OBJECTIVETo explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector.

METHODSTP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP, and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry. The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR, respectively. The 50% inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay. Finally, the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured, or in the cell culture lysates.

RESULTSThe HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations, and their transfection efficiency was about 95.0%. Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive, while vector-transfected cells were TP-negative. Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells. The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97, respectively, which were significantly higher than that in their parents cells (P < 0.01). The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) µmol/L and (707.66 ± 5.66) µmol/L, respectively (P < 0.05), while (0.59 ± 0.11) µmol/L in LS174T-TP cells and (239.20 ± 21.83) µmol/L in its parent cells, respectively (P < 0.05). The IC50 values of 5-Fu of HT29-TP cells and its parents cells were (5.42 ± 0.75) µmol/L and (14.19 ± 0.97) µmol/L, respectively (P < 0.05), while (4.41 ± 0.96)µmol/L in LS174T-TP cells and (16.42 ± 2.12)µmol/L in its parents cells, respectively (P < 0.05). The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5'-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds, respectively, higher than that in their parents cells, (P < 0.01 for all). Otherwise, just a little of 5-Fu was detected in the two TP-transfected cells lysate, about 0.9% to 4.2% of 5-Fu detected in the media of the same cultured cells, whereas no 5-Fu was detected in the two parent cell lysates.

CONCLUSIONSTransfection of TP cDNA into colorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells, enhance the conversion of 5-Fu from 5'-DFUR in the medium, and result in an enhanced anticancer effect on these two cell lines.

Antineoplastic Agents ; pharmacology ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; DNA, Complementary ; Floxuridine ; pharmacology ; Fluorouracil ; Genetic Vectors ; HT29 Cells ; Humans ; RNA, Messenger ; Thymidine Phosphorylase ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate the combination chemotherapy regimen with floxuridine, dactinomycin, etoposide, and vincristine (FAEV) as primary treatment for gestational trophoblastic neoplasia (GTN).

METHODSClinical data and outcome of the patients with GTN from 1 January 2004 to 31 December 2009 were retrospectively reviewed. Totally 38 eligible patients had received at least one cycle of FAEV chemotherapy as primary treatment. The primary end points were response rate and toxicity of FAEV regimen.

RESULTSTotally 38 patients and 205 cycles of FAEV chemotherapy were included. Twenty-eight of these patients (73.6%) achieved serologic complete remission (SCR). Regimens were changed in 10 patients because of 5 with no response and 5 with intolerable toxicity. The most serious adverse events were greater than or equal to grade 3 neutropenia (31.6%), febrile neutropenia (7.9%), and greater than or equal to grade 3 thrombocytopenia (5.3%). During the follow-up, none relapsed.

CONCLUSIONFAEV is an effective regimen with manageable toxicity for patients with GTN as primary treatment, especially for patients with non-metastatic low or high risk GTN.

Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Dactinomycin ; administration & dosage ; Etoposide ; administration & dosage ; Female ; Floxuridine ; administration & dosage ; Gestational Trophoblastic Disease ; drug therapy ; Humans ; Middle Aged ; Pregnancy ; Retrospective Studies ; Treatment Outcome ; Vincristine ; administration & dosage ; Young Adult

The survival of most patients with both unresectable hepatic and pulmonary metastases of colorectal cancer is poor. In this retrospective study, we investigated the efficacy of computed tomography (CT)-guided radiofrequency ablation (RFA) and systemic chemotherapy plus hepatic artery infusion of floxuridine (HAI-FUDR). Sixty-one patients were selected from 1,136 patients with pulmonary and hepatic metastases from colorectal cancer. Patients were treated with RFA and systemic chemotherapy plus HAI-FUDR (ablation group, n = 39) or systemic chemotherapy plus HAI-FUDR (FUDR group, n = 22). Patients in the two groups were matched by sex, age, number of metastases, and calendar year of RFA or FUDR. Survival data were evaluated by using univariate and multivariate analyses. Clinical characteristics were comparable between the two groups. All patients in the ablation group underwent RFA and chemotherapy. Median follow-up was 56.8 months. The 1-, 3-, and 5-year overall survival (OS) rates were 97%, 64%, and 37%, respectively, for the ablation group, and 82%, 32%, and 19%, respectively, for the FUDR group. The 1-, 3-, and 5-year survival rates after metastasis were 97%, 49%, and 26% for the ablation group, and 72%, 24%, and 24% for the FUDR group, respectively. The median OS times were 45 and 25 months for the ablation and FUDR groups, respectively. In the multivariate analysis, treatment allocation was a favorable independent prognostic factor for OS (P = 0.001) and survival after metastasis (P = 0.009). These data suggest that the addition of RFA to systemic chemotherapy plus HAI-FUDR improves the survival of patients with both unresectable hepatic and pulmonary metastases from colorectal cancer.
Aged ; Antineoplastic Combined Chemotherapy Protocols ; Catheter Ablation ; Colorectal Neoplasms ; therapy ; Combined Modality Therapy ; Cytoreduction Surgical Procedures ; Floxuridine ; Hepatic Artery ; Humans ; Infusions, Intra-Arterial ; Liver Neoplasms ; secondary ; Lung Neoplasms ; secondary ; Retrospective Studies ; Survival Rate ; Tomography, X-Ray Computed ; Treatment Outcome

