1 U.O. Hematology, Azienda Ospedaliera Careggi, University of Florence, Florence, Italy; 2 PharmaMar SAU, Research and Development, Comenar Viejo, Madrid, Spain
* Correspondence to: Dr A. Grossi, Istituto Leonardo da Vinci, Hematology, Via Colletta 22/r, 50100 Firenze, Italy. Tel: +39-338-5200467; Fax: +39-055-2487230; E-mail: alberto_grossi{at}libero.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: To confirm these findings we investigated APL-related VEGF inhibition and its cytotoxic effect on myeloid leukemic cells lines (K-562, HEL and HL60) and fresh leukemia blasts derived from 30 patients with acute myeloid leukemia (AML). The conventional active 4-demetoxi-daunorubicin (idarubicin; IDA) was included as a positive control.
Results: APL was found to be significantly (P < 0.001) more active than IDA in obtaining 50% growth-inhibition in K-562, HEL and HL60 cell lines. Results obtained with AML blast cells were superimposible. ID50 ranged from 0.024 to 0.610 µM for IDA (0.200 ± 0.176) and from 0.001 to 0.108 µM for APL (0.020 ± 0.031). Annexin V tests and cell cycle analysis performed on cell lines confirmed the stronger citotoxic capability of APL as apoptotic inducer and as a G1 blocker. The inhibitory effects of APL on VEGF release and secretion have been confirmed by ELISA tests performed on HEL: the VEGF concentration in cell surnatant was reduced from 169 to 36 pg/ml after 24 h of exposure to a pharmacological concentration of APL.
Conclusions: APL harbors a strong in vitro antileukemic activity at a concentration achievable in patients at non-myelotoxic doses. Our data also support the notion of an impact on VEGF secretion. Clinical studies with this new marine-derived compound in relapsed/resistant leukemia are underway.
Key words: aplidine, marine compounds, myeloid leukemia, VEGF inhibiton
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In contrast to the lack of bone marrow toxicity, a number of translational studies have demonstrated strong cytotoxic activity at pharmacological concentration against acute lymphoblastic leukemia blasts explanted from pediatric patients, with lack of cross-resistance with conventional antileukemic agents.
On this basis, this study aimed to characterize the cytotoxic activity of APL on myeloid leukemic cell lines and fresh leukemic cells derived from patients with acute myeloid leukemia (AML). Additionally, its effects on the cell cycle and impact on VEGF production and secretion have also been investigated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cultures
K-562 (human chronic myelogenous leukemia cell line), HEL (human promyelocytic leukemia cell line) and HL60 (human erythroleukemia cell line) cells were grown in RPMI (Gibco-BRL, Paisley, UK), supplemented with 10% fetal calf serum (Gibco-BRL) and antibiotics at 37°C in an atmosphere that contained 5% CO2.
AML primary blast cells
Bone marrow or peripheral blood samples were obtained at diagnosis from 30 adult patients (16 females and 14 males) with de novo or secondary AML, after obtainment of a signed informed consent. The diagnosis was based on FrenchAmericanBritish (FAB) criteria.
Mononuclear cells (MNCs) were isolated from peripheral blood using Ficoll gradient sedimentation (Gibco-BRL), washed in phosphate-buffered saline (PBS) (Gibco-BRL) and treated as described below. All samples contained >80% of leukemic blasts.
WST-1 cytotoxicity test
Cells lines and MNCs were seeded at a concentration of 25 000 and 50 000 cells/100 µl, respectively, in 96-well plates and exposed to increasing concentrations of idarubicin (IDA) and APL (0.0011 µM). The incubation time was 72 h for cell lines and 48 h for MNCs. Each concentration of the drugs was tested in quadruplicate; four wells were also prepared for untreated cells and medium. Cell viability was determined with a colorimetric assay, using Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim, Germany). The test is based on the cleavage of the tetrazolium salt WST-1 in formazan by mitochondrial dehydrogenases in viable cells [23]. The formazan dye was quantified by a scanning multiwell spectrophotometer by measuring the absorbance of the dye at 450 nm. After the incubation period, Cell Proliferation Reagent WST-1 (10 µl/well) was added and the absorbance was measured after 3 h. ID50 values (concentration of drug required to cause 50% growth inhibition of treated cells compared with control cells) were determined by plotting the percentage of cell survival versus the logarithm of anticancer drug concentration.
