[CANCER RESEARCH54, 2429-2432, May 1, 19941

apparent control of tumor growth in patients with advanced solidneoplasms (10—13).However, chemotherapy still remains the mostused tool to fight cancer. Unfortunately, a major barrier to achievingthe best possible response to cancer chemotherapy is the hematological toxicity of available agents, which limits optimal dosing (14). Tocircumvent this problem, hematopoietic rescue by colony-stimulatingfactors, interleukin 3, and/or autologous bone marrow transplantationis increasingly used (15—17). Handling of such procedures remains,

however, problematic because of negative side effects or incompletemarrow regeneration, respectively (15—17).The purpose of this studyis to determine whether melatonin can protect the bone marrow inmice transplanted with LLC3 and treated with the alkylating agentcyclophosphamide or the topoisomerase Il-reactive drug, etoposide.The in vitro effect of melatonin on apoptosis of bone marrow cells andsurvival of hematopoietic progenitor cells upon incubation with etoposide is also studied.

MATERIALS AND METhODS

Mice. Female C57BL/6 mice (2—3months old) were purchased fromCharles River Italia, Como, Italy. Age- and sex-matched C57BIi6'°'@athymic mice were purchased from The Jackson Laboratory, Bar Harbor, ME. Theanimals were kept under a 12-h light/dark cycle at 21 ± 1°Cwith free access

to food and water.Drugs and Reagents. Melatonin was graciously provided by Helsinn

Chemicals SA, Breganzona, Switzerland. Cyclophosphamide and etoposidewere purchased from Sigma Chemical Co., St. Louis, MO. Monoclonal ratanti-mouse GM-CSF antibody was purchased from Genzyme Co., Cambridge,MA

Lewis Lung Carcinoma. LLC was a kind gift of Dr. Laura Perissin,

Trieste, Italy. The tumor was maintained by serial passages in C57BL/6 miceas described (18). For the experiments, the primary tumor was excised fromdonor mice and teased by a loose-fitting teflon pestle; the cells were dissociated to obtain a single cell suspension in saline. After being washed, the cellswere counted, and viable cells (2 X 1O@)suspended in 200 p1 saline wereinjected i.m. in the left hind leg. Primary tumor size and number of lungmetastases were evaluated 20—22days after tumor inoculation. Primary tumorsize was evaluated by measuring the diameter of the leg at the tumor level witha caliper in two perpendicular directions. The tumor cross-section is consideredan ellipse, and its size was calculated according to the method of BartholeynsetaL (19)as

Tumor cross section (@2) = fl/4 [(ti t2) —(ci c2)}

where ti and t2 are the perpendicular axes of the tumorous leg and ci and c2are the perpendicular axes of the right control leg. Lung metastases wereenumerated in the dissected lungs by a dissection microscope.

Therapeutic Protocol. Melatonin was injected s.c. once a day at 4:00 p.m.at a dose of 1 mg/kg b.w. starting on day 8 after tumor transplantationthroughout the experiment. In some experiments, melatonin was injected only

during treatment with cyclophosphamide (days 8—12) or afterwards (day 13

through the end of the experiment). Cyclophosphamide or etoposide wasinjected i.p. once a day at 12:00 p.m. from day 8 through day 12. Two

3 The abbreviations used are: LLC, Lewis lung carcinoma; GM-CSF, granulocyte

macrophage colony-stimulating factor; b.w., body weight; MEM, minimal essential medium; GM-CFU, granulocyte-macrophage colony-forming units; S-CFU, spleen-colonyforming units.

2429

Hematopoietic Rescue via T-Cell-dependent, Endogenous Granulocyte-Macrophage

Colony-stimulating Factor Induced by the Pineal Neurohormone Melatonin in

Tumor-bearing Mice'

Georges J. M. Maestroni,2 Valeria Covacci, and A. ContiCenter for Experimental Pathology, Istituto Cantonale di Patologia, 6604 Locarno, Switzerland

ABSTRACT

We Investigated whether melatonin can affect tumor growth and/orhematopoiesis in mice transplanted with Lewis lung carcinoma andtreated with cyclophosphamide or etoposide. These agents were injectedLp. for 5 days at two different cumulative doses (cydophosphamide, 40and 160 mg/kg body weight; etoposide, 20 and 40 mg@kgbody weight)from day 8 through day 12 after tumor transpiantadon. Melatonin wasinjected s.c. at a dose of 1 mg/kg body weight/day, from day S throughoutthe experiments and from days 8 through 12 or from day 13 onwards.Melatonin did not influence tumor growth but selectively counteractedbone marrow toxicity when administered together with the cancer chemotherapy compounds without interfering with their anticancer action. Invitro, melatonin proved to counteract apoptosis in bone marrow cellsincubated with etoposide. Such protection was reflected by an Increasedfrequency ofgranulocyte/macrophage-colony forming units but not of thepluripotent spleen-colony forming units. The effect of melatonin wasneutralized by anti-granulocyte/macrophage-colony-stimulatlng factormonoclonal antibodies. When athymic, T-cell-deficlent mice were used asbone marrow donors, melatonin did not exert any protective effect. Thissuggested that melatonin Is able to stimulate the endogenous production of

granulocyte/macrophage-colony-stlmulating factor via bone marrowT-cells. Due to the well known lack of toxic and undesirable side effectsof melatonin, these findings might have a straightforward clinicalapplication.

