Docetaxel enhances S-phase cytotoxicity with dFdCyd resulting in synergy

Proc Amer Assoc Cancer Res, Volume 45, 2004 2965 The combination of dFdCyd (gemcitabine, 2’,2’-difluoro-2’-deoxycytidine) and docetaxel has been found to be clinically effective in varying types of cancer. Recently, startling clinical results (37-53% response rates) have been observed in several reported series of sarcoma patients in which dFdCyd preceded docetaxel during treatment. The reason for this apparent synergy is unclear since dFdCyd and docetaxel have differing mechanisms of cytotoxicity. dFdCyd requires phosphorylation for activity: the diphosphate derivative inhibits de novo deoxynucleotide production leading to inhibition of DNA synthesis, while the triphosphate can be incorporated into DNA leading to cell death. Docetaxel stabilizes microtubules preventing depolymerization, resulting in mitotic arrest or aberrant mitosis. Recent evidence suggested that docetaxel may also have a secondary mechanism of cytotoxicity that targets S-phase, but little is known about it. Previously, we determined that the sequence of drug addition is important in vitro with the sequence dFdCyd followed by docetaxel producing the greatest synergy in A549 non-small cell lung cancer, MCF-7 breast cancer, and SAOS-2 sarcoma cells. For example, the 24 hr IC50 of dFdCyd in A549 cells was decreased 2-fold (16 nM to 7.4 nM) with the subsequent addition of docetaxel (0.5 nM, docetaxel on the metabolism of dFdCyd and cell cycle progression under conditions which produced synergistic cytotoxicity in A549 cells. The addition of docetaxel (0.5 nM) after treatment with dFdCyd (20 nM, IC50) did not alter the accumulation of dFdCTP, nor did it increase its incorporation into DNA. Additionally, these conditions did not appear to alter cell cycle progression as measured by dual parameter flow cytometry (PI/BrdU). However, the addition of 2 nM docetaxel (IC75) to cells previously treated with dFdCyd (5-10 nM, SNI (an indicator for S-phase specific cell death) cells after 24 hr. In order to elucidate whether docetaxel was interacting with the effects of dFdCyd on deoxynucleotide pools or DNA synthesis, docetaxel was combined with either hydroxyurea or aphidicolin. Hydroxyurea followed by docetaxel did not produce synergy, only additivity. However, aphidicolin followed by docetaxel did produce synergy, indicating a possible interaction with DNA polymerases. Upon subsequent cell cycle analysis, it was observed that the addition of docetaxel to cells previously treated with aphidicolin increased both the amount of sub-G1 and SNI cells after 24 hr. This indicated that docetaxel was able to directly affect S-phase cells. Taken together, these studies suggest that docetaxel synergistically enhances the cytotoxicity of dFdCyd through S-phase specific interactions.