Caffeic Acid Phenetyl Ester, a Brazilian-Green-Propolis Derivative, Induces apoptosis in AML Cells, Promotes up Regulation of G-Protein Signaling and Hyper Secretion of IL-8
Authors: Scheucher Priscila S , Santos Guilherme A dos , Teixeira Hamilton L Gimenes , Thomé Carolina H , Lucena-Araujo Antonio R , Falcao Roberto P. , Rego Eduardo M. 
Published: October 2019

Abstract
Abstract 3274
Propolis is a generic name for an adhesive resin collected, processed and used by bees to plug gaps, smooth internal walls and protect the entrance of the hive from intruders. Chemically is a complex blend of resin and fragments of plant tissues, volatile substances and wax. It contains over 300 constituents including benzoic acids, flavonoids and cinnamic acid derivatives. The Brazilian green propolis (BGP) has been shown to have immunomodulatory and antitumor properties in vitro and in vivo. We selected the caffeic acid phenethyl ester (CAPE), which is one of the components of BGP, as a candidate molecule for further studies regarding the antineoplastic activity. Cytotoxicity induced by Brazilian green propolis alcoholic extract (BGPAE) and CAPE in the cell lineages of acute promyelocytic leukemia (APL) (NB4 and NB4R2), and bone marrow cells from patients with acute myeloid leukemia (AML) at diagnosis (primary cells) was evaluated by annexin V/propidium iodide staining and analyzed by flow cytometry. BGPAE and CAPE showed antiproliferative and apoptotic activity and this effect was dose and time dependent (Table 1 and 2). Accordingly, in primary cells, CAPE (32μg/ml) for 24 hours significantly increased the percentage of apoptotic cells compared to control. The median (25 and 75 percentiles) for the percentage of apoptotic cells was 17.32% (13-27%) in control and 37% (24-52%) in CAPE, p-value = 0.0008 The cell cycle was analysed by flow cytometry using propidium iodide staining and demonstrated that CAPE blocked the cell cycle at G2/M. In addition, CAPE induced activation of caspases 3 and 9, as determined by Western blot, thus suggesting the involvement of the intrinsic pathway of apoptosis. The modulation of gene expression induced by CAPE in NB4 was determined by microarray using the CodeLink Uniset Human I bioarray (Amersham/GE) and analyzing 10,000 genes. Adopting a False Discovery Rate of 4.72% and using the Significance Analysis of Miroarray (SAM) software, we detected an increased expression of: negative mediators of cell cycle, including the dual specific phosphatases CDC14A, CDC14B and the cyclin-dependent kinase (CDK) inhibitor CDKN1A (p21/CIP1); protein phosphatases; chemokines and molecules associated with signaling by G protein. Furthermore, CAPE induced a decrease in gene expression of: positive mediators of cell cycle (including CDK4 and CCNA2); genes related to the “spliceosome” and protein translation. The most important differences in gene expression were confirmed by real time PCR using Taqman® technology (Applied Biosystems). Based on the microarray analysis, we decided to further study the production of IL-8 induced by CAPE in NB4 cells. Using an ELISA assay, it was detected a time dependent increase in the production of IL-8 at CAPE concentrations of 16 and 32μg/ml that was significantly higher than in controls at 12 and 24h of treatment. In conclusion, our results demonstrate that CAPE was able to block the cell cycle and induce apoptosis in APL cells, and induce the production of IL-8 ALP cells.
Table 1 Effective doses 50% (ED-50) of BGPAE ( g/ml) for inhibition of proliferation and induction of apoptosis in NB4 and NB4R2 cell lines in 24, 48 and 72 hours of treatment time ED-50 CI 95% for DE-50 Proliferation NB4 24h 63,72 51,39 + 79,00 48h 43,00 37,20 – 49,70 72h 33,51 30,93 – 36,30 NB4R2 24h 30,28 25,42 – 36,06 48h 50,61 46,52 – 55,06 72h 50,40 45,15 – 56,27 Apoptosis NB4 24h 214,2 204,2 – 224,6 48h 201,0 189,0 – 213,8 72h 185,4 172,2 – 199,5 NB4R2 24h 139,0 132,7 – 145,5 48h 117,3 112,3 – 122,5 72h 101,5 95,18 – 108,2
Table 2 Effective doses 50% (ED-50) of CAPE (μg/ml) for inhibition of proliferation and induction of apoptosis in NB4 and NB4R2 cell lines in 24, 48 and 72 hours of treatment time ED-50 CI 95% for DE-50 Proliferation NB4 24h 8,106 6,977 – 9,418 48h 2,945 2,571 – 3,373 72h 1,648 1,451 – 1,872 NB4R2 24h 7,029 6,209 – 7,956 48h 4,342 4,017 – 4,694 72h 3,214 3,052 – 3,384 Apoptosis NB4 24h 51,81 44,67 – 60,10 48h 31,88 28,71 – 35,39 72h 21,38 18,99 – 24,07 NB4R2 24h 68,58 58,66 – 80,18 48h 34,92 30,39 – 40,14 72h 27,02 24,26 – 30,09

Disclosures:
No relevant conflicts of interest to declare.