Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.     

Synthesis and anticancer and antiviral activities of various 2'- and 3'-methylidene-substituted nucleoside analogues and crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride.

Various 2'- and 3'-methylidene-substituted nucleoside analogues have been synthesized and evaluated as potential anticancer and/or antiviral agents. Among these compounds, 2'-deoxy-2'-methylidene-5-fluorocytidine (22) and 2'-deoxy-2'-methylidenecytidine (23) not only demonstrated potent anticancer activity in culture against murine L1210 and P388 leukemias, Sarcoma 180, and human CCRF-CEM lymphoblastic leukemia, producing ED50 values of 1.2 and 0.3 microM, 0.6 and 0.4 microM, 1.5 and 1.5 microM, and 0.05 and 0.03 microM, respectively, but also were active in mice against murine L1210 leukemia. Of all the tested drug dosage levels (25, 50, and 75 mg/kg, respectively) compound 23 had no toxic deaths and compound 22 yielded only one toxic death at the highest dosage level. On the contrary, in the same study, 1-beta-D-arabinofuranosylcytosine (ara-C) resulted in 2/5, 5/5, and 5/5 toxic deaths, respectively. Both compounds 22 and 23 have shown better anticancer activity than ara-C, yielding higher T/C x 100 values and some long-term survivors (greater than 60 days). In addition, compounds 22 and 23 were found to have, respectively, approximately 130 and 40 times lower binding affinity for cytidine/deoxycytidine deaminase derived from human KB cells compared to ara-C, suggesting that the two 2'-methylidene-substituted analogues may be more resistant to deamination. Cytoplasmic deoxycytidine kinase (dCK) was required for compounds 22 and 23 action. Furthermore, compounds 14, 22, 23, and 24 also have antiherpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) activity in cell culture. In addition, the crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride (23-HCl) was determined by X-ray crystallography.     

Determinants of resistance to 2',2'-difluorodeoxycytidine (gemcitabine).

The inherent or induced resistance of tumors to cytostatic agents is a major clinical problem. In this review, we summarize the pre-clinical mechanisms of acquired and inherent resistance to the fluorinated deoxycytidine analog gemcitabine (2',2'-difluorodeoxycytidine, dFdC, Gemzar((R))), which has proven activity in non-small cell lung carcinoma, pancreatic and bladder cancer. Extensive research has been performed to elucidate the complex mechanism of action of this relatively new drug. Gemcitabine requires phosphorylation to mono-, di- and triphosphates to be active. Similar to the structurally and functionally related deoxycytidine analog ara-C, the first, crucial step in phosphorylation is catalyzed by deoxycytidine kinase (dCK). However, in contrast to ara-C, gemcitabine has multiple intracellular targets; up- or down-regulation of these targets may confer resistance to this drug. Resistance is associated with altered activities of enzymes involved in the metabolism of the drug, of target enzymes, and of enzymes involved in programmed cell death. However, the only strong correlations with gemcitabine sensitivity are dCK activity and dFdCTP pools, with a potential important role for ribonucleotide reductase.     

2'-O-nitro-1-beta-D-arabinofuranosylcytosine. A new derivative of 1-beta-D-arabinofuranosylcytosine that resists enzymatic deamination and has antileukemic activity

To overcome the susceptibility of the anticancer drug 1-beta-D-arabinofuranosylcytosine (ara-C) to enzymatic deamination, and hence deactivation, we prepared the 2'-O-nitro-1-beta-D-arabinofuranosylcytosine (termed nitrara-C) and evaluated it for biological activity. Nitrara-C was resistant to enzymatic deamination and inhibited the proliferation of several strains of human leukemic T and B lymphoblasts grown in culture. Moreover, it substantially extended the life spans of mice with L1210 leukemia. Studies with ara-C-resistant human leukemic lymphoblasts deficient in deoxycytidine kinase activity disclosed that the inhibitory activity of the new compound depends on its phosphorylation.     

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.     

