Caffeic Acid Phenethyl Ester Induces Apoptosis in Acute Myeloid Leukemia Cells - Possible Role of Chemokine/G-Protein Signaling.

Caffeic acid phenethyl ester (CAPE) is an active phenolic compound present in propolis obtained from honeybee hives. It is reported to present a spectrum of biological activities including antioxidant, anti-inflammatory and antitumoral. The antitumoral activity of CAPE as evaluated by several studies in vitro and in vivo seems to be related to distinct effects like inhibition of angiogenesis, invasion and metastasis and induction of apoptosis or differentiation of cancer cells. In the scenario of AML the demonstration of CAPE-induced apoptosis or cellular differentiation is restricted to the HL-60 cell line.

Our aim was to evaluate the effects of CAPE treatment on primary AML samples as well as APL cell lines NB4 and NB4-R2 (a cell resistant to ATRA-induced differentiation) and on AML cell line Kasumi-1 (representative of core binding factor leukemia with AML1-ETO rearrangement).

Proliferation and viability was evaluated by cell count with tripan blue in Neubauer chamber at fixed time intervals. Differentiation was evaluated by flow cytometer determination of CD11b expression. Apoptotic cells were defined as sub-G0 fraction and were evaluated by flow cytometer determination of propidium iodide- DNA fluorescence. Also apoptosis was detected by the annexin-V method. Leishman stained cytospins were used to confirm apoptosis or differentiation. CAPE did not induce differentiation in the cell lines NB4, NB4-R2 or Kasumi-1 and did not alter the differentiation induced by ATRA in NB4 cells.

CAPE inhibited the proliferation of AML cell lines in a time and dose dependent fashion. The ED50 in 24h treatment for NB4 cell line (tripan blue) was 32.1 mcg/ml. ED50 (at 24h) for induction of apoptosis in the more sensitive assay using annexin-V in NB4 cells after 24h was 7.5mcg/ml and for Kasumi-1 was 10.2mcg/ml. CAPE (32 mcg/ml) significantly induced apoptosis after 24h in cells from AML patients (n=10), mean (IC95%) of 40.5% (29.26 – 51.76) versus control treated cells 18.16% (12.27 – 24.05); p=0.0004

In order to evaluate the mechanisms of CAPE-induced apoptosis in NB4 cells we performed a microarray analysis after 12 hours treatment (32mcg/ml). The majority of downregulated genes fall into two categories: positive cell cycle regulators and ribosomal genesis / protein traduction. In the other hand, upregulated genes fall into several categories, we point out chemokines and G- protein signalization genes. (Table 1 and 2) The role of IL-8 and Gro chemokines, that signaling by G-protein coupled receptors, has been determined in tumor progression and invasiveness. We are currently investigating the possibility that CAPE exerts an inhibitory effect in chemokine signaling in APL.

In conclusion, CAPE-induced apoptosis in AML is associated with the regulation of specific genes. These properties are interesting and need further investigation.

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No relevant conflicts of interest to declare.