Melatonin protects against endometriosis via regulation of matrixmetalloproteinase-3 and an apoptotic pathway

Introduction

Endometriosis, a benign but invasive disorder, is commonin 10–15% of reproductive-age women. It is a cause ofconcern for infertility and ovarian cancer [1, 2]. Endome-

triotic lesions formation at an early stage is believed to beintricately associated with matrix metalloproteinases(MMPs) activity [3]. Therapy for endometriosis has been

a challenge and even today surgery is the gold standard.New therapeutics could open up with understanding on themechanism of MMP proteolytic cascades in endometriosis[4]. Although dissemination of menstrual fluid by retro-

grade menstruation has been used to explain endometriosis,the clues regarding invasion and attachment of menstrualdebris at ectopic sites were explained through MMPs

activity in remodeling of the endometrium [5–7].MMPs are a class of zinc-dependent endopeptidases that

actively participate in tissue remodeling and are precisely

regulated by their natural inhibitors known as tissueinhibitors of metalloproteinase (TIMPs) [8, 9]. Dysregula-tion of MMPs activity has been reported in different

pathogenic conditions notably arthritis and cancer [10–12].

Several MMPs including MMP-1, -2, -3, -7, and -9 have

received considerable attention as key players in thepathogenesis of endometriosis [13–18]. MMP-9 deservesspecial mention as its role in proteolysis and invasion inendometriosis has been confirmed by several authors [17,

18]. Despite the fact that MMP-3 or stromelysin-1 is centralto the proteolytic cascade because of its potency ofactivating other MMPs and because of its association with

other pathological conditions [19–22], the role of MMP-3 inendometriosis is still poorly understood.The early phase of endometriosis is difficult to study in

humans in vivo. The functions of MMPs have beenevaluated in murine model of surgically induced endome-triosis [15, 23]. We explored the role of activator protein-1

(AP-1) in regulating MMP-3 during endometriosis inmouse model. AP-1 is a known transcription factor forMMP-3 which is activated in both prooxidant and antiox-idant conditions [24, 25]. AP-1 is comprised of homo- or

heterodimeric proto-oncoproteins, fos and jun that bind totheir cognate consensus sequence of MMPs gene familyincluding MMP-3 [26, 27]. Additionally, the expression of

c-Fos is regulated by estrogen during the early phase of

Abstract: The role of matrix metalloproteinases (MMPs) in endometriosis, a

gynecological disease of women, is unclear. The study investigated the

activity of MMP-3 and its interplay with MMP-9 during the onset of

endometriosis. Additionally, the importance of MMP-3 on the apoptotic

pathway in endometriosis and effect of melatonin thereon were investigated.

A Significant increase in the activity of MMP-3 with the severity of

endometriosis in human was observed which was found similar in mice also.

During the early phase of endometriosis, MMP-3 but not MMP-9 was

increased and associated with the expression of transcription factor, c-Fos.

Moreover, urokinase plasminogen activator and tissue inhibitor of

metalloproteinase (TIMP)-3 were involved in MMP-3 regulation during

endometriosis. Furthermore, MMP-3 activity that was parallel to c-Fos

expression in endometriosis was reduced by melatonin pretreatment as

characterized by diminished activator protein (AP)-1 DNA-binding activity.

Because decreased apoptosis is an explanation for the perpetuation of

endometriosis, we tested the role of melatonin on apoptotic pathway in

preventing endometriosis. Significant regression of glandular epithelium was

observed in melatonin-treated when compared to untreated mice. Melatonin

treatment increased apoptotic cells in endometriotic zones. This was related

to reduced Bcl-2 expression along with increased Bax expression and

caspase-9 activation. In summary, early induction of MMP-3 was distinct

from MMP-9 during endometriosis, which was regulated by c-Fos and

TIMP-3. Melatonin suppressed MMP-3 activity and amplified apoptosis

while regressing endometriosis through a caspase-3 mediated pathway. Thus,

melatonin may be a therapeutic agent for resolving endometriosis.

Sumit Paul1, ParthaBhattacharya2, Pramathes DasMahapatra2 and SnehasiktaSwarnakar1

1Department of Physiology, Indian Institute of

Chemical Biology, Kolkata, India; 2Spectrum

Research Clinic, Kolkata, India

Key words: apoptosis, endometriosis,

inflammation, matrix metalloproteinase-3,

melatonin, tissue inhibitor of

metalloproteinase-3

Address reprint requests to Snehasikta

Swarnakar, Department of Physiology, Indian

Institute of Chemical Biology, 4, Raja S.C.

Mullick Road, Jadavpur, Kolkata 700032,

India.

E-mail: snehasiktas@hotmail.com

Received March 14, 2010;

accepted April 16, 2010.

