Gemcitabine (2', 2'-difluoro 2'deoxycytidine), an analogue of deoxycytidine (Commercial name: Gemzar), is a broad spectrum drug that affects a wide variety of cancers. In clinical medicine, it is ranked amongst top three drugs in cancer therapy . It is used to treat a wide variety of cancers, including breast and pancreatic cancers, non-small-cell lung carcinoma, acute myeloid leukemia as well as the cancer of ovaries . Compared with other drugs in cancer treatment, gemcitabine is actually less toxic, and it is being widely used for the treatment of cancers in children.
As a prodrug, gemcitabine is activated by several kinases. Upon entrance into the cell, it converted enzymatically to di- and tri-phosphorylated derivatives. These derivatives can perturb DNA synthesis by inhibition of ribonucleotide reductase enzymes within the cell, as well as incorporation into the DNA structure during replication [3, 4]. The effectiveness of gemcitabine as an anticancer drug is mainly mediated by deoxycytidine kinase (dCK) . Accordingly, its action is dependent to the normal activity of dCK. However, the mechanism of the interaction between gemcitabine and dCK is controversial .
Human variant of dCK can phosphorylate natural ribonucleotides such as deoxycytidine, deoxyguanosine and deoxyadenosine that are needed for DNA synthesis. It is also an important enzyme involved in phosphorylation of the nucleoside analogues (containing modified base, sugar or both moieties) . These analogues are being used as prodrug for treatment of cancers and some virus-related diseases. For the majority of these compounds, phosphorylation reaction catalyzed by dCK is the rate limiting step of the whole process [7, 8].
This enzyme is continuously expressed during the cell cycle, and it is found in all tissues including normal and cancerous cells. It has been shown that the expression level of dCK have deterministic role in gemcitabine activity, and that the defective dCK enzymes are resistance to the nucleosides analogues [5, 9].
It was also shown that dCK-deoxycytidine complex has similar structure with the corresponding structure when deoxycytidine is substituted by gemcitabine. Hence, gemcitabine has been suggested as an effective competitive substrate for binding to the normal structure of dCK [7, 10].
On the other hand, representative cancers are shown to be resistance to drugs . Clinical investigations have also demonstrated that some patients are resistance to gemcitabine -based treatment. Okazaki et al. has estimated that approximately 75% of patients of pancreatic cancer are resistance to gemcitabine . Investigations of Bergmen et al. have shown that drug resistance feature in patients has multifactorial origin, and the mechanism of resistance is mainly related to the complexity of drug metabolism . In line with these findings, it seems that future studies should pay special attention to the parameters that could predict resistance to gemcitabine. Ruiz van Haperen et al. have reported that resistance to gemcitabine in different cells is originated from the changes in the kinetics of the dCK activity due to the genetic modifications . Keeping in mind that the reaction catalyzed by the dCK is rate-limiting step in the metabolism of gemcitabine, it is proposed that inactivation of dCK is the main mechanism for gemcitabine –resistance feature [14, 15]. By studying various cell lines, it is found that inactivating mutations as well as decreasing the expression level of dCK leads to gemcitabine-resistance property in cell lines. Sebastiani et al. have shown that the expression level of dCK decreases with increasing the age of patients, demonstrating that there is a positive correlation between the age and resistance to gemcitabine . These findings together, indicate that the expression level and genetic mutations are two important factors in drug resistance feature . The second factor is the consequence of the single nucleotide polymorphism (SNP) found in human population. Indeed, several SNPs in the exon as well as intron region of immature hnRNA of dCK have reported by various research groups. A correlation between the SNPs and the cancer mortality in patients that has been treated by gemcitabine was also stablished [17–19]. Regarding the gemcitabine resistance mechanisms, Kocabas et al. have reported that patients bearing a presentative SNP are producing a double mutant of deoxycytidine (Ile24Val and Pro122Ser), are significantly resisted against gemcitabine therapy. Further enzymatic assay showed that the km parameter of the mutant deoxycytidine was significantly different from WT.
The aim of the current work is bioinformatics analysis and computational study on the dCK and its characterized double mutant (Ile24Val and Pro122Ser) to find a molecular explanation to the mechanism of gemcitabine resistance in patients containing this variant of the enzyme.