γ-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate

Background:

γ-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates for optimal nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. The modulation of GGH may therefore affect chemosensitivity of cancer cells, and exogenous folate levels may further modify this effect.

Methods:

We generated a novel model of GGH modulation in human HCT116 and MDA-MB-435 cancer cells and investigated the effect of GGH modulation on chemosensitivity to 5-fluorouracil (5FU) and methotrexate (MTX) at different folate concentrations in vitro and in vivo.

Results:

Overexpression of GGH significantly decreased chemosensitivity of MDA-MB-435 cells to 5FU and MTX at all folate concentrations as expected. In contrast, in HCT116 cells this predicted effect was observed only at very high folate concentration, and as the folate concentration decreased this effect became null or paradoxically increased. This in vitro observation was confirmed in vivo. Inhibition of GGH significantly increased chemosensitivity of both cancer cells to 5FU at all folate concentrations. Unexpectedly, GGH inhibition significantly decreased chemosensitivity of both cancer cells to MTX at all folate concentrations. In both GGH modulation systems and cell lines, the magnitude of chemosensitivity effect incrementally increased as folate concentration increased.

Conclusion:

Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects.     

Where do we stand with 5-fluorouracil?

For nearly four decades, 5-fluorouracil (5-FU) has been the mainstay of treatment for colorectal cancer. Due to the lack of other agents with significant activity, tremendous efforts have been undertaken to increase the efficacy of 5-FU by investigating alternative schedules of delivery and biomodulation. However, bolus 5-FU in combination with folinic acid (FA), either as the Mayo Clinic or Roswell Park protocol, still represents the standard treatment for adjuvant and first-line palliative chemotherapy of colorectal cancer. In a recent meta-analysis, infusional protocols of 5-FU demonstrated increased response rates (14% to 22%) and a marginal, but significant survival benefit of 3 weeks (11.3 to 12.1 months). In view of the much higher costs and complicated management of infusional 5-FU regimens, this marginal survival benefit does not yet allow protracted 5-FU application to be defined as standard therapy. However, protracted 5-FU infusion in combination with radiation can be considered standard therapy as adjuvant treatment of rectal cancer, since it has demonstrated a significant increase in survival. In the future, oral 5-FU prodrugs may be substituted for infusional 5-FU. Furthermore, current data indicate that 5-FU will also be an essential component of combination chemotherapy protocols with the new active agents oxaliplatin, irinotecan, and raltitrexed. Preclinical studies show synergistic antitumor activity of 5-FU with these agents, which corresponds well with clinical response rates of 50% in untreated and 15% to 25% in 5-FU-refractory patients. Moreover, 5-FU-based pro-drug-active drug systems serve as excellent models for tumor-targeted gene therapy.     

Concurrent allopurinol and 5-fluorouracil: 5-fluoro-2′-deoxyuridylate formation and thymidylate synthase inhibition in rat colon carcinoma and in regenerating rat liver

Summary The formation of FdUMP and the inhibition of TS were studied in a subcutaneously growing transplantable rat colon carcinoma and in regenerating rat liver following bolus administration of 5-FU, with or without HPP pretreatment.In tumor, peak levels of FdUMP at 30 min following bolus 5-FU, 100 mg/kg, averaged 4931±587 pmol/g. Pretreatment with HPP, 50 mg/kg, 24 h and 1 h before 5-FU, reduced the peak FdUMP level to 2085±387 pmol/g. The inhibition of TS by 5-FU treatment was greater than 95% by 30 min, and after 48 h residual enzyme inhibition averaged 40%. No effect on TS inhibition by 5-FU treatment could be observed as a result of HPP pretreatment. The levels of TS_tot increased linearly after 5-FU treatment and doubled within 48 h.In regenerating rat liver, neither FdUMP levels nor TS inhibition, studied at 1 h after bolus 5-FU, were affected by HPP pretreatment.Abbreviations BSA bovine serum albumin - DMH dimethylhydrazine - 5-FU 5-fluorouracil - HPP allopurinol (4-hydroxypyrazolopyrimidine) - CH₂FH₄ 5, 10-methylenepteroylmonoglutamic acid - FUMP 5-fluorouridine-5-monophosphate - FUTP 5-fluorouridine-5-triphosphate - FdUMP 5-fluoro-2-deoxyuridylate - TS thymidylate synthase (EC 2.1.1.45, dTMP synthase) - TS_f free, non-FdUMP-bound TS - TS_b ternary complex-bound TS - TS_tot total TS TS_f+TS_b - PRPP phosphoribosylpyrophosphate This study was supported by grants from the Swedish Cancer Society, The Medical Society of Göteborg, the University of Göteborg, the Wellcome Foundation, and NCI Grant 39629.     

