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

UNLABELLED: 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.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines.

Resistance to, the hydrophilic drug ara-C, might be meditated by decreased membrane transport. Lipophilic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara-C, while the compounds with the shortest fatty-acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara-C.

An Eimeria vaccine candidate appears to be lactate dehydrogenase; characterization and comparative analysis.

An Eimeria acervulina protein fraction was identified which conferred partial protection against an E. acervulina challenge infection. From this fraction a 37 kDa protein was purified and its corresponding cDNA was cloned and shown to encode a lactate dehydrogenase (LDH). Full length cDNAs encoding LDH from two related species, E. tenella and E. maxima, were also cloned. The homology between the primary amino acid sequences of these three Eimeria LDH enzymes was rather low (66-80%), demonstrating an evolutionary divergence. The Plasmodium LDH crystal structure was used to generate a 3D-model structure of E. tenella LDH, which demonstrated that the many variations in the primary amino acid sequences (P. falciparum LDH and E. tenella LDH show only 47% identity) had not resulted in altered 3D-structures. Only a single LDH gene was identified in Eimeria, which was active as a homotetramer. The protein was present at similar levels throughout different parasitic stages (oocysts, sporozoites, schizonts and merozoites), but its corresponding RNA was only observed in the schizont stage, suggesting that its synthesis is restricted to the intracellular stage.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines.

1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase. Altered membrane transport is also a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell, lipophilic prodrugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double bonds were esterified at the 5' position on the sugar moiety of ara-C. The compounds were tested in two pairs of ara-C resistant leukemic cell lines (murine L1210 and rat BCLO and their resistant variants L4A6 and Bara-C, respectively) and two pairs of cell lines with a resistance to gemcitabine, another deoxycytidine analog (human ovarian cancer A2780 and murine colon cancer C26-A and their resistant variants AG6000 and C26-G, respectively). L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the parent cell lines, ara-C was more active, but in the resistant variants several of the analogs were more active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were inactive. Structure-activity relation analysis showed that ara-C derivatives with shorter acyl chains and more double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and longer lasting than for the parent drug.

Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed.

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.

Differences in the induction of DNA damage, cell cycle arrest, and cell death by 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is an important target for chemotherapy and can be inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can be incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will influence activation of downstream mechanisms mediated via TS inhibition. Therefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG337, and MTA to determine the induction of DNA damage, cell cycle arrest, downstream protein expression, and cell death. At these concentrations, the specific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo and WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). Western blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treated Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nevertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells differences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS inhibition.

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.

Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines.

2',2'-Difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)-DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the CIs after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all CIs were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt-DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt-DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt-DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt-DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt-DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA.

Combination studies of antifolates with 5-fluorouracil in colon cancer cell lines.

The combined cytotoxic effects of the thymidylate synthase (TS) inhibitors 5-fluorouracil (5FU) and different antifolates were studied in seven colon cancer cell lines. Growth inhibition of the antifolates, Nolatrexed, Raltitrexed, GW1843U89, or MTA in combination with 5FU, was determined and multiple drug effect analysis showed that the drugs acted mostly additively. The only synergistic interaction was found for 5FU and Nolatrexed in the LS174T cell line. Also Raltitrexed and 5FU were slightly synergistic in WiDr/F cells grown at low folate levels, but for the other cell lines grown at high folate levels this combination was more antagonistic. GW1843U89 and 5FU were mainly additive, while 5FU and MTA showed antagonism in WiDr and additivity in LS174T. The effect of the drugs at their target was evaluated by in situ TS inhibition. We observed lower TS activity in all cells when two drugs were used instead of one. Statistical analysis revealed that none of the values of the combinations was higher or lower than could be expected from the product of the effect of single drugs. We concluded that the effects on TS inhibition were additive for all 5FU/antifolate combinations in all cell lines. DNA strand break formation, as a result of TS inhibition, was measured by means of a fluorometric analysis of DNA unwinding. Raltitrexed-induced DNA damage was significantly increased by 5FU in WiDr cells [single agent: 67% double stranded (ds) DNA, combination: 39% ds DNA, P<0.0001]. In LS174T a trend for antagonistic effects was observed for combinations of MTA, GW1843U89, or Raltitrexed and 5FU. The combinations showed additive effects in WiDr/F cells. The overall conclusion of the three assays in each of the cell lines indicated that 5FU and antifolate combinations were predominantly additive in colon cancer cells.

Characterization of human soft-tissue sarcoma xenografts for use in secondary drug screening.

