Correlation of thymidylate synthase,thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine

AIM:To determine the expression levels of three metabolicenzymes of fluoropyrimidines:thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidinedehydrogenase (DPD) in seven human gastrointestinalcancer cell lines,and to compare the enzyme levels withthe sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd).METHODS:TS,TP and DPD mRNA levels were assessedby semi-quantitative RT-PCR,TP and DPD protein contentswere measured by ELISA.Fifty percent inhibitoryconcentrations of growth (IC50),representing the sensitivityto drugs,were determined by MTT assay.RESULTS:IC50 values ranged from 1.28 to 12.26 uM for5-FU,and from 5.02 to 24.21 uM for FdUrd,respectively.Cell lines with lower DPD mRNA and protein levels tendedto be more sensitive to 5-FU (P<0.05),but neither IS norTP correlated with 5-FU IC50 (P>0.05).Only TS mRNA levelwas sharply related with FdUrd sensitivity (P<0.05),but TPand DPD were not (P>0.05).A correlation was foundbetween mRNA and protein levels of DPD (P<0.05),but notTP (P<0.05).CONCLUSION:DPD and TS enzyme levels may be usefulindicators in predicting the antitumor activity of 5-FU orFdUrd,respectively.     

Correlation of thymidylate synthase,thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine

AIM:To determine the expression levels of three metabolic enzymes of fluoropyrimidines:thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines,and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2′-deoxyuridine (FdUrd).METHODS:TS,TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR,TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50),representing the sensitivity to drugs,were determined by MTT assay.RESULTS:IC50 values ranged from 1.28 to 12.26μM for 5-FU,and from 5.02 to 24.21μM for FdUrd,respectively.Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P&lt;0.05), but neither TS nor TP correlated with 5-FU IC50 (P&gt;0.05).Only TS mRNA level was sharply related with FdUrd sensitivity (P&lt;0.05),but TP and DPD were not (P&gt;0.05).A correlation was found between mRNA and protein levels of DPD (P&lt;0.05),but not TP (P&lt;0.05).CONCLUSION:DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd,respectively.     

Characteristics of AZD9708, a novel, selective β2-adrenoceptor agonist with rapid onset and long duration of action

Here we describe the pre-clinical pharmacological profile of AZD9708, a novel long-acting β₂-adrenoceptor agonist that has potential as a once-daily therapy for asthma and chronic obstructive pulmonary disease (COPD).AZD9708 is a potent and selective agonist at the human β₂-adrenoceptor, with selectivity over human β₁- and β₃-adrenoceptors of >500 and >24 fold, respectively. AZD9708 relaxes carbachol-induced contraction of human bronchial rings with a time to 90% of maximal relaxation of 13–20 min, similar to that seen with formoterol and quicker than salmeterol. In anesthetized guinea pigs, AZD9708 provides significant protection against histamine-induced airway constriction at 24 h after intratracheal and nebulized doses. This is longer than with intratracheal salmeterol, which is bronchoprotective for approximately 8 h, and formoterol, which is bronchoprotective for 8 and 12 h following nebulized and intratracheal dosing, respectively.AZD9708 also shows the potential for a greater therapeutic margin than widely used β₂-adrenoceptor agonists such as formoterol. At a defined efficacy dose that provides 80% bronchoprotection (ED₈₀), formoterol leads to a decrease in blood potassium levels in guinea pigs, whilst AZD9708 is not associated with significant reductions in potassium levels at doses up to 7 times the ED₈₀. [¹⁴C]AZD9708 is associated with extensive protein binding in both human (mean 1.0% free) and rat (mean 2.6% free) plasma.This pharmacological profile indicates the potential of AZD9708 to become an important addition to the range of bronchodilators available for the treatment of patients with obstructive airways disease.     

