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. 2013 May 9:12:39.
doi: 10.1186/1476-4598-12-39.

6-Formylindolo (3,2-b)carbazole (FICZ) enhances retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells

Rodica P Bunaciu  1 , Andrew Yen
Affiliations

6-Formylindolo (3,2-b)carbazole (FICZ) enhances retinoic acid (RA)-induced differentiation of HL-60 myeloblastic leukemia cells

Rodica P Bunaciu et al. Mol Cancer. .

Abstract

Background: The aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts.

Methods: Using flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots.

Results: We demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRβ, a marker associated with retinoic acid syndrome was also increased.

Conclusions: Our data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.

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Figures

Figure 1
Figure 1
FICZ augments RA-induced differentiation. HL-60 cells were untreated (C, control) or treated with FICZ, RA, or RA plus FICZ for indicated times. FICZ augments RA-induced expression of differentiation markers. A. CD38 expression (assessed by flow cytometry with APC-conjugated antibody) at 6 h post treatment is not significantly modulated either by FICZ alone or in combination with RA. B. CD11b expression (assessed by flow cytometry with APC-conjugated antibody) was increased by the combination therapy compared to RA alone after 24 h (p = 0.012), C. 48 h (p = 0.042) and D. 72 h (p = 0.0029) of treatment. Flow cytometric assay of live cells was carried out setting the logical gate to exclude 95% of the untreated cells during CD38 and CD11b detection. E. Superoxide metabolites from inducible oxidative metabolism were measured using flow cytometry of DCF stained cells. Respiratory burst is not significantly enhanced at 48 h by combination therapy compared to RA alone (p = 0.08). F. Respiratory burst, measured by flow cytometry of DCF stained cells, is significantly enhanced at 72 h by co-treatment compared to RA alone (p = 0.001). G. Cell density was measured for control and treated cells at progressive times, 0, 24, 48 and 72 h. Cell growth shows that FICZ at the concentration used has no toxic effect. FICZ by itself does not decrease the cell number, however it enhances RA-induced growth inhibition. H. Cell cycle distribution was measured by flow cytometry of propidium iodide stained nuclei. The percent of cells in G0/G1 is shown. G0/G1 cell-cycle arrest was propelled by co-treatment compared with RA alone both at 48 h (p = 0.009) and I. 72 h(p = 0.035) as shown by flow cytometry of DNA-stained nuclei.
Figure 2
Figure 2
FICZ upregulates: AhR, AhR target genes and MAPK cascade components in the presence of RA. Cell lysates collected 48 h after initiation of the experiments were resolved on 12% polyacrylamide gels. 25 μg protein was loaded per well. In RA-treated cells, FICZ upregulates the amount of Cyp1A2, and p47phox, AhR protein expression and pERK1/2, pMEK1/2 and pS621 RAF phospho-protein in the presence of RA compared to cells treated with RA alone. Like pS621 RAF, terminal domain phosphorylated RAF is also upregulated. Total ERK1/2, MEK1/2, RAF are unchanged. The loading control was GAPDH.
Figure 3
Figure 3
FICZ upregulates RA-dependent signaling intermediates. Cell lysates collected at 48 h time point were resolved on 12% polyacrylamide gels. 25 μg protein was loaded per well. FICZ upregulates the amount of c-Cbl and IRF-1 expression and pY507 Lyn and pY1021 PDGFRβ phospho-protein. Total RARα expressed is not upregulated, nor is the GAPDH loading control.
Figure 4
Figure 4
AhR ligands modulate phosphorylation of RAF in the MAPK signaling cascade. Cell lysates collected 48 h after initiation of the experiments were resolved on 12% polyacrylamide gels. 25 μg protein was loaded per well. β-NF upregulates the amount of Cyp1A2, TD RAF and pS621RAF proteins in the presence of RA in a similar fashion to FICZ (comparing similarity of lanes 4 and 8), whereas α-NF does not. GAPDH is the loading control.
Figure 5
Figure 5
FICZ and other AhR ligands modulate MAPK signaling cascade regulatory molecules. Cell lysates collected after 48 h of RA treatment were resolved on 12% polyacrylamide gels. 25 μg protein was loaded per well. Similarly to FICZ, β-NF upregulates the amount of Fgr, Lyn, VAV1 protein and p-Y416 pan-SFK, pY507 Lyn, and pY458 p85PI3K phospho-protein, whereas the antagonist does not upregulate those markers Comparing lanes 4 and 8 FICZ and β-NF, the two AhR agonists, have apparently similar signaling profiles in RA-treated cells, which is not shared by α-NF, an AhR antagonist, lane 6). The agonists by themselves without RA do not have this effect. GAPDH is the loading control.
Figure 6
Figure 6
Summary figure.
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