Toxicity against gastric cancer cells by combined treatment with 5-fluorouracil and mitomycin c: implication in oxidative stress.

PURPOSE: Combined chemotherapy of 5-fluorouracil (5FU) and mitomycin c (MMC) is clinically used for gastric cancer, but the precise conditions and molecular mechanism of these agents when used together remain unclear. We examined the administration sequence of combining 5FU with MMC to maximize toxicity against a human gastric cancer cell line, and then investigated the possible molecular mechanisms underlying the observed toxic effects. METHODS: Human gastric cancer MKN45 cells were treated with a combination of 5FU and MMC, and the changes in cell viability and apoptosis-related proteins were determined by a tetrazolium dye-based cytotoxicity assay and Western blot analysis, respectively. The intracellular levels of reactive oxygen species (ROS) were monitored using a fluorescent probe or by a cytochrome c reduction assay. RESULTS: Pretreatment for 24 h with 5FU augmented the toxic effect of MMC in MKN45 cells. The synergic effect was mediated mainly via ROS formation and the p53-dependent apoptotic pathway, leading to mitochondrial dysfunction and caspase activation. In vitro experiments using extracts of the treated cells showed superoxide anion generation in a redox cycle of MMC, involving alterations in superoxide dismutase. CONCLUSIONS: Pretreatment with 5FU enhanced the MMC-induced toxicity against gastric cancer cells via alterations in antioxidant enzymes with resulting ROS generation. This observation will need confirmation in the clinical setting.

Orotate phosphoribosyltransferase gene polymorphism predicts toxicity in patients treated with bolus 5-fluorouracil regimen.

PURPOSE: We investigated whether the determination of orotate phosphoribosyltransferase (OPRT) and thymidylate synthase (TYMS) polymorphisms could predict the toxicity of 5-fluorouracil (5-FU) in colorectal cancer patients. EXPERIMENTAL DESIGN: The determination of OPRT and TYMS genotypes were done in genomic DNA extracted from blood by PCR amplification in 69 patients treated with bolus 5-FU as adjuvant chemotherapy. Associations between these polymorphisms and toxicity were evaluated retrospectively. RESULTS: The Ala allele in OPRT Gly213Ala polymorphism and the two tandem repeats (2R) in TYMS promoter polymorphism were associated with grade 3 to 4 neutropenia and diarrhea. The multivariate logistic regression models revealed that only TYMS promoter polymorphism had an independent value to predict grade 3 to 4 neutropenia [odds ratio, 19.2 for patients with the 2R allele compared with patients with homozygous with the three repeat (3R) alleles], whereas both OPRT and TYMS promoter polymorphisms were independent predictive factors for grade 3 to 4 diarrhea (odds ratio, 13.3 for patients with the Ala allele compared with patients in the Gly/Gly genotype and 11.1 for patients with the 2R allele compared with patients in the 3R/3R genotype). A significant difference was observed in the time to onset of severe toxicity, defined as grade 4 neutropenia and/or grade 3 to 4 gastrointestinal toxicities according to OPRT and TYMS promoter polymorphisms. CONCLUSION: OPRT Gly213Ala polymorphism seems to be a useful marker for predicting toxicity to bolus 5-FU chemotherapy. Prospective translational treatment trials including larger number of patients are needed to confirm our results.

Simple combinations of 5-FU pathway genes predict the outcome of metastatic gastric cancer patients treated by S-1.

We evaluated the expression of 5-FU pathway genes in prechemotherapeutic fresh frozen samples obtained from primary tumors to predict response and survival of 59 metastatic gastric cancer patients treated with S-1 monotherapy as first line treatment. Five 5-FU pathway genes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), thymidine phosphorylase (TP) and uridine phosphorylase (UP), were analyzed by the quantitative real-time reverse transcriptional PCR method. Median values of each gene were selected for cut-off values separating high and low gene expressions. In univariate analyses, low TS, high OPRT and low TP were significantly associated with a tumor shrinkage and a long survival, whereas DPD and UP gene expressions did not correlate with response and survival. Multivariate analyses revealed that independent variables were OPRT and TS for response and TS and TP for survival. When OPRT and TS were combined, a significantly increased accuracy rate of 91.5% was seen for response. Similarly, an increased hazard ratio of 10.29 was observed for survival in patients possessing low TS and low TP, compared with those with high TS or high TP. The simple combinations of 2 genes, OPRT and TS for response and TS and TP for survival, may allow identification of gastric cancer patients who will benefit from S-1 chemotherapy.