Apoptosis induced by preoperative oral 5′-DFUR administration in gastric adenocarcinoma and its mechanism of action

AIM: To study the apoptosis induced by preoperative oral 5'-DFUR administration in gastric adenocarcinoma and its mechanism of action. METHODS: Sixty gastric cancer patients were divided randomly into three groups (20 each group) before operation: group one: 5'-DFUR oral administration at the dose of 800-1200mg/d for 3-5d, group two: 500mg 5-FU 200 mg/d CF by venous drip for 3-5d, group three (control group). One or two days after chemotherapy, the patients were operated. Fas/FasL, PD-ECGF and PCNA were examined by immunohistochemistry and apoptotic tumor cells were detected by in situ TUNEL method. Fifty-four patients received gastrectomy, including 12 palliative resections and 42 radical resections. Six patients were excluded. Finally 18 cases in 5'-DFUR group, 16 cases in CF 5-FU group, and 20 cases in control group were analyzed. RESULTS: There was no significant difference in patient mean age, gender, white blood cell count, haematoglo-bin (HB), thromboplastin, perioperative complication incidence, radical or palliation resection, invasion depth (T), lymphonode involvement (N), metastasis (M) and TNM staging among the three groups. However, the PCNA index (PI) in 5'-DFUR group (40.51±12.62) and 5-FU CF group (41.12±15.26) was significantly lower than that in control group (58.33±15.69) (F=9.083, P=0.000). The apoptotic index (AI) in 5'-DFUR group (14.39±9.49) and 5-FU CF group (14.11±9.68) was significantly higher than that in control group (6.88±7.37) (F=4.409, P=0.017). The expression rates of Fas and FasL in group one and group three were 66.7% (12/18) and 50% (9/18), 43.8% (7/16) and 81.3% (13/16), 45.0% (9/20) and 85% (17/20), respectively. The expression rate of FasL in 5'-DFUR group was significantly lower than that in the other two groups (X2=6.708, P=0.035). Meanwhile, the expression rate of PD-ECGF was significantly lower in 5'-DFUR group (4/18, 28.6%) than in CF 5-FU group (9/16, 56.3%) and control group (13/20, 65.0%) (X2=7.542, P=0.023). The frequency of Fas expression was significantly correlated with palliative or radical resection (X2= 7.651, P= 0.006), invasion depth (X2= 8.927, P=0.003), lymphatic spread (X2 =4.488, P=0.034) and UICC stages (X2 =8.063, P=0,045) respectively. By the end of March 2005, 45 patients were followed up. The 0.5-, 1-, 2-, 3-year survival rates were 96%, 73%, 60%, 48%, respectively, which were related with T, N, M and Fas expression, but not with PD-ECGF and FasL expression. CONCLUSION: Preoperative oral 5'-DFUR administration may induce apoptosis of gastric carcinoma cells and decrease tumor cell proliferation index, but cannot improve the prognosis of patients with gastric cancer. Down-regulation of FasL and PD-ECGF expression mediated by 5'-DFUR may be one of its anti-cancer mechanisms. Fas expression correlates with the progression of gastric carcinoma and may be an effective prognostic factor.     

Apoptosis induced by preoperative oral 5′-DFUR administration in gastric adenocarcinoma and its mechanism of action

AIM: To study the apoptosis induced by preoperative oral 5'-DFUR administration in gastric adenocarcinoma and its mechanism of action. METHODS: Sixty gastric cancer patients were divided randomly into three groups (20 each group) before operation: group one:5'-DFUR oral administration at the dose of 800-1200 mg/d for 3 - 5 d, group two: 500 mg 5-FU + 200 mg/d CF by venous drip for 3 - 5 d,group three (control group). One or two days after chemotherapy, the patients were operated. Fas/FasL,PD-ECGF and PCNA were examined by immunohistochemistry and apoptotic tumor cells were detected by in situ TUNEL method. Fifty-four patients received gastrectomy, including 12 palliative resections and 42 radical resections. Six patients were excluded. Finally 18 cases in 5'-DFUR group, 16 cases in CF+5-FU group, and 20 cases in control group were analyzed. RESULTS: There was no significant difference in patient mean age, gender, white blood cell count, haematoglobin (HB),thromboplastin, perioperative complication incidence, radical or palliation resection, invasion depth (T), lymphonode involvement (N),metastasis (M) and TNM staging among the three groups. However,the PCNA index (PI) in 5'-DFUR group (40.51+/-12.62) and 5-FU+CF group (41.12+/-15.26) was significantly lower than that in control group (58.33+/-15.69) (F=9.083, P=0.000). The apoptotic index (AI) in 5'-DFUR group (14.39+/-9.49) and 5-FU+CF group (14.11+/-9.68)was significantly higher than that in control group (6.88+/-7.37) (F=4.409, P=0.017).The expression rates of Fas and FasL in group one and group three were 66.7% (12/18) and 50% (9/18), 43.8% (7/16) and 81.3% (13/16), 45.0% (9/20) and 85% (17/20), respectively. The expression rate of FasL in 5'-DFUR group was significantly lower than that in the other two groups (chi2=6.708, P=0.035). Meanwhile, the expression rate of PD-ECGF was significantly lower in 5'-DFUR group (4/18,28.6%) than in CF+5-FU group(9/16,56.3%)and control group (13/20,65.0%) (chi2=7.542, P=0.023). The frequency of Fas expression was significantly correlated with palliative or radical resection (chi2=7.651, P=0.006), invasion depth (chi2=8.927, P=0.003), lymphatic spread (chi2=4.488, P=0.034) and UICC stages (chi2=8.063, P=0.045) respectively. By the end of March 2005,45 patients were followed up. The 0.5-, 1-, 2-, 3-year survival rates were 96%,73%,60%,48%, respectively, which were related with T, N, M and Fas expression, but not with PD-ECGF and FasL expression. CONCLUSION: Preoperative oral 5'-DFUR administration may induce apoptosis of gastric carcinoma cells and decrease tumor cell proliferation index,but cannot improve the prognosis of patients with gastric cancer.Down-regulation of FasL and PD-ECGF expression mediated by 5'-DFUR may be one of its anti-cancer mechanisms.Fas expression correlates with the progression of gastric carcinoma and may be an effective prognostic factor.     

