VEGF expression is augmented by hypoxia­induced PGIS in human fibroblasts.

Prostacyclin synthase (PGIS or PTGIS) is an enzyme that catalyses the conversion of prostaglandin H2 (PGH2) to prostaglandin I2 (PGI2). PGI2 promotes cancer growth by activating peroxisome proliferator-activated receptor δ (PPARδ), and increases the expression levels of the pro-angiogenic factor vascular endothelial growth factor (VEGF). We found that the expression of the PGIS gene was enhanced in WI-38, TIG-3-20 and HEL human lung fibroblast cells and two cancer cell lines (NB-1 and G361) under hypoxic conditions. The main localization of PGIS changed from the cytoplasm to the nucleus by hypoxia in WI-38 cells. The induced PGIS had an enzymatic activity since the intracellular level of 6-keto-prostaglandin, a useful marker of PGI2 biosynthesis in vivo, was increased with the increasing levels of PGIS. Expression of VEGF was increased in parallel with PGIS induction under hypoxic conditions. PGIS knockdown resulted in the decreased expression of VEGF mRNA. Since VEGF is a known PPARδ target gene, we examined the effects of siRNAs targeting PPARδ on the expression of VEGF under hypoxic conditions. Knockdown of PPARδ suppressed the expression of VEGF under hypoxic conditions in WI-38 cells. These findings suggest that PGIS is induced by hypoxia and regulates the expression of VEGF in fibroblasts. Fibroblasts in the hypoxic area of tumors may have an important role in tumor growth and angiogenesis.

Up-regulation of insulin-like growth factor-binding protein 3 by 5-fluorouracil (5-FU) leads to the potent anti-proliferative effect of androgen deprivation therapy combined with 5-FU in human prostate cancer cell lines.

In this study, we investigated the synergistic mechanism of anti-androgen and 5-fluorouracil (5-FU) combination therapy against castration-resistant prostate cancer (CRPC). Four prostate cancer cell lines, LNCaP, 22Rv1, DU145 and PC3, were examined for their growth dependency on androgens and the insulin-like growth factor 1 (IGF1). We assessed the expression changes of certain growth factor receptors and regulating proteins when treated with 5-FU, and found that 5-FU increased the expression of the IGF-binding protein 3 (IGFBP3). Furthermore, 5-FU inhibited the phosphorylation of Akt and p70 S6K, while the knockdown of IGFBP3 reduced the levels of poly (ADP-ribose) polymerase cleaved by 5-FU in PC3 cells. Therefore, the up-regulation of IGFBP3 by 5-FU not only inhibits cell growth by reducing the IGF1 signal but also induces apoptosis in PC3 cells. The synergistic effect of bicalutamide and 5-FU on 22Rv1 cells was reduced by IGFBP3 gene silencing using small-interfering RNA. These results suggest that the up-regulation of IGFBP3 induced by 5-FU plays an important role in the potent anti-tumor effect of 5-FU combined with anti-androgens on CRPC. Androgen-deprivation therapy combined with 5-FU could therefore be an appropriate therapy for CRPC patients.

Hydroxyflutamide enhances cellular sensitivity to 5-fluorouracil by suppressing thymidylate synthase expression in bicalutamide-resistant human prostate cancer cells.

We investigated the antitumor effects of combination therapy with anti-androgens and 5-fluorouracil (5-FU), and examined the underlying mechanism of the treatment. Initially, we established the bicalutamide-resistant subline CDX25R from the androgen receptor (AR)-positive human prostate cancer cell line LNCaP through continuous exposure to bicalutamide. CDX25R cells lost the ability to respond to androgens, but still expressed AR. They showed significant resistance to bicalutamide, but had high sensitivity to hydroxyflutamide (OH-flutamide) compared with LNCaP cells. The CDX25R subline was thus considered to be a suitable model for prostate cancer that has developed resistance to first-line hormonal therapy but shows sensitivity to an alternative approach. Combined treatment with 5-FU and OH-flutamide had a synergistic effect on CDX25R cells. OH-flutamide decreased expression of the transcription factor E2F1, and subsequently of thymidylate synthase (TS), in CDX25R cells but not in AR-negative DU145 cells. This suggested that OH-flutamide enhanced the growth-inhibitory activity of 5-FU in CDX25R cells by reducing TS expression through the AR pathway. Combined therapy with 5-FU and OH-flutamide may, therefore, be appropriate for patients with prostate cancer that has acquired resistance to initial hormone therapy including bicalutamide.