Gene expression of ferredoxin reductase predicts outcome in patients with metastatic colorectal cancer treated by 5-fluorouracil plus leucovorin.

PURPOSE: Ferredoxin reductase (FDXR) is a putative contributor to p53-mediated apoptosis from 5-fluorouracil (5-FU) through the generation of oxidative stress in the mitochondria. However, the influence of FDXR gene expression levels on the outcome of 5-FU chemotherapy has been relatively little studied. The aim of this study is to investigate the association between FDXR gene expressions and the clinical outcome when treated by 5-FU chemotherapy, as well as the correlation of FDXR gene expressions and p53 mutation. METHODS: Pre-chemotherapeutic fresh frozen samples of 33 patients with metastatic colorectal cancer, who received bolus 5-FU and leucovorin (LV) as first line chemotherapy, were studied. FDXR gene expression and p53 mutation were evaluated by real-time RT-PCR and direct sequencing, respectively. RESULTS: FDXR gene expression was significantly higher in responding tumors compared with non-responding ones (P=0.0379). Patients with FDXR values above the cutoff value of 13.52 had a statistically longer survival than those with FDXR gene expressions below the cutoff value (P=0.0148). The 9 tumors with wild-type p53 had statistically higher FDXR gene expressions than the 14 tumors with mutant-type p53 which had sequence alterations within the "hot spot" codons, the L2-L3 loops, or frameshift (P=0.0463). CONCLUSIONS: FDXR gene expression did not affect clinical outcome in patients with wild-type p53 tumors, whereas, among patients with p53 mutant-type tumors, patients with tumors with low FDXR gene expression had a worse outcome than those with a high FDXR gene expression (P=0.0200). FDXR gene expression, which is regulated at least in part by p53, is associated with both response and survival when metastatic colorectal cancer is treated with 5-FU plus LV. In addition, analysis of p53 mutation combined with FDXR gene expression might be useful in estimating the outcome in 5-FU-treated patients.

Serum levels of interleukin-12 in Graves' disease and their dynamic changes after surgery.

PURPOSE: To investigate the role of interleukin-12 (IL-12) in Graves' disease, we measured the pre- and postoperative levels of serum IL-12 in patients undergoing surgery for Graves' disease. METHODS: The subjects of this study were 73 patients with Graves' disease, admitted for surgical treatment after taking antithyroid drugs for various durations. We collected blood from 11 of these patients, 1, 3, and 6 months postoperatively, to measure the serum IL-12 levels using a Human IL-12 +p40 Immunoassay Kit. RESULTS: The preoperative levels of serum IL-12 were higher in patients with Graves' disease than in healthy controls. Based on the preoperative data, there was a significant relationship between the levels of serum IL-12 and free T3. An analysis of the postoperative time course of these 11 patients showed that the levels of serum IL-12 decreased gradually from 1 month to 6 months, postoperatively. There was also a significant correlation between the levels of serum IL-12 and soluble IL-2R, and a significant negative correlation between the levels of serum IL-12 and thyroid-stimulating hormone receptor antibody. CONCLUSION: Measurement of the levels of serum IL-12 may be a valuable immunological marker in the time course of treatment for Graves' disease.

Serum interleukin-12 levels in patients with gastric cancer.

PURPOSE: To evaluate the immunological status of patients with gastric cancer before surgery, we investigated the relationship between serum interleukin-12 (IL-12) levels and clinicopathological factors. METHODS: We measured serum IL-12 levels in 127 patients with gastric cancer and 35 healthy controls, by a sandwich enzyme-linked immunosorbent assay using the Human IL-12 +p40 Immunoassay kit. RESULTS: The serum IL-12 levels in the patients with gastric cancer were significantly higher than those of the healthy controls (P < 0.05). There were no significant differences in disease stage or gross appearance among the cancer groups, but the serum IL-12 levels in patients with T4 disease were significantly lower than those in patients with T1, T2, or T3 (P < 0.01). There were no significant differences in serum IL-12 levels between patients with and those without lymph node, liver, or peritoneal metastasis. The serum IL-12 levels in patients with distant metastasis were significantly lower than those in patients without distant metastasis (P < 0.02). There were no significant differences in the serum IL-12 levels according to classification by histopathological findings. Analysis with the linear correlation coefficient showed no significant correlation between serum IL-12 and serum carcinoembryonic antigen, carbohydrate antigen (CA) 19-9, CA 72-4, alpha-fetoprotein, or immunosuppressive acidic protein. However, there was a significant relationship between serum IL-12 levels and soluble IL-2 receptor levels (r = 0.53, P < 0.01). CONCLUSION: Serum IL-12 levels in patients with far-advanced gastric cancer were significantly lower than those in patients with less-advanced gastric cancer. This is because macrophages in patients with far-advanced cancer would be hectic and unable to produce sufficient IL-12.