Combination of interferon-α and 5-fluorouracil induces apoptosis through mitochondrial pathway in hepatocellular carcinoma in vitro

Many clinical reports have proven that the combination therapy of interferon-alpha plus 5-fluorouracil is remarkably effective for advanced hepatocellular carcinoma (HCC). However, the mechanism of this therapy is not well understood. Here, we demonstrated that the combination therapy synergistically inhibited the growth of Fas-negative HCC cells, arrested cell-cycle progression and induced apoptosis. Moreover, the combination therapy significantly increased the protein expression of caspase-8, activated Bid and cytochrome c. Meanwhile, the expression of anti-apoptotic gene Bcl-xL was reduced and intracellular calcium elevated obviously during the early stage of treatment. Therefore, mitochondrial pathway was involved in the apoptosis of Fas-negative HCC cells induced by IFN-α/5-FU and Ca(2+) partially promoted the beneficial effect against HCC.     

Standard dose (Mayo regimen) 5-fluorouracil and low dose folinic acid: prohibitive toxicity?

Despite the perception that standard 5-fluorouracil/folinic acid (5-FU/FA) (425 mg/m2 per day and 20 mg/m2 per day intravenously once daily x 5 every 4 or 5 weeks) is well tolerated, we have been impressed by toxicity seen and frequent need for dose modification. We performed a retrospective analysis to quantitate the proportion of patients experiencing toxicity and attempted to identify associated clinical characteristics. One hundred thirty-four patients received 5-FU/FA at standard doses described by the Mayo regimen. Patient characteristics were as follows: female 35%, median age 66 years, Eastern Cooperative Oncology Group performance status less than or equal to 2, 96%. Sixty-eight percent received chemotherapy for metastatic disease. Forty-seven patients (35%+/-8%) experienced significant toxicity and were unable to receive the second cycle as scheduled: 76% required dose reduction, 11% discontinued therapy (including two toxic deaths), 11% discontinued therapy during the first cycle, and 2% required dose delay. Logistic regression was used to explore the following as predictors of toxicity: age, sex, performance status, adjuvant versus metastatic setting, prior chemotherapy, prior radiation, mean corpuscular volume, red blood cell distribution width, albumin, alkaline phosphatase, aspartate aminotransferase, bilirubin, and calculated creatinine clearance. No clinical characteristic was found to predict toxicity. Only high bilirubin approached statistical significance. We conclude that standard 5-FU/FA, when used in the general population, is associated with significant toxicity. Known clinical characteristics are not helpful in predicting toxicity. The lack of previous formal phase I evaluation of this regimen of 5-FU/FA raises concerns regarding its safety and generalizability in clinical practice.     

Influence of resistance to 5-fluorouracil and tomudex on [18F]‑FDG incorporation, glucose transport and hexokinase activity

Drug resistance is a major obstacle to cancer cure and may influence [18F]-fluorodeoxyglucose (FDG) incorporation. In this study, glucose transport, hexokinase activity and [18F]-FDG incorporation were measured in drug-resistant tumour cells generated by exposing H630 colon and MCF7 breast cancer cells to increasing concentrations of tomudex (raltitrexed) or 5-fluorouracil (5FU). Drug sensitivity was determined using the XTT assay: Tomudex-resistant (H630TDX and MCF7TDX) cells were more than 40,000-fold less sensitive to tomudex than were the parental wild-type, H630WT and MCF7WT cells, respectively. 5FU-resistant (H630R10) cells were 100-fold less sensitive than parental H630WT cells to 5FU. As previously reported for 5FU-resistant MCF7 breast cancer cells, [18F]-FDG incorporation was decreased in H630R10 colon cancer cells compared to the parental line. By contrast, both tomudex-resistant cell lines exhibited increased [18F]-FDG incorporation compared with the parental lines. H630R10 and MCF7TDX cells exhibited higher rates of glucose transport, measured as the initial rate of O-methyl-glucose (OMG) uptake, compared to wild-type cells; however, glucose transport was not significantly different between H630TDX cells and the parental cells. Hexokinase activity was lower in H630R10 and MCF7TDX cells compared with sensitive parental cells but unchanged in H630TDX cells. In conclusion, our results show that [18F]-FDG incorporation is influenced by resistance to antifolate and fluoropyrimidine-based anti-cancer drugs in a drug-dependent manner and the underlying mechanisms appear to be cell- and drug-dependent. Glucose transport may be a useful marker of resistance to 5FU.     