OBJECTIVESTo study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.

METHODSTP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.

RESULTSThe colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).

CONCLUSIONSThe stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Floxuridine ; metabolism ; Fluorouracil ; metabolism ; Humans ; Thymidine Phosphorylase ; genetics ; Transcription, Genetic ; Transfection

OBJECTIVETo investigate the inhibiting impact of 5'-deoxy-5-fluorouridine (5'-DFUR) on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.

METHODSTP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and the transfection efficiency was analyzed by flow cytometry. TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay. The volumes of 5-FU converted from 5'-DFUR in media, where TP-transfected and parental LOVO were cultured, were detected by HPLC.

RESULTSThe stable transfectants passed 5 generations were obtained and the transfection rate was 95%. Compared with parental cell, the RQ values of mRNA expression in TP-transfected LOVO was (282.5±86.8) folds higher significantly (P<0.01), also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells. The IC50 value of 5'-DFUR of TP-transfectants was (1087.7±89.1) μmol/L, less than (1607.3±56.8) μmol/L of parental cells significantly (P<0.01), while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L, P>0.05]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 μmol/L of 5'-DFUR respectively, 0, 2.10, 3.13, and 7.19 μmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture, but no 5-FU was found in the vector-transfectants culture.

CONCLUSIONTP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions, increase the 5-FU converted from 5'-DFUR, and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; DNA, Complementary ; genetics ; Floxuridine ; pharmacology ; Humans ; Thymidine Phosphorylase ; genetics ; Transfection

OBJECTIVETo detect the effect of interferon-α2a(IFN-α2a) on thymidine phosphorylase(TP) mRNA expression levels and the anticancer activity of 5-fluorouracil(5-FU) and 5'-deoxy-fluorouridine(5'-DFUR) in human colon carcinoma cell lines LOVO and SW480.

METHODSTwo human colon cancer cell lines LOVO and SW480 were cultured and treated with IFN-α2a at a series of dosage, and fluorescence quantitative PCR was carried out to detect the TP mRNA expression levels in these 2 cell lines. Then MTT assay and software Templet were used to determine the change of 50% inhibition concentration of 5-FU or 5'-DFUR combined with IFN-α2a on the two cell lines.

RESULTSThe TP mRNA expressions were up-regulated significantly by IFN-α2a at the doses of 500 U/ml and 5000 U/ml in LOVO(P<0.01). Compared with untreated cells(IFN-α2a 0 U/ml), no significance was found for TP mRNA expression levels in LOVO and SW480 treated by IFN-α2a at the dose of 50 U/ml (P>0.05). There was no significant difference for TP mRNA expression in SW480 between the dose of 0 U/ml and 500 U/ml of IFN-α2a(P>0.05), while a significant increace was detected at the dose of 5000 U/ml (P<0.01). No significant difference was found for the IC50 values after treatment of 5-FU combined with IFN-α2a (20 U/ml) on LOVO and SW480 compared with 5-FU alone, while the IC50 values after treatment of 5'-DFUR combined with IFN-α2a decreased significantly compared with 5'-DFUR alone(P<0.05).

CONCLUSIONThere is no direct inhibition effect of IFN-α2a on LOVO and SW480 in vitro, while it can up-regulate TP mRNA expression levels both in LOVO and SW480, and enhance the anticancer effect of 5'-DFUR on these 2 cell lines.

Cell Line, Tumor ; Colonic Neoplasms ; enzymology ; pathology ; Floxuridine ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; Recombinant Proteins ; pharmacology ; Thymidine Phosphorylase ; metabolism

OBJECTIVETo investigate the relationship between the expression of thymidine phosphorylase (TP) and the sensitivity of gastric carcinoma to 5-fluorouracil (5-FU) and its prodrugs.

METHODSGastric carcinoma cell line AGS was transfected with recombinant plasmid pEGFP-N1-TP or control plasmid pEGFP-N1 by lipofectamin 2000. The expression of green fluorescence labeled protein was observed under fluorescence microscope. Positive clones AGS-p and AGS-pTP were selected by G418 treatment. Expression of TP protein and mRNA was detected by immunocytochemistry and RT-PCR, respectively. Drug sensitivity to 5-FU and its prodrugs was assessed by MTT assay.

