Wilms' tumor protein and FLT3-internal tandem duplication expression in patients with de novo acute myeloid leukemia.

Bone marrow samples of 30 patients with de novo adult acute myeloid leukemia (AML) were analyzed for Wt1 and FLT3-internal tandem duplication (FLT3-ITD) expression measured by western blot and reverse transcription-polymerase chain reaction analysis, respectively. Wt1 was detected in 53·3% of AML patients (16/30), while FLT3-ITD in 23·3% (7/30). The high Wt1 expression correlated with the presence of FLT3-ITD (P = 0·014) and lower rate of complete remission (P = 0·023). The cumulative survival in AML patients was affected significantly by the presence of FLT3-ITD, being lower in the FLT3-ITD (+) group (6·0±2·4 months) compared to the FLT3-ITD (-) patients (17·9±3·3; P = 0·04). The expression of FLT3-ITD could probably activate Wt1 expression in AML blast cells and thus might contribute to its oncogenic function to provide cells with survival advantages in vivo. The detection of both molecular markers (Wt1 and/or FLT3-ITD) may be helpful in defining high risk AML patients that need special therapeutic strategies.

Erufosine: a membrane targeting antineoplastic agent with signal transduction modulating effects.

The ether lipid analog erufosine (erucylphospho-N,N,N,-trimethylpropylammonium, ErPC3) has high activity against leukemic cells without affecting the normal hematopoiesis. It belongs to the group of alkylphosphocholines (APC) that are inhibitors of protein kinase C and phospholipase C. However, the mechanism of action of erufosine remains rather unclear. We focused on combination effects with the tyrosine kinase inhibitor imatinib mesylate (gleevec, former STI-571 or CGP-57148) against two chronic myeloid leukemia (CML)-derived cell lines (K-562 and BV-173). The influence of erufosine on proteins involved in the phosphatidylinositol-3-phosphate pathway and on expression of the retinoblastoma protein Rb was studied, the latter being a key component for cell cycle entry and progression in mammalian cells. The consecutive treatment of K-562 and BV-173 cells with erufosine (2.5, 5, 15, 30 microM) and imatinib mesylate (0.05, 0.1 microM) led to synergism as measured by the MTT-dye reduction assay and this is reason to hypothesize that such combinations could be beneficial for relapsed patients with drug-resistant disease. Whole cell lysates from K-562 and BV-173 were investigated for the expression of Rb, PKB/Akt, pAkt, and p27 by Western blot. Erufosine caused decreases of pAkt and CML fusion protein p210 (BCR-ABL) protein expression, but induced the Rb protein expression in K-562 cells. A parallel increase in p27 level was observed after 24 and 48 h treatment. These alterations in signal transduction could be an explanation for the drug interaction found. Furthermore, Rb is a substrate of caspases and is cleaved during apoptosis as already evidenced for BV-173 cells. Our experimental findings suggest that erufosine acts through induction of changes in protein signaling and especially through Rb induction. This unique mode of action makes it an attractive partner for combination therapies, for example, in combination with imatinib mesylate for treatment of CML.

Cytotoxic efficacy of bendamustine in human leukemia and breast cancer cell lines.

PURPOSE: The purpose of this study was to characterise bendamustine's cytotoxic and apoptotic activity in a panel of leukemia and breast cancer cell lines in comparison to its clastogenicity in murine bone marrow. METHODS: The cytotoxic effect of bendamustine was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-dye reduction assay. Induction of apoptosis was evidenced by DNA gel electrophoresis, nuclear staining, Western blot poly-(adenosine diphosphate-ribose) polymerase (PARP) cleavage, and flow cytometry. As a measure of hematological toxicity, the formation of chromosomal aberrations was investigated in bone marrow cells isolated from mice treated with low non-toxic doses of bendamustine and lomustine. RESULTS: Bendamustine was preferably active against leukemic cells of lymphoid origin and was found to induce apoptosis in SKW-3 and BV-173 cells as shown by oligonucleosomal DNA and nuclear fragmentation, PARP cleavage, and formation of a sub-G1 fraction. Myeloid and breast carcinoma cell lines were resistant towards bendamustine with the exception of HL-60 cells which exhibit an intermediate sensitivity. Bendamustine was found to have a very low clastogenic effect as compared with equimolar doses of lomustine. CONCLUSION: Taken together, the mode of action of bendamustine includes induction of apoptosis. The specific spectrum of activity and the unexpectedly low clastogenicity support the hypothesis that bendamustine in not a typical alkylating agent but exerts an additional mode of action, possibly as a purine antimetabolite.

BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines.