We have established ten transplantable human soft-tissue sarcoma (STS) xenografts grown as subcutaneous tumours in the nude mouse. Nine xenografts originated from patients that needed chemotherapy in the course of their disease. The xenografts were tested for their sensitivity to maximum tolerated doses of five anti-cancer agents. Growth of treated tumours was expressed as a percentage of control tumour growth and a growth inhibition > 75% was measured for doxorubicin in 20% of the STS xenografts, for cyclophosphamide in 30%, for ifosfamide in 20%, for vincristine in 20%, whereas etoposide was not effective in the STS xenografts. In three out of ten STS xenografts MDR1 mRNA was detectable, but this was not related to the resistance against doxorubicin, vincristine or etoposide. Topoisomerase IIalpha mRNA expression levels did not reflect sensitivity to doxorubicin or etoposide. In all STS tissues, however, these levels were lower than topoisomerase IIalpha mRNA in a drug-sensitive human ovarian cancer xenograft. Glutathione concentrations and the activities of glutathione S-transferase, glutathione peroxidase and glutathione reductase were not related to resistance against the alkylating agents or doxorubicin. Of interest, in all STS tissues, glutathione S-transferase pi was the predominant isoenzyme present. In conclusion, chemosensitivity of the STS xenografts reflects clinical response rates in phase II trials on the same compounds in adult STS patients. Relatively low levels of topoisomerase IIalpha mRNA may partly account for intrinsic resistance against, for example, doxorubicin. Additional factors must contribute to moderate responsiveness to alkylating agents.

Toxicity profiles in vivo in mice and antitumour activity in tumour-bearing mice of di- and triorganotin compounds.

The in vivo toxicity profiles in mice and the antitumour activity in tumour bearing mice were screened for four di-n-butyltin and five triorganotin carboxylates, di-n-butyltin diterebate (5), bis(phenylacetate) (6), bis(deoxycholate) (7), bis(lithocholate) (8), tri-n-butyltin terebate (9), cinnamate (10), and triphenyltin terebate (11).At their maximum tolerated dosis (MTD), no antitumour effect (T/C ~1) was observed for the compounds 5, 7, 9, 10 and 11. The compounds 6 (T/C = 0.51) and 8 (T/C = 0.42) showed clear antitumour activity after single dose administration and might therefore be of interest for further antitumour activity studies.

A patient with adrenocortical carcinoma: characterization of its biological activity and drug resistance profile.

We describe a patient with a metastasized adrenocortical cancer who exhibited excessive production of both glucocorticoids and mineralocorticoids combined with suppressed androgen production. Unusual steroid metabolites found in the patient's urine have not been described previously in association with this tumor type. Investigation of the multidrug resistance phenotype in single-cell suspensions of the tumor revealed low expression of multidrug resistance protein but high expression of P-glycoprotein (Pgp) and lung resistance-related protein. Functional Pgp in these tumor cells was shown by the modulatory effect of PSC833 on daunorubicin accumulation. Mitotane, at a concentration achieved in this patient's plasma, completely reversed the Pgp-related resistance both in the Pgp-overexpressing KB8-5 cell line and in the patient's tumor cells. On the basis of these in vitro results, the patient was treated with a combination of multidrug resistance drugs (doxorubicin, vincristine, and etoposide) plus mitotane as a Pgp modulator. This treatment was ineffective, however. A chemosensitivity assay demonstrated that the tumor cells were highly resistant to the drugs used. The adrenocortical cancer cells expressed mutant p53, and no evidence for induction of apoptosis by these drugs was found.

Preclinical combination therapy with gemcitabine and mechanisms of resistance.

Cisplatin and gemcitabine both have activity in solid tumors, such as non-small cell lung, ovarian, and head and neck cancers. These drugs have the desired features needed to obtain synergistic activity, different side effect profiles, and mechanisms of action. Cisplatin acts by forming DNA-DNA cross-links (both intrastrand and interstrand) and DNA-protein cross-links; resistance to cisplatin is thought to be due to excision repair of the affected DNA. Gemcitabine acts by its incorporation into nucleic acids, leading to masked chain termination. By combining gemcitabine with cisplatin, it might be possible to achieve a better therapeutic effect than either drug alone and to bypass resistance to one or both drugs. Acquired resistance to gemcitabine was associated with a deoxycytidine kinase deficiency in vitro, but this was difficult to achieve in vivo. Proper scheduling may overcome intrinsic and transient resistance due to physiologic circumstances or aberrant biochemical properties. Preclinical in vitro and in vivo combination studies with cisplatin showed schedule- and model-dependent synergistic and additive effects between cisplatin and gemcitabine. Incorporation of gemcitabine into DNA might facilitate cisplatin-DNA adduct formation. Combining gemcitabine and cisplatin inhibited the DNA excision-repair process more than gemcitabine alone. This implies that if gemcitabine nucleotide is incorporated into the DNA strand, the action of the proofreading exonucleases is less efficient. In addition, both deoxyribonucleotide and ribonucleotide pools, essential for good functioning of DNA repair, are seriously depleted by gemcitabine. It is concluded that combining gemcitabine with cisplatin can be at least additive providing the right schedule is chosen, giving the best balance between acceptable toxicity and an enhanced antitumor activity.

