COMMENTARY

Combination of melatonin and a peroxisome proliferator-activatedreceptor-c agonist induces apoptosis in a breast cancer cell line

Nuclear receptors (NRs) and ligand-dependent transcrip-tion factors play multiple essential roles in development,homeostasis, reproduction, and immune function. NRs

influence transcription via several mechanisms and canboth activate or inhibit gene expression. The NRs includesteroidal transcription factors such as estrogen, glucocor-ticoids, retinoid acid receptor (RAR), retinoid X receptor

(RXR), and peroxisome proliferator-activated receptors(PPARs) [1]. Half of the NRs are so-called �orphan�receptors because the identity of their ligand, if any, is

unknown. Two prime examples are the PPARs and RXRs,which were discovered as orphan NRs, but for whichligands have now been tentatively suggested, although the

definitive identity of their physiological, endogenousligands is somewhat controversial [1]. From emergingevidence regarding the genomic actions of melatonin, it isnow clear that this indolamine is an endogenous ligand of

RXRs [2–4].PPARc and RXR play crucial roles in inducing apoptosis

in a variety of human cancer cells including colon [5] and

breast cancer [6, 7] cell lines. As several exogenous retinoicacid (RA) derivatives (e.g., 9-cis–trans RA and all-transRA) are considered ligands for RXRs, recent studies have

been performed using a combination of PPARc agonistsincluding rosiglitizone, ciglitizone, and troglitazone and RA

derivatives [5–7]. In light of recent evidence, we testedwhether the combination of a PPARc agonist and melato-nin would induce apoptosis in the MDA-MB-231 human

breast cancer cell line.Cells were cultured at 37�C in a water-saturated atmo-

sphere with 5% CO2-95% air. The culture medium wasrenewed each day. Cells were treated either with vehicle

alone, 100 lm troglitazone, 1 mm melatonin, or withtroglitazone + melatonin for 72 hr. The 3-(4,5-dimethy-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)

assay, used to assess cell viability, is based on the abilityof viable cells to convert MTT into a blue formazanproduct. In contrast to normal cells, the nuclei of apoptotic

cells have highly condensed chromatin that is uniformlystained by Hoechst 33342; use of this method allows one toobserve the morphological changes in the nuclei of apop-totic cells [8]. The addition of melatonin or troglitazone

alone to cultured cells caused 16% and 47% reductions inthe number of cells, respectively (Fig. 1). The combinationof the two agents resulted in a 84% reduction in cell

number. After exposure to melatonin and troglitazone for72 hr, remaining cells underwent apoptosis which wascharacterized morphologically by chromatin condensation

and nuclear fragmentation (Fig. 1). Clearly, the combina-tion of the PPARc agonist and melatonin caused a very

Melatonin Control

Melatonin + PPAR agonistPPAR agonist

Fig. 1. Fluorescence microscopy ofhuman breast cancer cells (MDA-MD-231) labeled with Hoechst 33342 aftertreatment with vehicle (control) or withmelatonin, troglitazone (PPARc agonist)or both melatonin and troglitazone for72 hr. All photomicrographs were takenusing an Olympus BX60 epifluorescencemicroscope (with Olympus U Plan Fluo-rite 20X objective and U-MNU filter set).Clearly, the PPARc agonist in combina-tion with melatonin greatly reduced thecell number. Also, the combination treat-ment induced a large percentage (virtuallyall) of the cells to undergo apoptosis (lightblue-bright cells).

J. Pineal Res. 2009; 46:115–116Doi:10.1111/j.1600-079X.2008.00635.x

� 2008 The AuthorsJournal compilation � 2008 Blackwell Munksgaard

Journal of Pineal Research

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significant reduction in cell number and increased apoptosisof the cancer cells.RXR can regulate transcription in a heterodimeric

complex and generally does not involve gene transcription.Via its role as a required heterodimeric partner, RXRcontrols the function of many other NRs, thus integrating aunique transcriptional network dependent on RXR

responses. RXR forms heterodimers with virtually allNRs. Both in vitro and in vivo approaches have revealedthat NRs require RXR as a heterodimerization partner for

their function. Ligand-activated NRs dissociate fromco-repressors and recruit co-activators, and can bothactivate or inhibit gene expression [9]. Although RAR

controls many metabolic genes, many others are not RARtargets. Eck-Enriquez et al. [10] reported a potent apoptoticeffect of a combination of melatonin and RA on humanbreast cancer cells while Crowe et al. [6] used a selective

RXR-ligand (AGN194204) in combination with a PPARcagonist and found that AGN194204 potentiates the anti-proliferative and apoptotic responses to the PPARc ago-

nist. In reference to cell viability, a variety of mechanismsmight be synergistically activated by this combination,including reduced telomerase activity in human cancer cells

[11, 12].Convincing data have, additionally, documented the

emerging role of epigenetic perturbations in both cancer

initiation and progression of human malignancies includingbreast cancer [13]. Because epigenetic changes may bereversible, they represent an active area for new druginvestigation and are promising targets for cancer therapy.

Recent evidence revealed that melatonin influences cellsvia a variety of epigenetic mechanisms including NRs,co-regulators, histone acetylating, and DNA methylating

enzymes [14, 15].In conclusion, the marked inhibitory effects of the

combination of melatonin and PPARc ligands on inhibition

of established breast cancer cell lines require further study.The evaluation of melatonin as an RXR ligand thatcooperates with PPARs may result in important therapeutic

breakthroughs.

Ahmet Korkmaz1, Hiroshi Tamura1, Lucien C. Manchester1,Gary B. Ogden2, Dun-Xian Tan1 and Russel J. Reiter1

1Department of Cellular and Structural Biology, University

of Texas Health Science Center; 2Department of BiologicalScience, St. Mary�s University, San Antonio, TX, USA

E-mail: korkmaz@uthscsa.edu;

drakorkmaz@gmail.com

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Commentary