Molecular determinants of folate levels after leucovorin administration in colorectal cancer.

PURPOSE: Oral leucovorin (LV) is used with uracil/tegafur (UFT) in the treatment of colorectal cancer (CRC). In order to find the factors related to the efficacy of LV in enhancing the antitumour effect of UFT, we investigated the relationships between the reduced folate levels in the CRC tissue after LV administration and the gene-expression levels of folate-metabolizing enzymes and folate transporters. METHODS: The subjects were 60 CRC patients, scheduled to undergo surgery. The control group (n = 30) did not receive LV. Three groups (n = 10 for each) received a single dose of oral LV at 25 mg, 4, 12 or 18 h before surgery (LV 4 h, LV 12 h or LV 18 h groups, respectively). The reduced folate levels in plasma and tissues were measured by high-performance liquid chromatography (HPLC) or a thymidylate synthase-FdUMP binding assay, respectively. The intratumoral expression levels of 34 genes were quantitatively evaluated with a real-time polymerase chain reaction (RT-PCR) assay. RESULTS: The reduced folate levels persisted for a longer period of time in the CRC tissue than in the plasma after LV administration. A multivariate logistic regression analysis revealed that high folylpolyglutamate synthase (FPGS) gene expression, low gamma-glutamyl hydrolase (GGH) gene expression and low ATP-binding cassette sub-family C, number 1 (ABCC1) gene expression in CRC tissues were predictive factors for a high reduced folate level after LV administration. CONCLUSIONS: The expression level of FPGS, GGH and ABCC1 in CRC tissues could predict the reduced folate level after LV administration, and these factors may determine the efficacy of LV treatment.

Different histological types of non-small cell lung cancer have distinct folate and DNA methylation levels.

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC.

Gene expression signature for recurrence in stage III colorectal cancers.

BACKGROUND: Colorectal cancer patients with lymph node metastases (stage III) show poorer prognosis than those without. Predicting development of recurrence may guide the need for intensive follow-up and/or adjuvant chemotherapy in such patients. The authors' objective was to identify a set of discriminating genes that could predict recurrence in stage III colorectal cancer. METHODS: Thirty-six stage III colorectal cancer patients were studied. Tumor samples were obtained from surgically resected specimens. Thirteen patients developed recurrence, whereas 23 patients did not. Gene expression profiles were determined using human HG-U133 Plus 2.0 Gene Chip (Affymetrix, Santa Clara, Calif). RESULTS: The authors identified 45 discriminating genes between patients with and without recurrence. By using this gene set, they established a new model to predict recurrence with an accuracy of 90.9%. The discriminating genes included calcineurin-binding protein 1 (CABIN1), whose expression differed remarkably between patients with and without recurrence (P=.0073). The authors further examined the DNA copy number of CABIN1 and were able to show a significant relation with recurrence (P<.012). Patients having CABIN1 gene loss demonstrated a higher risk of recurrence (odds ratio, 18.8). DNA copy number of CABIN1 alone could predict recurrence with an accuracy of 80.0%. CONCLUSIONS: The results of the current study demonstrated that gene expression profiling is useful in predicting recurrence in stage III colorectal cancer. The authors identified CABIN1 among discriminating genes that may play a key role in the development of recurrence. These results may help to establish an individualized therapy for stage III colorectal cancer.

Oral fluoropyrimidine S-1 combined with leucovorin is a promising therapy for colorectal cancer: Evidence from a xenograft model of folate-depleted mice.

The oral fluoropyrimidine S-1 has marked efficacy in treating metastatic colorectal cancer patients. In the present study, we explored the therapeutic potential of combined in vivo treatment in the human colon cancer cell lines COL-1, KM12C and KM20C with consecutive oral S-1 and leucovorin (LV) in 2-week therapeutic periods. This combination had increased anticancer activity compared to S-1 alone in the xenografts tested. Moreover, oral S-1/LV treatment showed a more potent anticancer effect on COL-1 xenografts than infusional 5-fluorouracil (5-FU)/LV, with comparable loss of body weight. The reduced folate level in the tumors was initially low, but rapidly rose and persisted for a long period of over 24 h after a single LV administration. This resulted in the formation of much higher levels of the ternary complex with thymidylate synthase (TS) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) derived from 5-FU, leading to a prolonged inhibition of TS activity in combined administration with oral S-1. These results suggest that the co-administration of LV with S-1 might improve therapeutic efficacy in the treatment of colorectal cancer patients.

