2, and RASSF2 were analyzed in 107 specimens of PF by quantitative methylation-specific polymerase chain reaction. All patients were placed into one of 3 groups: group A (n = 42), patients with depth of cancer invasion at muscularis propria (MP) or less than MP; group B (n = 45), depth of cancer invasion beyond the MP; and group C (n = 20), histologically diagnosed peritoneal metastasis or cancer cells cytologically defined in the peritoneal cavity. Patients in both groups A and B were diagnosed as having no cancer cells by peritoneal cytology and histology. RESULTS: The methylation status of the 6 genes was found to be significantly different among the 3 groups (group A, 0-5%; group B, 0-15%; group C, 15-45%; P < 0.01). Furthermore, the rate of positive methylation in any of the 6 genes was significantly different in each group (group A, 7%; group B, 20%; group C, 75%; P < 0.001). Three of 9 patients in group B with positive methylation in any of 6 genes experienced peritoneal recurrence. On the other hand, only 1 of 36 patients without gene methylation experienced peritoneal recurrence (P < 0.05). CONCLUSIONS: DNA methylation in PFs is a possible marker detecting occult neoplastic cells on the peritoneum. Methylation analysis along with a cytological examination might therefore improve the positive detection of cancer cells in PF of gastric cancer.

Immunohistochemical and molecular genetic analyses of multiple sporadic gastrointestinal stromal tumors.

A 77-year-old Japanese male patient was admitted to our hospital complaining of general fatigue and melena. A gastroduodenal endoscopic examination revealed no definitive localized lesions. However, both a large amount of cruor and blood flow from the small intestine into the ascending colon was observed during the colonoscopic examination. At least three tumors, believed to originate from the small intestine, were detected by abdominal computed tomography. Based on these findings, multiple and hemorrhagic small intestinal tumors were diagnosed and surgical treatment of the tumors planned. During the celiotomy, twelve tumors were found in the small intestine. Intestinal wedge or partial resection was applied. All excised specimens demonstrated morphology of a submucosal tumor and the largest tumor had a delle with coagulation on the mucosal face. In the histological findings, hematoxylin and eosin staining showed spindle cell morphology. The immunohistochemical examination revealed that the tumor cells were diffusely positive for KIT and CD34. The myenteric plexus layer of the small intestine was focal-positive for KIT and showed no intestinal cells of Cajal hyperplasia. The tumor sequencing results revealed an identical missense mutation in codon 642 of c-kit exon 13 leading to the replacement of lysine by glutamic acid and a silent germ-line mutation in exon 12 of the PDGFRA gene concerning whole blood, normal mucosa and tumors. We concluded that the current subject was categorized as having multiple sporadic-type gastrointestinal stromal tumor with identical mutational types. Although the patient did not receive any adjuvant chemotherapy, there has been no sign of recurrence over the 3 years since the surgery.

Aberrant gene methylation in the lymph nodes provides a possible marker for diagnosing micrometastasis in gastric cancer.

BACKGROUND: This study was designed to determine whether gene methylation is a novel diagnostic marker for micrometastasis to the lymph nodes (LNs) in gastric cancer. METHODS: The gene methylation of CHFR, p16, RUNX3, E-cadherin, MGMT, hMLH1, and ABCG2 genes were analyzed in 49 primary gastric cancer tissues, corresponding to noncancerous tissues and matched LNs by quantitative methylation-specific PCR (q-MSP). RESULTS: CHFR, RUNX3, MGMT, and hMLH1 were more frequently methylated in primary cancer compared with the noncancerous mucosa. Further analyses investigated whether the methylation of the four cancer-specific genes was preserved in LN tissues using the 29 control cases, in which LN metastasis had been histologically confirmed. The methylation of both lesions (M/M pattern) in at least one gene, which was judged to be positive for cancer cells in LNs, was observed in 25 of 29 cases (86%). Quantitative RT-PCR (qRT-PCR) of CEA, CK19, and CK20 mRNA was conducted using the same samples. The mRNA expression of at least one of the three genes was observed in 100% of the specimens. The results of the control analysis were used to attempt to predict micrometastasis by q-MSP and qRT-PCR in the 20 test cases without histological LN metastasis. Six cases (30%) showed the M/M pattern in at least one of the four genes. Three of 20 cases (15%) exhibited both the M/M pattern and positive mRNA. CONCLUSIONS: The methylation analysis revealed the clinical feasibility of detecting occult neoplastic cells in the regional LNs.

