Constitutive and stimulated production of VEGF by human megakaryoblastic cell lines: effect on proliferation and signaling pathway.

Release of vascular endothelial growth factor (VEGF) and other candidate angiogenic factors such as basic fibroblast growth factor and transforming growth factor beta, may play a role in sustaining neoplastic cell proliferation and tumor growth. We evaluated VEGF expression and synthesis in the two erythromegakaryocytic cell lines B1647, HEL and one megakaryocytic cell line MO7 expressing erythroid markers. In this study RT-PCR was performed to evaluate VEGF expression and that of its receptor KDR; VEGF production was assayed by Elisa test and western blot analysis; sensitivity to VEGF was tested by thymidine incorporation. VEGF and its receptor KDR were expressed in B1647 and HEL, both as mRNAs and as proteins, while only KDR transcript was found in MO7 cells. Only B1647 and HEL cells showed a strong spontaneous proliferating activity. In fact, measurable amounts of VEGF were present in the unstimulated cell medium, thus suggesting an autocrine production of VEGF by B1647 and HEL cells, but not by MO7, which was inhibited in mRNA-silencing conditions. This production could not be further boosted by other growth factors, whereas it was inhibited by TGF-beta1. Finally, analysis of Shc signal transduction proteins following stimulation with VEGF indicated that only p46 was tyrosine phosphorylated. These data indicate that leukemic cells may be capable of autocrine production of VEGF which, in turn, maintains cell proliferation, possibly mediated by Shc p46 phosphorylation.

VEGF inhibition and cytotoxic effect of aplidin in leukemia cell lines and cells from acute myeloid leukemia.

BACKGROUND: Aplidine (APL) is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that is under clinical phase II development. In contrast to the lack of bone marrow toxicity reported in phase I/II studies, it has been shown to induce cytotoxicity at very low concentration against lymphoblastic leukemia blast, as well as having an impact in the vascular endothelial growth factor (VEGF)/VEGF receptor 1 loop. 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 super imposible. ID(50) ranged from 0.024 to 0.610 microM for IDA (0.200+/-0.176) and from 0.001 to 0.108 microM 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 G(1) 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.