J. Pineal Res. 2010; 49:156–168Doi:10.1111/j.1600-079X.2010.00780.x

� 2010 Indian Institute of Chemical Biology, CSIR, IndiaJournal compilation � 2010 John Wiley & Sons A/S

Journal of Pineal Research

156

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endometriosis, a common estrogen-dependent disorder [28,29]. Morsch et al. [30] reported that c-Fos gene expressionis higher in endometriotic implants compared to that of

normal endometrium.Melatonin, N-acetyl-5methoxy tryptamine, is mainly

secreted from pineal gland and is well known for its effecton seasonal reproduction and also circadian rhythm [31,

32]. Along with its metabolites, melatonin is capable ofscavenging radicals as well as stimulating antioxidantenzymes and has potent anti-inflammatory attributes [33–

36]. We reported earlier that melatonin arrests peritonealendometriosis in mice via downregulation of MMP-9 [18].However, the effects of melatonin on regulation of MMP-3

especially in endometriosis are unknown. Considerabledata suggest that melatonin can induce apoptosis and cellcycle arrest in various tumor cell lines [37, 38]. Caspases, afamily of cysteinyl aspartate-specific proteases, are known

as the executioners of apoptotic cell death. Althoughinitiator caspases (caspase-8, -9) undergo autoactivation,effector caspases (caspases-3, -6, -7) are activated following

processing at specific cleavage sites by other activatedcaspases. In the intrinsic apoptotic pathway, autoactivationof initiator caspase-9 is dependent on cytochrome-c. How-

ever, alternate mechanism of activation of caspase-9involves caspase-8 via Fas-ligand, an extrinsic pathwayfor apoptosis [39, 40]. A mouse model of endometriosis is a

helpful tool to evaluate melatonin�s action on MMP-3 aswell as the apoptotic pathway during protection of endo-metriosis.

Our study is the first to show the upregulation of secreted

and synthesized MMP-3 in human and mouse endometri-otic tissues. The results of this study provide a mechanisticbasis for MMP-3 upregulation in endometriosis and the

potential utility of endogenous antioxidant melatonin forregressing endometriosis.

Materials and methods

Chemicals

Gelatin from porcine skin, Triton X-100, protease inhib-itors mixture, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, and ethylenediaminetetraacetic acid

(EDTA) were obtained from Sigma (Sigma Aldrich Inc.,St. Louis, MO, USA). Prestained protein molecularweight marker and 100-base pair DNA ladder were

purchased from Fermentas (Fermentas Inc., Washington,DC, USA). Human and mouse reactive polyclonal anti-MMP-3, anti-MMP-9, anti-TIMP-3, anti-urokinase plas-

minogen activator (uPA), anti-TNF-a, anti-c-Fos, anti-Bcl2, anti-Bax, anti-Fas-L, anti-caspase 8,-3,-9, and anti-b-actin antibodies were purchased from Santa Cruz(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Bradford reagent was purchased from Bio-Rad (Hercules,CA, USA). Primers of MMPs and TIMP-3 were preparedby Primer-3 software and were purchased from Sigma

oligos (Sigmaoligos.com, Bangalore, India). TRIzol re-agent, Taq DNA polymerase, reverse transcriptase, andoligo (dT)15 primer were purchased from Invitrogen

(Carlsbad, CA, USA). All other chemicals were purchasedfrom a local company.

Collection of human samples

Patients with symptoms suggestive of endometriosis attend-

ing the gynecology unit of Spectrum Research Clinic,Kolkata, India, were included in the study. Endometriosiswas identified during laparoscopy in women consulting forinfertility and/or pelvic pain. Endometriotic tissues were

collected after approval of the Human Ethics Committeeand consent from patients. Briefly, we collected endome-triotic samples with severe endometriosis (stage III–IV) and

mild–moderate endometriosis (stage I–II). The stages ofendometriosis were indexed according to the score ofrevised American Fertility Society (rAFS) [41]. After

collection, all biopsies were stored at )70�C for futureexperiments.

Induction of peritoneal endometriosis in BALB/c miceand its protection by melatonin

Mature female BALB/c mice of 6–8 wk-old bred in-house

with free access to food and water were used in allexperiments. All experiments were carried out followingthe guidelines of animal ethics committee of the institute.

Endometriosis was surgically induced as previously de-scribed by Vernon and Wilson [23]. Briefly, the distalportion of one uterine horn was surgically removed and

cut into four equal size squares of uterine tissue usingaseptic technique. The other uterine horn was tied with silksutures served as a sham-operated control (Endo 0). Fourbiopsies were sutured to the peritoneal wall using silk

suture, and the abdominal incision was closed. Mice wereovariectomized 1 wk prior to induction of endometriosisand implanted subcutaneously with estrogen pellets.

Experiments were designed to minimize animal sufferingand to use the minimum number associated with validstatistical evaluation. Mice, four in each group (n = 24),

were sacrificed on 6, 48, 72 hr, 7th (Endo 7), 15th (Endo15), and 21st day (Endo 21) of postinduction of endome-triosis. Different doses of melatonin (16, 32, and 48 mg/kgb.w.) in 15% ethanol were administered intraperitoneally

(i.p) to separate group of mice (three in each group)30 min prior to induction of endometriosis and twice dailyfor the next 3 days to test their protective effects in

endometriosis (n = 18). Animals were anesthetized byketamine (12 mg/kg b.w.) and sacrificed by cervical dislo-cation. All animal studies were approved and conducted in

accordance with Institutional Animal Care Committeeguidelines.

Therapeutic model of peritoneal endometriosis inmice

Peritoneal endometriosis was induced in mice as described

previously. Mice, three in each group (n = 18), withperitoneal endometriosis were given melatonin (48 mg/kgb.w.) or vehicle intraperitoneally once daily for the follow-

ing 10 and 20 days from the 15th day of postendometriosisinduction. Earlier experiments were repeated three times.Regression of endometriotic lesions in melatonin-treated

and vehicle-treated mice was monitored after sacrificingthem on the 10th (Endo 25) and 20th (Endo 35) day.

