Tranilast strongly sensitizes pancreatic cancer cells to gemcitabine via decreasing protein expression of ribonucleotide reductase 1.

Gemcitabine (Gem) is a dFdC analogue with activity against several solid tumors. Gem is intracellularly phosphorylated by dCK, leading to the production of the metabolite dFdCDP. dFdCDP exhibits the cytotoxic effect by inactivating ribonucleotide reductase larger subunit 1 (RRM1), which is a rate limiting enzyme for de novo DNA synthesis. To date, RRM1 expression is believed to determine sensitivity to Gem in pancreatic and non-small cell lung cancer. In the present study, we found that an anti-allergic drug, tranilast strongly enhanced the sensitivity of pancreatic cancer cell line KP4 to Gem. In growth inhibition assay, 100 microM of tranilast plus 1 microM of Gem more strongly suppressed the growth of KP4 at 12.7-fold in IC50 than single Gem treatment, while this compound no longer affected the sensitivity to other drugs such as 5-fluorouracil, irinotecan or paclitaxel. FACS and TUNEL analysis demonstrated the increased apoptotic population in KP4 cells under tranilast plus Gem, compared with single Gem treatment. In Western blot analysis, tranilast treatment decreased RRM1 expression at protein level with dose-dependency in KP4 cells. Proteasome inhibitor MG132 disturbed the reduction of RRM1 expression in tranilast treated KP4 cells, indicating protein degradation by the activated proteasome. Transfection using siRNA against RRM1 increased the sensitivity of KP4 to Gem, suggesting that RRM1 suppression is an important step in increasing Gem efficacy. Finally, we demonstrated that tranilast reduced RRM1 protein and increased Gem efficacy in 4 other pancreatic cell lines. In a future, a novel chemotherapeutic strategy by Gem along with tranilast might improve Gem efficacy against pancreatic cancer.

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.

Hypoxia-inducible factor-1alpha expression predicts the response to 5-fluorouracil-based adjuvant chemotherapy in advanced gastric cancer.

Hypoxia frequently occurs in various solid tumors, thereby accelerating cancer progression and treatment resistance. Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in tumor hypoxia by up-regulating the gene expression related to angiogenesis, cancer invasion and anti-apoptosis. The present study immunohistochemically investigated HIF-1alpha expression in 63 gastric cancer specimens. Those specimens were obtained from 44 patients that received 5-FU chemotherapy post-operatively whereas the remaining 19 patients did not. The immunostaining pattern of HIF-1alpha was classified into 3 patterns: diffuse-positive within the tumor (DP), positive at the invasive front of the tumor (FP) and negative (N). Thirty-six of 63 (57.1%) patients exhibited DP, 24 (38.1%) revealed FP and the remaining 3 (4.8%) patients were judged as N. The HIF-1alpha expression pattern grouped into DP and FP/N correlated with the clinicopathological factors and survival. As a result, the HIF-1alpha expression did not show a significant correlation with the clinicopathological factors, such as the depth of invasion, lymph node metastasis and tumor stage, nor patient survival in the 63 patients. However, in the 44 patients that underwent chemotherapy, patients with the FP/N pattern showed longer survival than those with the DP pattern. On the other hand, no significant difference in survival was found between the 2 patterns among 19 patients without the chemotherapy. These results indicated that the diffuse expression of HIF-1alpha in gastric tumors might lead to drug resistance against adjuvant chemotherapy using 5-FU. In conclusion, the assessment of the HIF-1alpha expression in the resected tissues might predict the drug response to adjuvant 5-FU chemotherapy in advanced gastric cancer patients.

Ribonucleotide reductase subunit M1 is a possible chemoresistance marker to gemcitabine in biliary tract carcinoma.

Biliary tract carcinoma is a relatively rare tumor with a poor prognosis. Surgical resection is the only potential cure. However, the efficacy of chemotherapeutic agents is disappointing in advanced or recurrent cases. Gemcitabine (GEM) appears to be one of the most promising chemotherapeutic agents in biliary tract carcinoma, and ribonucleotide reductase subunit M1 (RRM1) plays an important role in GEM resistance. The aim of this study was to evaluate the correlation between the expression of RRM1 and the response to GEM in biliary tract carcinoma in vitro and in vivo. The drug sensitivity to GEM was assessed by MTT assay. The expression of RRM1 was evaluated by quantitative RT-PCR, a Western blot analysis and immunohistochemistry. RNAi assay was used to investigate the down-regulation of the expression of RRM1. After the RRM1-specific RNAi transfection, a cell growth assay was performed to evaluate the drug sensitivity to GEM, and flow cytometry and TUNEL assay were performed to evaluate apoptosis. The results showed that in 6 biliary tract carcinoma cell lines, a tendency for a positive correlation between the expression of RRM1 and IC50 for GEM exists (R=0.620, p=0.19). The transfection of the RRM1-specific RNAi suppressed the expression level of RRM1 in a dose-dependent manner. After the transfection of RRM1-specific RNAi into G-415, the drug sensitivity to GEM markedly increased (p<0.001), and apoptosis was highly induced according to flow cytometry and the TUNEL assay. In an analysis of cancer tissue specimens obtained from the 12 patients treated with GEM for biliary tract cancer, RRM1 immunostaining was strongly positive in all of the PD cases (3/3), while weakly positive in all of the PR cases except for one (4/5). In conclusion, RRM1 may be one of the key marker molecules for GEM chemotherapy that overcomes advanced and recurrent biliary tract carcinoma.

