[Thymidylate synthase gene promoter polymorphism in patients with advanced head and neck cancer treated by radio- and 5-fluorouracil chemotherapy]. / A timidilátszintáz gén promoterpolimorfizmusának vizsgálata kis dózisú 5-fluorouracil kemoterápiával és sugárterápiával kezelt elôrehaladott stádiumú fej-nyaki rákos betegeken.

AIM: Thymidylate synthase (TS) is a rate-limiting enzyme in the DNA synthetic pathway, and represents a cellular target of antimetabolite drug 5-fluorouracil. It catalyzes the reductive methylation of deoxyuridine-5'-monophosphate to deoxythymidine-5'-monophosphate. Inhibition of TS will result in depletion of both dTMP and, subsequently, dTTP. As a consequence, dUTP is misincorporated into DNA, resulting in DNA breakage and cell death. Developing resistance against 5-fluorouracil (FURA) based drugs might be due to the failure of inhibition of thymidylate synthase enzyme function. In the promoter region of the TS gene there is a tandem repeat sequence (2R or 3R), which was found to be polymorphic and influences the gene expression. Effectiveness and tolerability of Tegafur treatment might be influenced by expression of the TS gene. Our purpose was to determine the effectiveness and tolerability of concomitant radiotherapy and low-dose chemotherapy using Tegafur in respect of promoter polymorphism of thymidilate synthase gene. MATERIAL AND METHODS: TS promoter polymorphism (2R/2R, 2R/3R, 3R/3R) was determined by polymerase chain reaction using genomic DNA, in 47 patients with advanced head and neck cancer. RESULTS: Thirty patients out of 47 showed complete response, and genotyping of these patients revealed 2R/2R in 22 (73.3%), 2R/3R in 2 (6.7%) and 3R/3R in 6 (20%). Seventeen out of 47 patients reacted with partial response, and 2R/2R or 2R/3R were revealed in 5 (29.4%) and 3 (17.6%) patients, respectively, and 3R/3R genotype was identified in 9 patients (53%). CONCLUSION: We did not find any correlation between patient's data and response to therapy, but strong correlation was found between the latter and the patient's genotype. This facts indicate that the analysis of promoter polymorphism of thymidylate synthase gene might be a useful target to examine before FURA-based chemotherapy, and might allow to go into the direction of individualized treatment of head and neck cancer. We suppose that tumor response depends on genomic features of the patients.

[Radiotherapy in combination with low-dose chemotherapy in locally advanced head and neck cancer]. / Kis dózisú kemoterápiával kombinált sugárterápiával szerzett eredményeink lokálisan elôrehaladott stádiumú fej-nyaki rákban.

A previous preclinical study revealed that the maximal additive effect between chemotherapy (CT) and irradiation (RT) occurred at a low level of CT. Therapy was therefore designed with an oral drug daily given in combination with RT in order to determine the efficacy and toxicity. Locoregionally advanced head and neck tumor patients were treated with simultaneous RT and CT. RT was administered 5 times per week at 2 Gy per fraction in a total dose of 70 Gy. Throughout the treatment 30 mg/kg Tegafur was given daily orally. In the period between 2000 and 2002, 50 patients were enrolled. Complete remission was attained in 60%, with an overall response rate of 94%. Acute mucositis of grade 2 or 3 was observed in 56% (28 patients), and gastrointestinal and hematologic toxicity of grade 2 or 3 occurred in 8% (4 patients). Because of side-effects, the duration of treatment was at most 2 weeks longer. Toxicity was eliminated quickly by careful supportive therapy. In conclusion, it is considered that oral low-dose CT in combination with RT is an efficient and simple mode of treatment for locally advanced head and neck tumor patients with a poor prognosis.

Effective chemoradiotherapy without additive toxicity in locoregionally advanced head and neck cancer.

A previous preclinical study revealed that the maximum additive effect between chemotherapy (CT) and irradiation (RT) occurred at a low level of CT. Therapy was therefore designed with an oral drug daily given in combination with RT in order to determine the efficacy and toxicity. Locoregionally advanced head and neck tumor patients were treated with simultaneous RT and CT. RT was administered 5 times per week at 2 Gy per fraction to a total dose of 70 Gy. Throughout the treatment 30 mg/kg Tegafur was given daily orally. In the period between 2000 and 2002, 50 patients were enrolled. In 60%, complete remission was attained with an overall response rate of 94%. Acute mucositis of grade 2 or 3 was observed in 56% (28 patients), while gastrointestinal and hematological toxicity of grade 2 or 3 occured in 8% (4 patients). Because of side-effects, the duration of treatment was at most 2 weeks long. Toxicity was eliminated quickly by careful supportive therapy. In conclusion, it is considered that oral low-dose CT in combination with RT is an efficient and simple mode of treatment for locally advanced head and neck tumor patients with a poor prognosis.