RESULTSCell clones with the expression of green fluorescent protein (AGS-p) and a clone with TP and green fluorescent fusion protein (AGS-pTP) were established. Immunostaining of TP protein was strongly positive in AGS-pTP and negative in AGS-p and AGS. The expression of TP mRNA was significantly higher in AGS-pTP (0.8090 ± 0.0450) than that in AGS (0.0490 ± 0.0046) and AGS-p (0.0610 ± 0.0069; P < 0.01). The sensitivity to doxifluridine and capecitabine in AGS-pTP was significantly increased, as compared with that in AGS-p. IC50 values of AGS-pTP to doxifluridine and capecitabine were estimated 1.7 folds and 2.2 folds as much as that of AGS-p, respectively. The sensitivity to 5-FU was not different between AGS-pTP and AGS-p.

CONCLUSIONSEnhancement of TP expression improves the sensitivity of gastric carcinoma cells to doxifluridine and capecitabine. But it does not affect the sensitivity to 5-FU.

Antimetabolites, Antineoplastic ; pharmacology ; Capecitabine ; Cell Line, Tumor ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Floxuridine ; pharmacology ; Fluorouracil ; analogs & derivatives ; pharmacology ; Humans ; Plasmids ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sensitivity and Specificity ; Stomach Neoplasms ; metabolism ; pathology ; Thymidine Phosphorylase ; biosynthesis ; genetics ; Transfection

OBJECTIVETo develop a new prodrug of 5-fluorouracil-polyethylenimine-beta-cyclodextrin-floxuridine (PEI-beta-CyD-Fd) and to test its antitumor activity.

METHODSFloxuridine was conjugated to polyethylenimine-beta-cyclodextrin to form prodrug PEI-beta-CyD-Fd. The structure of synthesized PEI-beta-CyD-Fd was confirmed by (1)H-NMR, FT-IR and UV. MTT assay and cell wound healing assay were performed on human hepatic carcinoma cell line HepG2.

RESULTThe drug loading was 2 %. The MTT assay and cell wound healing assay indicated that PEI-beta-CyD-Fd significantly inhibited proliferation and migration of HepG2 cells.

CONCLUSIONThe synthesized prodrug PEI-CyD-Fd has a significant antitumor activity on HepG2 cells.

Antimetabolites, Antineoplastic ; chemical synthesis ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Floxuridine ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Polyethyleneimine ; pharmacology ; Prodrugs ; chemical synthesis ; pharmacology ; beta-Cyclodextrins ; pharmacology

Exogenous factors, including environmental substances and drugs, are known to induce scleroderma-like reactions. Various scleroderma-like reactions induced by anti-cancer drugs have recently been reported. This is the first report that doxifluridine (Didox), an oral prodrug of the antineoplastic agent 5-fluorouracil (5-FU), induced a localized sclerderma-like reaction. A 51-year-old woman was referred to our clinic with multiple pearly, shiny patches on both her breasts, her pelvis and her back. After surgical excision and radiation therapy due to her right breast cancer, she took Didox for 7 months. A skin biopsy specimen revealed that the dermal collagen thickening extended even to the subcutaneous tissue. The routine laboratory tests were within the normal ranges and the tests for antinuclear antibody (ANA), anti SS-A antibody, anti SS-B antibody and anti U1-RNP antibody were all negative. After discontinuation of Didox, the lesions gradually improved. Based on these finding, we diagnosed this case as a localized scleroderma-like reaction induced by doxifluridine and we should pay attention to detect this adverse effect of the long term use of doxifluridine.
Antibodies, Antinuclear ; Biopsy ; Breast ; Breast Neoplasms ; Collagen ; Female ; Floxuridine ; Fluorouracil ; Humans ; Hydroxamic Acids ; Middle Aged ; Pelvis ; Reference Values ; Skin ; Subcutaneous Tissue

Doxifluridine is a pyrimidine derivative and is activated to 5-fluorouracil by pyrimidine phosphorylase. Multiple acral hyperpigmented macules have been reported in patients treated with systemic 5-fluorouracil or some of its prodrugs. However, there have been no reports of this adverse event being induced by doxifluridine. Herein we present a 42-year-old woman with multiple pigmented lesions on the palm and sole after chemotherapy with oral doxifluridine.
Adult ; Female ; Floxuridine ; Fluorouracil ; Humans ; Hyperpigmentation ; Immunosuppressive Agents ; Prodrugs ; Pyrimidines