Regression of endometriosis after melatonin

157

Tissue extraction

Tissues were suspended in phosphate buffer saline (PBS)

containing protease inhibitors, minced, and incubated for10 min at 4�C [18]. The suspension was centrifuged at12,000 g for 15 min, and supernatant was collected as PBSextracts. The pellet was further extracted in lysis buffer

(10 mm Tris–HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, and protease inhibitors) and centrifuged at 12,000 gfor 15 min to obtain Triton X-100 (TX) extracts. Proteins

were estimated either by Lowry method or by Bradfordassay [42, 43].

Histological studies

Endometriotic tissues and uteri were cut into 2–3 mm2

pieces. The tissue samples were fixed in 10% formalin for

48 hr, dehydrated in ascending alcohol series, and embeddedin paraffin wax. Approximately 5-lm-thick serial sectionswere stained with hematoxylin and eosin or subjected to

terminal deoxynucleotidyltransferase-mediated dUTP endlabeling (TUNEL) assay by using a commercial reagent kit(DeadEnd� Fluorometric TUNEL System, Promega,

Madision, WI, USA). Fixation, permeabilization, andstaining runs were carried out in exact parallel to ensurecomparative significance between groups. Images were cap-

tured at 40X under an Olympus microscope (1 · 70) usingCamedia software (Chicago, MI, USA) (E-20P 5.0 Mega-pixel) and processed using Adobe Photoshop version 6.0.

Casein and gelatin zymography

For assay of MMP-3 and -9 activities, tissue extracts were

electrophoresed in 8% SDS-PAGE containing 1 mg/mlcasein or gelatin respectively, under nonreducing conditions[44]. For human and mouse tissues, 30- and 70-lg proteins

respectively were loaded equally in all the lanes. The gelswere washed twice in 2.5% Triton X-100 and thenincubated in calcium assay buffer (40 mm Tris–HCl, pH7.4, 0.2 M NaCl, 10 mMCaCl2) for 21 hr at 37�C. Gels

were stained with 0.1% Coomassie blue followed bydestaining. The zones of gelatinolytic or caseinolyticactivities came as negative staining. Quantification of

zymographic bands was performed using densitometrylinked to proper software (Lab Image, Kapelan Gmbh,Leipzig, Germany) [44].

Reverse zymography

For assay of TIMP-3, tissue extracts were electrophoresedin 12% SDS-PAGE containing 1 mg/ml gelatin undernonreducing conditions. For MMP source, we used en-dometriotic tissue extracts of control mice containing 800-

lg total proteins that copolymerized with the gelatin-SDS-polyacrylamide gel. Various endometriotic tissue extractswere screened to confirm the presence of MMP-2 and

absence of MMP-9 before they were copolymerized withthe gelatin-SDS-polyacrylamide gel for analyzing TIMPs[45]. Endo samples (70-lg protein) were loaded equally in

all the lanes. The gels were washed and incubated asdescribed earlier.

Western blot

Tissue extracts (50 lg/lane) were resolved using 8% reduc-

ing SDS-PAGE and transferred to nitrocellulose mem-branes [18]. The membranes were blocked for 2 hr at roomtemperature in 3% BSA solution in 20 mm Tris–HCl, pH7.4 containing 150 mm NaCl and 0.02% Tween 20 (TBST)

followed by overnight incubation at 4�C in 1:200 dilution ofthe respective primary antibodies in TBST containing 0.2%BSA. The membranes were washed five times with TBST

and then incubated with alkaline phosphatase-conjugatedsecondary antibody (1:2000) for 1.5 hr. The bands werevisualized using 5-bromo-4-chloro-3-indolyl phosphate/

nitro blue tetrazolium substrate solution.

Reverse transcriptase-PCR (RT-PCR)

Total cellular RNA was extracted with TRIzol reagent(Invitrogen BioServices India Pvt. Ltd, Bangalore, India)according to the manufacturer�s protocol and quantified by

measuring the absorbance at 260 nm. ComplementaryDNA was synthesized using 1 lg of total RNA in a 20-lL reaction buffer using Superscript II Reverse Transcrip-

tase (Invitrogen BioServices India Pvt. Ltd) with anoligo(dT)15 primer (Invitrogen BioServices India Pvt.Ltd). The cDNA (1 lL) was then amplified in a 20-lLreaction buffer for 35 cycles of denaturation (94�C for 30 s),annealing (58�C for 30 s), and extension (72�C for 60 s)using the following primers: for MMP-3 sense, 5¢-GGATTGTGAATTATACACCGGAT-3¢ and antisense,

5¢-GGATAACCTGCTAGCTCCTCGT-3¢ (expected prod-uct 297 bp); for MMP-9 sense, 5¢-ACCTTCCAGTAGGGGCAACT-3¢ and antisense 5¢-TGAATCAGCTGGCTTTT

GTG-3¢, for GAPDH, sense, 5¢-TGGGGTGATGCTGGTGCTGAG-3¢, and anti-sense, 5¢-GGTTTCTCCAGGCGGCATGTC-3¢ (expected product 497 bp) for uPA sense 5¢-CACGCAAGGGGAGATGAA-3¢ and anti-sense, 5¢-ACAGCATTTTGGTGGTGACTT3¢ (expected product size341 bp) and for TIMP-3, sense 5¢-CTTGTCGTGCTCCTGAGCTG-3¢, and antisense-5¢-CAGAGGCTTCCGTGTGA

ATG-3¢. The PCR products were analyzed by electropho-resis in 2% agarose gels and visualized by ethidiumbromide staining. PCR product sizes were estimated by

100-bp DNA ladder in each case [44].

