Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0699790 (colon cancer)
 28,837 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.
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PMID:Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates. 1095 84

The effects of 5-fluorouracil (5-FU) on cell growth were investigated using a primary culture of human fibroblasts, MRC-5, and three established human colon cancer cell lines, DLD-1, LoVo and SW620. Detailed flow cytometric analyses revealed differential growth inhibition among these cell lines including three modes of cell growth modulation: (a) loss or accumulation of S phase cells; (b) G2/M block; and (c) G1-S arrest. From analyses on the amount of 5-FU incorporated into cellular RNA and the activity of thymidylate synthase (TS), suppression of TS and depletion of dTTP, a possible consequence of the former, was considered to be the major action of 5-FU in these cells. Differences in the cellular responses to the nucleotide pool imbalance appeared to make the cell growth modulation diverse. Loss of S phase cells and G1-S phase arrest were evident in p53 wild-type cells, MRC-5 and LoVo. Cells proficient in DNA mismatch repair, SW620 and MRC-5, showed marked modulations in S-G2/M progression. These findings suggest that multiple factors, including p53 and DNA mismatch repair, participate in diverse cell growth modulations in cells treated with 5-FU. Cellular resistance to 5-FU correlated well with a loss of modulations in S-G2/M progression, rather than with a defect of G1-S arrest, which suggests the significance of DNA mismatch repair as a factor affecting the sensitivity of cells to 5-FU.
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PMID:Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines. 1100 May 83

New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent cell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers. The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to observe cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools. Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase. P53 expression increased. Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers. Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g. the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions. Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting application is the possibility to perform drug combination studies. The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line). Neither of the drugs was independently able to produce similar effects. In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening.
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PMID:The multilayered postconfluent cell culture as a model for drug screening. 1103 3

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.
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PMID:In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase. 1105 26

5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coli UPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines were used (HT29, KM12 and SW1116). The intracellular levels of 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-FU incorporated into RNA after 5-FU treatment in cells infected with adenovirus containing the UPRT gene (AdCA-UPRT) were significantly higher than those of non-infected cells. This was accompanied by marked inhibition of thymidylate synthase (TS) in all cell lines. Furthermore, HT29, KM12 and SW1116 infected with AdCA-UPRT were, respectively, 13.1-, 30.2- and 70.5-fold more sensitive to 5-FU than non-infected cells. Most importantly, treatment with AdCA-UPRT and 5-FU effectively inhibited the growth of HT29-xenografted subcutaneous tumours in nude mice. Therefore, AdCA-UPRT/5-FU treatment had the potential to enhance the actions of 5-FU at both the DNA and RNA levels. Treatment augmented the sensitivity of human colon cancer cells to 5-FU both in vitro and in vivo. We conclude that adenoviral-mediated transfer of the E. coli UPRT gene into colon cancer cells can achieve biochemical modulation of 5-FU and this provides a new approach in the treatment of colorectal cancer.
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PMID:Adenoviral-mediated transfer of Escherichia coli uracil phosphoribosyltransferase (UPRT) gene to modulate the sensitivity of the human colon cancer cells to 5-fluorouracil. 1109 17

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2'-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5' upstream cis-acting regulatory element (nucleotides 80-109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of beta-actin, alpha-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.
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PMID:Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells. 1113 11

The folate-dependent enzymes are attractive targets for cancer chemotherapy. Methotrexate (MTX), which inhibits dihydrofolate reductase, has been widely used for the treatment of solid tumors and hematological cancers. Raltitrexed ("Tomudex") ), which inhibits thymidylate synthase, is a novel anticancer agent active against colorectal cancer and some other solid tumors. We studied the optimal schedule of raltitrexed and MTX in combination against four human colon cancer cell lines Colo201, Colo320, LoVo, and WiDr. These cells were simultaneously exposed to raltitrexed and MTX for 24 h, or sequentially exposed to raltitrexed for 24 h followed by MTX for 24 h, or vice versa. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentrations of drug that produced 80% and 50% cell growth inhibition (IC(80) and IC(50)) were analyzed by the isobologram method (Steel and Peckham, 1979). Cytotoxic interactions between raltitrexed and MTX were schedule-dependent. The simultaneous exposure to raltitrexed and MTX showed additive effects in Colo201, LoVo and WiDr cells and antagonistic effects in Colo320 cells. The sequential exposure to raltitrexed followed by MTX produced additive effects in all four cell lines. The sequential exposure to MTX followed by raltitrexed produced synergistic effects in Colo201, LoVo and WiDr cells and additive effects in Colo320 cells. These findings suggest that the sequential administration of MTX followed by raltitrexed produces more than the expected cytotoxicity and may be the optimal schedule at the cellular level. Further in vivo and clinical studies will be necessary to determine the toxicity and to test the antitumor effects of sequential administration of MTX followed by raltitrexed proposed on the basis of the in vitro synergism.
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PMID:Schedule-dependent synergism and antagonism between raltitrexed ("Tomudex") and methotrexate in human colon cancer cell lines in vitro. 1117 47