Apoptosis (Annexin-V-FLUOS staining kit)
Apoptosis was evaluated in cell lines and MNCs after 48 and 24 h, respectively, of incubation with the drugs (10, 50 and 100 nM). The cells were then collected, washed in PBS and incubated for 15 min with Annexin-Vfluorescein [24, 25
] and propidium iodide (Roche Diagnostics) to distinguish apoptotic cells from necrotic cells. Samples were analysed by flow cytometry using 488 nm excitation.
Cell cycle analysis
Cell lines were cultured in serum-free RPMI medium for 24 h. Serum and drugs (0.011 µM) were then added for 24 h. After incubation, the cells were fixed in 95% cold ethanol and kept at 4°C overnight. DNA was then stained with propidium iodide (50 ng/ml) (Roche Diagnostics) in the presence of Nonidet P40 0.01% and Rnase (Sigma). Samples were incubated for 1 h and DNA histograms analyzed using a FACScan flow cytometer (Becton Dickinson, Sparks, MD, USA).
VEGF mRNA and protein levels
HL60 and HEL cells were used to evaluate APL effects on VEGF release. Cells were seeded in 24-well plates at a concentration of 500 000 cells/ml and treated with APL 10, 20 and 50 nM. After different times, cells and surnatants were collected to determine VEGF expression and release. Levels of VEGF secreted in the medium were measured with a Quantikine immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. In addition, total RNA was extracted from treated cells to evaluate VEGF expression by semiquantitative RTPCR. VEGF/ß-actin primers were used in a ratio of 4:1 in PCR mixture {primers: VEGFS 5'-CGA AGT GGT GAA GTT CAT GGA TG-3' and VEGFAS 5'-TTC TGT AGT CTT TCC TGG TGA G-3' (three isoforms: 404, 535 and 607 bp) [26]; ß-actin BA5 5'-TGG ACT TCG AGC AAG AGA TG-3' and BA3 5'-GAT CTT CAT TGT GCT GGG TG-3' (320 bp) [27
]}. Signal intensities of the products were quantified on 3% agarose gel by densitometric analysis of non satured bands using Chemi Doc acquisition system (Bio-Rad, Baltimore, MD, USA) and Quantity One Quantification Software (Bio-Rad). The variations in cDNA synthesis in the samples were normalized by their relative quantities of ß-actin.
Statistical analysis
Results were analyzed by a standard paired Student's t-test. A P value of <0.01 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Further confirmation was provided by apoptotic assays in which APL clearly shows an extremely potent and rapid apoptotic activity in myeloid cells. In 24 h (AML MNCs) and 48 h (cell lines) experiments this effects was dose-dependent up to 50 nM, while at higher concentration the curve was plateauing due to cell necrosis.
As previously demonstrated [17], APL causes important perturbations of the cell cycle, inducing a block at different phases of the cycle, probably activating cell cycle check points, but its mechanism of action is still unclear. In our studies APL was an effective cycle blocker agent, active already at nanomolar concentrations, and the cell cycle arrest was evident and rapid. In fact, after 24 h incubation APL was able to block K-562, HL60 and HEL cells in G1 phase at concentration of 100 nM, while an S phase delay was observed at 10 nM. IDA obtained the same results at concentrations 10 times higher than those of APL.