Thymidylate synthase, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase mRNA and protein expression levels in solid tumors in large scale population analysis.

It has been reported that the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil (5-FU)-based therapy in cancer patients. We investigated the differences in the mRNA and protein expression of these enzymes in various tumor tissues. A total of 17,613 specimens of head and neck, gastric, colorectal, breast, lung and pancreatic cancer were collected from multiple facilities in Japan, and the mRNA and protein expression levels of the above enzymes were examined in 4,830 and 12,783 of these specimens, respectively. The mRNA levels were analyzed using RT-PCR in laser-captured microdissected formalin-fixed paraffin-embedded specimens, while the protein levels were analyzed by enzyme-linked immunosorbent assays. The median values of the relative TS, DPD and OPRT mRNA levels were 2.06, 0.803 and 1.17, respectively, while the median protein levels were 22.1, 134.8 and 3.81 ng enzyme/mg protein, respectively. The carcinomas were classified into two sets of four groups each using the overall median levels of TS and DPD or TS and OPRT as cutoff values. Approximately 60% of the gastric cancers exhibited elevated mRNA and protein expression levels of DPD, while >65% of the colorectal cancers showed low levels of DPD expression. Overall, 75% of the head and neck cancers exhibited high expression levels of DPD. Among the lung and pancreatic cancers, 50-74% showed low TS/high DPD expression. In conclusion, the mRNA expression and protein levels of TS, DPD and OPRT differed according to the type of cancer. The results of this large-scale population analysis are expected to be useful as reference data for predicting the relationship between the respective enzyme levels and the efficacy of 5-FU-based chemotherapy.

Molecular basis for the induction of an angiogenesis inhibitor, thrombospondin-1, by 5-fluorouracil.

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein.

Down regulation of c-Myc and induction of an angiogenesis inhibitor, thrombospondin-1, by 5-FU in human colon cancer KM12C cells.

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.

Anti-angiogenic effect of 5-Fluorouracil-based drugs against human colon cancer xenografts.

In addition to the direct cytotoxic effects of chemotherapy agents on tumor cells, the anti-angiogenic activities attained by these agents by targeting proliferating endothelial cells in tumor blood vessels has attracted much research interest. In this study, we examined the antitumor activity of 5-Fluorouracil (5-FU)-based drugs (S-1 [1M tegafur, 0.4M 5-chloro-2,4-dihydroxypyridine and 1M potassium oxonate] and capecitabine) on human colorectal cancer xenografts and evaluated their anti-angiogenic effects. Both drugs showed significant antitumor activities against COL-1 xenografts at a sub-maximum tolerated dose (sub-MTD), which was lower than the maximum tolerated dose (MTD). At the sub-MTD, a significant reduction in the microvessel number and the enhancement of tumor-associated microvessel endothelial cell apoptosis was seen in xenografts treated with S-1. In addition, we found that thrombospondin-1 (TSP-1) expression, known to be a mediator of the anti-angiogenic effects of metronomic chemotherapy, was significantly up-regulated in xenograft tumor tissues and plasma in animals treated with S-1 at a sub-MTD. Capecitabine also showed a trend toward the induction of TSP-1. These results suggest that 5-FU-based drugs inhibit tumor progression through different modes of action, including cytotoxic activity derived from 5-FU and the inhibition of angiogenesis through the induction of TSP-1.

Combination of O6-methylguanine-DNA methyltransferase and thymidylate synthase for the prediction of fluoropyrimidine efficacy.