Genome-wide screen of promoter methylation identifies novel markers in melanoma.

DNA methylation is an important component of epigenetic modifications, which influences the transcriptional machinery aberrant in many human diseases. In this study we present the first genome-wide integrative analysis of promoter methylation and gene expression for the identification of methylation markers in melanoma. Genome-wide promoter methylation and gene expression of eight early-passage human melanoma cell strains were compared with newborn and adult melanocytes. We used linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression to discover DNA methylation markers. This approach identified 76 markers, of which 68 were hyper- and eight hypomethylated (LME, P < 0.05). Promoter methylation and differential gene expression of five markers (COL1A2, NPM2, HSPB6, DDIT4L, MT1G) were validated by sequencing of bisulfite-modified DNA and real-time reverse transcriptase PCR, respectively. Importantly, the incidence of promoter methylation of the validated markers increased moderately in early and significantly in advanced-stage melanomas, using early-passage cell strains and snap-frozen tissues (n = 18 and n = 24, respectively) compared with normal melanocytes and nevi (n = 11 and n = 9, respectively). Our approach allows robust identification of methylation markers that can be applied to other studies involving genome-wide promoter methylation. In conclusion, this study represents the first unbiased systematic effort to determine methylation markers in melanoma and revealed several novel genes regulated by promoter methylation that were not described in cancer cells before.

MEDME: an experimental and analytical methodology for the estimation of DNA methylation levels based on microarray derived MeDIP-enrichment.

DNA methylation is an important component of epigenetic modifications that influences the transcriptional machinery and is aberrant in many human diseases. Several methods have been developed to map DNA methylation for either limited regions or genome-wide. In particular, antibodies specific for methylated CpG have been successfully applied in genome-wide studies. However, despite the relevance of the obtained results, the interpretation of antibody enrichment is not trivial. Of greatest importance, the coupling of antibody-enriched methylated fragments with microarrays generates DNA methylation estimates that are not linearly related to the true methylation level. Here, we present an experimental and analytical methodology, MEDME (modeling experimental data with MeDIP enrichment), to obtain enhanced estimates that better describe the true values of DNA methylation level throughout the genome. We propose an experimental scenario for evaluating the true relationship in a high-throughput setting and a model-based analysis to predict the absolute and relative DNA methylation levels. We successfully applied this model to evaluate DNA methylation status of normal human melanocytes compared to a melanoma cell strain. Despite the low resolution typical of methods based on immunoprecipitation, we show that model-derived estimates of DNA methylation provide relatively high correlation with measured absolute and relative levels, as validated by bisulfite genomic DNA sequencing. Importantly, the model-derived DNA methylation estimates simplify the interpretation of the results both at single-loci and at chromosome-wide levels.

CPT-11 (SN-38) chemotherapy may be selectively applicable to biliary tract cancer with low hMLH1 expression.

Biliary tract cancer is of highly malignancy with a poor 5-year survival. However, established chemotherapeutic regimens have not yet been established. Previously, we have reported that hMLH1, a mismatch repair (MMR) gene was frequently (57%) found to be lacking in surgically resected biliary tract carcinomas and the patients lacking the expression of hMLH1 revealed a poorer prognosis than those patients who possessed it. The MMR gene has been considered to be associated with sensitivity to various chemotherapeutic agents that act on DNA. A loss of MMR expression has been reported to increase sensitivity to topoisomerase inhibitors such as etoposide (ETP) or camptothecins (CPT). In the present study, whether or not hMLH1 deficiency resulted in a higher sensitivity to irinotecan (CPT-11) active form (SN-38) was investigated using a short interfering (Si)RNA system. A quantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to measure the levels of hMLH1 expression in seven cancer cell lines, and this was compared with the drug sensitivity (IC50) to SN-38. The hMLH1 expression was correlated with the IC50 for SN-38, although the relationship was not statistically significant (R = 0.717, p = 0.0715). SiRNA double strand RNA (dsRNA) was transiently transfected into KMG-C (gallbladder cancer) cells. hMLH1 mRNA expression was repressed by hMLH1 dsRNA in a dose-dependent manner in comparison to the control dsRNA. The cell growth of the hMLH1 dsRNA transfected group was decreased by approximately 50% by SN-38 exposure. Flow cytometry was also carried out to examine the effect of the SN-38 treatment on the cell cycle. Following hMLH1 dsRNA transfection, the subG1 fraction was increased in comparison with the control in a dose-dependent manner. In conclusion, a low expression of hMLH1 in biliary tract cancer may aid in predicting its responsiveness to CPT-11 (SN38).