Preparation of nuclear extracts and electrophoreticmobility shift assay (EMSA)

Tissues were minced, hand-homogenized, and centrifuged

at 1000 g for 5 min at 4�C. After washing with ice coldPBS, pellets were suspended in 200 lL low-salt buffer(10 mm HEPES pH-7.9, 1.5 mm MgCl2 and 10 mm KCl),incubated for 10 min on ice, followed by vigorous mixing

after addition of 20 lL of 10% NP-40. Nuclei werecollected by centrifugation and resuspending in 50 lLhigh-salt buffer (20 mm HEPES pH-7.9, 420 mm NaCl,

1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol). Both bufferswere supplemented with protease inhibitors and 0.5 mm

DTT. Nuclei were incubated for 15 min on ice, vortexed

periodically, and centrifuged at 12,500 g for 10 min toobtain the nuclear extracts. The AP-1 (sense: 5¢- CTA GTG

Paul et al.

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ATG AGT CAG CCG GAT C- 3¢ and antisense: 5¢- GATCCG GCT GAC TCA TCA CTA G-3¢)-specific oligonu-cleotides were used for EMSA. Binding reactions were

performed for 30 min on ice with 50-lg nuclear extract and(c-32P) ATP-labeled oligonucleotide. Binding complexeswere electrophoresed in 7% nonreducing polyacrylamide-gels, dried, and radioactive signals were detected by

autoradiography [46].

Statistical analysis

Data were fitted using Sigma plot. Data obtained fromthree independent experiments were represented as the

mean ± S.E.M. P < 0.05 was accepted as level of signif-icance. The statistical analysis of the data was carried outusing GraphPad Instat 3 software. Comparison betweengroups was made using one-way analysis of variance

(ANOVA) followed by Student–Newman–Keuls test.Quantification of zymographic bands was performed usingdensitometry linked to proper software (Lab Image, Kap-

elan Gmbh, Germany).

Results

We first examined the activities of MMP-3 in eutopicendometrium of women with and without endometriosis

(Fig. 1A). We observed moderate (approximately 2.5-fold)upregulation of secreted proMMP-3 activity in eutopicendometrium of diseased women when compared to normal

(Fig. 1B). We then compared the activity of proMMP-3 inhuman endometriotic tissues possessing varying degrees ofseverity. Our results showed a strong correlation between

disease progression and the activity of both secreted andsynthesized proMMP-3. PBS and TX extracts were used toexamine secreted and synthesized MMP-3 activities inendometriotic tissues, respectively. It is revealed from

Fig. 1C,D that the proMMP-3 activities were graduallyelevated from mild to severe endometriosis with maximumupregulation of approximately 8.5-fold in case of secreted

and synthesized also in comparison with normal.Fig. 2A,B shows that from 7th day onwards, activity of

secreted proMMP-3 gradually increased to approximately

fourfold and attained approximately ninefold increment onthe 21st day of postendometriosis induction when com-pared to control in mouse model of endometriosis. Fig. 2Bfurther shows that synthesized proMMP-3 activity was

elevated by approximately fivefold on the 15th day, whichfurther increased to approximately ninefold on 21st daywhen compared to control. Fig. 2C further evidenced the

overexpression of proMMP-3 in 15th day when comparedto 0 day endometriotic tissue extracts. Our results alsorevealed the higher expression of MMP-3 mRNA in mouse

endometriotic tissues (Fig. 2D).The temporal regulation of MMP-3 and -9 activities in

early phase of endometriosis was tested using casein

(Fig. 3A) and gelatin zymography (Fig. 3C). We observeda rapid upregulation of proMMP-3 activity both at the levelof secretion and synthesis (Fig. 3B) in endometriotic lesions

(A) (B)

(D)(C)

Fig. 1. Severity-dependent increase in secretory and synthesized proMMP-3 activity in endometriotic tissues of normal women and womenwith endometriosis. Casein zymography of endometrial tissue extracts from women with (n = 28) and without (n = 14) endometriosisrepresenting secretory proMMP-3 activity (A). Densitometric analysis of secreted proMMP-3 activity of endometrial tissues (B). Caseinzymography was performed using biopsies extracted either in phosphate buffer saline or Tx to monitor the activity of proMMP-3 at the levelof secretion and synthesis in human samples possessing varying severities of endometriosis (C). Densitometric analysis of secreted andsynthesized proMMP-3 activity of endometrial tissues possessing varying severity (D). Activities were measured by using Lab Imagedensitometry program. Values are ± S.E.M. of the above zymogram and three other representative zymograms from independentexperiment. *P < 0.001 versus the appropriate control using ANOVA followed by Student–Newman–Keuls test.

Regression of endometriosis after melatonin

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from 48 hr onwards while no induction of proMMP-9activity either at the level of secretion or synthesis (Fig. 3D)during the first 72 hr in mouse model of endometriosis.