Nucleoside conjugates. 10. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitins

Three 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitins from L-, D-, and DL-alpha-dipalmitoylphosphatidic acids have been synthesized and their antitumor activity against two ara-C2 resistant L1210 lymphoid leukemia sublines in mice were evaluated. These new prodrugs of ara-C include ara-CDP-L-dipalmitin, ara-CDP-D-dipalmitin, and ara-CDP-DL-dipalmitin. The L and DL isomers produced significant increase in life span (greater than 400%) and four to five long-term survivors (greater than 45 days) out of six animals bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C (I)], while the D isomer displayed a marginal activity (ILS 100-121%). In contrast, the L isomer was completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C (II)]. However, the results demonstrate that the L and DL isomers of ara-CDP-dipalmitin are promising new prodrugs of ara-C with improved efficacy.     

A new mechanism of methotrexate action revealed by target screening with affinity beads

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-beta-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.     

Synthesis and biological activity of a gemcitabine phosphoramidate prodrug

A gemcitabine (2',2'-difluorodeoxycytidine, dFdC) phosphoramidate prodrug designed for the intracellular delivery of gemcitabine 5'-monophosphate was synthesized. The prodrug was about an order of magnitude less active than gemcitabine against wild-type cells, and the nucleoside transport inhibitor dipyridamole reduced prodrug activity. The prodrug was more active than gemcitabine against two deoxycytidine kinase-deficient cell lines. The results suggest that the prodrug is a potent growth inhibitor that can bypass dCK deficiency at higher drug concentrations.     

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.     

Nucleoside conjugates. 11. Synthesis and antitumor activity of 1-beta-D-arabinofuranosylcytosine and cytidine conjugates of thioether lipids

Five 1-beta-D-arabinofuranosylcytosine conjugates and two cytidine conjugates of thioether lipids (1-S-alkylthioglycerols) linked by a pyrophosphate diester bond have been prepared and their antitumor activity against an ara-C2 sensitive (L1210/0) and two ara-C resistant L1210 lymphoid leukemia sublines in mice were evaluated. These prodrugs of ara-C include ara-CDP-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (8a), ara-CDP-rac-1-S-octadecyl-2-O-palmitoylthioglycerol (8b), and ara-CDP-rac-1-S-octadecyl-2-O-methyl(or -ethyl, -hexadecyl)thioglycerols (8c-e). The cytidine conjugates include CDP-rac-1-S-octadecyl-2-O-palmitoyl(or -methyl)- 1-thioglycerols (9a and 9b). Sonicated solutions of the conjugates existed in the form of micellar disks (size 0.01-0.04 microns). Single doses (200-400 mg/kg) of 8a and 8b produced significant increase in life span (257-371%) in mice bearing ip implanted L1210/0 leukemia. In contrast, conjugates 8c-e were less effective (ILS 19-75%) and cytidine conjugates (9a and 9b) were ineffective. Even though 8a and 8b were found to be curative in a high percentage of mice bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C(I)], they were completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C(II)]. However, the present results, together with the previous, demonstrate that 8a and 8b are promising new prodrugs of ara-C with improved efficacy.     

Induction of deoxycytidine kinase by 5-azacytidine in an HL-60 cell line resistant to arabinosylcytosine.