Tumor-stromal cell interaction under hypoxia increases the invasiveness of pancreatic cancer cells through the hepatocyte growth factor/c-Met pathway.

The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

Aberrant promoter hypermethylation in biliary tract carcinoma.

Biliary tract carcinoma is a relatively rare tumor with a poor survival rate. The molecular biological mechanisms underlying the development of biliary tract carcinomas are not well understood. Promoter methylation is an important epigenetic mechanism for suppressing tumor-suppressor gene activity. There is limited information regarding the abnormal methylation of cancer-related genes in biliary tract carcinoma; however, a few insights have been obtained into the role of epigenetic silencing in the progression of biliary tract carcinoma. In this review, we summarize recent data on gene silencing by promoter hypermethylation, and we discuss the implications for biliary tract carcinomas.

Deficient expression of the DPD gene is caused by epigenetic modification in biliary tract cancer cells, and induces high sensitivity to 5-FU treatment.

5-FU is the drug most frequently used to treat biliary tract cancer, while dihydropyrimidine dehydrogenase (DPD) is known to be a principal factor in 5-FU drug resistance. However, whether DPD activity and mRNA levels correlate with response to 5-FU is unknown for biliary tract cancers. The precise mechanism of DPD regulation also remains to be elucidated. In the present study, we quantitatively analyzed DPD mRNA in 8 biliary tract cancer cell lines using real-time RT-PCR, and assessed whether DPD mRNA levels correlate with DPD activity or the sensitivity to 5-FU. Finally, we examined the epigenetic gene silencing of DPD using one of the 8 lines, a gallbladder cancer cell line with deficient DPD expression, KMG-C. Strong correlation was found between DPD activity and DPD mRNA expression in the 8 cancer cell lines (R=0.797, P=0.0148). DPD mRNA expression and DPD activity exhibited positive correlation with the IC50 for 5-FU (R=0.658, R=0.644, respectively), although these relationships were not statistically significant. In the KMGC cells with deficient DPD mRNA levels, restoration of DPD expression was observed by 5-Aza-2' deoxycytidine (5-aza-C) treatment in a dose-dependent manner, suggesting gene suppression by promoter hypermethylation. Combined bisulfite restriction analysis was performed to analyze the methylation on CpG islands around the 5'-flanking region and intron 1 of the DPD gene, however, no methylated CpG sites were identified in these regions. In addition, the restored DPD expression level was more strongly induced by the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), than 5-aza-C treatment. These findings suggest that other mechanisms, including histone modification, may be important for DPD suppression. In conclusion, these results may aid the selection of 5-FU chemotherapy following determination of DPD expression in biliary tract cancers. Furthermore, epigenetic gene silencing appears to be an important mechanism of DPD suppression in biliary tract cancer.

The significance of aberrant CHFR methylation for clinical response to microtubule inhibitors in gastric cancer.

BACKGROUND: We studied the correlations between CHFR (checkpoint with FHA and RING finger) gene methylation and responses to microtubule inhibitors (MI) in gastric cancer. METHODS: We examined 9 gastric cancer cell lines and 46 gastric cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by methylation-specific polymerase chain reaction (MSP). CHFR mRNA expression was estimated by quantitative reverse transcription-PCR. The MI-induced growth inhibition was assayed by a standard MTT method. RESULTS: CHFR expression was silenced by aberrant promoter methylation in 3 of 9 gastric cancer cell lines. The level of CHFR mRNA expression was closely correlated with IC(50) in the MI-treated cells (R=0.889, P=0.005). In 46 patients with gastric cancers, 24 (52%) presented aberrant CHFR methylation. Among them, 12 patients had received treatment with MI because of advanced-stage tumor or tumor recurrence after surgery. The responders to the MI treatment were 29% in patients with CHFR methylation and 20% in those without the methylation. However, 6 (86%) of 7 patients with methylated CHFR tumor showed some regression or no progression, whereas 4 (80%) of 5 patients with unmethylated CHFR tumor manifested progressive deterioration. CONCLUSIONS: These observations indicated that CHFR methylation may be a clinically useful approach to predict the responsiveness of gastric cancers to treatment with MI.