Melatonin down-regulates HIF-1a expression through inhibition ofprotein translation in prostate cancer cells

Introduction

Melatonin is an indoleamine synthesized in the pineal

gland. Major areas of melatonin research include its effectson seasonal reproduction [1], circadian rhythms [2], andfree radical scavenging [3–5]. Other new properties of

melatonin also have been reported [6, 7]. An emerging fieldof research on melatonin is its oncostatic and antiprolifer-ative effects [8–10]. Melatonin inhibits cell growth andmetastatic properties in cell culture, mainly in endocrine

tumors [11–13]. Furthermore, several reports showed mel-atonin decreases tumor growth in vivo [14, 15]. Themechanisms involving the antitumoral properties of mela-

tonin are still not clear [8].Hypoxia inducible factor 1 (HIF-1) is a basic-loop helix

PAS (PER-ARNT-SIM) transcription factor composed of

two subunits, HIF-1a and HIF-1b. The HIF-1a subunit isdegraded rapidly through the ubiquitin–proteasome path-way under normoxic conditions and stabilized underhypoxic conditions, while HIF-1b is constitutively

expressed [16, 17]. The proteasomal degradation of HIF-1a occurs through protein hydroxylation on Pro-402 andPro-564 by specific HIF-prolyl hydroxylase in the presence

of iron and oxygen. The hydroxylated protein then interactswith von Hippel-Lindau tumor suppressor protein whichfunctions as an E3 ubiquitin ligase [18, 19]. Under hypoxia,

HIF-1a is accumulated and translocated to the nucleuswhere it heterodimerizes with HIF-1b and activates thetranscription of more than 40 genes important for adapta-

tion and survival under hypoxia [16, 20, 21]. In addition,oxygen-independent signaling pathways activated bygrowth factors [insulin-like growth factor-1 (IGF-1), IGF-2,

and epidermal growth factor] and cytokines can induceHIF-1a accumulation [20, 22–25]. Overexpression of HIF-1a has been demonstrated in more than 70% of human

cancers and their metastases compared to their adjacentnormal tissue, including prostate, breast, lung, and headand neck cancers [26]. The development of cancer thera-peutics targeting HIF-1 activity appears to be attractive.

Recently, it has been suggested that the antiangiogeniceffect of melatonin relates to the antineoplastic propertiesof melatonin [27–29]. A preliminary clinical study showed

that a decline in serum levels of vascular endothelial cellgrowth factor (VEGF), which is the most potent endoge-nous angiogenic factor and the target of transcription

factor HIF-1, in patients treated with melatonin [27]. Invitro, melatonin inhibits the expression of VEGF and HIF-1a in cancer cells under hypoxic conditions induced by

cobalt chloride [29]. Yet, the mechanism of inhibitoryaction of melatonin on VEGF and HIF-1a is unclear. Inthis study, we investigated the effects of melatonin on theexpression of HIF-1a and VEGF in prostate cancer cells.

Abstract: Melatonin, the main secretory product of the pineal gland, has

been shown to exert an oncostatic activity in cancer cells. Recently, several

studies have shown that melatonin has antiangiogenic properties. However,

the mechanism by which melatonin exerts antiangiogenenic effects is not

understood. Hypoxia inducible factor (HIF)-1 is a transcription factor which

mediates adaptive response to changes in tissue oxygenation. HIF-1 is a

heterodimer formed by the association of a constitutively expressed HIF-1bsubunit and a HIF-1a subunit, the expression of which is highly regulated. In

this study, pharmacologic concentrations of melatonin was found to inhibit

expression of HIF-1a protein under both normoxic and hypoxic conditions

in DU145, PC-3, and LNCaP prostate cancer cells without affecting HIF-1amRNA levels. Consistent with the reduction in HIF-1a protein levels,

melatonin inhibited HIF-1 transcriptional activity and the release of vascular

endothelial growth factor. We found that the suppression of HIF-1aexpression by melatonin correlated with dephosphorylation of p70S6K and

its direct target RPS6, a pathway known to regulate HIF-1a expression at the

translational level. Metabolic labeling assays indicated that melatonin

inhibits de novo synthesis of HIF-1a protein. Taken together, these results

suggest that the pharmacologic concentration of melatonin inhibits HIF-1aexpression through the suppression of protein translation in prostate cancer

cells.

