Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and in human xenografts

Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP-4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP-4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP-4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP-4125 exposure, while these pools decreased directly after removal of gemcitabine. In conclusion: CP-4125 is an interesting new gemcitabine derivative.     

N-Acyl-phosphoramidates as potential novel form of gemcitabine prodrugs

Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency. Thus, proper prodrugs carrying the 5′-pdFdC moiety may help to overcome this problem. A series of new derivatives of gemcitabine possessing N-acyl(thio)phosphoramidate moieties were prepared and their cytotoxic properties were determined. N-Acyl-phosphoramidate derivatives of gemcitabine have similar cytotoxicity as gemcitabine itself, and have been found accessible to the cellular enzymes. The nicotinic carboxamide derivative of gemcitabine 5′-O-phosphorothioate occurred to be the best inhibitor of bacterial DNA polymerase I and human DNA polymerase α.     

Deamination rates of 1-beta-D-arabinofuranosylcytosine, deoxycytidine and 5-methyl-2'-deoxycytidine in seven hematopoietic cell lines

Enzymatic deamination activity was determined with tritium-labelled substrates in seven established hematopoietic cell lines, in order to compare deamination rates in intact vs. broken cells with cytosine arabinoside, deoxycytidine and 5-methyldeoxycytidine. Deaminase activity was found in all the cell lines, although it was very low in mouse leukemia L1210 cells. The deamination activity of intact cells varied from 1.0 to 38.3 pmoles/micrograms protein/30 min, being highest in the human null-cell ALL line (NALL-1), the human promyelocytic leukaemia line (HL-60) and the human T-ALL line (JM). The variation in specific activities in the broken cells was between 0.9 and 30.2 pmoles/micrograms protein/30 min. The deamination rate of deoxycytidine was in general higher than that of 5-methyldeoxycytidine or cytosine arabinoside.     

Increased Cytotoxicity of 2′,2′‐Difluoro‐2′‐Deoxycytidine in Human Leukemic Cell‐Lines After a Preincubation with Cyclopentenyl Cytosine

The in vitro modulating effect of Cyclopentenyl cytosine (CPEC) on the metabolism of gemcitabine was studied in lymphocytic and myeloid leukemic cell‐lines. In MOLT‐3 cells, that were pretreated with CPEC, the incorporation of 2′,2′‐difluoro‐2′‐deoxycytidine triphosphate (dFdCTP) into DNA was significantly increased by 57–99% in comparison with cells that were only treated with gemcitabine. The increased incorporation of dFdCTP into DNA in CPEC pretreated cells was paralleled by an increase in apoptotic and necrotic cells of 17–34%. In HL‐60 cells that were preincubated with CPEC, increased concentrations of the mono‐/di‐ and triphosphate form of gemcitabine were observed, as well as an increased incorporation of dFdCTP into DNA (+ 773%). This increased incorporation was paralleled by a significant increase in apoptosis and necrosis. We conclude that CPEC enhances the incorporation of dFdCTP into DNA and thus increases the cytotoxicity of gemcitabine in lymphocytic and myeloid leukemic cell‐lines.     

Cellular resistance against troxacitabine in human cell lines and pediatric patient acute myeloid leukemia blast cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC50: >3000 nM), and the cladribine resistant CEM (IC50: 150 nM) and HL-60 (IC50: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC50; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.     

Cell specific cytotoxicity and structure-activity relationship of lipophilic 1-B-D-arabinofuranosylcytosine (ara-C) derivatives.

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5'-position with fatty acids (16-22 C-atoms, 0-3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.     

Metformin‐induced alterations in nucleotide metabolism cause 5‐fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma

5‐Fluorouracil (5‐FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5‐year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti‐cancer activity, in combination with 5‐FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5‐FU in WHCO1 and WHCO5 cells, by more than five‐ and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5‐FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a “dilution” of 5‐FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre‐treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5‐FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.     

Cellular Resistance Against Troxacitabine in Human Cell Lines and Pediatric Patient Acute Myeloid Leukemia Blast Cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC₅₀: >3000 nM), and the cladribine resistant CEM (IC₅₀: 150 nM) and HL-60 (IC₅₀: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC₅₀; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Cell Specific Cytotoxicity and Structure-Activity Relationship of Lipophilic 1-B-D-Arabinofuranosylcytosine (Ara-C) Derivatives

Abstract

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5′-position with fatty acids (16–22 C-atoms, 0–3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2′,2′-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.     