Interestingly, in experimental mouse model there wasupregulation of MMP-3 prior to MMP-9 and the previousremained high till day 21 suggesting the role of MMP-3 in

initial phases as well as in progression of endometriosis. Insupport, we found drastic surge in MMP-3 transcription asearly as 6 hr while changes in MMP-9 transcription wereobserved only during the late phase (Fig. 3E).

The changes in the MMP-3 and -9 protein expressionsduring the onset (Fig. 4A) and progression (Fig. 4B) ofendometriosis were similar to gene expression. A rapid

surge in MMP-3 expression with a peak at 48 hr wasobserved that remained high till day 21 of postendometr-iosis (Fig. 4C). On the other hand, MMP-9 expression

showed no detectable changes in control in the first 72 hr(Fig. 4C). We next examined the expressions of AP-1/c-Fostranscription factor during endometriosis (Fig. 4A,B). To

understand the sequential interplay of MMP-3, MMP-9and c-Fos with the progression of endometriosis, theirexpressions were plotted against time (Fig. 4C). A parallelinduction of c-Fos and MMP-3 during the early stage of

endometriosis was distinctly observed (Fig. 4C).The expressions of key markers of inflammation and

oxidative stress were monitored to dissect the early and late

pathologic events that caused differential temporal regula-tion of MMP-3 and -9. We observed significant differencesin the expressions of TNF-a with progression of endome-

triosis. During the first 72 hr of endometriosis no changesin TNF-a expression was observed (Fig. 4A) while asignificant increase with progression of the disease was

observed from day 7 onwards and marked the beginning ofincreased oxidative stress (Fig. 4B).Our results revealed overexpression of uPA gene in the

first 72 hr of postendometriosis (Fig. 4D). Moreover, c-Fosexpression surged down gradually from day 7 postendo-metriosis while uPA remained overexpressed till day 21postendometriosis, indicating its role in modulating MMP-

9 activity and expression as well (Fig. 4B). Furthermore,the functional role of TIMP-3 on MMP-3 activity duringendometriosis was examined. Surprisingly, the initial 72 hr,

which was marked by increased MMP-3 expression andactivity, showed no detectable change in the expression ofTIMP-3 (Fig. 4A). The activity of TIMP-3 decreased

significantly on 15th day postendometriosis compared toEndo 0. However, TIMP-3 underwent a drastic reductionin its expression from day 15 onwards, indicating the

involvement of TIMP-3 in modulating MMP-3 activityduring the later stage of endometriosis (Fig. 4B,E).Casein zymography using PBS and TX extracts of

endometriotic tissues from melatonin-pretreated and vehi-

cle-treated mice with peritoneal endometriosis were per-formed. Our results show that there was significantregression of endometriosis with increased doses of mela-

tonin pretreatment that paralleled with the reduction in theactivity of both secretory (Fig. 5A) and synthesized(Fig. 5B) proMMP-3. Melatonin at a dose of 48 mg/kg

(A) (B)

(D)(C)

Fig. 2. MMP-3 activity and expression inendometriotic tissues in mouse model.Mice were sacrificed on the 7th (Endo7),15th (Endo15), and 21st (Endo 21) daysfollowing endometriosis induction as de-scribed in Materials and Methods. Endo 0served as a sham-operated control. Caseinzymography was performed to monitorthe activity of proMMP-3 at the level ofsecretion and synthesis (A). Densitometricanalysis of secreted and synthesizedproMMP-3 activity at different time ofendometriosis induction (B). Activitieswere measured by using Lab Image den-sitometry program. Values are ± S.E.M.of the above zymogram and three otherrepresentative zymograms from indepen-dent experiment. Western blot analysis ofEndo 0 and Endo 15 samples using poly-clonal anti-MMP-3 antibody (C). RT-PCR analysis of MMP-3 mRNA expres-sion in sham-operated control and 15 dayendometriotic tissues of mouse (D).GAPDH was used as a positive control.*P < 0.001 versus the appropriate con-trol using ANOVA followed by Student–Newman–Keuls test.

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b.w. successfully prevented peritoneal endometriosis byreducing both secreted and synthesized proMMP-3 activityby �80% (Fig. 5C,D).

The expressions of MMP-3, TIMP-3, and TNF-a in PBSextracts of endometriotic samples were tested to understandmelatonin�s action (Fig. 5E). Melatonin pretreatmentblocked the expression of proMMP-3 by approximately

fourfold (Fig. 5E) compared to 15th day postendometriosismice. Moreover, it attenuated the expressions of TNF-athereby reducing inflammation (Fig. 5E) while stimulated

the expression of TIMP-3 by approximately twofold whencompared to nontreated ones (Fig. 5E). Electrophoreticmobility shift assay revealed that AP-1 DNA-binding

activity was induced between 6 and 72 hr (Fig. 5F lane 3–5) with a rapid surge at 48 hr paralleling the peak activity ofMMP-3 (Fig. 4C). Fig. 5F showed the higher expression ofMMP-3 mRNA in 15-day mouse endometriotic tissues that

was significantly inhibited by melatonin while TIMP-3expression followed reverse order. Additionally, significantreduction in AP-1 DNA-binding activity on melatonin-

treated samples revealed the regulatory role on AP-1inhibition in regression of early stages of endometriosis(Fig. 5G lane 6).Typical endometriotic lesions with an epithelial layer

and a stromal layer having numerous inflammatory cellswere observed in Endo15 when compared to Endo 0samples in mouse model of endometriosis (Fig. 6A,B) as

adjudged by histology. Considerable regression of thehighly glandular epithelium as well as number of inflam-matory cells within the stromal layer (Fig. 6C) was

observed in melatonin-treated Endo15 samples comparedto untreated ones.To further substantiate the role of melatonin on regres-

sion of endometriosis, we performed TUNEL assay. Sham-

(A) (B)