Combination therapy of intra-arterial 5-fluorouracil and systemic pegylated interferon α-2b for advanced hepatocellular carcinoma

Background  

This study evaluated the efficacy of combined 5-fluorouracil (5-FU) and pegylated interferon (PEG-IFN) α-2b in patients with advanced hepatocellular carcinoma (HCC).     

Is salvage chemotherapy for metastatic breast cancer always effective and well tolerated? A phase II randomized trial of vinorelbine versus 5-fluorouracil plus leucovorin versus combination of mitoxantrone, 5-fluorouracil plus leucovorin

Metastatic breast cancer remains an incurable disease and the median overall survival has not significantly improved over the past two decades. Aims of the present randomized phase II trial were to analyse activity and toxicity of chemotherapies with single agent or with combination regimens in previously treated patients with advanced breast cancer. Ninety-nine eligible patients were randomized to receive the following chemotherapies: Arm A – 30mg/m² i.v. weekly; Arm B – leucovorin 100mg/m² i.v. followed by 5-fluorouracil 370mg/m² i.v. days 15, q 28days; Arm C – mitoxantrone 12mg/m² i.v. only day 1+leucovorin 100mg/m² i.v. followed by 5-fluorouracil 370mg/m² i.v. days 13, q 28days. Patients characteristics are comparable in the three groups. The median number of chemotherapy courses administered was 7, 6 and 5 in arm A, B and C, respectively. Objective responses were 24%, 30% and 21% and the median duration of responses were 2, 2.5 and 5.5 months in the arm A, B and C, respectively. Median overall survivals were 9.5, 9 and 9 months in the three arms. No difference was noted comparing the survivals of responding or non responding patients. General toxicity was not mild, with 27.5% of patients experiencing WHO grade 3–4 toxicities.Our results are similar in the three groups of patients and comparable to those reported by other authors. Chemotherapy applied to patients with second or subsequent recurrence allow objective responses in a small percentage of patients. Moreover responders have a negligible prolongation of survival.     

Do 5-fluorouracil therapies alter CYP2C19 metaboliser status?

Purpose

To report a case of altered CYP2C19 metaboliser status following 5-fluorouracil treatment.

Methods

A 78-year-old male with stage III colorectal adenocarcinoma was prescribed with weekly iv 5-fluorouracil and folinic acid (FU/FA). Fourteen weeks after starting FU/FA, the patient was enrolled in a clinical study to investigate the role of tumour burden on drug metabolising enzyme activity. CYP2C19 genotype was determined and the activity of CYP2C19 was measured using proguanil (PG) as a probe substrate. A metabolic ratio (PG/CG) for CYP2C19 activity was determined on three separate occasions, 7 days apart.

Results

The patient was homozygous wild type (CYP2C19*1/*1), and on the first test, the metabolic ratio was concordant with the extensive metaboliser genotype. However, at day 14 and day 21, the metabolic capacity of this enzyme had decreased, and the subject had become a poor metaboliser (PG/CG > 30). The patient developed grade III hand and foot syndrome at day 10 of the study during the period of null CYP2C19 activity.

Conclusions

Although 5FU is not a substrate for hepatic drug metabolising CYP enzymes, it may interfere with the synthesis of CYP2C19. Decreased activity of a related enzyme, CYP2C9, following 5FU has been reported previously. Down regulation of CYP2C9 and CYP2C19 synthesis by 5FU therapies may explain the adverse effect of 5FU on the clinical disposition of warfarin and phenytoin.     

The confused cancer patient: a case of 5-fluorouracil–induced encephalopathy

The fluorinated pyrimidine 5-fluorouracil (5-fu) is an anticancer agent used in most adjuvant and palliative treatment regimens for colorectal cancer. Neurotoxicities are considered extremely rare side effects of 5-fu. Here, we report a case of 5-fu–induced encephalopathy, manifesting as seizures and delirium, in an era of oxaliplatin-containing chemotherapy. If ammonia levels are elevated, lactulose may be considered in the initial management of neuropsychiatric complications from 5-fu.     

Capecitabine versus 5-fluorouracil in colorectal cancer: where are we now?