Churg-Strauss syndrome manifesting as perforation of the small intestine: report of a case.

We report a case of Churg-Strauss syndrome (CSS) causing perforation of the small intestine. A 51-year-old woman was admitted with an asthma attack and paralysis of both legs. Intravenous predonisolone (40 mg) was given to relieve her asthma. Laboratory data on admission showed leukocytosis with hypereosinophilia and a high level of serum IgE. Neurological examination also revealed mononeurutis multiplex. Based on these findings, we diagnosed CSS, and oral corticosteroids were continued. On the 20th day after admission, she suffered sudden abdominal pain. Abdominal X-ray showed free air in the abdomen, suggesting perforation of the gastrointestinal tract. Emergency laparotomy revealed generalized peritonitis caused by a perforated ulcer of the ileum. The resected specimens contained a perforation and multiple nonperforated ulcers with an irregular shape on the mucosal surface. Histopathological examinations revealed angiitis of the small vessels surrounded by eosinophilic infiltration and granuloma, consistent with CSS. Considering the high risk of perforation of the gastrointestinal tract, including the small intestine, during corticosteroid treatment in patients with CSS, any abdominal pain or discomfort must be investigated carefully.

Thymidylate synthase and dihydropyrimidine dehydrogenase gene expression in relation to differentiation of gastric cancer.

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are important enzymes of DNA de novo synthesis and the salvage pathway in cancer cells, respectively. Intratumoral TS and DPD gene expressions were evaluated to determine the correlation between the expression of the 2 genes in both normal stromal tissues and tissues with different degrees of malignant differentiation in primary gastric cancer. The study population consisted of 78 consecutive patients with advanced gastric cancer who underwent surgical treatment. Laser-captured microdissection of malignant or normal stromal tissues was performed in formalin-fixed, paraffin-embedded specimens. After extraction of RNA, TS and DPD gene expressions were measured by the real-time reverse transcriptional PCR method. Apart from degree of differentiation, TS and DPD in malignant tissue showed no correlation with clinicopathologic factors. TS in malignant tissue was higher in differentiated type cases than undifferentiated type cases (p < 0.01). However, DPD in malignant tissue of undifferentiated type cases was statistically higher than that of differentiated type cases (p < 0.05). In normal stromal tissue, neither TS nor DPD had any correlation with clinicopathologic factors. TS in malignant tissue was statistically higher than in normal stromal tissue in both differentiated and undifferentiated types (p < 0.0001). DPD in differentiated type malignant tissue was statistically lower than in normal stromal tissue (p < 0.001), but no difference was seen in undifferentiated type cases. TS and DPD gene expressions in primary gastric cancer differ according to degree of differentiation and between malignant and normal stromal tissue.

Changes in liver enzymes after surgery in anti-hepatitis C virus-positive patients.

The aim of this study was to assess the influence of surgical intervention on changes in liver enzymes in patients with antibodies to hepatitis C virus (HCV). Of 623 patients who underwent laparotomy in our department during the 2 years between January 2000 and December 2001, a group of 39 (6.3%) who were positive for the HCV antibody were enrolled in this study. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and cholinesterase (ChE) were the standard liver tests performed. The antibody to HCV was measured in serum using an ELISA kit that can detect antibodies against the combined epitopes. The postoperative elevated values of AST and ALP in the anti-HCV-positive group were significantly higher than those in the anti-HCV-negative group ( p < 0.05). The postoperative decreased values of ChE in the anti-HCV-positive group were significantly greater than those in the anti-HCV-negative group ( p < 0.02). The postoperatively decreased ratios of ChE in the anti-HCV positive group were significantly greater than those in the anti-HCV negative group ( p < 0.0001). Using multivariate logistic regression modeling, testing positive for the antibody to HCV was independently and significantly associated with abnormal levels of ALT and ALP ( p = 0.035 and 0.018, respectively). Monitoring liver enzymes such as ChE, ALT, and ALP might be effective for evaluating liver function after surgery in anti-HCV-positive patients.

Situs inversus totalis with malignant lymphoma of the stomach: report of a case.