Expression of the hMLH1 gene is a possible predictor for the clinical response to 5-fluorouracil after a surgical resection in colorectal cancer.

The loss of a DNA mismatch repair occurs in approximately 15% of sporadic colorectal cancer (CRC) and is usually caused by the lack of expression of the hMLH1 gene due to promoter methylation. Despite undergoing adjuvant 5-fluorouracil (5-FU) therapy after a curative surgical resection, some patients with advanced-stage CRC develop recurrence. In the present study, we investigated whether the hMLH1 mRNA expression or promoter methylation is a prognostic factor in CRC patients treated with adjuvant 5-FU. The hMLH1 mRNA expression levels were measured by quantitative reverse transcription PCR in cancer and normal epithelial cells that were obtained from 94 CRC patients using a laser capture microdissection. Then, the methylation status of the hMLH1 promoter in the CRC tissues was examined by methylation-specific PCR. The hMLH1 mRNA expression levels were significantly lower in the cancer cells than in the normal mucosa (p<0.01) and the hMLH1 mRNA expression levels in the cancer cells were significantly lower in the CRC tissues with methylated versus unmethylated hMLH1 (p<0.01) in the 94 patients. Among the 35 patients receiving adjuvant 5-FU, the disease-free survival rate was significantly better in the patients demonstrating a low hMLH1 mRNA expression in the cancer cells in comparison to that of the patients with a high hMLH1 mRNA expression (p<0.01). Moreover, a multivariate analysis revealed that hMLH1 mRNA expression was a significant independent prognostic factor for tumor recurrence in CRC patients treated with adjuvant 5-FU. However, hMLH1 methylation was not correlated with the survival in these 35 patients. These data suggest that the hMLH1 mRNA quantitation in colorectal cancer cells may be helpful for evaluating the prognosis of CRC patients receiving 5-FU-based adjuvant chemotherapy after a surgical resection.

Helicobacter pylori infection is an independent risk factor for Runx3 methylation in gastric cancer.

Runx3, a member of the human runt-related transcription factor family, is known as a possible tumor suppressor gene for gastric cancer. Runx 3 expression is frequently suppressed by the promoter hypermethylation in gastric cancer cell lines and tissues. However, the precise mechanism of the induction of Runx3 methylation, which is considered to be a critical step in gastric carcinogenesis, remains to be elucidated. In the present study, we evaluated runx3 gene methylation in 57 resected early gastric cancer specimens. Then, we correlated Runx3 methylation in the cancer tissue specimens with clinicopathological factors as well as the mucosal backgrounds, such as intestinal metaplasia surrounding the cancer cells and Helicobacter pylori (H. pylori) infection. Runx3 methylation was observed in 30 of the 57 (52.6%) cancer specimens, whereas methylation was detected in 10 of the 57 (17.5%) corresponding non-cancerous mucosae. In comparison to the clinicopathological factors, Runx3 methylation was significantly correlated with both age and tumor location. A multivariate analysis demonstrated that age and tumor location as well as H. pylori infection were independent risk factors for Runx3 methylation. We demonstrated for the first time that H. pylori infection contributes to Runx3 methylation in gastric cancer tissues. When a persistent infection by H. pylori continues in the middle/lower stomach for a long period, Runx3 methylation may be induced and the subsequent loss of Runx3 expression may therefore affect gastric carcinogenesis.

Role of ASC in hypoxia-mediated cell death in pancreatic cancer.

