Troglitazone Exerts a Cell-Dependent Effect on the Extracellular Secretion of VEGF165 Independently from the Rapamycin Sensitivity or the Level of eIF4E Expression in AML-Derived Cells.
Authors: Friday Ellen , Glass Jonathan , Turturro Francesco 
Published: April 2020

Abstract
Coexpression of vascular endothelial growth factor-165 (VEGF165) and its receptors as components of an autocrine loop has been well documented in hematopoietic malignancies. Therapeutic strategies that aim at blocking VEGF-induced signaling are currently being investigated for the treatment of malignancies. Furthermore, eukaryotic initiation factor 4E (eIF4E) allows a fast constitutive translation of mRNAs with short unstructured 5′-UTRs. Longer highly structured 5′-UTR mRNAs such as VEGF, which are less preferentially translated in normal conditions, become highly translated via an increase of eIF4E during malignant transformation. The thiazoladinedione troglitazone (TRO) has recently been reported to either increase or decrease the secretion of VEGF165 in several non hematopoietic cell lines tested. In the current study, we evaluated the action of TRO in HL-60 and U937 cells, derived from acute myelogenous leukemia (AML) known to be VEGF165 secretors, and to express VEGF receptors. The level of VEGF165 measured by ELISA as pg/ml/106 cells, and expressed as percentage of increase/decrease over control (no treatment), was reduced to 40% ± 15, and 14% ± 4.5 as compared to controls in HL-60 cells treated with 10m and 50m TRO for 24 h, respectively (P = 0.04 and 0.01). The number of HL-60 viable cells (Trypan blue) concurrently decreased to 55% ± 3.4 and 45% ± 2.3 as compared to controls after treatment with 10m and 50m TRO for 24 h, respectively (P = 0.01 and 0.006). On the other hand, the same doses of TRO did not significantly reduce the level of secreted VEGF165 and the number of viable cells in U937 cells treated for 24 h. Surprisingly, the level of VEGF165 was increased compared to controls (126% ± 2.5) when U937 cells were incubated with 50m TRO. This observation was in agreement with previous findings in the same cell line (Arterioscler Thromb Vasc Biol, 2001;21:560). To investigate whether the action of TRO on the level of VEGF165 secretion and cell viability was mediated through mTOR/eIF4E pathway in HL-60 cells, we measured the level of total eIF4E by image analysis software of Western blots and expressed as percentage of increase/decrease over controls (treated vs not treated). HL-60 cells present high levels of eIF4E (41% ± 7.2) compared to normal controls (circulating mononucleated cells from healthy donors). There was a slight decrease in level of eIF4E in cells treated with TRO and this was not statistically significant. Furthermore, the level of secreted VEGF165 was unchanged and the number of viable cells was not significantly reduced (80% ± 3 vs control, P = 0.06), when HL-60 cells were treated with 500 nM Rapamycin (mTOR/eIF4E pathway inhibitor) for 24 h. Our data show that TRO may exert an action on the VEGF-proliferative-autocrine loop that is cell dependent and independent from the mTOR/eIF4E pathway in HL-60 cells. These results deserve further investigation to elucidate the mechanism in an attempt to define a new therapeutic modality.