Jong-Wook Park*, Mi-SunHwang*, Seong-Il Suh andWon-Ki Baek

Chronic Disease Research Center, School of

Medicine, Keimyung University, Daegu, Korea

Key words: angiogenesis, HIF-1a, melatonin,

p70S6K, prostate cancer, translation,

vascular endothelial cell growth factor

Address reprint requests to Won-Ki Baek,

Department of Microbiology, Keimyung

University School of Medicine, Dongsan-Dong,

Daegu 700-712, Korea.

E-mail: wonki@dsmc.or.kr

*These authors contributed equally to this

work.

Received January 2, 2009;

accepted February 18, 2009.

J. Pineal Res. 2009; 46:415–421Doi:10.1111/j.1600-079X.2009.00678.x

� 2009 The AuthorsJournal compilation � 2009 Blackwell Munksgaard

Journal of Pineal Research

415

Material and methods

Cell line and reagents

DU145 and PC3 cells were cultured in Dulbecco�s modifiedEagle�s medium (DMEM) containing 10% heat-inactivated

fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mLpenicillin, and 100 ug/mL streptomycin. LNCaP cells werecultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin,

100 ug/mL streptomycin, 1% sodium pyruvate and 1%HEPES. The cells were maintained at 37�C in a humidified5% CO2 atmosphere. For hypoxia treatment, the cells were

incubated with 200 lm CoCl2. Melatonin was obtainedfrom Sigma (St. Louis, MO, USA). Proteasome inhibitorMG132 was purchased from Calbiochem (San Diego, CA,

USA). Prolyl hydroxylase inhibitor N-(methoxyoxoacetyl)-glycin methyl ester (DMOG) was obtained from CaymanChemical (Ann Arbor, MI, USA). Protein synthesis inhib-itor cycloheximide was purchased from Sigma. IGF-1 was

purchased from R & D Systems (Minneapolis, MN, USA).

Immunoblotting

Anti-HIF-1a (BD Biosciences), anti-HIF-1b (Santa CruzBiotechnology, Santa Cruz, CA, USA), Anti-phospho-

p70S6K (Thr421/Ser424) (Cell Signaling Technology, Dan-vers, MA, USA), anti-phospho-RPS6 (Ser235/236) (CellSignaling Technology) antibodies were used at a dilution of

1:1000. Anti-actin antibody (Sigma) was used at a dilutionof 1:5000. To prepare cell extracts, cells were harvested incold phosphate-buffered saline (PBS) and lysed in TNNbuffer [150 mm NaCl, 50 mm Tris (pH 7.4), 0.5% NP-40]

containing protease and phosphatase inhibitors (50 lg/mLphenylmethylsulfonyl fluoride, 10 lg/mL aprotinin, 5 lg/mL leupeptin, 0.1 lg/mL NaF, 1 mm DTT, 0.1 mm sodium

orthovanadate and 0.1 mm b-glycerophosphate) for 20 minat 4�C. Lysates were cleared by centrifugation, and proteinswere separated by gel electrophoresis. Immunoblots were

performed as described previously [30]. Briefly, membraneswere blocked in Tris-buffered saline–0.05% Triton X-100(TBS-T)–4% (w/v) milk for 1 hr at room temperature (RT).

Membranes were then incubated with primary antibodiesdiluted in TBS-T–4% (w/v) milk for 1 hr at RT orovernight at 4�C (in the case of phospho-specific antibod-ies). Subsequently, membranes were washed with TBS-T

and incubated with horseradish peroxidase secondaryantibody (1:2000; Amersham Biosciences, Fairfield, CT,USA) diluted in TBS-T–4% (w/v) milk. Membranes were

washed in TBS-T, and bound antibody was detected byenhanced chemiluminescence (Amersham Biosciences). Themembrane was exposed to X-ray film or analyzed with LAS

3000 (Fujifilm Co., Tokyo, Japan) image analyzer usingMultiGauge software.

