Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems

The elevated cyclooxygenase-2 (COX-2) expression has been shown to affect the carcinogenesis and tumor progression processes, including cell proliferation, motility and angiogenesis. COX-2 is overexpressed in approximately 80% of sporadic colorectal carcinomas and COX-2 enzyme is the best defined target of non-steroidal anti-inflammatory drugs (NSAIDs). In the chemotherapy of colorectal carcinomas 5-fluorouracil (5-FU) has been the most important of the basic drugs for more than 40 years. In order to improve the effectiveness of 5-FU therapy different biological modifiers i.e. inhibitors of its catabolism or activators of anabolism have been studied recently. The rate-limiting enzyme of 5-FU catabolism is dihydropyrimidine dehydrogenase (DPD) since more than 80% of the administered 5-FU is catabolized by DPD. Tumoral DPD has become of clinical interest because elevated intratumoral DPD can decrease the tumor response to 5-FU therapy. The main purpose of our experiments was to investigate the effect of COX inhibitors on the efficacy of 5-FU on high and low COX-2 expressing HCA-7 and HT-29 human colon adenocarcinoma cell lines, respectively, and also on xenografts derived from HT-29 cells. The cytotoxic and antitumor effects of 5-FU in the presence of low doses of indomethacin (non-selective COX-2 inhibitor) and that of NS-398 (highly selective COX-2 inhibitor) on HT-29 and HCA-7 cells and also on the HT-29 xenograft were investigated. In addition, our intention was to understand the mechanism(s) by which NSAIDs could enhance the cytotoxic effect of 5-FU. Our data indicated that the elevated COX-2 expression of HCA-7, the collagen-induced HT-29-C cells and of the HT-29 xenograft were associated with reduced 5-FU sensitivity. Based on the fact that at the same time DPD activity was also increased it might be conceivable that a possible explanation for the decrease of 5-FU sensitivity is the co-existence of high COX-2 and DPD activity. Indomethacin or NS-398 enhanced in a simultaneous and significant manner the sensitivity and cytotoxic effect of 5-FU on high COX-2 expressing cells and xenografts through the modulation of DPD - decrease of its mRNA expression and/or enzyme activity. Based on our results it could be presumable that 5-FU efficacy is limited by the COX-2 associated high DPD expression and activity in patients with colorectal cancer as well, therefore further clinical studies are warranted to decide if NSAIDs in the therapeutic protocol might improve the antitumor potency of 5-FU. Réti A. Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems.     

Relationship between p53 status and 5-fluorouracil sensitivity in 3 cell lines

Mouse lymphoma L5178Ytk+/- (MOLY) cells and human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. MOLY and WTK-1 cells have a p53 mutation, while TK6 cells, which were derived from the same parental line as WTK-1 cells, do not. In this study, we tested the clastogen 5-fluorouracil (5-FU) in the Tk assay and the in vitro micronucleus (MN) assay in MOLY, TK6, and WTK-1 cells to clarify whether differential responses were related to p53 gene status. We also determined the effect of 5-FU on the frequency of apoptotic cells and on cell cycle distribution in each cell line. Furthermore, we measured the activity of the 5-FU metabolizing enzymes (thymidylate synthetase (TS), dihydrouracil dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidine phosphorylase (TP)) in each cell line. We treated MOLY cells with 1.0-8.0 microg/mL 5-FU for 3 h and TK6 and WTK-1 cells with 1.56-25 and 3.13-50 microg/mL, respectively, for 4 h. In MOLY cells, the mutation frequency (MF) and MN frequency increased. In WTK-1 cells, the MN frequency but not the MF increased. In TK6 cells, neither the MF nor the MN frequency increased. Furthermore, the IC50 of 5-FU was lower in MOLY cells than in the human cells. The response to 5-FU treatment differed in other ways as well. At the same level of cytotoxicity, the frequency of apoptotic cell was highest in TK6 cells. The cell cycle was delayed just after treatment in MOLY cells while the delay appeared 24 h later in TK6 and WTK-1 cells. Nothing in our analysis, however, revealed marked differences between the cell lines that could account for the severe cytotoxic and mutagenic responses that 5-FU elicited only in MOLY cells. 5-FU is phosphorylated by OPRT and TP and detoxified by DPD. MOLY cells have higher OPRT activity and markedly lower DPD and TP activity than TK6 and WTK-1 cells. The content of TS, however, the target enzyme of 5-FU, was similar in all cell lines, suggesting that 5-FU was more readily phosphorylated and less readily detoxified in MOLY cells than in TK6 and WTK-1 cells. MOLY cells were more sensitive to 5-FU than WTK-1 cells even though both have a mutated p53 gene, suggesting that the different responses to 5-FU were due to differences in 5-FU metabolism rather than the p53 status.     