Finally, our data confirm that APL modulates VEGF release, probably inhibiting VEGF secretion. Broggini et al. [20] previously demonstrated the capability of APL to inhibit VEGF secretion, down-regulate VEGFR-1, and inhibit VEGF mRNA synthesis. Our results support the first hypothesis: in fact APL induces a strong dose-dependent reduction of VEGF protein release in both cell lines used (HEL and HL60), but mRNA levels did not appear reduced by a 24-h incubation. In contrast, VEGF transcript accumulation was observed in cell cytoplasm, suggesting a possible block of extracellular secretion. As previously reported [29
], APL probably induces rapidly an oxidative stress in the cell that is linked to glutathione depletion and causes the activation of a series of kinases including the c-Jun terminal kinase (JNK), p38 mitogen-activated kinase, Src and epidermal growth factor receptor. Sustained activation of JNK has been found to be crucial for the induction of apoptosis by the drug [30
]; however, the activation of these kinases must have additional consequence that remains mostly unknown. VEGF is usually synthesized and secreted in response to cellular hypoxia as a result of the stabilization of the transcription factor hypoxia inducible factor (HIF)-
that activates its promoter. The possibility exists that the cellular oxidative status, perhaps mediated at least partially by these enzymes, causes the blockade of VEGF secretion by affecting the secretory pathway, and in a delayed response can also inhibit VEGF expression via the blockade of HIF-
stabilization or action.
In conclusion, this study has shown that APL is an effective agent displaying both cytotoxic and anti-angiogenic activities when tested in vitro against AML cells, confirming previous results reported in pediatric acute lymphoblastic leukemia blasts. The full experimental results generated with APL in leukemia models supports the notion of a therapeutic potential of APL in patients with relapsed acute leukemia; phase I trials in pediatric patients as well as phase II studies in adult relapsed leukemias are underway.
![]() |
Acknowledgements |
---|
Received for publication December 18, 2004. Revision received May 19, 2005. Accepted for publication May 20, 2005.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Rinehart KL, Holt TG, Fregeau NL et al. Bioactive compounds from aquatic and terrestrial sources. J Nat Prod 1990; 53: 771792.[ISI][Medline]
3. Rinehart KL, Gloer JB, Cook JC et al. Structures of the didemnins, antiviral and cytotoxic depsipeptides from a Caribbean tunicate. J Am Chem Soc 1981; 103: 18571859.[CrossRef][ISI]
4. Hendriks HR, Fiebig HH, Giavazzi R et al. Antitumor activity of ET743 against human tumor xenografts from melanoma, non-small-cell lung and ovarian cancer. Ann Oncol 1999; 10: 12331240.[Abstract]
5. Jimeno JM, Faircloth GT, Cameron L et al. Progress in the acquisition of new marine-derived anticancer compounds: development of Ecteinascidin-743 (ET-743). Drugs Future 1996; 21: 11551165.
6. Taamma A, Misset JL, Riofrio M et al. Phase I and pharmacokinetics study of ET-743, a new marine compound, administered as a 24-hour continuous infusion in patients with solid tumors. J Clin Oncol 2001; 19: 12561265.
7. Twelvws C, Hoeckman H, Bowman A et al. Phase I and pharmacokinetic study of Et-743 evaluating a 3-hour intravenous infusion in patients with solid tumors. Clin Cancer Res 1999; 5: 3790s.
8. Faircloth JG, Rinehart K, Nunez de Castro I, Jimeno J. Dehydrodidemnin B, a new marine derived antitumor agent with activity against experimental tumor models. Ann Oncol 1996; 7: 34.
9. Depenbrock H, Peter R, Faircloth GT et al. In vitro activity of aplidine, a new marine-derived anti-cancer compound, on freshly explanted clonogenic human tumor cells and haematopoietic precursor cells. Br J Cancer 1998; 78: 739744.[ISI][Medline]
10. Urdiales JL, Morata P, Nunez de Castro I, Sanchez-Jimenez F. Anti-proliferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett 1996; 102: 3137.[CrossRef][ISI][Medline]
11. Lobo C, Garcia-Pozo SG, De Castro IN, Alonso FJ. Effect of dehydrodidemnin B on human colon carcinoma cell lines. Anticancer Res 1997; 17: 333336.[ISI][Medline]
12. Geldof AA, Mastbergen SC, Hemrar RE, Faircloth GT. Cytotoxicity and neurotoxicity of new marine anticancer agents evaluated using in vitro assay. Cancer Chemother Pharmacol 1999; 44: 312318.[CrossRef][ISI][Medline]
13. Raymond E, Ady-Vago N, Ribrag V et al. Phase I and pharmacokinetics study of Aplidine, a marine derived compound, given as 24 h infusion every 2 weeks in patients (PTS) with advanced solid tumors and non-Hodgkin lymphomas (NHL). Ann Oncol 2000; 11: 134.
