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Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 μM CAFdA was 20 pmol/107cells in HL/CAFdA20 and 3 pmol/107cells in HL/CAFdA80 compared with 63 pmol/107cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.
The mainstay of leukemia chemotherapy has included purine and pyrimidine nucleoside analogs for nearly 50 years. Clofarabine (2-chloro-2′-arabinofluoro-2′-deoxyadenosine, CAFdA) is a relatively new purine nucleoside analog.[1-5] The rationale behind its design was to combine the structural features of cladribine and fludarabine.[5, 6] CAFdA is toxic to non-proliferating human lymphocytes and rapidly proliferating cells. Preclinical studies showed that CAFdA had a high degree of efficacy. In 2004, the US Food and Drug Administration approved CAFdA (Clolar, Genzyme, Cambridge, MA, USA) for the treatment of pediatric patients with acute lymphoblastic leukemia (ALL). The anti-cancer activities of CAFdA toward various types of tumors are thus being investigated in other age groups of leukemia.[7-10]
On administration, CAFdA is transported into leukemic cells through two types of nucleoside transporters: human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. The transport via hENT is a sodium-independent mechanism involving primarily bidirectional facilitative diffusion driven by a gradient in nucleoside concentration. In contrast, the hCNTs are sodium-dependent active transporters for their ability against a concentration gradient. Inside the cell, CAFdA is phosphorylated to its monophosphate derivative, not only by the cytoplasmic enzyme deoxycytidine kinase (dCK) but also by the mitochondrial enzyme deoxyguanosine kinase (dGK). Further intracellular phosphorylation results in the production of the active metabolite CAFdA triphosphate (CAFdATP). CAFdATP is incorporated into DNA, thereby terminating DNA elongation and eventually inducing apoptosis.[12-17] CAFdA also induces apoptosis via direct mitochondrial damage.
Pharmacological understanding of action of CAFdA is crucial to its optimal administration. Moreover, the biochemical and molecular mechanisms of drug resistance may be a clue to overcoming treatment failure. In the present study, two resistant to CAFdA were developed and their mechanisms of resistance were investigated. The study focused on factors that were involved in intracellular CAFdATP production and in drug-induced apoptosis. An effective strategy to sensitize the cells to CAFdA was also proposed.
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In the present study, two CAFdA-resistant leukemic clones were successfully developed. The mechanism of drug resistance was multifactorial, but it was ultimately associated with reduction in intracellular CAFdATP production and antiapoptosis. The decreased CAFdATP production was attributable to reduced transport capacity (Fig. 1b,c) and decreased dCK and dGK (Fig. 2f–h). The extent of the resistance appeared to be associated with the level of CAFdATP production (Fig. 1a). The reduced CAFdATP production attenuated the incorporation of CAFdA into the nuclei and mitochondria (Fig. 3a,b). In contrast, antiapoptosis was associated with increased Bcl2 and decreased Bim (Fig. 5a). Importantly, the alterations of hCNT3, dGK, Bcl2 and Bim have not been previously reported in the context of CAFdA resistance. In these respects, and in contrast to previous studies, the present study investigated the mechanism of CAFdA resistance intensively and extensively.[10-14]
The resistant variants developed here exhibited cross-resistance against similar nucleoside analogs (Table 1). Because the activation pathway is basically the same, such cross-resistance may be caused by the decrease in transport capacity and the reduced dCK. Two variants were also resistant to doxorubicin. This might be due to the overexpression of Bcl2, because doxorubicin-induced apoptosis was reported to be inhibited in cancer cells showing Bcl2 and/or Bcl-xL overexpression.[31, 32] However, the cells were not resistant to etoposide. Etoposide-induced apoptosis is mediated by proapoptotic protein Bax, which was unchanged in the resistant variants. It is suggested that the mechanism of the cross-resistance shown would be specific in part to nucleoside analogs and in part to their antiapoptotic nature.
The cellular uptake is mediated via hENT 1, 2 and hCNT 3. King et al. report that CAFdA is carried into oocytes by hCNT3, which attributes >5 times more than hENT1 and hENT2, suggesting that hCNT3 is crucial to the cellular uptake of CAFdA. Our results of uptake ability using tritited CAFdA also suggested that hCNT3 would attribute significantly to transport nucleoside analogs. Transcript levels of all three transporters were reduced in the two CAFdA-resistant cell lines (Fig. 1d–f). Nevertheless, the reduction in hCNT3 mRNA appeared to be more profound compared with the reductions in hENT1 and 2. Thus, the results suggested the importance of hCNT3 in relation to CAFdA resistance, which has not been reported previously.
In the two variants, the reduction in dGK was demonstrated at protein and transcript levels (Fig. 2b,f,h). Ara-C and cladribine are phosphorylated exclusively by dCK, while dGK also participates in the phosphorylation of CAFdA and 9-beta-d-arabinofuranosyl guanine.[34, 35] It was previously shown that, in cladribine-resistant cells, the activity of dCK decreased, but the activity of dGK was unchanged. However, Månsson et al. demonstrated that the cell line (MOLT-4) that was developed to be resistant to 9-beta-d-arabinofuranosylguanine showed a 42% decrease in dCK enzyme activity and a 26% decrease in dGK enzyme activity, the results of which indicated a more crucial role of dGK to metabolize 9-beta-d-arabinofuranosyl guanine than dCK. The present results demonstrated (Fig. 2f–h) were compatible with their findings, which strongly suggested that dGK has an important role in the phosphorylation pathway of CAFdA.
The cellular activation mechanism of ara-C is similar to that of CAFdA. Ara-C is influxed into leukemic cells mainly through hENT1, not using hCNT3, and phosphorylated to the active metabolite ara-C triphosphate (ara-CTP) by dCK, but not by dGK. Ara-CTP is incorporated into DNA, thereby terminating DNA synthesis. Ara-C does not inhibit ribonucleotide reductase nor induce apoptosis directly. The combination of CAFdA and the Bcl2 inhibitor ABT737 gave synergistic effects in three cell lines (Fig. 5b,c). Bcl2 protein expresses less in HL60 cells than in resistant cell lines, but ABT737 is effective in three cell lines. ABT737 has an effect on both BclxL and Bcl2. The CI was lower in both resistant variants, which might correspond to Bcl2 protein (Fig. 5a), suggesting the contribution of this increased antiapoptotic factor to the development of CAFdA resistance. In a literature search, a combination of Bcl2 inhibitor and nucleoside analog has been suggested for targeting solid tumor and hematological malignancies.[36-40] Hann et al. report that ABT737 induced apoptosis and had synergistic effects with etoposide against primary small cell lung cancer xenografts. The combination of ABT737 and etoposide caused significant decreases in tumor growth rates. Ugarenko et al. reported that ABT737 was used in combination with doxorubicin to overcome doxorubicin-resistant, Bcl2-overexpressing HL-60 cells. The addition of ABT-737 enhanced the cytotoxicity of doxorubicin-induced DNA adducts and subsequently induced classical apoptosis. The present study clearly demonstrated that ABT737 also enhanced the cytotoxicity of a nucleoside analog against cancer cells.
There has been one report of an investigation of the mechanisms of CAFdA resistance by Månsson et al.; however, they mainly demonstrate a decrease in dCK and do not deal with dGK, transporters and apoptosis-related factors.[15, 41] The present study showed conclusively that the mechanism of cellular resistance to CAFdA in the two variants was multifactorial, but primarily involved reduced intracellular CAFdATP and antiapoptosis. Moreover, CAFdA and the Bcl2 inhibitor ABT737 showed synergistic effects in the two variants with Bcl2 overexpression, which suggests that this combination treatment has clinical potential.