We investigated the correlation between the response to fluoropyrimidines as first-line therapy and the expressions of genes in patients with primary colorectal cancer (CRC). The study group comprised 92 patients with metastatic CRC. Total RNA was isolated from laser-captured tumour cells in surgically resected primary lesions, and gene expression was quantitatively evaluated by real-time RT-PCR assay. Low thymidylate synthase (TS), low gamma-glutamyl hydrolase, high reduced folate carrier 1, high O6-methylguanine-DNA methyltransferase (MGMT) and low cyclin E expressions were associated with a good response (P=0.0030, 0.0250, 0.0120, 0.0030 and 0.0020, respectively) on univariate analysis. On multivariate logistic regression analysis, TS and MGMT remained independent predictors of the response. The clinical response rates were 63.2% in the low TS or high MGMT group and 14.3% in high TS and low MGMT group (P<0.0001). The combination of high TS and low MGMT expression is a significant predictor of a poor response to fluoropyrimidine treatment.

Folylpolyglutamate synthase and gamma-glutamyl hydrolase regulate leucovorin-enhanced 5-fluorouracil anticancer activity.

Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2'-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU.

Orotate phosphoribosyltransferase expression level in tumors is a potential determinant of the efficacy of 5-fluorouracil.

Although the intratumoral expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are known to affect the antitumor activity of 5-fluorouracil (5-FU), the importance of orotate phosphoribosyltransferase (OPRT) has remained unclear. This study investigated the relationship between intratumoral OPRT expression and the antitumor activity of 5-FU using human NCI60 cell lines with similar levels of TS and DPD messenger RNAs, as well as 31 tumor xenografts. The OPRT mRNA level was positively correlated with the 5-FU efficacy in these cell lines. In vitro, the 50% growth-inhibitory concentrations of 5-FU were closely correlated with the OPRT mRNA levels in cancer cell lines with similar levels of TS mRNAs when combined with a DPD inhibitor. Moreover, downregulation of OPRT with small-interfering RNA decreased the sensitivities of the cultured tumor cells to 5-FU. These results suggest that the OPRT expression level in tumors is an additional determinant of the efficacy of 5-FU.

Beneficial effects of suplatast tosilate (IPD-1151T) in a rat cystitis model induced by intravesical hydrochloric acid.

OBJECTIVE: To examine the effects of suplatast tosilate (IPD-1151T), a Th2 cytokine inhibitor recently recognized to improve the symptoms in patients with interstitial cystitis (IC), in a rat model of HCl-induced chronic cystitis, to elucidate the possible mechanisms by which the drug improves the symptoms of IC. MATERIALS AND METHODS: Chronic cystitis was induced by intravesical instillation of 0.2 mL of 0.4 m HCl in female adult rats. After a once-daily oral administration of IPD-1151T (0.1-100 mg/kg) or prednisolone (5 mg/kg) for 7 days, cystometry was performed under urethane anaesthesia. The bladder from HCl-induced cystitis rats was also assessed histopathologically. RESULTS: On cystometrography there was frequent voiding in cystitis rats. Administration of IPD-1151T for 7 days after intravesical HCl instillation dose-dependently increased the micturition volume and intercontraction intervals. Treatment with prednisolone had similar therapeutic effects. Histological analyses in the bladder from cystitis rats revealed oedema and infiltration of inflammatory cells such as mast cells and eosinophils in the lamina propria and the transitional epithelial thickening. These histological changes and the number of mast cells and eosinophils were reduced by administration of IPD-1151T or prednisolone. CONCLUSION: The present results indicate that IPD-1151T improves bladder function and pathological changes in HCl-induced cystitis rats, as previously observed in patients with IC. The rat cystitis model induced by HCl could provide useful information for studying proposed therapies for IC which might involve T cell-dependent inflammatory responses as one of its potential pathophysiologies.

Contrasting effects of extended low dose versus standard dose shorter course UFT chemotherapy on microscopic versus macroscopic established tumors: implications for optimal postoperative adjuvant chemotherapy.