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr-abl rearrangement (K-562, LAMA-84, CML-T1 and BV-173) and two other leukaemic cell lines (HL-60 and SKW-3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR-ABL-positive cell lines than the two reference APC. It induced programmed cell death in HL-60 and SKW-3 cells after exposure for 24 h, and in bcr-abl expressing cells after a prolonged incubation period (48 h). LAMA-84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast-like phenotype. Experiments with a cell-free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT-PCR we confirmed that K-562 and LAMA-84 cells carry the b3a2 fusion type, and CML-T1 and BV-173 the b2a2 variant. BV-173 cells had the lowest level of bcr-abl mRNA which correlated with their increased sensitivity. Transfection of K-562 cells with antisense oligonucleotides directed against bcr-abl caused a specific suppression of K-562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR-ABL-positive blasts as compared with miltefosine and perifosine. The expression of BCR-ABL cannot prevent apoptosis but delays erucylphosphocholine-induced programmed cell death. Transfection with bcr-abl directed antisense oligonucleotides reduces the clonogenicity of K-562 cells.

Human urinary bladder carcinoma cell lines respond to treatment with alkylphosphocholines.

Alkylphosphocholines (APC) constitute a new group of antineoplastic agents without haematological toxicity. Their first clinically available derivative hexadecylphosphocholine (miltefosine) is locally used to control skin metastases of breast cancer. Since intravesical chemotherapy represents a form of topical treatment we investigated whether a new APC with a long alkyl chain would be active against 5637 and EJ bladder cancer cell lines. Their antineoplastic activity was inversely related to the alkyl chain length of the respective APC. Erucylphosphocholine and its congener with modified phosphocholine head erucylphospho-N,N,N-trimethylpropanolamine were the most effective derivatives. APC with alkyl chains over 16 carbons in length induced programmed cell death in both cell lines, as determined by oligonucleosomal DNA fragmentation and morphology. The distinct antineoplastic effects lead us to predict that urinary bladder instillation of APC will be of therapeutic benefit for patients with urinary bladder neoplasia.

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells.

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.

Alkylphosphocholines induce apoptosis in HL-60 and U-937 leukemic cells.

Alkylphosphocholines (APC) represent a new group of ether-lipid-related compounds with remarkable activity against transformed cells in vitro and good tolerability in vivo. Their mechanism of action remains unknown. The aim of the present study was to investigate the effects of a series of APC on three human leukemic cell lines: K-562, HL-60, and U-937. The tetrazolium dye-reduction (MTT) assay and cell counting were used to determine the cytotoxicity of the APC used. DNA gel electrophoresis and enzyme-linked immunosorbent assay (ELISA) detection of oligonucleosomes were performed to identify and quantify DNA fragmentation. Electron and phase-contrast microscopy were used to detect morphologic changes specific for programmed cell death. HL-60 and U-937 cells were found to be sensitive, but K-562 cells were relatively resistant to APC exposure. APC with long alkyl chains exerted stronger cytotoxicity than did those with short alkyl chains. DNA fragmentation was found after treatment with APC in HL-60 and U-937 cells but not in K-562 cells. In HL-60 cells the increase in mono- and oligonucleosome formation as measured by ELISA was correlated with the length of the alkyl chains at 14 h of exposure to APC but plateaued at 20 h. The morphologic alterations in HL-60 and U-937 cell lines, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies, confirmed the induction of apoptosis after APC exposure. It is concluded that programmed cell death plays an important role in the cytotoxicity of APC against certain human leukemic cell lines. The antineoplastic profiles of APC with long alkyl chains render them attractive for further therapeutic application.

Efficacy of anticancer alkylphosphocholines in Trypanosoma brucei subspecies.

Tetradecylphosphocholine (TPC), hexadecylphosphocholine (HPC), hexadecylphospho(N-N-N-trimethyl)hexanolamine (HPC6), octadecylphosphocholine (OPC), and octadecyl-[2-(N-methylpiperidinio)ethyl]-phosphate (OMPEP) were investigated for antitrypanosomal activity in vitro and in vivo. OMPEP showed the best trypanocidal efficacy in vitro; it was superior to the model compound HPC and comparable to the reference compound alpha-DFMO. HPC showed moderate activity in vivo in terms of increased life expectancy (up to 35% in the acute NMRI-mouse model or 49% if combined with phenylbutazone) and increased packed cell volume, if administered daily. However, HPC and the other alkylphosphocholines failed to prolong survival time of treated mice if given intermittently. Phenylbutazone had no own trypanocidal effect but increased the efficacy of alkylphosphocholines in vitro and in vivo: the combination of HPC and phenylbutazone acted apparently synergistic.

Synthesis and tumor inhibiting activity of lanthanum (III) complexes with some 1-aminocycloalkancarboxylic acids.

Complexes of La(III) with 1-aminocyclopentane-, -hexane, -heptane and -4-ethylcyclohexanecarboxylic acids were obtained. The compounds were characterized by elemental analyses, IR spectroscopy and conductivity measurements. The following general formula was derived: LaL3Cl3 x 5 H2O, where L is the corresponding 1-aminocycloalkanecarboxylic acid. The pharmacological studies showed that all complexes manifested higher cytostatic and cytotoxic effects in comparison with lanthanum chloride. Much higher cytotoxic (anti-P388/D1) and cytostatic (anti-L-1210 and anti-melanoma-B16) activity was found for the lanthanum complex with 1-aminocyclopentanecarboxylic acid.