WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential

Abstract

PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5-1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC 50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025-0.05 lM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBTpositive NB4 cells at definitely higher levels than with 1 lM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086

“…Apoptosis was revealed by the presence of nuclear fragmentation in stained cells and by the DNA ladder assay as reported previously (Laurenzana et al, 2005). Neutral lipids were determined (i) histochemically (Constantinou et al, 1998) on cell monolayers which were quickly fixed with À201C methanol, stained with Oil Red O (Sigma-Aldrich) and counterstained with haematoxylin; or (ii) spectrophotometrically (Cary 50 Scan, Varian, Victoria, Australia) at 510 nm by recording absorbance of cell-bound Oil Red O following extraction with isopropanol (Wang et al, 2001).…”

“…Harvested cells were resuspended in the lysis buffer (Laurenzana et al, 2005) and separated on 12.5% SDS -PAGE. Western-blot membranes were probed using primary antibodies against cathepsin D, p53, PTEN, and a-tubulin (Santa Cruz Biotech, Santa Cruz, CA, USA) and ERa (Ventana Medical Systems, Tucson, AZ, USA).…”

Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells: The crucial role of PTEN

“…Culture conditions, cell counting, and viability were as reported previously [14]. Leukemic blasts from bone marrow aspirate and/or peripheral blood of 19 newly diagnosed AML patients with M0 through M5 subtypes were obtained after informed consent and according to the Ethics Hospital Committee guidelines (Helsinki declaration).…”

“…Indeed, epigenetic modulators are functional to set up an environment permissive for cell differentiation and/or apoptosis, but may not always be sufficient themselves to trigger these processes without the cooperation of other proactive signals. It is of great interest, therefore, to identify and characterize new agents capable of inducing cytostasis, differentiation, and apoptosis in AML cells, and supporting the action of epigenetic modulators and conventional chemotherapy so as to decrease effective therapeutic doses and untoward consequences to the host.We previously showed that WEB-2170 da ligand of platelet-activating factor receptor (PAF-r) originally synthesized from an anxiolytic triazolobenzodiazepine [11,12]d could also promote murine erythroleukemia cell maturation [13] and trigger apoptosis in ATRA-sensitive and -resistant APL cell lines, and in blasts from patients with APL [14]. The present study aimed to understand mechanisms of WEB-2170-induced cytostasis and apoptosis in the APL cell line NB4, as well as to assess drug efficacy in other AML cell lines and, eventually, ex vivo in blasts from patients with different (M0 through M5) AML subtypes.…”

“…We previously showed that WEB-2170 da ligand of platelet-activating factor receptor (PAF-r) originally synthesized from an anxiolytic triazolobenzodiazepine [11,12]d could also promote murine erythroleukemia cell maturation [13] and trigger apoptosis in ATRA-sensitive and -resistant APL cell lines, and in blasts from patients with APL [14]. The present study aimed to understand mechanisms of WEB-2170-induced cytostasis and apoptosis in the APL cell line NB4, as well as to assess drug efficacy in other AML cell lines and, eventually, ex vivo in blasts from patients with different (M0 through M5) AML subtypes.…”