Thymidylate synthase predicts for clinical outcome in invasive breast cancer

Thymidylate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Whether TS or DPD could be used as valuable parameters for 5-FU sensitivity in clinical patients are largely unknown. We analyzed TS and DPD expression in breast carcinomas to evaluate the clinicopathological significance of these enzymes in patients with invasive breast cancer receiving 5-FU-based chemotherapy. A total of 197 patients with invasive ductal carcinoma were included in our study. Both the TS and DPD expression were analyzed using immunohistochemical method for all the surgical samples. Sixty-three out of 197 (31.97%) patients are positive for TS expression, and 77 out of 197 (39.09%) patients are positive for DPD expression. TS expression was not correlated with DPD expression. Patients with TS-positivity had aggressive phenotype including large tumor size, low differentiation and nodal metastasis. DPD expression is not related with phenotype or prognosis. Multivariate analysis demonstrated that TS expression was an independent prognostic factor for both disease-free and overall survival. The current study demonstrated that TS but not DPD expression was associated with both progression and prognosis in breast cancer receiving 5-FU-based chemotherapy. TS expression in the primary tumor might be useful as a predictive parameter for the efficacy of 5-FU-based chemotherapy for breast cancer.     

The prognostic significance of thymidine phosphorylase, thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in breast carcinomas

Thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been indicated as possible predictive markers for epithelial malignancies. All these three enzymes are actively involved in 5-FU metabolism. In this report, we investigated mRNA expression of these factors with real-time quantitative PCR in a series of 86 micro-selected breast carcinomas and 8 micro-selected tumour-adjacent normal breast epithelial specimens. Highly variable mRNA expressions of these factors were observed in both normal and cancerous samples. TP and TS mRNA expressions in breast carcinomas were elevated, but only TS mRNA expression showed a trend for statistical difference, compared with the expression in normal breast epithelial samples. Although the DPD mRNA expression range in tumours was also elevated, the average mean was reduced in tumours compared to that in normal samples. No association between mRNA expressions of TP, TS and DPD and clinicopathological features such as histological grade, tumour size, node status, S-phase fraction, ploidy, and clinical stage was found. A negative association between DPD mRNA expression and age was, however, revealed. Ten-year follow-up analysis showed no association between TP and DPD mRNA expression and clinical outcome. An high level of TS mRNA expression, however, was associated with a shorter clinical survival, indicating its potential role as a clinical marker in breast carcinoma.     

Pharmacogenetic studies on the prediction of efficacy and toxicity of fluoropyrimidine-based adjuvant therapy in colorectal cancer

The cytotoxic effect of 5-fluorouracil (5-FU) is mediated by the inhibition of thymidylate synthase (TS), however, at the same time 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity and on pharmacogenetic factors influencing these enzymes. Our aims were (1) to determine the distribution of DPD activity, the frequency of DPD deficiency and the DPD (IVS14+1G>A) mutation in the peripheral blood mononuclear cells of colorectal cancer (CRC) patients, and study the relationship between DPD deficiency and toxicity of 5-FU; (2) to investigate the influence of TS polymorphisms and DPD activity on the survival of CRC patients receiving 5-FU-based adjuvant therapy. The frequency of DPD deficiency was determined by radiochemical methods in the peripheral blood mononuclear cells (PBMCs) of 764 CRC patients treated with 5-FU. The relationship between the TS polymorphisms, DPD activity and the disease-free and overall survival was studied in 166 CRC patients receiving 5-FU-based adjuvant therapy. TS polymorphisms were determined in the DNA samples separated from the PBMCs, by PCR-PAGE and PCR-RFLP-PAGE (restriction fragment length polymorphism) methods. Low DPD values (<10 pmol/min/106 PBMCs) were demonstrated in 160/764 patients (20.9%), and of those DPD deficiency (<5 pmol/min/106 PBMCs) was verified in 38 patients (4.9%). In the latter group severe (>Gr 3) toxicity was found in 87%. The prevalence of the DPD IVS14+1G>A mutation among the 38 DPD-deficient patients was 7.8% (3/38) and was accompanied by severe Gr 4 toxic symptoms (neutropenia, mucositis, diarrhea). TS polymorphisms showed a relationship with the survival of CRC patients. It is important to mention that by combining the 3-3 genotypes of 5'-TSER and 3'-TSUTR polymorphisms the obtained 8 genotype combinations showed significantly different Kaplan-Meier survival curves. The evaluation of these curves with Cox regression analysis resulted in two prognostically different groups: "A" good prognosis (RR<1) and "B" bad prognosis (RR>1). The disease-free- and overall survival of these two groups were significantly different. DPD activity also showed correlation with the survival; patients with DPD activity <10 pmol/min/106 PBMCs showed significantly longer disease-free and overall survival. The determination of DPD activity proved to be a more valuable parameter in the evaluation of serious 5-FU-related toxicity compared to the IVS14+1G>A mutation analysis. According to the Cox multivariate analysis the combination of germline TS polymorphisms and DPD activity is/an independent prognostic marker of survival in CRC patients treated with adjuvant 5-FU therapy.     

Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.     

A rationale for the clinical development of the thymidylate synthase inhibitor ZD9331 in ovarian and other solid tumours

ZD9331 is an antifolate drug that potently and specifically inhibits thymidylate synthase (TS). In contrast with TS inhibitors such as raltitrexed, it cannot be polyglutamated, leading to antitumour activity independent of folylpolyglutamyl synthetase (FPGS) activity.The growth inhibition IC50 values for ZD9331 and raltitrexed were determined for a panel of 18 human tumour cell lines, that included six colon and six ovarian. The colon lines largely displayed overlapping sensitivities to both drugs with only one of the six lines being drug resistant. In contrast, the ovarian cell lines displayed non-overlapping sensitivities with four being highly resistant to raltitrexed and only one was cross-resistant to ZD9331. Studies were undertaken to explain these results. The colon and ovarian cell lines were characterised for TS activity, and TS and FPGS mRNA expression. TS activity correlated with sensitivity to ZD9331 (r=0.50; p=0.097) and raltitrexed (r=0.74; p=0.0063). Provided the data from the highly drug-resistant cell lines (BE and 41 M) were omitted, TS mRNA expression levels also correlated with ZD9331 (r=0.77; p=0.013) and raltitrexed IC50 (r=0.84; p=0.0031). FPGS mRNA expression correlated with higher sensitivity to raltitrexed relative to ZD9331 (higher ZD9331/raltitrexed IC50 ratios) (r=0.62; p=0.048). Similarly, cell lines with IC50 ratios>median expressed a 1.8-fold higher median level of FPGS mRNA (p=0.0087) compared with those with ratios相似文献   

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.     

胸苷酸合成酶表达调控的分子机制

胸苷酸合成酶(thymidylate synthase,TS)是生物体内催化胸苷酸合成所必需的酶．多年来一直作为肿瘤化疗的重要靶酶。对TS基因调控机制的研究表明：基因扩增、转录、翻译和翻译后过程都参与了TS表达的调控。先前的研究表明：TS可与自身的mRNA结合形成TS-mRNA复合物,使mRNA翻译受阻,5-氟尿嘧啶(5-fluorouracil,5-FU)等抗代谢药物可与TS蛋白结合,结合后的复合物不能与TS mRNA作用,导致体内TS的表达升高,是肿瘤细胞产生抗药性的重要分子机制之一。现对TS基因表达调控研究进展、翻译调控与抗药性产生的分子机制进行综述。     