ASC, an apoptosis-associated speck-like protein, can regulate apoptosis in response to various types of cell death stimuli. In this study, we investigated the role of ASC in hypoxia-mediated cell death in pancreatic cancer. ASC was inducible under a 1% O2 hypoxic condition in pancreatic cancer cells, which was HIF1α-dependent but p53-independent. Two representative chemotherapeutic agents for pancreatic cancer, 5-fluorouracil and gemcitabine, promoted cell death in a p53-dependent manner; however, 1% hypoxia caused chemoresistance to these drugs. Western blot analysis of this condition showed that expression of Bax, a pro-apoptotic gene, decreased, while the anti-apoptotic genes IAP-2 and survivin increased. These results suggest that, although hypoxia induces both pro-apoptotic and anti-apoptotic genes, the total balance seems to be anti-apoptotic dominant, which might explain chemoresistance in pancreatic cancer. To overcome this anti-apoptotic dominant condition, we infected adenovirus-expressing ASC into pancreatic cancer cells. The expressed ASC induced cell death even under 20% normoxia, and was enhanced by hypoxia. Our data demonstrate the possible mechanism of chemoresistance under hypoxia in pancreatic cancer cells, thereby suggesting the potential use of ASC as a new treatment strategy for pancreatic cancer.

Aberrant methylation of p16 predicts candidates for 5-fluorouracil-based adjuvant therapy in gastric cancer patients.

BACKGROUND: Aberrant methylation of some cancer-related genes has been reported to correlate with sensitivity to chemotherapeutic agents. The present study was designed to determine whether DNA methylation in six cancer-related genes affects recurrence of gastric cancer in patients who received 5-fluorouracil-based adjuvant chemotherapy. METHODS: The methylation status of six genes, MGMT, CHFR, hMLH1, p16INK4a, E-cadherin, and Runx3, was analyzed in 56 surgically resected gastric cancer tissue specimens by methylation-specific polymerase chain reaction. Of the 56 patients who underwent surgical resection, 38 received 5-fluorouracil (5-FU)-based adjuvant chemotherapy postoperatively (adjuvant group), whereas the other 18 (32%) did not (surgery group). RESULTS: There were no significant differences between the two groups with respect to sex, cancer differentiation, depth of tumor invasion, lymph node metastasis, lymphatic invasion, vascular invasion and tumor stage. Among the genes, methylation of p16INK4a showed a significant correlation with longer survival in the 38 patients of the adjuvant group, but not in the 18 patients of the surgery group. A multivariate analysis identified p16INK4a methylation to be an independent factor predicting a longer recurrence-free period under 5-FU-based adjuvant chemotherapy. CONCLUSIONS: The present study demonstrated for the first time that gastric cancer patients with p16INK4a methylation specifically benefit from 5-FU-based adjuvant chemotherapy.

The hypoxic environment in tumor-stromal cells accelerates pancreatic cancer progression via the activation of paracrine hepatocyte growth factor/c-Met signaling.

BACKGROUND: Pancreatic cancer is one of the representative solid tumors, in which the hypoxic microenvironment plays a crucial role in malignant progression. We previously demonstrated that tumor-stromal interaction under hypoxia enhances the invasiveness of pancreatic cancer cells through hepatocyte growth factor (HGF)/c-Met signaling. METHODS: We investigated the immunohistochemical expression of hypoxia inducible factor-1alpha (HIF-1alpha) c-Met, and HGF in both cancer and stromal cells using 41 pancreatic cancer tissue specimens, and tried to identify any correlations with the clinical features and survival. RESULTS: Positive staining for HIF-1alpha was observed in both pancreatic cancer and the surrounding stromal cells in more than 30% of the cases, and it significantly correlated with lymph node metastasis (P < .05). A significant correlation was observed between the expression of HIF-1alpha and HGF in stromal cells (P < .05). In addition, the c-Met expression in cancer cells was found to significantly correlate with the HGF expression in not only cancer but also stromal cells. The disease-free survival rates of the patients with HIF-1alpha in cancer, stromal, c-Met in cancer, and an HGF expression in stromal cells was significantly worse than those without such expressions (P < .05). CONCLUSIONS: These data suggest that the HGF/c-Met signaling via HIF-1alpha ?may therefore negatively affect the prognosis in patients with pancreatic cancer, and targeting tumor stroma under hypoxia might thus be potentially useful as a novel therapy for this cancer.

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.

Ethanol injection for ablation of an intractable digestive tract fistula: report of a case.

We successfully occluded an intractable digestive tract fistula by injecting it with absolute ethanol after all other treatments failed. A 48-year-old man suffered from a complex and relapsing digestive tract fistula after curative surgery for advanced colon cancer invading the pancreas and duodenum. After conservative management by fasting, drainage, and irrigation failed, fibrin glue infusion achieved only transient occlusion. We performed surgical repair and he was discharged from hospital, at which time fistulography showed no fistula. However, 1 month later fistulography showed that the fistula had recurred and involved the transverse colon, stomach, and intrahepatic bile duct via the jejunum. Finally, we gave five injections of absolute ethanol into the fistula, which resulted in complete occlusion within 6 months. Considering its clinical efficacy, safety, and cost efficiency, we think that ethanol sclerotherapy is a feasible treatment for intractable digestive tract fistula when conservative therapy fails.