Identification of the molecular determinants of 5-fluorouracil (5-FU) and irinotecan (CPT-11) efficacy and toxicity is critically important for the development of more efficient and less toxic treatment strategies for patients with colon cancer. We have identified molecular predictors of response to chemotherapy with 5-FU and survival in patients with advanced colorectal cancer. Low gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) are associated with response and survival. Preliminary data suggest that gene expression levels of topoisomerase I, p21, bcl-2, and ICE may be predictive of response to therapy with CPT-11. Increased toxicity seen in patients treated with CPT-11 may be explained by polymorphism in the UGT1A1 gene, which is responsible for glucuronidation of the active metabolite of CPT-11.
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PMID:Determinants of prognosis and response to therapy in colorectal cancer. 1117 41

Raltitrexed (Tomudex) is a novel thymidylate synthase inhibitor with significant activity against advanced colorectal cancer. We studied the cytotoxic interactions of raltitrexed and 5-fluorouracil (5-FU) in four human colon cancer cell lines on various schedules. The cell growth inhibition after 5 days was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxic interactions at the IC80 level were evaluated by the isobologram method. Simultaneous exposure to raltitrexed and 5-FU for 5 days produced additive to synergistic effects in Colo201 cells, and produced additive effects in Colo321, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 24 h produced additive effects in Colo201, LoVo, and WiDr cells, and produced antagonistic effects in Colo320 cells. Sequential exposure to raltitrexed for 24 h followed by 5-FU for 24 h produced additive effects in Colo201, Colo320, and LoVo cells, and produced antagonistic effects in WiDr cells. The reverse sequence produced additive effects in Colo201 cells, and produced antagonistic effects in Colo320, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 4 h and sequential exposure to raltitrexed for 4 h followed by 5-FU for 4 h with a 20-h interval produced additive effects, while the reverse sequence produced antagonistic effects in LoVo and WiDr cells. These findings suggest that the simultaneous administration of raltitrexed and 5-FU or the sequential administration of raltitrexed followed by 5-FU may be the optimal sequence, while the reverse sequence may be inappropriate. Preclinical and clinical studies of the simultaneous administration of raltitrexed and 5-FU and the sequential administration of raltitrexed followed by 5-FU are required to better understand the antitumor, toxic, and pharmacokinetic interactions of this combination in order to develop the combination chemotherapy of raltitrexed and 5-FU.
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PMID:Schedule-dependent interaction between raltitrexed and 5-fluorouracil in human colon cancer cell lines in vitro. 1121 72

Raltitrexed (Tomudex) is a specific inhibitor of thymidylate synthase with clinical activity in colorectal cancer. The combination of raltitrexed and 5-iodo-2'-deoxyuridine (IdUrd, a cytotoxic pyrimidine analog) resulted in increased IdUrd incorporation into DNA and exhibited in vitro synergism against colon and bladder human carcinoma cell lines. We designed a phase I trial to determine the MTD, pharmacokinetics, and biologic effects of escalating doses of the combination of IdUrd given as a 24-hour infusion after a raltitrexed 15-minute infusion every three weeks. Thirty-four patients received 95 courses of raltitrexed and IdUrd at doses ranging from raltitrexed 1 mg/m2 and IdUrd 750 mg/m2 to raltitrexed 2.5 mg/m2 and IdUrd 10,400 mg/m2. The median number of cycles administered was 2 (range 1-10). Dose limiting hematologic toxicity occurred at doses of raltitrexed 2.5 mg/m2 and IdUrd 10,400 mg/m2. In addition, we determined the mean plasma concentrations C(SS) of IdUrd, the iodouracil level at 22 hours and the IdUrd clearance. Raltitrexed did not appear to affect the pharmacokinetics of IdUrd in the dose range tested. The recommended phase II dose is raltitrexed 2 mg/m2 and IdUrd 10,400 mg/m2 repeated every three weeks. Evidence of potential antitumor activity was observed: 1 patient (with colon cancer) had a partial response while 15 others had stable disease.
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PMID:Phase I trial of sequential administration of raltitrexed (Tomudex) and 5-iodo-2'-deoxyuridine (IdUrd). 1143 31


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