解毒消癞饮逆转氟尿嘧啶诱导的肝癌干细胞耐药的机制研究

目的探讨解毒消瘾饮乙酸乙酯提取物（EE—JXY）能否降低肝癌细胞株Huh7对小剂量氟尿嘧啶（5-FU）化疗耐药的影响,并从肿瘤干细胞的角度探讨可能的机制。方法EE-JXY联合小剂量5-FU体外干预人肝癌细胞株Huh7。MTT法检测细胞存活率;流式细胞仪分析Huh7肝癌细胞株中侧群（sidepopulation,SP）细胞的比例;PCR检测肝癌干细胞相关基因ATP结合盒转运蛋白G2（ABCG2）和八聚体结合转录因子4（Oct4）mRNA的表达。采用单因素方差分析对数据进行统计。结果d,N量5-FU对Huh7细胞的增殖有抑制作用[24h存活率（84．54±2．88）％,96h存活率（58．36±3．52）％],但SP细胞的比例也从24h的4．72％升高至96h的12．07％,同时诱导了ABCG2和Oct4mRNA表达的上调。EE．JXY能够增强小剂量5-FU对Huh7细胞增殖的抑制能力[24h存活率从（84．54±2．88）％下降到（31．23±2．42）％,96h存活率从（58．36±3．52）％下降到（25．37±0．99）％],SP含量从24h的4．72％下降至1．69％,96h的12．07％下降至9．97％,同时也抑制了ABCG2和Oct4 mRNA的表达。结论肝癌Huh7细胞对小剂量5．FU存在化疗抵抗。EE—JXY能通过降低肝癌干细胞相关基因ABCG2 mRNA和Oct4 mRNA水平及SP的比例,增强Huh7细胞对小剂量5-FU的化疗敏感性,这可能是解毒消瘤饮降低肿瘤复发和转移的机制之一。     

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 γ-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.     

Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry.

BACKGROUND: The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections. METHODS: Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections. RESULTS: The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of phantom contouring and two-color staining. Sections of the T-24 and ZR-75-1 cell lines placed on the same slide as the tumor section were used as internal standards for fluorescence measurements. The TP/DPD ratios measured in three cell lines correlated well with the cytotoxicity of 5'-deoxy-5-fluorouridine measured in vitro, indicating that the measurements are related to the biological activity of the drug. CONCLUSIONS: Plotting the data as contour maps of the topologic distribution of fluorescence intensities in tumor sections allows subsequent histopathologic examination, which may reveal features of the tumors leading to high or low ratios of these enzymes. In addition, this method can be used for any drug target/metabolic system where the key components are known and suitable antibodies are available.     

Synergistic Anticancer Effects of Polyphyllin I and Evodiamine on Freshly-Removed Human Gastric Tumors

Objective

The present study was designed to examine the anticancer effect of Traditional Chinese Medicine of polyphyllin I (PPI) and evodiamine (EVO) on freshly–removed gastric tumor tissues.

Methods

Sixty freshly–removed gastric tumor tissues were collected. Their sensitivity to PPI, EVO, platinum (Pt), 5-FU, irinotecan (CPT-11) were determined by histoculture drug response assay (HDRA). Those samples were also formalin-fixed and paraffin-embedded, which were used to examine the mRNA expression levels of aprataxin(APTX), excision repair cross-complementing 1(ERCC1), thymidylate synthase(TS) and topoisomerase I(TOPO1) by quantitative RT-PCR. The association of the gene expression levels and in vitro sensitivity were analyzed.

Results

PPI, EVO, Pt, 5-FU and CPT-11 had anticancer effects on the freshly-removed gastric tumor tissues with average inhibition rates of 20.64%±14.25% for PPI, 21.14%±13.43% for EVO, 50.57%±22.37% for Pt, 53.54%±22.03% for 5-FU, and 39.33%±24.79% for CPT-11, respectively. Combination of PPI and Pt, EVO and Pt, EVO and 5-FU had higher inhibition rates than any single drug of them (P<0.001, P = 0.028, P = 0.017, respectively). The mRNA expression levels of ERCC1 were correlated with Pt sensitivity (rho = −0.645, P<0.001); the mRNA expression levels of TS were correlated with 5-FU sensitivity (rho = −0.803, P<0.001). There were also weak but significant correlations between APTX mRNA expression levels and CPT-11 sensitivity (rho = −0.376, P = 0.017) or EVO sensitivity (rho = −0.322, P = 0.036). ERCC1 mRNA expression levels was markedly suppressed by the presentation of PPI (P = 0.001) and slightly suppressed by the presentation of EVO (P = 0.04); whereas, TS mRNA expression levels was markedly decreased by the presentation of EVO (P = 0.017) and slightly decreased by the presentation of PPI (P = 0.047).

Conclusion

PPI and EVO both could inhibit the activity of freshly-removed gastric tumor, and they could enhance the anticancer effect of Pt and 5-FU by reducing the mRNA expression levels of ERCC1 and TS.