Modulation of intrinsic tumoral radioresistance by 2-2' difluorodeoxycytidine (Gemcitabine) : characterization and study of mechanisms of action
(2008)
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- Recent research in cancer therapy has focused on combining radiation with chemotherapy in an attempt to improve patient outcome. This approach is validated by the concept of radiation enhancement. In this concept chemotherapy drugs have the potential to enhance the anti-tumour efficacy of ionizing radiation. Among several classes of drugs which have demonstrated in vitro radioenhancement properties, nucleoside analogs such as Gemcitabine represented attractive challenges. Since it has been recognized that more than one factor is important in causing a radio-enhancing effect and that it was difficult to elucidate mechanisms of radioenhancement by comparing different cell lines, we have considered that unknown cell line characteristics might unintentionally influence the results. To avoid this bias, the originality of our work has been to face this challenge using the same experimental tool. Our in vitro studies have demonstrated that dFdC enhanced the cellular radiation response. This radioenhancement was recognized not to be significantly influenced by the p53 status of studied cell lines. The originality of this work has been not only to confirm the fact that dFdC's radio-enhancement was observed in different culture conditions but also that the amplitude of such phenomenon was cell line - dependent. Moreover, it depended on the proliferation index applied in experimental conditions. For the first time, we have published that radioenhancement interested Human Head and Neck squamous carcinoma cell lines. For these cell lines and others, dFdC's radioenhancement was evident at non-cytotoxic concentrations and in a similar range (micro molar) as the assessed dFdC plasma concentration observed in clinical routine. Our observations confirmed also that such a phenomenon depended on drug incubation time, drug concentration as well as on the timing of irradiation. Lastly, we have placed in a prominent position the fact that dFdC's radioenhancement could depend on the intrinsic cellular radio sensitivity level since dFdC did radioenhance preferentially the more radioresistant cells. In addition, using a unified experimental procedure, our results have partially resolved the molecular mechanisms of interaction between dFdC and ionizing radiation. Taking into account our results and those published in the literature, we suggest that dFdC-mediated radioenhancement occurs through a combination of mechanisms that have been identified. These main determinants of Gemcitabine's enhancement of radiation response included: inhibition of DNA repair (influence on radiation-induced genomic lesions and more particularly on its interferences with the repair of radiation-induced DNA damage inducing secondary chromosome breaks).This implication of Gemcitabine in the radiation-induced DNA repair pathway conducted us to understand and validate the necessity of giving Gemcitabine before irradiation in view of obtaining such enhancement. cell cycle modulation dFdC and ionizing radiation could interact with the cell cycle by a para synchronous movement of cells through the cell cycle and a DNA synthesis inhibition for cells in the S-phase of the cycle. Nevertheless,our report indicates that in our two studied cell lines, enhancement of radiation response by dFdC did not result from a modulation of the G1-S checkpoint. Nucleotide pool perturbations Our research has allowed us to propose that dFdC-induced radioenhancement was not systematically associated with dNTP pool depletion.If the Gemcitabine-induced dNTP depletion could play a role in enhancing the radiation response for specific cell lines, it did not constitute a predominant mechanism of action. Intracellular dFdCTP form As DNA inhibition repair is well implicated in the radioenhancement process, we consider that such inhibition is mediated either by dFdCDP-induced inhibition of ribonucleotide reductase inducing dATP pool depletion or direct inhibition of DNA polymerase by dFdCTP. However, the "effective dFdCTP concentration" (i.e. the concentration required to produce a given biological effect such as radioenhancement) appears to be quite different from cell line to cell line. The degree of radioenhancement seems to be related to the extent of dFdCTP incorporated into DNA.These observations could explain why dFdC's radioenhancement is time- and dose-dependent.These hypotheses have been strengthened by our results which suggest a correlation between intracellular dFdCTP concentration, dCK activity and dFdC's radioenhancement. In addition, intracellular concentration of this tri-phosphorylated form of dFdC depends on dCK activity. In this context, we have also shown under experimental conditions used in this thesis, a significant correlation between dCK activity and dFdC's radioenhancement. Finaly, we consider that associated to the primordial role played by the intracellular dFdCTP, other dFdC's dependent mechanisms which could be considered as modulator of this pathway exist:  Potential dFdCDP inhibition of ribonucleotide reductase activity inducing intracellular dATP pool depletion and interference in the DNA repair replication.  Additive effect in the stimulation of independent mechanisms of dCK activity which remove the well documented saturation phenomenon of this enzymatic activity. Our results have demonstrated that dCK activity could be modulated independently by dFdC as well as by radiation. This additive stimulation may increase the intracellular level of dFdCTP and hence increase the overall effect of the combined treatment. All of these four determinants were complementary. However, their levels of implication in the radioenhancing process were certainly cell line dependent. Although effects of dFdC on cell cycle redistribution and dNTP pools may contribute to, or even be necessary for dFdC-mediated radioenhancement, they do not ultimately determine whether or not dFdC treatment is going to result in enhanced radiosensitivity. At the present time, all our investigations reported in this thesis allow us to suggest that Gemcitabine must be considered not only as a potential "radiosensitizer" but as "radio-enhancer"as well
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Rosier, J.-F. (2008). Modulation of intrinsic tumoral radioresistance by 2-2′ difluorodeoxycytidine (Gemcitabine) : characterization and study of mechanisms of action. https://hdl.handle.net/2078.5/112096