(C) (D)

(E)

Fig. 3. Differential regulation of MMP-3and MMP-9 during onset of endometri-osis. Casein zymography was performedto monitor the activity of proMMP-3 atthe level of secretion and synthesis duringthe early phase of experimental endome-triosis induced in mouse (n = 36). Ani-mals were sacrificed after 6 (Endo 6 hr),48 (Endo 48 hr), and 72 (Endo 72) hrafter endometriosis induction in mousemodel (n = 36). (A). Densitometric anal-ysis of secreted and synthesized proMMP-3 activity at different time of endometri-osis induction (B). Gelatin zymographywas performed to monitor the activity ofproMMP-9 at the level of secretion andsynthesis during the early phase of exper-imental endometriosis induced in mouse(C). Densitometric analysis of secretedand synthesized proMMP-9 activity atdifferent time of endometriosis induction(D). Activities were measured by usingLab Image densitometry program. Valuesare ± S.E.M. of the above zymogram andthree other representative zymogramsfrom independent experiment. RT-PCRwas performed to analyze the expressionsof MMP-3 and MMP-9 mRNA during theprogression of endometriosis in mousemodel (E). GAPDH was used as a positivecontrol. *P < 0.001, **P < 0.01, andNS, nonsignificant versus Endo 0 usingANOVA followed by Student–Newman–Keuls test.

Regression of endometriosis after melatonin

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operated control uteri (Endo 0) of mouse showed no typicalstaining of DNA fragmentation when compared to Endo 15samples (Fig. 6D). As evidenced from Fig. 6E,F melatonin

promoted apoptosis in 15-day postendometriosis.Although, some apoptotic cells were visible in the untreatedgroup (Fig. 6E), which, however, can be explained to theauto healing (in absence of melatonin) response that

occured with time in surgically induced endometriosis inmouse model.

To elucidate the mechanism of melatonin-induced apop-tosis, the expressions of key apoptotic markers wereexamined. It is evident from Fig. 6G that �no melatonin�response was mediated via the death receptor pathway assupported by the increased expression of Fas-L and activecaspase-8. Moreover, there was no change in the expressionratio of Bcl2 and Bax in auto healing response. On

contrary, to the vehicle treatment, melatonin-mediatedapoptosis occurred via the classical pathway which was

(A) (B)

(C)

(D) (E)

Fig. 4. Potential mechanism of MMP-3and -9 regulation during onset and pro-gression of endometriosis in mice. Phos-phate buffer saline extracts of tissues from(A) early phase and (B) late phase weresubjected to Western blot and probed withpolyclonal anti-MMP-3, anti-MMP-9,anti-TNF-a, anti-TIMP3, anti-urokinaseplasminogen activator, and anti-b actinantibodies. Nuclear extract was preparedfrom tissues sacrificed at several timeintervals to monitor change in c-Fosexpression by using polyclonal anti-c-Fosantibody. The number listed under eachlane represents its fold intensity comparedto control value. The protein expressionsof proMMP-3, proMMP-9, and c-Fos atdifferent time points were quantified fromtheir corresponding Western blots bydensitometric analysis and was plottedagainst time (C). Fold expressions weremeasured by using Lab Image densitom-etry program. Values are ± S.E.M. of theabove zymogram and three other repre-sentative blots from independent experi-ment. RT-PCR analysis was performed toanalyze urokinase plasminogen activatorgene expression during early phase ofexperimentally induced endometriosis inmouse model (D). GAPDH was used as apositive control. Reverse zymography toassess TIMP-3 activity in endometriotictissues (E).

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associated with reduced Bcl2 expression and Bax induction

with time (Fig. 6H). Fig. 6G shows that active caspase-3and active caspase-8 were upregulated in vehicle-treatedendometriotic tissues. However, Fas-L expression remainedsimilar in melatonin-treated tissues compared to vehicle

ones (Fig. 6G,H).Having found protective effect of melatonin on endome-

triosis, we then tested therapeutic effect of melatonin from

day 15 onwards in surgically induced endometriosis in mice.Endometriosis was halted in a time-dependent manner on atherapeutic regimen of melatonin administration that

continued 20 days, and proMMP-3 activity at the level ofsecretion and synthesis was decreased in extracts ofendometriotic tissues(Fig. 7A,B). Melatonin progressively

decreased the secreted proMMP-3 activity by �40% and�80% (Fig. 7C), while the synthesized proMMP-3 activity

was blocked by �35% and �80% (Fig. 7D) on the 25th-

and 35th-day, postendometriosis respectively when com-pared to vehicle-treated ones.

