Abstract
Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.
Highlights
Nucleoside analogs are synthetic, chemically modified nucleosides that due to their resemblance can be incorporated into RNA and DNA to inhibit their synthesis and subsequently inhibit cell division [1]
Expression levels were normalized to the level of expression of ß-actin. short interfering RNAs (siRNAs) downregulation in the SW1573 cell line yielded similar results, showing an 87% reduction of UCK1 and 79% reduction of UCK2 mRNA, the siRNA negative sample showed an increase in UCK2 expression (Fig 2B)
A549 cells were moderately sensitive to 11.7 μM RX-3117 (63.7% cell growth), but the cells transfected with siUCK2 were completely protected (123.6% cell growth), while this was not the case in cells treated with scrambled siRNA negative control (63.7% cell growth; not shown) or siUCK1 (62.4% cell growth)
Summary
Nucleoside analogs are synthetic, chemically modified nucleosides that due to their resemblance can be incorporated into RNA and DNA to inhibit their synthesis and subsequently inhibit cell division [1]. This has potential therapeutic benefits such as the inhibition of cancer cell growth and combatting viral infections [2]. Examples of successful cytidine analogs in anti-cancer applications are cytarabine and gemcitabine [2, 3], the latter drug is predominantly used for treatment of patients with non-small cell lung cancer (NSCLC) [4]. The inter- and intra-tumor heterogeneity can imply for resistance to drugs in patients. There is a need for novel anti-cancer drugs which vary in their mechanism of cellular action and can overcome the resistance
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