Fluorouracil (5-FU) remains the most widely used agent for colorectal cancer. Capecitabine is a rationally designed 5-FU pro-drug developed to mimic the continuous infusion of 5-FU while avoiding complications and inconvenience of intravenous administration. Capecitabine is absorbed intact from the gastrointestinal tract, converted enzymatically to active 5-FU, and released directly into the tumor. Capecitabine’s efficacy and safety are shown in multiple phase III trials across different disease stages and therapy lines. Three randomized phase III trials demonstrated the equivalence of capecitabine plus oxaliplatin (XELOX) versus 5-FU/leucovorin (LV)/oxaliplatin (FOLFOX). The safety of capecitabine compared with 5-FU depends on the regimen of 5-FU used. The adverse event rate with oxaliplatin in combination with infusional 5-FU is similar to that of capecitabine plus oxaliplatin but is associated with more neutropenia and venous thrombotic events; capecitabine plus oxaliplatin-based regimens tend to be associated with more grade 3 diarrhea and hand-foot skin reaction. Combination therapy with capecitabine and irinotecan (CapeIRI) versus 5-FU/LV and irinotecan (FOLFIRI) had more variable results; some former schedules resulted in excessive treatment-related toxicity. More recent data show that lower capecitabine and irinotecan doses, different schedules, and combination with targeted agents (e.g, bevacizumab) have resulted in more favorable outcomes.     

Association of adverse events and survival in colorectal cancer patients treated with adjuvant 5-fluorouracil and leucovorin: Is efficacy an impact of toxicity?

BackgroundAdverse events associated with 5-fluorouracil (5FU) based adjuvant therapy in colorectal cancer (CRC) patients may predict survival. We studied whether haematological (leucopenia, neutropenia, thrombocytopenia) or non-haematological (mucositis, diarrhoea, nausea/vomiting, hand-foot syndrome or other toxicity) adverse events were associated with disease-free survival (DFS) or overall survival (OS) in a large patient material treated with 5-fluorouracil based adjuvant chemotherapy.Patients and methodsData from two prospective randomised adjuvant trials were combined to achieve a dataset of 1033 radically operated stage II and III CRC patients treated with either monthly 5FU and leucovorin (LV) as bolus injections (Mayo or modified Mayo) or bi-monthly with bolus and continuous infusion (LV5FU2 or simplified LV5FU2). Toxicities were recorded at each treatment cycle according to NCI-C CTC (the Common Toxicity Criteria of the National Cancer Institute of Canada). The worst toxicity grade was taken into account. The median follow-up time of patients was 6.05 years.Results47% of patients developed neutropenia, 54% nausea/vomiting and 43% mucositis. Any grade neutropenia was associated with improved DFS (hazard ratio (HR) 0.81), any grade nausea/vomiting with improved DFS (HR 0.79) and OS (HR 0.62) and mucositis with improved DFS (HR 0.74) and OS (HR 0.72). Patients experiencing no predefined toxicity had the worst outcome.ConclusionSpecific adverse events related to adjuvant fluorouracil chemotherapy are associated with improved DFS and OS in early stage CRC patients.     

Irinotecan combined with bolus 5-fluorouracil and folinic acid for metastatic colorectal cancer: is this really a dangerous treatment?

Rothenberg et al called for caution in the palliative use of irinotecan, 5-fluorouracil (5-FU) bolus and leucovorin (IFL schedule), because of early treatment related deaths in C89803 and N9741 studies. The objective of our multicenter phase II study was to evaluate the efficacy and safety of the combination of 5-FU bolus, folinic acid (FA) and irinotecan as first-line chemotherapy for metastatic colorectal cancer. From December 1999 to June 2002 138 patients (pts) were treated. The chemotherapy regimen was as follows: irinotecan 125 mg/m2 i.v. over 90 min and 5-FU 500 mg/m2 preceded by FA 20 mg/m2, both given by bolus, weekly, for 4 weeks every 6 weeks. Treatment continued until disease progression or unacceptable toxicity. Total number of administered cycles was 404. Average dose intensity was 75%. 47 out of 131 evaluable pts achieved a complete (n = 6) or partial (n = 41) response, leading to an overall response rate (RR) of 36% [95% confidence interval (CI) 24% to 48%], stable disease was registered in 50 (38%). The estimated median time to progression and survival were 6.5 months (95% CI 5.2-9.4) and 16.6 months (95% CI 15.1-19.3) respectively. Two-year survival was 35%. Toxicity was well manageable. In 18 (13.8%) pts the chemotherapy was stopped because of toxicity. Treatment related death was not observed. Close clinical monitoring, early recognition of toxicity, immediate therapeutic intervention are recommended for pts receiving IFL. In our experience this regimen has manageable toxicity and appropriate level of dose intensity and seems to be a good option for first-line therapy in metastatic colorectal cancer.