Discussion

Endometriosis is an inflammatory disease that is charac-terized by growth of uterine tissues outside the uterine

cavity [4]. Endometriotic lesions usually show increasedexpression of proteolytic enzymes [17], and the progressionof endometriosis is estrogen dependent [29]. Previously, we

have documented the upregulation of MMP-9 activity inboth human and mouse endometriosis [18]. Numerousstudies described the presence of MMP-9 or -3 separately in

endometriosis. However, the importance of MMPs andtheir balance with TIMP in remodeling of endometrium

(A)

(C) (D)

(B)

(E)(F)

(G)

Fig. 5. Role of melatonin in modulationof MMP-3 activity and expression duringprotection against endometriosis in mousemodel. Mouse endometriotic tissues trea-ted with different doses of melatonin weresubjected to casein zymography to moni-tor secreted (A) and synthesized (B)proMMP-3 activity. Histographic repre-sentation of arbitrary activity of secreted(C) and (D) synthesized proMMP-3against melatonin doses. Activities weremeasured by using Lab Image densitom-etry program. Values are ± S.E.M. of theabove zymogram and three other repre-sentative zymograms from independentexperiment. *P < 0.001, **P < 0.01, andNS, nonsignificant versus Endo 0 usingANOVA followed by Student–Newman–Keuls test. Phosphate buffer salineextracts of melatonin-pretreated and un-treated mouse endometriotic tissues weresubjected to Western blot and probed withpolyclonal anti-MMP-3, anti-TIMP-3monoclonal anti-b actin antibody (E).Fold changes were measured by using LabImage densitometry program from theabove blots and three other representativeblots from independent experiments ineach case. RT-PCR analysis of MMP-3,TIMP-3 mRNA expression in sham-operated control and 15-day endometri-otic tissues of mouse with or withoutmelatonin pretreatment (F). GAPDH wasused as a positive control. EMSA wasperformed to detect the DNA-bindingactivity of AP-1 from the nuclear extractsof early phase endometriosis in mousemodel (0–72 hr) with or without melato-nin treatment. Lane 1 represents freeprobe and lane 6 represents melatonin-pretreated mouse possessing endometri-osis for 48 hr (G).

Regression of endometriosis after melatonin

163

have not been elucidated clearly [14, 17, 18]. MMP-3 is anexcellent candidate for participation in tissue remodelingand invasion in the peritoneal cavity and more importantly,an activator of latent MMPs including progelatinase

(proMMP-9) as well as procollagenases (proMMP-1 and -8) [19–22]. In accordance, Cox et al. [15] documented thehigh expression of MMP-3 gene in ectopic endometrialtissues in rat. As the coordinated activity of different

(A) (B) (C)

(D)

(G) (H)

(E) (F)

Fig. 6. Protective role of melatonin on endometriosis via regulation of apoptotic pathway. Hematoxylin and eosin staining of sham-operated control mouse uteri labeled as Endo 0 (A), endometriotic tissues of surgically induced mouse endometriosis of day 15 labeled asEndo15 (B), melatonin-pretreated Endo15 (C). The blue arrows represent glandular epithelium; the green arrows represent the stromallayer, and the pink arrows for infiltrating inflammatory cells. TUNEL assay to monitor apoptosis in endometriotic tissues of mouse model.Sham-operated control mouse uteri (D) endometriotic tissues of surgically induced mouse endometriosis of day 15 labeled as Endo15 (E)melatonin pretreated Endo 15 (F) Blue arrow represents DNA fragmentation in stromal layer of endometriotic tissues in green fluorescence,and purple arrow represents epithelial layer of endometriotic tissues. Micrographs were recorded at 40· magnification using both FITC andrhodamine filter. Fluorescein -12-dUTP (green) was used as primary stain and propidium iodide (red) as counterstain. Phosphate buffersaline extracts of tissues from melatonin untreated (G) and melatonin pretreated (H) mouse endometriotic tissues were subjected to Westernblot and probed with polyclonal anti-Bcl2, anti-Bax, anti-FasL, anti-caspase-8, 3, -9, and anti-b actin antibodies.

Paul et al.

164

MMPs is critical, the regulatory effect of MMP-3 may be

important in the overall regulation of MMP-9 in theendometrium. It may be possible that MMP-9 upregulationin ectopic uterine tissue occurs via a cascade of events

involving MMP-3 because MMP-3 activity appeared priorto MMP-9 in surgically induced mouse endometriosis.Assuming MMP-3 involvement in initial phases of endo-

metriosis, we propose that MMP-3 might be responsible forthe upregulation of MMP-9 which in turn utilizes distinctmechanisms for aggravation of the disease, whose signaling

is further controlled by TIMP-1 and TIMP-3 expressions.The biological importance of TIMP-3 has become increas-ingly apparent mainly as a regulator of inflammation owingto blocking the release of TNF-a [47]. Moreover, recent

evidences indicate the important role of TIMP-3 inpromoting cell death by apoptosis [48–50]. We observedsignificant downregulation of TIMP-3 from day 15 to day

21 postendometriosis induction which was responsible forenhancing MMP-3 activity at a post-translational phase.Additionally, the expression of uPA in endometriotic

samples might play a role on MMP-3 upregulation viaplasmin [21, 51].