Situs inversus totalis is a rare congenital anomaly that often occurs concomitantly with other disorders. We report a case of situs inversus totalis with malignant lymphoma of the stomach, which was successfully treated by surgery followed by chemotherapy and irradiation. The patient was a 51-year-old woman who present with colicky pain in the left upper quadrant of her abdomen. Chest X-ray showed a right-sided heart, and ultrasonography and computed tomography (CT) of the abdomen showed a situs inversus totalis with multiple gallstones in the gallbladder. Tree-dimensional reconstructed CT of the abdomen showed no other malformations coexisting with situs inversus totalis, but a barium upper gastrointestinal series found an inverted stomach and an elevated tumor with ulceration in the center, localized in the antrum of the stomach. First, we performed a cholecystectomy, followed by a total gastrectomy with dissection of the lymph nodes and splenectomy, and Roux-en-Y reconstruction. Histopathological examination confirmed a diagnosis of malignant lymphoma of the stomach (diffuse large B-cell type) with metastasis to the regional lymph nodes. Chemotherapy using the CHOP regimen was given three times, starting 1 month postoperatively. A followup CT scan showed enlargement of one lymph node around the abdominal aorta and irradiation was delivered to the area of the inverted Y in the abdomen. At the time of writing, 10 months after surgery, the patient is well with no signs of recurrence and leading a normal life. Careful preoperative assessment is very important for determining the most appropriate surgical procedure in patients with situs inversus totalis associated with a malignancy.

Changes in the levels of serum-soluble interleukin-2 receptor after surgical stress.

PURPOSE: Surgical stress induces alterations in numerous physiological functions, including the cell-mediated immune response. It is known that interleukin-2 receptor (IL-2R) is released from its specific affinity membrane receptor on activated T lymphocytes and then is detected as a form of the alpha-chain of the IL-2R in the bloodstream. The levels of serum-soluble IL-2R (sIL-2R) reflect the quantity of activated T lymphocytes. This study investigated the changes in the serum sIL-2R levels and the relationship of such changes with other cytokines and the number of lymphocytes after abdominal surgery. METHODS: Twenty-four patients who were scheduled to undergo abdominal operations were enrolled in this study. Blood samples of these cases were collected before surgery, and on postoperative days (POD) 1, 3, 7, and 14. The levels of serum sIL-2R were measured by an enzyme-linked immunosorbent assay. RESULTS: The levels of serum sIL-2R achieved the maximal values on POD 1, and gradually decreased until POD 14. The levels of serum sIL-2R on POD 1, 3, and 7 were significantly higher than the preoperative levels. There was a significant and positive correlation between the levels of serum sIL-2R and serum IL-6. There were significant and positive correlations between the levels of sIL-2R and the number of white blood cells and neutrophils. Conversely, there was a significantly negative correlation between the levels of serum sIL-2R and the number of lymphocytes. CONCLUSIONS: As high levels of serum sIL-2R were recognized after abdominal operations, the proliferation of T lymphocytes might still be highly activated in a state of surgical stress, though it is popularly acceptable that surgical stress induces a suppression of cell-mediated immunity.

Adrenal metastasis from rectal cancer: report of a case.

We report a case of heterochronic adrenal metastasis from colorectal carcinoma in a 51-year-old woman. A left adrenal metastasis was found by computed tomography and magnetic resonance imaging 8 months after an anterior resection for advanced rectal carcinoma, and a left hepatectomy for a solitary liver metastasis. The level of serum carcinoembryonic antigen was still within the normal range. A left adrenalectomy was performed, and histopathological examination revealed adenocarcinoma, compatible with the rectal carcinoma resected 8 months earlier. The patient died of lung metastases 6 months after the adrenalectomy. A review of autopsy series in the world literature revealed that adrenal metastasis from colorectal cancer is not rare. Therefore, the possibility of adrenal metastasis should be considered in the follow-up of patients after primary surgery for colorectal cancer, even though the liver and lung are the main metastatic sites.

Combination of dihydropyrimidine dehydrogenase and thymidylate synthase gene expressions in primary tumors as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer.

Dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) gene expressions in metastatic colorectal cancer have been reported to be predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy. In this study, we investigated the association between both DPD and TS expressions in primary colorectal tumor and the antitumor effect in patients with metastatic colorectal cancer when treated with a fluoropyrimidine-based protocol. DPD and TS expressions were measured by reverse transcription-PCR in surgically resected materials of primary colorectal tumors from 37 patients who went on to receive oral treatment of uracil and tegafur and leucovorin for either synchronous or metachronous metastatic diseases. Relative mRNA amounts of DPD or TS were expressed as the ratios of targeted gene to glyceraldehyde-3-phosphate dehydrogenase reverse transcription-PCR products. Median values of DPD mRNA expressions were 0.30 and 0.65 for responding tumors and nonresponding ones, respectively, with a statistical significance (P < 0.0001). No responding tumor had a DPD mRNA expression >/= 0.5. A total of 19 tumors had low DPD mRNA expressions of <0.5, and 63% of them showed response. There was no responding tumor with both high DPD and high TS (TS mRNA expression >/= 1.0). However, the response rate was 75% in tumors with both low DPD and low TS. The median survival time was 16.3 months in patients with both low DPD and low TS versus 8.4 months in patients with high DPD or high TS mRNA expression. In conclusion, the combination of DPD and TS mRNA expressions in the primary tumor might be useful as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer.