Reverse transcriptase–polymerase chain reaction

Total RNA was isolated from cultured cells using Trizolreagent (Invitrogen, Carlsbad, CA, USA). Reverse trans-

criptase–polymerase chain reaction (RT–PCR) reactionswere performed as described previously [31]. The RT–PCR

products were separated on agarose gels and visualized byethidium bromide staining under ultraviolet transillumina-tion. The primer sequences were as follows: (forward

primer) 5¢-CTC AAA GTC GGA CAG CCT CA-3¢ and(reverse primer) 5¢-CCC TGC AGT AGG TTT CTG CT-3¢for HIF-1a, (forward primer) 5¢-CGT CTT CAC CACCAT GGA GA-3¢ and (reverse primer) 5¢-CGG CCA TCA

CGC CAC AGT TT-3¢ for GAPDH.

Transient transfection and luciferase assay

The hypoxia response element (HRE) driving firefly lucif-erase expression plasmid pGL2-TK-HRE were a kind gift

from Dr. Giovanni Melillo (National Cancer Institute,Frederick, Maryland). DU145 cells were seeded in a 24well-plate at a density of 5 · 104 cells/well the day beforethe transfection. The cells were transiently cotransfected

with pGL2-TK-HRE plasmids and pRL-TK plasmids(Promega, Madison, WI, USA), a renilla luciferase expres-sion plasmid, using Lipofectamine 2000 (Invitrogen)

according to the manufacturer�s instructions. The trans-fected cells were cultured 20 hr, followed by incubationwith melatonin for 6 hr. The cells were washed with PBS

and analyzed with the Dual-Glo luciferase assay system(Promega). The relative luciferase activity was determinedby the ratio of firefly/renilla luciferase activity.

Metabolic labeling

DU145 cells were seeded in 60 mm culture dishes at a

density of 4 · 105 cells/dish. After overnight incubation, thecells were washed twice with methionine/cystein-freeDMEM and treated with melatonin in methionine/

cystein-free DMEM. After 3 hr, 35S-methionine/cystein(NEN life Sciences, Boston, MA, USA) was added to afinal concentration of 200 lCi/mL. The cells were incu-

bated for 1 hr, and harvested. Equal amount of theextracted protein were subjected to immunoprecipitationusing anti-HIF-1a antibody (Santa Cruz Biotechnology).The immunoprecipitates were washed and separated by

SDS–PAGE. The gel was dried and exposed to X-ray film.

Quantitation of VEGF production

Media were collected from 1.5 · 105 cells in 6-well cultureplates and centrifuged at 800 r.p.m. for 4 min at 4�C to

remove cellular debris and then stored at )70�C. VEGF inthe medium was measured by using the Quantikine humanVEGF ELISA kit from R & D Systems according to the

manufacturer�s instruction.

Statistical analysis

The data represent mean ± S.D. from three independentexperiments. Statistical analysis was performed by Stu-dent�s t-test at a significance level of P < 0.05.

Results

We examined the effects of melatonin on HIF-1a expressionin DU145, PC-3 and LNCaP prostate cancer cells under

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normoxic and hypoxic conditions. The hypoxic conditionswere induced by the treatment of hypoxia mimetic agent,cobalt chloride (CoCl2). Both in normoxic and hypoxic

conditions, the pharmacologic concentration (1 mm) ofmelatonin showed significant inhibitory effects on HIF-1aprotein expression in all three tested prostate cancer cellswhereas the physiologic concentration (1 nm) of melatonin

did not induce changes in HIF-1a levels (Fig. 1A). But, thelevels of HIF-1a mRNA were not changed by melatoninsuggesting that melatonin did affect post-transcriptional

regulation (Fig. 1B). Next, we examined the HIF-1 tran-scriptional activity using a reporter gene assay to testwhether the reduced HIF-1a expression resulted in a

decrease in HIF-1 transcriptional activity. To this end,DU145 cells were transiently transfected with a constructcontaining Luciferase gene under the control of the hypoxiaresponsive element (HRE) from the inducible nitric oxide

synthase promoter, and treated with pharmacologic con-centration of melatonin. Treatment of 1 mm melatonininhibited basal and hypoxia-induced promoter activity, as

expected (Fig. 2A). Moreover, in DU145 cells, VEGF levelsin conditioned medium were reduced by the treatment of1 mm melatonin both in normoxic and hypoxic conditions

