Protein phosphatase 2A (PP2A) has a potential role in CAPE-induced apoptosis of CCRF-CEM cells via effecting human telomerase reverse transcriptase activity.
Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC(50) of 1 muM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC(20) of inhibitors and IC(50) of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC(50) of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE.
Avci
Cigir Biray
CB
Department of Medical Biology, Ege University School of Medicine, Bornova, Izmir, Turkey.
Sahin
Fahri
F
Gunduz
Cumhur
C
Selvi
Nur
N
Aydin
Hikmet Hakan
HH
Oktem
Gulperi
G
Topcuoglu
Nejat
N
Saydam
Guray
G
D017209:Apoptosis / Q000187:drug effects*; D002109:Caffeic Acids / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000494:pharmacology; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D019098:Telomerase / Q000378:metabolism*
D002109:Caffeic Acids; D004791:Enzyme Inhibitors; C509186:TERT protein, human; D019098:Telomerase; D054648:Protein Phosphatase 2; C055494:caffeic acid phenethyl ester; D010626:Phenylethyl Alcohol