Induction of 2'-deoxycytidine kinase (dCK) by 5-azacytidine (5-Aza-C) in a dCK-deficient HL-60 cell line resistant to 1-beta-D-arabinofuranosylcytosine (Ara-C) (HL-60/Ara-C) was examined by measurement of the incorporation of [3H]deoxycytidine ([3H] dCyd) into cellular DNA, the kinetic properties of purified dCK, the cytotoxic potency (IC50 values), and the DNA methylation patterns of 5-Aza-C-treated and untreated cells. Following a 72-hr exposure to 1 microM 5-Aza-C, the incorporation of [3H]dCyd into DNA was increased 6-fold and the total dCK activity was increased 12-fold, with a peak for both on day 6. The onset of dCK induction was dependent on the length of exposure time. The IC50 values for cell growth inhibition by Ara-C on day 3 were 0.08 microM for HL-60 cells, 12.5 microM for HL-60/Ara-C cells, and 0.55 microM for HL-60/Ara-C cells pretreated with 5-Aza-C for 40 hr. The Km and Vmax of dCyd for HL-60 dCK were similar to those for 5-Aza-C-induced HL-60/Ara-C dCK. The restriction enzymes Hpall, which cleaves CCGG sequences but cannot cleave at sites methylated at the internal cytosines (5'-CMeCGG), and Mspl, which cleaves sequences with internal methylated cytosine but cannot cleave at sites methylated at external cytosines (5'-MeCCGG), were used for DNA methylation pattern determination. The newly synthesized DNA of HL-60 wild-type cells was cleaved by Mspl to a greater extent than that of HL-60/Ara-C cells. After exposure to 1 microM 5-Aza-C for 40 hr, methylation patterns of newly synthesized DNA reverted in HL-60/Ara-C cells to a clevage pattern similar to that in HL-60 wild-type cells. When compared with untreated control, DNAs from 5-Aza-C-treated resistant cells were cleaved to a greater extent by Mspl than by Hpall, suggesting that internal cytosine-residue methylation was relatively uninhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells

Continuous cultivation of T-lymphoid H9 cells in the presence of 3′-azido-2′,3′-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2′,3′-didehydro-3′-deoxythymidine as well as different deoxycytidine analogs such as 2′,3′-dideoxycytidine, 2′,2′-difluoro-2′-deoxycytidine (dFdC) and 1-ß-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5×10⁴-, 5×10³-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2′,3′-dideoxy-3′-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 μM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10⁶ cells after 4 hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.     

Potentiation of ara-C induced cytotoxicity by hydroxyurea in LoVo colon carcinoma cells

The present study was undertaken to determine whether cytotoxicity by 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells, which are resistant to high concentrations of ara-C, would be enhanced by concurrent exposure to hydroxyurea (HU). Since mechanisms underlying the effects of HU on ara-C induced cytotoxicity are unclear, we also evaluated the effect of HU on the incorporation of ara-C into DNA, as well as potential consequences of misincorporation. Our results demonstrate that HU synergistically enhances cytotoxicity by ara-C in these cells. This effect was not present when HU was combined with aphidicolin, an agent that resembles ara-C in competing with dCTP for binding to polymerase alpha but that is not incorporated into DNA. Further, cells exposed to HU and ara-C incorporated up to 5-fold as much ara-C into DNA as cells solely treated with ara-C. While the extent of inhibition of DNA synthesis was comparable with cells exposed to HU and aphidicolin as those treated with HU and ara-C, recovery of DNA synthesis was delayed more significantly by the latter combination. These findings suggest that HU synergistically potentiates ara-C induced cytotoxicity by enhancing incorporation of ara-C in LoVo cell DNA.     

Synthesis and biologic studies of arabinosylcytosine 5'-methylphosphonate

Arabinosylcytosine (ara-C), a clinically useful antitumor agent, is ineffective against cells that have deleted deoxycytidine kinase, the enzyme necessary for conversion of ara-C to its active nucleotide form. To circumvent this resistance, arabinosylcytosine-5'-methylphosphonate (ara-CMeP) was synthesized as an analogue of ara-CMP that would be membrane-permeable, resistant to serum phosphatase attack, and resistant to nucleoside deaminase inactivation. Ara-CMP was inhibitory to leukemia P388 in vitro but required concentrations 90-fold greater than that of ara-C for comparable cell inhibition. Both ara-CMeP and ara-CMP were competitive inhibitors of dCMP kinase from leukemia L1210 with K_i values of 4.0 × 10^?3 and 4.4 × 10^?3 M respectively. However, ara-CMP is a substrate for dCMP kinase, whereas ara-CMeP was not. Thus, the inability of ara-CMeP to be phosphorylated precludes its usefulness as a functional analogue of ara-CMP.     

Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5'-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations.     

The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis. Simultaneous and sequential exposure (preexposure to 3-AP for 24h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC(25) concentration for 24h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR. Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR. In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.