Successful treatment of advanced hepatocellular carcinoma by combined administration of 5-fluorouracil and pegylated interferon-a

We report a case of hepatocellular carcinoma (HCC) treated successfully by transarterial chemoembolization (TACE) followed by combination therapy of 5-fluorouracil (5-FU) and pegylated interferon-α (PEG-IFN-α). In the present case, the patient had massive and advanced HCC with a diameter of over 8 cm located in segment 7 (S7) of the liver. Furthermore, the tumor invaded into the major branch of the portal vein (Vp3). After TACE, combined administration of 5-FU and PEG-IFN-α was performed for 5 mo. HCC was totally eradicated and the serum levels of tumor markers were markedly decreased by the treatment. Although it has been reported that the combined use of conventional IFN-a and 5-FU showed striking effects on HCC in some cases, this case may suggest the more promising effect of PEG-IFN-α with a long-lasting effect, in the combined use with 5-FU for the treatment of massive advanced HCC.     

γ-Hydroxybutyric acid and 5-fluorouracil, metabolites of UFT, inhibit the angiogenesis induced by vascular endothelial growth factor

UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, γ-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5^′-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT. This revised version was published online in June 2006 with corrections to the Cover Date.     

Successful treatment of advanced hepatocellular carcinoma by combined administration of 5-fluorouracil and pegylated interferon-α

We report a case of hepatocellular carcinoma (HCC) treated successfully by transarterial chemoembolization (TACE) followed by combination therapy of 5-fluorouracil (5-FU) and pegylated interferon-alpha (PEG-IFN-alpha). In the present case, the patient had massive and advanced HCC with a diameter of over 8 cm located in segment 7 (S7) of the liver. Furthermore, the tumor invaded into the major branch of the portal vein (Vp3). After TACE, combined administration of 5-FU and PEG-IFN-alpha was performed for 5 mo. HCC was totally eradicated and the serum levels of tumor markers were markedly decreased by the treatment. Although it has been reported that the combined use of conventional IFN-alpha and 5-FU showed striking effects on HCC in some cases, this case may suggest the more promising effect of PEG-IFN-alpha with a long-lasting effect, in the combined use with 5-FU for the treatment of massive advanced HCC.     

Thymosin β10 as a predictive biomarker of response to 5-fluorouracil chemotherapy in cholangiocarcinoma

