OBJECTIVETo determine the contents of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in pancreatic cancer to provide a basis for the clinical use of capecitabine in pancreatic cancer patients.

METHODSThe contents of TP and DPD in pancreatic cancer and adjacent normal tissues from 20 patients were determined by ELISA and the TP to DPD ratios in the cancer and adjacent normal tissue were compared.

RESULTSTP content was 5- to 283-fold higher in tumor tissue (mean 74-fold) than in the adjacent normal tissue (P < 0.01). DPD in the cancer tissue increased significantly. So did the TP to DPD ratio, when compared to that in normal pancreatic tissue (P < 0.01).

CONCLUSIONThe increased TP to DPD ratio in pancreatic cancer suggests that capecitabine could be activated by the cancer, these capable of selectively kill the tumor cells.

Dihydrouracil Dehydrogenase (NADP) ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Pancreas ; enzymology ; Pancreatectomy ; Pancreatic Neoplasms ; enzymology ; surgery ; Thymidine Phosphorylase ; metabolism

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 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 explore the relation between the expressions of PD-ECGF and VEGF and the evolution of capillary hemangioma, so as to provide theoretical basis for treatment.

METHODSFourty cases with capillary hemangioma, proved by pathologic method, were randomly selected and divided into proliferative (n=22) and involuted groups (n=18), according to the Mulliken standard. 8 specimens from 8 children with prepuce operation were used as control group. All the specimens were fixed, embedded and underwent HE staining. The expression of PD-ECGF, VEGF and CD34 in endothelial cells were detected by immunohistochemistry. The microvessel-density (MVD) was also calculated. The results were analyzed by SPSS12.0.

RESULTSThe positive expression rates of PD-ECGF and VEGF were 95.45% (21/22) and 86.36% (19/22) in proliferative hemangioma, 77.78% (14/18) and 66.67% (12/ 18) in involuted hemangioma, 37.50% (3/8) and 37.50% (3/8) in normal skin. MVD in proliferative and involuted hemangioma and normal skin was 93.68 +/- 20.56, 51.94 +/- 20.73 and 17.50 +/- 5.30, respectively. There was a significant difference in PD-ECGF expression and MVD between the proliferative and involuted groups, or between the hemangioma and control groups (P < 0.05). The VEGF was significantly different between the proliferative and involuted groups, or between the proliferative and control groups (P < 0.05), but not between the involuted and control groups (P > 0.05). The expression of VEGF, PD-ECGD and MVD showed a positive relationship.

CONCLUSIONSPD-ECGF and VEGF have a synergetic effect in the proliferation of micro-vessels. PD-ECGF may enhance the activity of thymidine phosphorylase. They play an important role in the proliferation and involution of hemangioma.

Child, Preschool ; Female ; Hemangioma, Capillary ; metabolism ; pathology ; Humans ; Infant ; Male ; Neoplastic Syndromes, Hereditary ; metabolism ; pathology ; Thymidine Phosphorylase ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) in superficial bladder cancer and its significance.

METHODSPD-ECGF mRNA expressions were determined by RT-PCR in 28 cases of superficial bladder cancers and 6 cases of normal bladder mucosa. The relation between PD-ECGF mRNA expression and tumor invasion to lamina propria or recurrence after transurethral resection was also analyzed.

RESULTSSome degree of PD-ECGF mRNA expression was present in all the samples. The PD-ECGF mRNA level was 3.1-fold higher in pT(1) tumors than in normal bladder mucosa (t = 2.13, P < 0.05) and 2.2-fold higher in pT(1) tumors than in pT(a) tumors (t = 2.66, P < 0.05); G(3) tumors expressed 3.3-fold higher PD-ECGF mRNA than normal bladder mucosa (t = 2.44, P < 0.05) and 2.5-fold higher than G(1 - 2) tumors (t = 3.36, P < 0.01). Eleven cases recurred during the mean follow-up period of 18 months. Three-fold higher PD-ECGF mRNA expression was showed in cases who recurred after transurethral resection than that in cases who did not recur (t = 4.49, P < 0.01). The specificity and sensitivity of predicting tumor recurrence were 82.4% and 81.8% respectively using 0.095 as a cutoff value of PD-ECGF mRNA level in this group of superficial bladder cancer.

CONCLUSIONPD-ECGF mRNA expression correlates with tumor dedifferentiation and plays an important role in the early invasion in superficial bladder cancer. To analyze the PD-ECGF mRNA level contributes to the evaluations of tumor differentiation and invasion to lamina propria as well as recurrence prediction in superficial bladder cancer.

Carcinoma, Transitional Cell ; metabolism ; pathology ; Follow-Up Studies ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Phosphorylase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.
Human ; Neoplasms/drug therapy ; Pentosyltransferases/*antagonists and inhibitors ; Pyrimidines/*metabolism ; Structure-Activity Relationship ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Thymidine Phosphorylase/antagonists and inhibitors ; Uracil/*analogs and derivatives/chemical synthesis/metabolism ; Uridine Phosphorylase/*antagonists and inhibitors

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.
Human ; Neoplasms/drug therapy ; Pentosyltransferases/*antagonists and inhibitors ; Pyrimidines/*metabolism ; Structure-Activity Relationship ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Thymidine Phosphorylase/antagonists and inhibitors ; Uracil/*analogs and derivatives/chemical synthesis/metabolism ; Uridine Phosphorylase/*antagonists and inhibitors

OBJECTIVETo investigate the relationship between mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) and invasion of bladder transitional cell carcinoma (BTCC).

METHODSThe mRNA expression of PD-ECGF in BTCC was detected by RT-PCR. The target PCR bands were analyzed by NIH Image 1.62 software.

RESULTSThe mRNA level of PD-ECGF in BTCC was 3.86 times as high as that of normal bladder mucosa (t = 2.36, P < 0.05). The expression level of stage Ta, T1 and T2-4 tumor was 1.33, 4.02 and 7.59 times as high as that of normal bladder mucosa, respectively. That of Grade 3 tumor was 2.27 times as high as that of Grade 1 - 2 tumor (t = 3.52, P < 0.01).

CONCLUSIONThe mRNA expression of PD-ECGF was positively correlated with the invasiveness and grade of BTCC. The results suggest that the mRNA level of PD-ECGF might be used as an indicator of tumor progression and a guide for clinical treatment of bladder transitional cell carcinoma.

Adult ; Aged ; Carcinoma, Transitional Cell ; metabolism ; pathology ; Cell Differentiation ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; RNA, Messenger ; analysis ; Thymidine Phosphorylase ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology

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 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