(Fig. 2B). In LNCaP cells, melatonin (1 mm) significantlyreduced hypoxia-induced VEGF production, although the

basal levels of VEGF were very low and the inhibitoryeffects of melatonin were not significant (Fig. 2C). Takentogether, these results suggest that pharmacologic concen-

tration of melatonin inhibits VEGF production through thereduction of HIF-1a protein expression in prostate cancercells.To evaluate how melatonin reduces HIF-1a protein

levels, we checked the half-life of HIF-1a after treatingmelatonin. DU145 cells were treated with protein transla-tion inhibitor cycloheximide (CHX) alone or in combina-

tion with melatonin. As shown in Fig. 3A, 1 mm melatonindid not induce a significant change in protein half-life ofHIF-1a suggesting that melatonin does not affect HIF-1adegradation. Next, we examined the effect of melatonin inHIF-1a protein synthesis using metabolic labeling assay.DU145 cells were labeled with S35-methionine/cystein in thepresence or absence of 1 mm melatonin for 60 min. The

metabolic labeling assay was done in the presence of prolylhydroxylase inhibitor DMOG to block degradation ofnewly synthesized HIF-1a protein in DU145 cells. In this

assay melatonin treatment significantly reduced newlysynthesized HIF-1a protein accumulation while a proteintranslation inhibitor CHX abrogates de novo protein

synthesis of HIF-1a (Fig. 3B). To confirm the inhibitoryeffect of melatonin on HIF-1a protein synthesis, we

Fig. 1. Melatonin inhibits HIF-1a protein expression in prostate cancer cells. (A) DU145, PC-3, and LNCaP prostate cancer cells weretreated with the indicated concentrations of melatonin in the absence or presence of CoCl2 for 3 hr. Cell lysates were collected for Westernblot analysis. To show the change of HIF-1a protein levels in the absence or presence of CoCl2, X-ray films exposured to the same blot forshort (upper) or long (lower) times are presented. (B) DU145, PC-3, and LNCaP prostate cancer cells were treated with the indicatedconcentrations of melatonin in the absence or presence of CoCl2 for 3 hr. Total RNAs were isolated and subjected to RT–PCR analysis.

Melatonin inhibits HIF-1a expression

417

examined the accumulation rate of HIF-1a protein whenDU145 cells were treated with melatonin in the presence ofprolyl hydroxylase inhibitor DMOG or proteasome inhib-

itor MG132. We expected that if melatonin inhibits HIF-1aprotein translation, melatonin treatment would decreasethe accumulation of HIF-1a protein even in the presence ofDMOG or MG132 since prolyl hydroxylation of HIF-1aprotein and proteasomal degradation are important forHIF-1a degradation in normoxic condition. As expected,DMOG or MG132 treatment induced accumulation of

HIF-1a both in melatonin untreated or melatoninco-treated DU145 cells. But the accumulation rates weresignificantly lower in melatonin co-treated cells

(Fig. 3C,D). These results, taken together with CHXexperiments, suggest that pharmacologic concentration ofmelatonin inhibits HIF-1a protein synthesis rather thanenhancing HIF-1a protein degradation.