Introduction and aim. 5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic drug in the treatment of cholangiocarcinoma (CCA). Since development of drug resistance to 5-FU in CCA patients is the primary cause of treatment failure, a better understanding of the mechanism of drug resistance of this cancer is essential to improve the efficacy of 5-FU in CCA therapy.Material and methods. A 5-FU resistant CCA cell line (M214-5FUR) for a comparative chemo-resistance study was established. Real time RT-PCR was used to determine gene expression levels. Cell cytotoxicity was measured by the MTT assay. Protein expression levels were detected by the immunofluorescene method.Results. It was found that 5-FU resistance was associated with the overexpression of Tβ10 in CCA cell lines. 5-FU treatment at various concentrations induced the expressions of Tβ10 and ABC transporters (ABCB1, ABCG2 ABCA3) in two CCA cell lines, KKU-M055 and KKU-M214. M214-5FUR, a 5-FU-resistant cell line, exhibited a 5-FU resistant phenotype with a 16-fold extremely high expression of Tβ10 and ABC transporters, as compared to the parental cells, KKU-M214. siRNA targeted to Tβ10 significantly reduced expression of ABC transporters tested in the M214-5FUR cells (P < 0.05).Conclusions. The present novel findingsof Tβ10 connected with drug resistance as shown in this study provides a new insight for the therapeutic value of Tβ10 as a predictive biomarker of 5-FU chemoresistance. Inhibiting Tβ10 may be a valuable adjunct for suppression of ABC transporters and sensitizing chemotherapy treatment, especially 5-FU in CCA patients.     

Establishment of a nonradioactive assay for 2′-5′ oligoadenylate synthetase and its application in chronic hepatitis C patients receiving interferon-α

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Re-evaluation of antitumor effects of combination chemotherapy with interferon-α and 5-fluorouracil for advanced hepatocellular carcinoma

AIM: To evaluate the efficacy of combination chemotherapy with interferon-α(IFNα) and 5-fluorouracil(5-FU) in patients with advanced hepatocellular carcinoma (HCC). METHODS: Twenty-eight HCC patients in advanced stage were enrolled in the study. They were treated with IFNa/ 5-FU combination chemotherapy. One cycle of therapy lasted for 4 wk. IFNα(3×10~6 units) was subcutaneously injected thrice weekly on days 1, 3, and 5 for 3 wk, and 5-FU (500 mg/d) was administered via the proper hepatic artery for 5 consecutive days per week for 3 wk. No drugs were administered during the 4~(th) wk. The effect of combination chemotherapy was evaluated in each patient after every cycle based on the reduction of tumor volume. RESULTS: After the 1~(st) cycle of therapy, 16 patients showed a partial response (PR, 57.1%) but none showed a complete response (CR, 0%). At the end of therapy, the number of patients who showed a CR, PR, or no response (NR) was 1, 10, and 17, respectively. The response rate for therapy (CR+PR) was 21.5%. Biochemical tests before therapy were compared between responsive (CR+PR) and non-responsive (NR) patients, but no significant differences were found for any of the parameters examined, indicating that no reasonable predictors could be identified in our analysis. CONCLUSION: Attempts should be made to discriminate between responders and non-responders by evaluating tumor size after the first cycle of IFNα/5-FU combination chemotherapy. For non-responders, therapy should not proceed to the next cycle, and instead, different combination of anticancer drugs should be explored.     

Inhibition of fluorouracil catabolism in cancer patients by the antiviral agent (E)-5-(2-bromovinyl)-2′-deoxyuridine

The thymidine analogue (E)-5-(2-bromovinyl)-2-deoxyuridine (BVdUrd), which is an antiviral agent effective against herpes simplex virus type 1 and varicella zoster virus, has also proved to be a potent inhibitor of dihydrouracil dehydrogenase, the major degrading enzyme of the anticancer drug fluorouracil (FUra). To evaluate the effect of BVdUrd on the pharmacokinetics of FUra in cancer patients, BVdUrd was administered orally at a daily dose of 250 mg (five patients) or 3×250 mg (five patients). FUra was infused at doses of 110–400 mg over 10 min. Blood and urine samples were collected regularly during a period of up to 50 h. BVdUrd was rapidly absorbed, peak plasma levels of 0.6–7.1 g/ml being achieved after 1–2 h. Following a rapid decline, plasma BVdUrd levels remained higher than 10 ng/ml for up to 2 days after BVdUrd administration. The total body clearance of FUra was approximately 6 l/h andt _1/2 markedly increased to 4–7 h. The mean urinary excretion of FUra was 50%. No differences in FUra kinetics were observed between patients receiving one or three oral doses of BVdUrd. We conclude that the concomitant use and subsequent interaction of FUra and the antiviral agent BVdUrd resulted in an impressive inhibition of FUra breakdown and marked increase of renal clearance. The findings indicate that the simultaneous use of FUra and drugs resembling this antiviral agent in patients may result in unexpected toxicity. Further experience with BVdUrd and new analogues might enable the development of new FUra treatment schedules and treatment designs e.g. combinations with leucovorin.Abbrevations BVdUrd (E)-5-(2-bromovinyl)-2-deoxyuridine - FUra 5-fluorouracil - BVUra (E)-5-(2-bromovinyl)uracil - C _TB total body clearance     