Herein, the overexpression of c-Fos, which is dependenton estrogen response in early phase of endometriosis, was

associated with MMP-3 expression. The promoter region ofMMP-3 gene has AP-1 responsive element for binding to c-Fos, an estrogen-induced early response gene [26–28]. In

this study, gel shift assay revealed direct dependence of AP-1 activation on MMP-3 expression during the initiation ofendometriosis. Thus, the process described in this study

may represent an additional mechanism by which MMP-3is upregulated in AP-1 dependent signaling pathway atearly phase of the disease. Moreover, the role of inflam-

matory molecules involved in signal for MMP-3 upregula-tion in endometriosis has not been documented so far. Toaddress the issue, we tested TNF-a expression along withMMP-3 during progression of endometriosis in mouse

model. TNF-a is a multifunctional cytokine and plays animportant role in uterine physiology [52, 53]. Several

evidences documented the increased expressions of TNF-ato the aberrant expressions of MMPs [54]. TNF-a canindeed be targeted to relieve inflammatory load in endo-metriosis, as studies documented the reduced development

of endometriosis by blocking its action [55, 56]. Wespeculate that MMP-3 activity may be enhanced duringinflammation in endometriotic lesions.

Targeting MMPs to regress endometriosis seems to be aviable option [57, 58]. We addressed the question as towhether melatonin has any effect on MMP-3 activity and

expression during regression of endometrial lesions. Mela-tonin efficiently attenuated proMMP-3 activity both at thelevel of synthesis and secretion during regression ofendometriosis in both protective and therapeutic model of

endometriosis in mice. This report also documents theinduction of apoptosis by melatonin that regressed endo-metriosis in mouse model. It has been shown that melatonin

inhibits cell growth of several types of cancers and moreimportantly at high concentration, melatonin displaysoncostatic actions in neuroblastoma cell lines. These

actions of melatonin are independent of its receptor-mediated property that functions at nanomolar concentra-tion but relate to the antioxidant properties requiringmillimolar concentration in some cases [35, 37]. Melatonin

therapy showed no side effects when high doses wereadministered to rodents over long periods of time [59].Pharmacological doses of this indole used in human cancers

have improved survival [60]. Herein, we also show that theapoptotic pathway was activated by melatonin followingthe classical caspase 3-dependent pattern that was indepen-

dent of Fas-L-mediated pathway. In addition, melatoninexhibited extrinsic pathway for apoptosis. This extrinsicpathway was only found in absence of melatonin in

protective model of endometriosis in mice. We furtherevaluated an active role of melatonin on Bax inductionduring regression of endometriotic lesions in mice. Thisscenario is in agreement with our results achieved by

histology and TUNEL assay. Our findings indicate signif-icantly higher expression of TNF-a at later stages of disease

(A) (B)

(C) (D)

Fig. 7. Effect of melatonin on MMP-3activity during healing of endometriosis inmouse model. Endometriotic tissues ofmice with or without melatonin treatmentfor 10 (Endo 25) and 20 days (Endo 35)were subjected to casein zymography toassess secretory (A) and synthesized (B)proMMP-3 activity. Histographic repre-sentation of secreted (C) and synthesized(D) proMMP-3 activity with time oftreatment with melatonin or vehicle.Activities were measured by using LabImage densitometry program. Valuesare ± S.E.M. of the above zymogram andthree other representative zymogramsfrom independent experiment. *P < 0.001and NS, nonsignificant versus appropriatecontrol using ANOVA followed by Stu-dent–Newman–Keuls test.

Regression of endometriosis after melatonin

165

progression. Additionally, melatonin acted as an anti-inflammatory agent as evidenced by reduced expression ofTNF-a. Melatonin�s action on therapeutic model of mouse

endometriosis as well as preventive model was found to beeffective in arresting endometriosis. Moreover, the upreg-ulation of TIMP-3 by melatonin may also suggest its anti-inflammatory role in regressing endometriosis. This data is

in support to the fact that mice deficient in TIMP-3 havesevere inflammation in liver [47]. The fact that melatoninupregulated the expression of TIMP-3 and regressed

endometriosis by induction of apoptosis suggests a role ofTIMP-3 in melatonin-mediated apoptosis. Furthermore,TIMP-3 downregulation during the late phase of endome-

triosis provides a clue to the delayed apoptotic responseduring auto healing of endometriosis in this study.Although it is beyond the scope of this study, it is possiblethat melatonin can act as an anti-estrogenic agent in

regression of endometriotic lesions in mice. Several studieshave demonstrated the anti-estrogenic role of melatonin[61, 62].

Using mouse model of endometriosis, we showed thatMMP-3 activity and expression were increased at earlyphase of endometriosis that was parallel to the expression

of c-fos. Interestingly, we also found maximum upregula-tion of MMP-3 that reached its peak on day 3 postendo-metriosis and then declined. The upregulation of MMP-9

occurred day 3 onwards thereby suggesting its importanceon late phase of endometriosis. Our study documents thatmelatonin can act beyond the conservative role of anantioxidant and inhibits over expression of c-Fos, thereby

regressing endometriosis at early and late stages as well.Previously described properties of melatonin together withthe fact that it is highly effective as an apoptotic agent

suggesting its potential therapeutic value in relievingendometriosis. We hypothesize that melatonin signals theamplification of apoptosis via classical mitochondrial

pathway, which is lacking in absence of melatonin thatresults in decelerated healing of endometriotic lesions.

Acknowledgements

This study was supported by grants MLP13 and IAP 001 ofCouncil of Scientific and Industrial Research, India, and

GAP209 of Indian Council of Medical Research, NewDelhi, India. SP is recipient of senior research fellowshipfrom Council of Scientific and Industrial Research, India.

Conflict of interest

Authors have nothing to disclose.

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