Relationship between the tumour tissue pharmacokinetics and the antiproliferative effects of anthracyclines and their metabolites.

The intrinsic activity of anthracyclines and their metabolites was measured in order to determine whether the tumour exposure to the compounds reflects the difference in their ability to inhibit tumour growth. (Dox), 4'-epidoxorubicin (Epi-Dox), daunorubicin (Dauno), N-1-leucyl-doxorubicin (Leu-Dox) and their metabolites were analysed for their antiproliferative effects in three human malignant cell lines: MCF7, RPMI 8226 and A2780. The antitumour efficacy of equitoxic, maximum tolerated doses of the present drugs was assessed in nude mice bearing subcutaneous (s.c.) well-established A2780 doses of the parent drugs was assessed in nude mice bearing subcutaneous (s.c.) well-established A2780 human ovarian cancer xenografts. The same doses were given to tumour-bearing mice to determine the distribution of the anthracyclines and their metabolites in A2780 tumour tissue during the first 48 h after injection. In vitro antiproliferative effects of the anthracyclines and their metabolites revealed a comparable activity for the parent drugs and daunorubicinol, whereas the other metabolites were at least 10-fold less active. The growth inhibition obtained in A2780 xenografts was 87% for Dox, 82% for Epi-Dox, 74% for Dauno and 97% for Leu-Dox. In vivo, the exposure of tumour tissue to the drug, calculated as the area under the concentration-time curve (AUC), was related to the extent of growth inhibition after correction of the AUC values for the intrinsic activity of the anthracycline. For each of the anthracyclines, the sum of the corrected AUC values (nmol/g/min) of the active compounds was calculated as 8812 for Dauno; 9320 for Epi-Dox; 10 986 for Dox and 15 163 for Leu-Dox. The sequence of increasing AUC values corresponded with the sequence of increasing growth inhibition by the four anthracyclines observed in A2780 xenografts.

Synergistic interaction between cisplatin and gemcitabine in vitro.

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.

Tumor size and origin determine the antitumor activity of cisplatin or 5-fluorouracil and its modulation by leucovorin in murine colon carcinomas.

5-Fluorouracil (FUra) is one of the few effective agents in the treatment of patients with colorectal cancer. Its effects on the target enzyme thymidylate synthase (TS) can be modulated by leucovorin (LV) or cisplatin (CDDP). Tumor size and differentiation of tumor characteristics can influence therapeutic efficacy. We therefore studied the relationship between tumor size (cutoff point 200 mm3) and the antitumor activity of FUra and its modulation by LV in murine Colon 26 and Colon 38 tumors. The doubling time of tumors measuring > 200 mm3 was about 160% longer. The antitumor effect of FUra in these large tumors was decreased and could not be modulated by LV. In addition, three subtypes of Colon 26 (Colon 26-A, Colon 26-B, and Colon 26-10) were identified and characterized for tumor-induced weight loss, TS activity, response to chemotherapy, and histological features. Mice bearing Colon 38 and Colon 26-10 did not lose weight as a result of tumor growth. Colon 26-A caused a weight loss of up to 19%, whereas mice with Colon 26-B tumors remained within 10% of their initial weight and tolerated at least 2.5 times more tumor load than did mice bearing Colon 26-A, which induces cachexia. Among untreated tumors, TS catalytic activity was highest in Colon 26-B (5536 pmol mg protein(-1) h(-1)) and lowest in Colon 38 (799 pmol mg protein(-1) h(-1)); Colon 26-A and Colon 26-10 had intermediate activities (about 2500 pmol mg protein(-1) h(-1)). 5-Fluoro-2'-deoxyuridine monophosphate (FdUMP) binding was comparable in the three Colon 26 subtypes but was lower in Colon 38. The antitumor activity of FUra could be modulated by LV in Colon 38, Colon 26-10, and Colon 26-A but could not in Colon 26-B, with complete responses (CR) being obtained in Colon 26-10 and Colon 38. The latter two were highly sensitive to CDDP, followed by Colon 26-A and Colon 26-B (CRs: 50%, 40%, 25%, and 0, respectively). Furthermore, necrosis was noted in Colon 26-B and Colon 38 but not in Colon 26-A. In conclusion, (1) the antitumor activity of FUra in large tumors is decreased and cannot be modulated by LV and (2) characteristics and sensitivity to chemotherapeutics can vary substantially in closely related tumors of the same origin.