Re-evaluation of antitumor effects of combination chemotherapy with interferon-α and 5-fluorouracil for advanced hepatocellular carcinoma

AIM: To evaluate the efficacy of combination chemotherapy with interferon-alpha (IFNalpha) and 5-fluorouracil (5-FU) in patients with advanced hepatocellular carcinoma (HCC). METHODS: Twenty-eight HCC patients in advanced stage were enrolled in the study. They were treated with IFNalpha/5-FU combination chemotherapy. One cycle of therapy lasted for 4 wk. IFNalpha (3 x 10(6) units) was subcutaneously injected thrice weekly on days 1, 3, and 5 for 3 wk, and 5-FU (500 mg/d) was administered via the proper hepatic artery for 5 consecutive days per week for 3 wk. No drugs were administered during the 4(th) wk. The effect of combination chemotherapy was evaluated in each patient after every cycle based on the reduction of tumor volume. RESULTS: After the 1(st) cycle of therapy, 16 patients showed a partial response (PR, 57.1%) but none showed a complete response (CR, 0%). At the end of therapy, the number of patients who showed a CR, PR, or no response (NR) was 1, 10, and 17, respectively. The response rate for therapy (CR+PR) was 21.5%. Biochemical tests before therapy were compared between responsive (CR+PR) and non-responsive (NR) patients, but no significant differences were found for any of the parameters examined, indicating that no reasonable predictors could be identified in our analysis. CONCLUSION: Attempts should be made to discriminate between responders and non-responders by evaluating tumor size after the first cycle of IFNalpha/5-FU combination chemotherapy. For non-responders, therapy should not proceed to the next cycle, and instead, different combination of anticancer drugs should be explored.     

Activity and safety of pegylated liposomal doxorubicin, 5-fluorouracil and folinic acid in inoperable hepatocellular carcinoma： A phase Ⅱ study

AIM: To improve the results of New therapeutic strategies in hepatocellular carcinoma (HCC). We have conducted a phase Ⅱ study with pegylated liposomal doxorubicin (PLD), 5-fluorouracil (5FU) and folinic acid (FA). METHODS: Thirty-one patients with hystologically- confirmed, inoperable HCC, received combination chemotherapy with PLD 25 mg/mq on d 1, 5FU 1200 mg/mq in 48 h continuous infusion, and oral FA 30 mg on d 1 and 2 every 3 wk until disease progression or intolerable toxicity. RESULTS: The median age was 65 years (range 41-82) and 28 patients were hepatitis C virus seropositive (90%). The majority of patients were Child-Pugh Class B (55%). Two patients showed a partial response (PR), and 16 had stable disease (SD). With a median follow-up of 14 mo, the median time to progression of all evaluable patients was 4 mo (95% CI 1.7-7). Median overall survival was 9 mo (95% CI 3-24 mo). After 1 year, 9 of 18 PR/SD patients were alive. Chemotherapy was well tolerated. CONCLUSION: PLD/FU/FA combination seems capable of achieving durable stabilization of HCC. The manageable toxicity supports a role for combination with other anticancer agents.     

Scleroderma and chronic myeloid leukemia: a sheer coincidence, a consequence of long lasting d-penicillamine therapy or a plausible relationship of both diseases?

Systemic sclerosis is a chronic multisystem disorder of unknown etiology characterized by the involvement of skin and visceral organs caused by an accumulation of collagen. It has been reported that the incidence of solid and hematological malignancy increased in systemic sclerosis. Multiple myeloma and chronic lymphocytic leukemia are the most common hematological malignancies seen in patients with systemic sclerosis. Chronic myeloid leukemia (CML) has only rarely been reported so far. We here report a case with CREST (calcinosis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactly, telangiectasia) who developed CML 7 years after the onset of CREST. Ours is the second case with CML developing after the onset of CREST in the literature. We also briefly discuss the possible tendency to hematological malignancy in systemic sclerosis.