It is reported that p70S6K has signaling roles in HIF-1atranslation regulation [22, 32]. To test whether the inhibi-tion of HIF-1a expression by melatonin is associated with

modulation of p70S6K signaling pathway, we examined theeffects of melatonin on the phosporylation status ofp70S6K and its downstream targets RPS6 and eIF4B using

Western blot. In DU145, PC3, and LNCaP cells 1 mm

melatonin treatment inhibited phosphorylation and activityof p70S6K in parallel with decrease of HIF-1a suggesting

melatonin downregulates HIF1a protein translationthrough p70S6K inhibition (Fig. 4A). Several growthfactors are known to induce HIF-1a expression throughincreasing its translation [22–24, 32, 33]. A prediction is

that if melatonin inhibits HIF-1a expression throughinhibiting protein translation, melatonin could decreasethe growth factor-induced expression of HIF-1a. To test

whether this was the case, we examined the effects ofmelatonin on IGF-1 induced HIF-1a expression in DU145cells. After serum starvation, DU145 cells were pretreated

with melatonin for 1 hr and stimulated with IGF-1 for 6 hr.IGF-1 induced expression of HIF-1a and this induction wasinhibited by pretreatment of 1 mm melatonin whereas the

physiologic concentration (1 nm) of melatonin did affectneither HIF-1a protein levels nor phosphorylation ofp70S6K which are induced by IGF-1 (Fig. 4B).LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor,

reduced IGF-1-induced HIF-1a expression in parallel withinhibition of PI3K activity and phosphorylation ofp70S6K. These results suggested that melatonin�s action

on HIF-1a inhibition in pharmacologic dose is associatedwith p70S6K inhibition.

Discussion

Melatonin is an indolic hormone produced by the pinealgland. In the past two decades, numerous studies reported

melatonin has anticancer properties in vitro and in vivo.However, the anticancer mechanism of melatonin is not stillclearly understood. Several mechanisms proposed for

anticancer action of melatonin and its metabolites relateto their ability to reduce DNA damage caused by unstableoxygen and nitrogen-based reactants [34–36], affecting the

uptake and metabolism of fatty acids including linoleic acid[37], inhibiting telomerase activity [38], and reducing

Fig. 2. Melatonin inhibits transcriptional activity of HIF-1 andproduction of vascular endothelial cell growth factor (VEGF). (A)DU145 cells transiently cotransfected with pGL2-TK-HRE plas-mids and pRL-TK plasmids were treated with vehicle and 1 mm

melatonin in the absence or presence of CoCl2 for 6 hr. Theluciferase activity was assayed using the Dual-Glo luciferase assaysystem. The relative luciferase activity was determined by the ratioof firefly/renilla luciferase activity and normalized to the value ofcontrol. *Significant difference from the vehicle treated group inthe absence of CoCl2 (P < 0.05). #Significant difference fromvehicle treated group in the presence of CoCl2 (P < 0.05). DU145cells (B) and LNCaP cells (C) were seeded on 6-well culture plate at1.5 · 105 cells/well and 5 · 105 cells/well, respectively. After 24 hr,the cells were treated with 1 mm melatonin for 10 hr in the absenceor presence of CoCl2. The VEGF protein levels in the culturemedium were analyzed by enzyme-linked immunosorbent assay asdescribed in Material and Methods. *Significant difference fromthe vehicle treated group in the absence of CoCl2 (P < 0.05).#Significant difference from vehicle treated group in the presence ofCoCl2 (P < 0.05).

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endothelin-1 synthesis [39]. Moreover, several investigatorsreported that melatonin inhibits angiogenesis directly orindirectly. In a preliminary clinical study showed exogenousmelatonin can reduce VEGF levels in vivo [27]. In vitro,

pharmacologic concentrations of melatonin inhibited pro-liferation of vascular endothelial cells [28]. Recently, it hasbeen reported that melatonin decrease the expression of

VEGF and HIF-1a induced by CoCl2 in cultured cancercells suggesting that melatonin exerts its antiangiogeniceffects through the inhibition of HIF-1a [29]. However, the

mechanism of its anti-angiogenic action is not fullyunderstood.

We show here that pharmacologic concentrations of

melatonin reduced VEGF expression in normoxia andhypoxic conditions induced by CoCl2. The reduction ofVEGF expression induced by pharmacologic concentrationof melatonin was associated with a decrease in HIF-1aprotein levels. These results suggest melatonin may sup-press angiogenesis by inhibiting HIF1-a expression.

