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.

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.

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.

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.

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.

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.

Distal colorectal cancers with microsatellite instability (MSI) display distinct gene expression profiles that are different from proximal MSI cancers.

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

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.

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.

[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 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.

hRFI overexpressed in HCT116 cells modulates Bcl-2 family proteins when treated with 5-fluorouracil.

Exogenous overexpression of hRFI, originally isolated in our laboratory, inhibits not only death receptor-mediated apoptosis but also the mitochondrial apoptosis induced by several chemotherapeutic agents including 5-fluorouracil (5-FU). Recently, it has become clear that hRFI targets and degradates caspase-8 and -10 in death receptor-mediated apoptosis by E3 ubiquitin activity in a ring finger domain homologous to that of X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, the cellular mechanism of the inhibition of mitochondrial apoptosis by hRFI has not been fully elucidated. We prepared HCT116 overexpressing hRFI (HCT116/hRFI) cells and comprehensively analyzed the expression changes of 51 apoptosis-related genes with or without 5-FU treatment between HCT116/hRFI and mock cells using microfluidic low-density arrays. As a result, we identified four genes (Bcl-2, Bcl-XL, cIAP2, and CFLAR) whose expression was four or more times higher in HCT116/ hRFI cells than in HCT116/LacZ cells, and found that Bcl-2 and the ratio of Bcl-2/Bax or Bcl-2/Bak were upregulated when HCT116/hRFI cells were treated with 5-FU. Furthermore, we also validated the up-regulation of Bcl-2 and Bcl-XL in HCT116/hRFI cells treated with 5-FU by Western blot analysis. Such evidence suggests that the modulation of Bcl-2 family proteins seen in 5-FU treatment plays an important role in the anti-apoptotic function of HCT116/hRFI cells.

Expression of cancer cachexia-related factors in human cancer xenografts: an immunohistochemical analysis.

We immunohistochemically evaluated the involvement of five cancer cachexia-related factors, including leukemia-inhibitory factor (LIF), zinc-alpha2-glycoprotein (ZAG), interleukin 6 (IL-6), proteolysis-inducing factor (PIF) and tumor necrosis factor alpha (TNF alpha) in causing cancer cachexia. Twenty-six xenografts implanted into mice were examined for the expression of the cancer cachexia-related factors, in relation to the body weight loss of the hosts. Five xenografts were categorized in the cachectic group, and the remaining 21 xenografts belonged to the non-cachectic group. LIF was extensively expressed in both the cachectic and non-cachectic groups. ZAG and IL-6 were expressed in one of the cachectic and some non-cachectic xenografts. PIF and TNF alpha were detected in one and two non-cachectic xenografts, respectively, but in none of the cachectic ones. Any of five factors examined were not conclusive for causing cancer cachexia in the murine xenograft model. Further analysis is needed in order to elucidate the mechanisms responsible for cancer cachexia.

Preparation of anti-orotate phosphoribosyltransferase antibody and its application to immunochemical detection in human tumor cells.

Orotate phosphoribosyltransferase (OPRT) is a key enzyme in the anabolism of 5-fluorouracil (5-FU), and its expression in tumors is thought to increase the efficacy of 5-FU against the tumor. To detect the OPRT protein by immunoblot and/or immunohistochemical methods, we prepared highly specific antibody to the peptides contained in the human OPRT amino acid sequence. The anti-OPRT polyclonal antibodies, obtained by immunizing rabbits with the OPRT peptides, had a high specificity for the OPRT protein in human tumor xenografts when it was analyzed by immunoblotting, and furthermore, there was a positive correlation between OPRT activity and protein content in 12 human tumors (R2 = 0.632). These results suggest that immunohistochemical detection of tumoral OPRT protein expression with our anti-OPRT antibodies may provide a useful method for predicting the clinical response to 5-FU-based chemotherapy.

[Preparation of anti-OPRT antibody for immunochemical detection].

Orotate phosphoribosyl transferase (OPRT, EC 2.4.2.10) is a key enzyme in the anabolism of 5-fluorouracil (5 FU), and its expression in tumor is thought to increase the efficacy of 5-FU against the tumor. To detect the OPRT protein by immunoblotting and/or immunohistochemical methods, we tried to prepare highly specific antibody against the peptide including human OPRT amino acid sequence. The anti-OPRT polyclonal antibody obtained by immunization of OPRT peptides to rabbits had high specificity for the OPRT protein in human tumor xenografts in immunoblot and immunohistochemical analysis. Furthermore, there was a positive correlation between the activity and proteins of OPRT (R2=0.632) in 12 human tumors. These results suggest that immunohistochemical detection of tumoral OPRT protein expression using our anti-OPRT antibody may provide useful methods to predict the clinical response to 5-FU-based chemotherapy.

[Establishment of enzyme-linked immunosorbent assay for quantification of orotate phosphoribosyltransferase in gastric carcinoma].

A number of enzymes have been shown to be involved in the process of activation and/or degradation of 5-fluorouracil, and they are potential candidates for predicting factors of chemosensitivity to 5-fluorouracil. Among them, orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first-step activation process of 5-fluorouracil and therefore it has been shown to be an important enzyme for the prediction of sensitivity to 5-fluorouracil and its related derivatives. We developed a new enzyme-linked immunosorbent assay (ELISA) system to accurately assess intratumoral activity of orotate phosphoribosyltransferase. A new sandwich ELISA system was established using anti-OPRT polyclonal antibodies obtained from the rabbit immunized with the recombinant human peptides of the OPRT molecule. OPRT levels were measured in 8 human cancer xenografts transplanted in nude mice and 58 gastric cancer tissues using both a newly established ELISA and a conventional enzyme assay using radiolabeled 5-fluorouracil as a substrate. OPRT levels in 8 human cancer xenografts measured by this ELISA were significantly correlated with the OPRT enzyme activities (r2=0.782). Furthermore, OPRT activities measured in 58 gastric cancer tissues by enzyme assay were significantly correlated with those measured by the newly-established ELISA (r2=0.664). The ELISA system developed for the measurement of OPRT required a minimal amount of carcinoma tissue samples, which could be an easy-of-use assay system to predict sensitivity to 5-fluorouracil in gastric carcinoma. These results suggest that this newly-developed sandwich ELISA system for the quantification of OPRT is technically simple, feasible, and may be a useful tool to predict sensitivity to fluoropyrimidine-based anticancer chemotherapy in patients with gastric carcinoma and other cancers.

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.

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.

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.

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.