Chemosensitizing effect of vitamin C in combination with 5-fluorouracil in vitro.

The antiproliferative effect and apoptosis-inducing action of 5-fluorouracil (5-FU) in combination with vitamin C were tested in vitro against the chemosensitive mouse lymphoma, the chemoresistant HEp-2 and a human lung fibroblast cell line. Vitamin C itself had no antiproliferative effect on the fibroblasts, but increased the anticancer effect of 5-FU dose-dependently. In the case of the chemoresistant cell line, only a high concentration of vitamin C increased the cytotoxicity of 5-FU. A combination of 5-FU and vitamin C exerted a significantly enhanced apoptotic effect on the mouse lymphoma cell line, whereas for the HEp-2 cell line this effect was less marked and was achieved only at a high concentration of vitamin C. These findings suggest that the administration of a high dose of vitamin C in combination with 5-FU chemotherapy enhances the chemoresponsiveness of cancer cells and serves as a potential sensitizer, especially in chemo-resistant cell lines. One of the mechanisms by which vitamin C potentiates cytostatics could be apoptosis induction.

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines.

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.

Ki-67, cyclin D1, p53 and bcl-2 expression in advanced head and neck cancer.

In order to analyse the radiosensitivity of tumours and to evaluate the possibility of improving the treatment results with regard to genetic alterations, we examined 33 patients with advanced head and neck tumours after 66-70 Gy irradiation. Between 1998 and 2001, 33 patients with advanced head and neck squamous cell carcinoma (HN-SCC) were observed. They received 66-70 Gy to the primary tumour site and pathological lymph nodes. One month later, physical examination and CT were performed to verify the effect of radiotherapy, and the patients were followed until their death. The histological grading and Ki-67, cyclin D1, p53 and bcl-2 status were examined from the aspect of their potential prognostic value in all patients. The average survival was 13 months; 25% of the patients survived for at least 20 months. Seventy-two percent of the patients demonstrated Ki-67 positivity, 69% p53 positivity and 40% cyclin D1 positivity; there were only 12% bcl-2-positive cases. A significant correlation was not found between the tumour response or the duration of survival and the Ki-67, p53 or cyclin D1 positivity. Only bcl-2-positive cases exhibited significantly better outcome. These parameters indicate the proliferating (Ki-67 and cyclin D1) and apoptotic (p53 and bcl-2) activities of advanced HN-SCC cells. Our results proved that they proliferate rapidly and have impaired repair or apoptotic functions. The heterogeneity of our results did not allow us to conclude that the above parameters are of clinically reliable prognostic value, but the obviously high kinetic activity of HN-SCC underlines the potential efficacy and need for accelerated irradiation in these cases.

Combined effect of cisplatin and 5-fluorouracil with irradiation on tumor cells in vitro.

The concomitant administration of chemotherapy and radiation is an alternative tool in cancer therapy. The antiproliferative and the radiosensitizing effect of Cisplatin and 5-Fluorouracil (5-FU) were studied on mouse lymphoma cells transfected with human MDR1 gene (mdr) and its parent cell line (par) combined with and without radiation. HEp2 radioresistant cell culture was used in our experiments as a model of radioresistance. The growth rate and antiproliferative effect was measured by the MTT method. Significant inhibition of tumor cell growth was observed at a low concentration of Cisplatin and 5-FU combined with radiation on the mouse lymphoma cell lines. However an extremely high dose of Cisplatin and 5-FU resulted in moderate growth inhibition in the case of the HEp2 cell line. We assume that the radiosensitizing effect of 5-FU and Cisplatin can be considered as a synergistic antitumor effect at low doses of chemotherapy and radiation in a radiosensitive cell line. In the case of a radio-and chemoresistant cell line, high doses of radiation and chemotherapeutic agent achieved moderate tumour growth inhibition without significant synergistic effect. In addition the simultaneous application of both treatments can result in remarkable toxicity.