14. Maroun J, Belanger, Lesleys S et al. Phase I study of Aplidine (APL) in a 1 hour daily infusion x5 Q 3 weeks in patients (PTS) with advanced solid tumors and low and intermediate grade non Hodgkin lymphomas (NHL): a National Cancer Institute of Canada-Clinical Trials Group (NCIC-CTG) study. Ann Oncol 2000; 11: 134.
15. Raymond E, Paz-Ares L, Izquierdo MA et al. Activity of aplidine, a new marine compound, against medullary thyroid carcinoma (MTC) phase I trials as screening tool for rare tumors. Ann Oncol 2002; 13: 22.
16. Crews CM, Collins JL, Lane WS et al. GTP-dependent binding of the antiproliferative agent didemnin to elongation factor 1-alpha. J Biol Chem 1994; 269: 1541115414.
17. Erba E, Bassano L, Di Liberti G et al. Cell cycle phase perturbations and apoptosis induced by aplidine. Br J Cancer 2002; 86: 15101511.[CrossRef][ISI][Medline]
18. Erba E, Ronzoni S, Bergamaschi D et al. Mechanism of antileukemic activity of Aplidine. Proc Am Assoc Cancer Res 1999; 40: 3.
19. Marchini S, Contegno F, D'Incalci M et al. Gene expression profile in human leukemic MOLT-4 cells treated with the marine compound aplidine. Proc Am Assoc Cancer Res 2000; 41: 833.
20. Broggini M, Marchini SV, Galliera E et al. Aplidine, a new anticancer agent of marine origin, inhibits vascular endothelial growth factor (VEGF) secretion and blocks VEGF-VEGFR-1 (flt-1) autocrine loop in human leukemia cells MOLT-4. Leukemia 2003; 17: 5259.[CrossRef][ISI][Medline]
21. D'Incalci M, Colombo T, Ubezio P et al. The combination of Yondelis and cisplatin is synergistic against human tumour xenografts. Eur J Cancer 2003; 29: 19201926.
22. Bresters D, Broekhuizen A, Faircloth G et al. In vitro cytotoxicity of Aplidine and cross-resistance with other cytotoxic drugs in childhood leukemic and normal bone marrow and blood samples; a rational basis for clinical development. Leukemia 2003; 17: 13381343.[CrossRef][ISI][Medline]
23. Scudiero DA, Shoemaker RH, Paull KD et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth anf drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988; 48: 48274833.[Abstract]
24. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptotic flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184: 3951.[CrossRef][ISI][Medline]
25. Koopman G, Reutelingsperger CP, Kuijten GA et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cell undergoing apoptosis. Blood 1995; 84: 14151420.[ISI]
26. Möhle R, Green D, Moore M et al. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997; 94: 663668.
27. Wada H, Saikawa Y, Niida Y et al. Selectively induced high MRP gene expression in multidrug-resistant human HL60 leukemia cells. Exp Hematol 1999; 27: 99109.[CrossRef][ISI][Medline]
28. Jimeno J, Lopez Martin JA, Ruiz Casado et al. Progress in the clinical development of new marine derived anticancer compounds. Anticancer Drugs 2004; 15: 321329.[CrossRef][ISI][Medline]
29. Cuadrado A, García-Fernández LF, González L et al. Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of epidermal growth factor receptor, Src, Jun N-terminal kinase and p38 kinase. J Biol Chem 2003; 278: 241250.
30. Cuadrado A, González L, Suárez Y et al. JNK activation is critical for AplidinTM-induced apoptosis. Oncogene 2004; 27: 46734680.[CrossRef]
|