Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360).

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 µM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 µM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 µM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 µM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

Metabolism and accumulation of the lipophilic deoxynucleoside analogs elacytarabine and CP-4126.

Cytarabine (ara-C) and gemcitabine (dFdC) are commonly used anticancer drugs, which depend on the equilibrative (ENT) and concentrative-nucleoside-transporters to enter the cell. To bypass transport-related drug resistance, lipophilic derivatives elacytarabine (CP-4055), ara-C-5'elaidic-acid-ester, and CP-4126, (CO 1.01) gemcitabine-5'elaidic-acid-ester, were investigated for the entry into the cell, distribution, metabolism and retention. The leukemic CEM-cell-line and its deoxycytidine-kinase deficient variant (CEM/dCK-) were exposed for 30 and 60 min to the radiolabeled drugs; followed by culture in drug-free medium in order to determine drug retention in the cell. The cellular fractions were analyzed with thin-layer-chromatography and HPLC. Elacytarabine and CP-4126 were converted to the parent compounds both inside and outside the cell (35-45%). The ENT-inhibitor dipyridamole did not affect their uptake or retention. Inside the cell Elacytarabine and CP-4126 predominantly localized in the membrane and cytosolic fraction, leading to a long retention after removal of the medium. In contrast, in cells exposed to the parent drugs ara-C and dFdC, intracellular drug concentration increased during exposure but decreased to undetectable levels after drug removal. In the dCK- cell line, no metabolism was observed. The concentrations of ara-CTP and dFdCTP reached a peak at the end of the incubation with the drugs, and decreased after drug removal; peak levels of dFdCTP were 35 times higher than ara-CTP and was retained better. In contrast, after exposure to elacytarabine or CP-4126, ara-CTP and dFdCTP levels continued to increase not only during exposure but also during 120 min after removal of the elacytarabine and CP-4126. Levels of ara-CTP and dFdCTP were higher than after exposure to the parent drugs. In conclusion, the lipophilic derivatives elacytarabine and CP-4126 showed a nucleoside-transporter independent uptake, with long retention of the active nucleotides. These lipophilic nucleoside analogues are new chemical entities suitable for novel clinical applications.

Cellular pharmacology of multi- and duplex drugs consisting of ethynylcytidine and 5-fluoro-2'-deoxyuridine.

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.

Selection of the best blood compartment to measure cytidine deaminase activity to stratify for optimal gemcitabine or cytarabine treatment.

Cytidine deaminase (CDA) plays a crucial role in the degradation of cytidine analogs, such as gemcitabine and cytarabine. Several studies showed that a low CDA activity is associated with more toxicity but a higher efficacy, while a high activity will lead to a lower efficacy but less toxicity. A stratified dosing strategy based on the relative CDA activity would increase efficiency. In order to predict these events, a reliable measurement of CDA with a validated method is crucial. We aimed to determine which phenotype assay would be most suitable; a spectrophotometric assay using cytidine as a substrate, or an HPLC assay using gemcitabine as a substrate. In serum and whole blood of 26 volunteers, both assays showed an excellent correlation (R>0.999), but not in plasma nor in red blood cells. Moreover, there was no difference between males and females. In conclusion, the spectrophotometric assay seems the most simple and cost-effective test. It should be performed in serum, while it should be normalized on protein content as measured by the Bicinchoninic Acid.

Decay of γ-H2AX foci correlates with potentially lethal damage repair in prostate cancer cells.

To determine the relationship between ionizing radiation-induced levels of γ-H2AX foci and cell survival in cultured prostate cancer cell lines, three prostate cancer cell lines: LNCaP (wt TP53), DU145 (mut TP53) and PC3 (TP53 null), were studied. For γ-H2AX foci induction, cells were irradiated with a single dose of 2 Gy and foci levels were studied at 30 min and 24 h after irradiation. Cell survival was determined by clonogenic assay, directly and 24 h after irradiation with doses ranging from 0 to 8 Gy. Irradiation was performed with a Siemens Stabilipan 250 KeV X-ray machine at a dose rate of approximately 3 Gy/min. Survival curves were analyzed using the linear-quadratic model S(D)/S(0)=exp-(αD+ßD2). LNCaP cells clearly demonstrated potentially lethal damage repair (PLDR) which was assessed as increased survival levels after delayed plating as compared to cells plated immediately after irradiation. DU145 cells demonstrated only a slight PLDR and PC3 cells did not show PLDR at all. Levels of γ-H2AX foci were significantly decreased in all cell lines at 24 h after irradiation, compared to levels after 30 min. The LNCaP cells which demonstrated a clear PLDR also showed the largest decay in the number of γ-H2AX foci. In addition, the PC cells which did not show PLDR had the lowest decay of γ-H2AX foci. A clear correlation was demonstrated between the degree of decay of γ-H2AX foci and PLDR.

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

Oxaliplatin activity in selected and unselected human ovarian and colorectal cancer cell lines.

Oxaliplatin is used for treatment of colon cancer in combination with 5-fluorouracil or irinotecan. Oxaliplatin has similar, but also different resistant mechanisms as cisplatin. We studied the activity of oxaliplatin in ovarian and colon cancer cells with different resistance patterns to cisplatin. The 40-fold cisplatin-resistant cell line ADDP was only 7.5-fold resistant to oxaliplatin. The gemcitabine-resistant AG6000 cell line, 9-fold resistant to cisplatin, was not cross-resistant. LoVo-175X2, with mutant p53 showed no resistance compared to the empty vector control. However, LoVo-Li, with inactive p53, was 3.6-fold resistant corresponding to decreased accumulation and Pt adducts. Accumulation and DNA adducts formation showed no significant correlation with oxaliplatin sensitivity. Cell cycle distribution after exposure to oxaliplatin showed arrest in G2/M (A2780) or in S-phase (LoVo-92) for wt-p53 cells. ADDP and LoVo-Li showed G1 arrest followed by S-phase arrest and no changes in distribution, respectively. The cell cycle related proteins Cyclins A and B1 and (p)CDC25C were marginally affected by oxaliplatin. Expression of hCTR1 was decreased in ADDP, LoVo-Li and AG6000, OCT1 decreased in ADDP and AG6000 and OCT3 in LoVo-175X2, compared to the parental cell lines. In ADDP and LoVo-175X2 ATP7A and B were decreased but were increased in AG6000. From this study it can be concluded that changes in cell cycle distribution were cell line dependent and not related to changes in expression of Cyclin A or B1. Oxaliplatin accumulation was related to hCTR1 and, at low concentration, ATP7A to DNA adducts formation while the retention was related to hCTR1, OCT2 and ATP7B.