Given such differences as relative tumor burden, the optimal dose and schedule for postoperative adjuvant chemotherapy of microscopic disease might be expected to differ significantly from therapy of advanced higher volume disease. We investigated this hypothesis by determining the optimal dose and schedule of the 5-FU pro-drug, UFT, for treatment of early versus later stage disease models of the Lewis lung carcinoma (LLC). Postoperative adjuvant therapy of early stage disease was modeled by intravenous injection of LLC cells and initiating therapy one day later, thus simulating the presence of micrometastases at the time of surgery. As a model of 'late' stage disease, a LLC fragment was implanted subcutaneously and UFT therapy was initiated when the tumor was firmly established and had grown to >5 mm in size. A number of UFT dosing protocols were evaluated such as short-term (daily, for 7 days) maximum tolerated dosing (MTD), e.g. 31 mg/kg/day, or a much longer-term (e.g., daily, for up to 60 days) repetitive dosing using doses such as 24 mg/kg/day (the MTD) or lower. The long-term consecutive administration of UFT at relatively low minimally toxic dose levels is a superior dosing regimen in the postoperative adjuvant chemotherapy model; in contrast, the short-term higher dose protocols were superior for treatment of more advanced, established cancer. In addition, the efficacy of UFT in an adjuvant setting is more effective when drug administration is continued for longer periods and when treatment is initiated at progressively earlier time points, after disease establishment.

Genome-wide screening of loci associated with drug resistance to 5-fluorouracil-based drugs.

Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome-wide correlation analysis between DNA copy number and antitumor activity against 5-fluorouracil (5-FU)-based drugs (S-1, tegafur + uracil [UFT], 5'-DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nucleotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5-FU-based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5-FU-based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response.

[Benefits of TS-1 plus leucovorin combination therapy for colorectal cancer: evaluation of therapeutic effect of TS-1 and leucovorin combination therapy using rodent model fed a low folate diet].

We evaluated the therapeutic effect of TS-1, a novel oral fluoropyrimidine, in combination with leucovorin with in vivo experiments using a murine tumor xenograft model fed a low folate diet. The reduced folate levels in the tumors of mice fed a low folate diet were significantly lower than those in the tumors of mice fed a normal diet. In addition, the basal reduced folate levels in the tumors of mice fed a low folate diet were comparable to those in human colorectal cancer tissues. Furthermore, when leucovorin was orally administered, the reduced folate levels in the tumors of mice fed a low folate diet were more than two-fold higher than those of mice fed the normal diet. The leucovorin-induced potentiation of TS-1 antitumor activity was obviously higher in COL-1 tumor-bearing mice fed a low folate diet than in mice fed a normal diet. The remarkable increase in reduced folate levels following the administration of leucovorin contributed to the leucovorin-induced potentiation of TS-1 antitumor activity in this low-folate-diet animal model. These results suggest that rodent models fed a low folate diet are suitable for in vivo evaluation of reduced folate drugs like leucovorin. In this model, the combination of leucovorin with TS-1 resulted in a higher antitumor efficacy than TS-1 alone or the combination of UFT and leucovorin, suggesting that TS-1 and leucovorin combination therapy may be an effective regimen for patients with colorectal cancer.

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.

Additive antitumor effect of concurrent treatment of 4-hydroxy tamoxifen with 5-fluorouracil but not with doxorubicin in estrogen receptor-positive breast cancer cells.

PURPOSE: The sequential addition of tamoxifen (TAM) to chemotherapy seems superior to its concurrent addition in patients with breast cancer. This study was conducted to clarify the hypothesis that there are differential interactions among TAM and chemotherapeutic agents. METHODS: Estrogen receptor (ER)-alpha-positive or -negative breast cancer cells were treated with 4-hydroxy TAM (4OHT), 5-fluorouracil (FU) and/or doxorubicin (Dox). Changes in the expression levels of genes related to sensitivity and resistance to TAM, 5-FU or Dox were tested. RESULTS: Concurrent treatment of 4OHT with 5-FU but not with Dox additively inhibited the growth of ER-alpha-positive cells. 5-FU did not change the expression levels of any tested genes related to either sensitivity or resistance to TAM. Although Dox did not change the expression levels of any genes related to the sensitivity to TAM, Dox significantly increased the expression levels of some genes related to TAM resistance, Eph A-2, ER-beta, Fos and vascular endothelial growth factor. 4OHT significantly decreased thymidilate synthase (TS) activity. CONCLUSIONS: Although the antitumor effect of concurrent 4OHT and 5-FU was additive, that of concurrent 4OHT and Dox was less than additive in ER-alpha-positive cells. The increased expression of genes related to TAM resistance by Dox might be responsible for the interaction. Decreased TS activity by 4OHT might increase the antitumor activity of 5-FU. These findings may provide a preclinical rationale for concurrent use with 5-FU and TAM.