Pyrimidine salvage pathways in adult Schistosoma mansoni

Adult Schistosoma mansoni can utilize radiolabelled cytidine, uridine, uracil, orotate, deoxycytidine and thymidine for the synthesis of its nucleic acids. In this respect, cytidine is the most efficiently utilized pyrimidine precursor. Cytosine, thymine and orotidine are transported into the parasites but not metabolized. High performance liquid chromatography analysis of the nucleobase, nucleoside and nucleotide pools from in vivo metabolic studies and assays of enzyme activities in cell-free extracts indicate the presence of nucleoside and nucleotide kinases which phosphorylate the various nucleosides to their respective nucleoside mono-, di- and triphosphates. Uridine, thymidine and deoxyuridine can also be cleaved to their respective nucleobases by uridine phosphorylase. Uracil can be converted directly to UMP by orotate phosphoribosyltransferase or by the sequential actions of uridine phosphorylase and uridine kinase. Nucleoside 5'-monophosphates were dephosphorylated by active phosphohydrolases. All enzymes tested were found in the cytosol fraction with the exception of the phosphohydrolases which were associated mainly with the particulate fraction. No deamination of cytosine, cytidine, deoxycytidine, CMP or dCMP was detected either in vivo or in vitro. The active metabolism of cytidine and absence of deamination and phosphorolysis of cytidine derivatives in schistosomes raise the possibility of using cytidine analogues for the selective treatment of schistosomiasis.     

Human deoxycytidine kinase. Sequence of cDNA clones and analysis of expression in cell lines with and without enzyme activity

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.     

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Micro-Array Analysis of Resistance for Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunits

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ß-D-arabinofuranosylcytosine (ara-C), 2-chloro-2’deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.     

Deoxycytidine deaminase-resistant stereoisomer is the active form of (+/-)-2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication.

2',3'-Dideoxy-3'-thiacytidine (+/-)-SddC) was found to have potent activity against human hepatitis B virus as well as human immunodeficiency viruses in culture. The (-)form ((-)-SddC) which is resistant to deoxycytidine deaminase was found to be the more active antiviral stereoisomer than the (+)-form ((+)-SddC). The (+)-SddC is susceptible to deamination by deoxycytidine deaminase and is 25- and 12-fold more toxic than (-)-SddC in CEM cells in terms of anti-cell growth and anti-mitochondrial DNA synthesis, respectively. Similar results were obtained using a mixture of their 5-fluoro analogs ((+/-)-FSddC). Unlike 2',3'-dideoxycytidine, which is a potent inhibitor of mitochondrial DNA synthesis and results in such delayed toxicity as peripheral neuropathy with long term usage, (-)-SddC does not affect mitochondrial DNA synthesis. The (-)form is phosphorylated to (-)-SddCMP and is subsequently converted to (-)-SddCDP and (-)-SddCTP. One additional major metabolite which has been tentatively assigned the name "(-)-SddCMP sialate" was also identified. No significant difference in terms of the profiles of the metabolites was found between 4 and 24 h. There is an appreciable amount of (-)-SddCTP detectable 24 h after removal of the drug. (-)-SddCTP was also found to be approximately 3-fold more potent than (+)-SddCTP in inhibiting human hepatitis B virus DNA polymerase. This is the first nucleoside analog with the unnatural sugar configuration demonstrated to have antiviral activity.     

Zooxanthellactone, a novel gamma-lactone-type oxylipine from dinoflagellates of Symbiodinium sp.: structure, distribution, and biological activity

A novel fatty acid derivative named zooxanthellactone (ZL) was isolated from several strains of symbiotic microalgae, dinoflagellates of the genus Symbiodinium. The metabolite is structurally related to docosahexaenoic acid (DHA) and seems to be biosynthesized by oxidation and subsequent lactonization. The absolute stereochemistry was determined from the specific rotation of the perhydro derivative. The distribution of ZL within several Symbiodinium isolates was quantitatively analyzed by HPLC techniques and suggested a relationship between the productivity of this metabolite and the Symbiodinium phylogeny. The cytotoxicity of ZL was evaluated by using human squamous cell carcinoma cell lines in comparison with that of DHA and other common fatty acids, suggesting that the long unsaturated chain was important rather than the gamma-lactone moiety.