EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models.

EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt's lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10-20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5-10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy

Measurement of intracellular calcium after EGF treatment, in the presence or absence of DMH4.

<p>THP1 or Raji cell lines were cultured as 10⁶ cells/well and incubated, or not, with 20<b>μ</b>M DMH4 for lh30min and then treated with 20ng/ml of EGF in a time-course at various time intervals. Fluorometry was then used in order to measure intracellualr calcium concentrations which were significantly and rapidly increased, in both cell lines, but were not affected by VEGFR2 inhibition using 20<b>μ</b>M DMH4. Results are representatives of three independent experiments (n = 3), for each time point and treatment condition, reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.</p

Effect of EGFR-induced VEGF on phosphorylation of VEGFR2, AKT and ERK.

<p>A total of 10⁶ THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF. <b>(A)</b> EGF stimulation increased very early the phosphorylation levels of p-VEGFR2 in THP1 (Left) or Raji (Right) cell lines, which peaked at ~5–10 min after treatment. <b>(B)</b> In order to measure p-Akt, p-ERK and p-p38, VEGFR2 was blocked using 20μM DMH4 inhibitor for 1h30min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Phosphorylation of AKT and ERK, but not p38, was diminished when the cell lines were pre-incubated with DMH4. <b>(C)</b> Similarly, phosphorylation of AKT and ERK, but not p38, was inhibited when cells were pre-incubated for 1h with 2μg/ml anti-VEGFR2 antibodies. <b>(D)</b> siRNA (20nM) against VEGFR2, left after transfection for 72h, caused a decrease in the phosphorylation levels of AKT and ERK, but not p38. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blots corresponding to ERK1 and ERK2 or their phosphorylated isoforms. However, when ERK1 antibody is used, only 1 band appears on the blot. Results are representatives of four independent western blot experiments (n = 4).</p

EGFR and VEGFR2 are expressed in THP1 and Raji cell lines.

<p>A total of 10⁶ cells/well were cultured. (A) VEGFR1 and VEGFR3 were found to be absent in both cell lines, using RT-PCR. (B) Cells were shown to express EGFR and VEGFR2 mRNA and (C) proteins using RT-PCR and western blot; respectively. Jurkat cell line was used as a negative control whereas monocytes were used as a positive control.</p

Measurement of ROS production after EGF treatment, in the presence or absence of DMH4 or NAC.

<p>THP1 or Raji cell lines were cultured as 10⁶ cells/well and pretreated for 1h30min with DMH4 (1μM or 20μM) or for 30min with N-acetylcysteine (NAC, 5μM) before treatment with 20ng/ml of EGF in a time-course. Following that, H₂O₂ production was measured using fluorometry. (A) Relative fluorescence curves at the basal level, after stimulation with EGF or after inhibition by DMH4 over a period of time. (B) Quantification of the relative fluorescence curve intensities. ROS production was significantly reduced by VEGFR2 inhibition, using 20μM DMH4. (C) In order to measure p-Akt, p-ERK and p-p38, cells were pretreated with 5μM of the ROS inhibitor N-acetylcysteine (NAC) for 30 min and then treated with EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Western blot analysis showed a significant decrease in pAKT, pERK and p-p38. Therefore, VEGFR2 is responsible for EGF-induced ROS production. Results are representatives of three independent experiments (n = 3), reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.</p

Effect of EGFR-induced VEGF on AKT, ERK and p38.

<p>A total of 10⁶ THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF and the phosphorylation levels of AKT, ERK and p38 were analyzed by Western blot. <b>(A)</b> EGF was treated for the indicated time intervals. Phosphorylation levels of p-AKT, p-ERK and p-p38 increased significantly after EGF stimulation in both THP1 (Left) and Raji (Right) cell lines. <b>(B)</b> In order to measure p-Akt, p-ERK and p-p38, EGFR was blocked using 5μM AG1478 inhibitor for 20min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. The expression of pAKT, pERK, and p38 was found to be downregulated, using western-blot. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blot corresponding to ERK1 and ERK2 or their phosphorylated isoforms. Results are representatives of four independent experiments (n = 4), for each time point and treatment condition.</p

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T 125 4 protein transporters, steady-state extraction of I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “Lsystem and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the efficient T4 transport from blood to CSF. The current study highlights a carrier-mediated transport mechanism for T4 movement from blood to brain at the basolateral side of B-CSF-B/CP, as an alternative route to BBB