CP‐4055 and CP‐4126 are active in ara‐C and gemcitabine‐resistant lymphoma cell lines

The clinical activity of cytarabine (ara-C) and gemcitabine can be impaired by a deficient cellular drug uptake due to a reduced expression of nucleoside transporters in cancer cells (Galmarini et al, 2001; Takagaki et al, 2004; Hubeek et al, 2005; Pastor-Anglada et al, 2005). Various attempts have been made to overcome this problem. CP-4055 and CP-4126, the 5′-elaidic acid esters of ara-C and gemcitabine, respectively, have been synthesized to overcome the resistance caused by limited cellular uptake. Once inside the cell, these compounds are metabolized to free nucleoside analogues by hydrolytic cleavage of the ester bond, releasing the fatty acid. Intracellular free nucleoside analogues are then phosphorylated by different kinases to their diphosphate and triphosphate derivatives in order to induce cell death (Bergman et al, 2004). The cytotoxic activity of CP-4055 (previously named P-4055) against cancer cells has previously been evaluated in different studies in vitro and in animal tumour models and CP-4055 and CP-4126 are currently studied in clinical trials (Breistol et al, 1999; Aamdal et al, 2007; Adams et al, 2008). As CP-4055 and CP-4126 have the ability to cross cell membranes independently of nucleoside transport proteins, their administration should overcome drug resistance in cancer cells with a deficiency in nucleoside transport (Adema et al, 2008). To test this hypothesis, we compared the sensitivity profiles of CP-4055 and CP-4126 with those of ara-C and gemcitabine using lymphoma cell lines with proficient (CEM) or deficient 5CEM-araC/8C) nucleoside transport (originated by B. Ullman and obtained from J Mackey, Alberta University, Canada) (Ullman, 1989). Parental CEM cells presented a similar sensitivity to ara-C, gemcitabine, CP-4055 and CP-4126 (Fig 1A). The mean drug concentration resulting in 50% growth inhibition (GI50) relative to untreated cells after 72 h treatment values for all compounds were approximately 0·01 μmol/l. In contrast, CEM/araC/8C cells were highly resistant to ara-C and gemcitabine (Fig 1B) with GI50 values placed at the high μmol/l or low mmol/l range. Compared to CEM cells, CEM/araC/C8 cells were 56 700-fold and 2800-fold more resistant to ara-C and gemcitabine respectively. On the other hand, CEM/araC/8C cells were more sensitive to the action of CP-4055 and CP-4126 compared with ara-C or gemcitabine (Fig 1A). Not only were the GI50 values for both compounds in the nmol/l range, but these drug resistant cells were only 75-fold and 40-fold more resistant to CP-4055 and CP-4126 respectively. Cell growth inhibition curves after treatment of nucleoside transport proficient (CEM; solid lines and black icons, both A and B) and deficient (CEM/araC/8C or CEM with NBTI inhibitor respectively; dashed lines and white icons, A and B) cells. Both cell lines were plated at 20 × 103 cells/well in the presence of increasing concentrations of ara-C (; ), gemcitabine (; ), CP-4055 (; ) and CP-4126 (; ). Cell growth inhibition activity of the four compounds was then determined from concentration-effect curves generated in triplicated paired parental/resistant cell lines side-by-side in 72-h cytotoxicity assay (MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay). The results shown are representative of three experiments performed in triplicate. In order to confirm these results, the equlibrative nucleoside transport activity in CEM cells was inhibited with nitrobenzylthioinosine (NBTI; 30 μmol/l for 30 min) and then these cells were exposed to the four compounds for 72 h. As expected, NBTI treatment rendered CEM cells resistant to ara-C and gemcitabine (Fig 1B). GI50 values for both compounds were in the μmol/l range, showing resistant ratios of 720- and 1200-fold to ara-C and gemcitabine respectively. In contrast, NBTI treatment hardly affected the growth inhibitory activity of CP-4055 and CP-4126 as GI50 values continued to be in the nmol/l range. These values were only increased 5·5- and sixfold compared to the NBTI non-treated cells. All together, these data demonstrated that CP-4055 and CP-4126-compounds can overcome the in vitro drug resistance to ara-C and gemcitabine due to deficient nucleoside analogue transport. This data corroborated previous in vitro studies described by Breistol et al (1999). We then evaluated apoptotic response by the four compounds in both cell lines. In CEM cells (Table I), ara-C, gemcitabine, CP-4055 and CP-4126 treatment induced similar levels of apoptosis. In contrast, while no apoptosis induction was observed after treatment of CEM/araC/8C cells with ara-C or gemcitabine, CP-4055 and CP-4126 induced high levels of annexin-V positive cells. Thus, the increase in cytotoxic activity observed after treatment of CEM/araC/8C cells with CP-4055 or CP-4126 was principally because of an increase in apoptosis induction compared to the parental drugs. We finally analysed cell cycle perturbations induced by the four drugs in both cell lines (Table I). Treatment of CEM cells with ara-C, CP-4055 and gemcitabine showed a classical arrest of cell cycle in the S-phase. Differently, CP-4126 induced a G1-cell cycle arrest. In CEM/araC/8C cells, ara-C, gemcitabine and CP-4126 induced a slight S-phase arrest while CP-4055 induced a slight G1 arrest. All together, these results demonstrated that in parental cells, the four compounds induced similar cell cycle perturbations that would result in similar apoptotic levels. However, drug-resistant cells presented a differential apoptotic response without major differences in drug-induced cell cycle perturbations indicating that after treatment with ara-C and gemcitabine, drug-resistant cells were slightly perturbed in terms of cell cycle but remained alive. In contrast, at this incubation time, CP-4055 and CP-4126 killed by apoptosis a high proportion of resistant cells; those left alive should die in the next 24 h or showed another type of resistance to these compounds. In conclusion, our data demonstrate that the fatty acid derivatives CP-4055 and CP-4126 circumvent drug resistance to nucleoside analouges caused by deficient membrane nucleoside transport. Given the potency of CP-4055 and CP-4126 against drug resistant tumor cells, further animal experiments are necessary to evaluate the in vivo potential of these compounds in drug resistant models. This strategy offers the opportunity for the treatment of cancer patients who do not respond to nucleoside analogues because of a deficiency in membrane transport.

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