Induction of thymidine phosphorylase by interferon and taxanes occurs only in human cancer cells with low thymidine phosphorylase activity.

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism. It has been reported to be a prognostic factor for tumor angiogenesis and to activate some prodrugs of 5-fluorouracil (5-FU) to 5-FU. There is also evidence that TP is induced by interferons (IFNs) and xenobiotics, such as cyclophosphamide and taxanes, in experimental human cancer cells and xenografts. We investigated the induction of TP expression by IFNalpha and Paclitaxel in vitro and in vivo in human tumor cells with low and with high TP activity. TP activity in KB, NUGC-3, and KOC2S cells, which had low TP activity, was increased 2 to 4 fold by IFNalpha, but was still lower than in non-treated SHIN-3 and HRA cells, which have high TP activity. IFNalpha did not promote TP activity in SHIN-3 and HRA cells, but expression of TP mRNA increased 2 to 4 fold in response to IFNalpha in all cells tested. These results suggest that the expression of TP protein would be regulated post-transcriptionally by another factor after IFN-induced amplification of TP mRNA. A single dose of Paclitaxel to nude mice xenografted with KB and KM20C tumors, expressing low TP activity, increased TP activity about 4 to 7 fold compared to non-treated tumors. In contrast, TP expression in MX-1 and H-31 tumors was originally high and did not change by the treatment of Paclitaxel. The activities of uridine phosphorylase in all tumors used showed no changes in response to IFNalpha or Paclitaxel. We determined the level of STAT1alpha, an IFN-inducible transcription factor of the TP gene, and found that it was low in low TP expressing tumor cells and markedly increased to about 4 fold by IFN, almost reaching the level in high TP expressing cells whose STAT1alpha level was unchanged by IFN. When TP activity and STAT1alpha expression in clinically resected colorectal cancers were simultaneously measured, almost all tumors had high expression of both TP and STAT1alpha. In conclusion, our results suggest that IFN and Paclitaxel affect human cancer cells with low TP activity but not those with high TP activity and that the STAT1alpha expression may reflect TP activity, at least in experimental human cancer cells.

Serum soluble interleukin-2 receptor (IL-2R) and immunohistochemical staining of IL-2R/Tac antigen in colorectal cancer.

BACKGROUND: In the present study, we investigated the significance of serum soluble interleukin-2 receptor (IL-2R) as a tumor marker, and examined the existence and localization of cells positive for IL-2R/Tac antigen in colorectal cancer tissues and their regional lymph nodes. METHODS: The study included 155 patients with colorectal cancer. Levels of serum soluble IL-2R were measured by an enzyme-linked immunosorbent assay. In the tissues obtained from 18 patients, immunohistochemical staining was performed, with the use of the avidin-biotin-peroxidase complex technique, in which mouse anti-human IL-2R antibody was used. RESULTS: The preoperative levels of serum soluble IL-2R in patients with colorectal cancer were significantly higher than those of normal controls ( P = 0.0065). The levels of serum soluble IL-2R in patients with metastatic lymph nodes were also significantly higher than the levels in those without metastatic lymph nodes ( P = 0.0258). Concerning tumor markers, there were significant differences in serum soluble IL-2R levels between patients who were positive and those who were negative for carcinoembryonic antigen (CEA) and between these who were positive and those who were negative for immunosuppressive acidic protein (IAP). In the immunohistochemical staining of IL-2R, 16 of the 18 patients (88.8%) showed IL-2R-positive cells in the colorectal cancer tissues. In regard to the metastatic lymph nodes, all of 5 patients (100%) showed IL-2R-positive cells. On the other hand, IL-2R-positive cells were not recognized in normal colorectal tissues and non-metastatic lymph nodes. CONCLUSION: These results suggest that activated T lymphocytes infiltrating into cancer tissues to play an antitumor role may release a large amount of the alpha-chain of IL-2R, resulting in the high levels of serum soluble IL-2R in patients with colorectal cancer.