The expression level of HIF-1a is regulated through both

protein degradation and protein synthesis. In this study, we

found that melatonin inhibited HIF-1a expression inprostate cancer cells in hypoxic conditions and even innormoxic conditions. Melatonin reduced HIF-1a proteinlevels without affecting either the half life of HIF-1a protein

and mRNA levels of HIF-1a in DU145 cells. Melatonininhibited the expression levels of HIF-1a protein despite ofthe inhibition of proteasomal protein degradation, and

metabolic labeling assays indicated that melatonindecreases de novo synthesis of HIF-1a protein. Theseresults suggest that melatonin inhibits expression of HIF-1aprotein through inhibiting protein translation rather thanenhancing protein degradation. Many studies have shownthat activation of oncogenes, inactivation of tumor sup-

pressor genes, and stimulation of various growth factorsincrease HIF-1a protein expression through enhancingHIF-1a translation in cancer cells [20]. Therefore, giventhe regulation of HIF-1a protein in the protein translation

step, our results suggest that melatonin has a potentialaction in inhibiting HIF-1a expression induced by activa-tion of oncogenes or inactivation of tumor suppressor

genes, and stimulation of various growth factors in cancer

Fig. 3. Melatonin does not affect HIF-1a degradation but inhibits HIF-1a protein translation. (A) DU145 cells were pretreated with vehicleor 1 mm melatonin for 30 min and cycloheximide (CHX) was added for indicated times. Equal amounts of protein from each sample wereresolved by SDS–PAGE, and Western blot was performed as described in Material and methods. Lower panel shows quantification of theHIF-1a signal by LAS 3000 image analyzer following normalization to actin levels. HIF-1a levels from CHX untreated cells are arbitrarilygiven the value of 100%. (B) Metabolic labeling was performed in normoxic DU145 cells as described in Material and methods. DU145 cellswere pretreated with 1 mm melatonin for 2 hr, and 35S-methionine/cystein was added to medium for 1 hr. DMOG (5 mm) or CHX (50 lm)were co-treated with 35S-methionine/cystein at indicated samples. (C & D) DU145 cells were co-treated with 1 mm melatonin and 10 lm

MG132 (C) or 5 mm DMOG (D) for indicated times. Equal amounts of protein from each sample were resolved by SDS–PAGE, andWestern blot was performed. Lower panels show quantification of the HIF-1a signal by LAS 3000 image analyzer following normalizationto actin levels. HIF-1a levels from untreated cells are arbitrarily given the value of 100%.

Melatonin inhibits HIF-1a expression

419

cells. Consistent with this speculation, melatonin inhibitedIGF-1 induced HIF-1a expression.It has been reported that p70S6K plays a role in HIF-1a

expression [22, 32]. Activation of p70S6K is thought to

enhance the translation of mRNA containing 5¢-terminaloligopyrimidine tract (TOP) sequences in their 5¢-UTR [40,41]. Interestingly, the 5¢-UTR of HIF-1a mRNA contains

5¢-TOP sequences [33, 42]. We have demonstrated thatmelatonin suppresses phosphorylation of p70S6K and itsdownstream targets RPS6 and eIF4B in parallel it decreases

HIF-1a protein level in all three tested prostate cancer celllines. This correlation between p70S6K phosphorylationand the expression level of HIF-1a protein was also

observed when DU145 cells were treated with growth factorIGF-1 and melatonin. Taken together, our results suggestthat melatonin-induced suppression of p70S6K might beinvolved in the inhibition of HIF-1a protein translation.

In summary, the findings show that melatonin in highconcentrations inhibits HIF-1a protein expression throughthe suppression of protein translation in prostate cancer cells.

Acknowledgment

This work was supported by grant No. R13-2002-028-02001-0 from the Basic Research Program of KOSEF forMedical Research Centers.

Author contributions

Jong-Wook Park contributed to acquire and interpretationof data, and drafting of this manuscript. Mi-Sun Hwangcontributed to acquire and interpretation of data, anddrafting of this manuscript. Seong-Il Suh contributed to

design of experiments and data interpretation. Won-KiBaek contributed to concept, design of the experiments,data interpretation, and revising the draft.

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