INTRODUCTION

Melatonin is an indoleamine synthesized from serotonin in thepineal gland. Its synthesis and release follows a circadian rhythm withthe highest blood concentration occurring at night in all species (1).Melatonin regulates fertility in seasonally breeding animals (2),whereas its role in other species, including human, is less clear (2). Inprevious work, we have shown that melatonin can augment the

immune response and correct immunodeficiency states which mayfollow acute stress, viral diseases, aging, or drug treatment (reviewedin Ref. 3). Relevant to the present study, we observed that melatoninwas able to antagonize the effect of high dose cyclophosphamide on

antibody production (4). This finding has been then confirmed andextended to other immune parameters by other authors (5, 6). Suchinteresting effects of melatonin seem to depend on activated CD4+,T-cells which upon melatonin stimulation show an enhanced synthesisand/or release of opioid peptides, interleukin 2, and y-interferon (3,5—8).A large body of evidence indicates also that melatonin mayinhibit carcinogenesis and tumor growth in a variety of experimentaland clinical situations (9). On the basis of our animal studies (3), wehave investigated the clinical effect of melatonin in association withlow-dose interleukin 2 in cancer patients and found that this association represents a well-tolerated strategy capable of determining an

Received 12/22/93; accepted 3/1/94.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This study has been supported by the Ciba-Geigy JubilAums Stiftung and by the

pharmaceutical concem Angelini, Rome, Italy.2To whom requests for reprints should be addressed.

on April 19, 2021. © 1994 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Table I Effect of melatonin and cancer chemotherapy agents in LLC-bearingmiceCyclophosphamide

(CY) and etoposide were injected at the doses indicated (mg/kg b.w.) in C57BIJ6 mice from day 8 through day 12 after LLC transplantation. Melatonin(ME)wasinjected from day 8 throughout the experiments. The values represent the mean ±SD. Leukocytes and platelets were evaluated 16 days after tumor transplantation, whereastheremaining

parameters were measured at the end of the experiments (days20—22).Platelets/pi

Primary TumorGroups (n) Leukocytes/pi (X 10@) GM-CFU/Femur (cm2) No. ofmetastasesControl―

(10) 10300 ±1070 253 ±69.5 8459 ±1674PBS (64) 17128 ±3264― 3@ ±79.6 10303 ±2907 4.09 ±0.87 16.8 ±6.7ME

(46) 12960 ±2667 287 ±74.1 9835 ±4067 3.97 ±0.73 12.8 ±5.2CY16O(10) 5650 ±980 161 ±43.3 2471 ±490 0 0

CYI6O + ME (12) 11083 ±2882'@ 316 ±59.7' 5927 ±1898― 00CY4O(20) 8310 ±1343 209 ±45.7 6569 ±1791 2.8 ±0.88 3.95 ±1.47

CY4O + ME (20) 16875 ±2754c 344 ±51.9c 12644 ±2479― 2.9 ±1.48 4.3 ±4.4ET4O(14) 8943 ±251 149 ±23.8 4089 ±1528 1.65 ±0.76 1.4 ±1.4

ET4O + ME (14) 10912 ±1352c 316 ±77' 6459 ±2826'@@ ±0.87 1 ±0.9ET2O(22) 8673 ±I 184 238 ±44.4 3916 ±1343 2.56 ±0.6 3.09 ±2.2

ET2O + ME (27) 13215 ±2845― 272 ±44.8 8052 ±3407b 2.44 ±1.25 2.8 ±2.5

Table 2 Timing effect of melatonin in relation to cyclaphosphamidetreatmentMelatoninwas injected either together with cyclophosphamide from day 8 throughday12

after tumor transplantation (ME and CY) or only after CY treatment starting on day13tillthe end of the experiment (CY —ME). Cyclophosphamide was injected at 160mg/kgb.w.

The values represent the mean ±SD and have been measured on day18.Platelets/pi

Groups (n) Leukocytes4@; (X 10@) GM-CFU/10@cellsPBS―

(7) 19260 ±2971 205 ±28.2 68.1 ±4.1CY(7) 4957±1013 115±24.3 12.7±4.7

ME and CY (7) 10028 ±1@3b 226 ±25c 42.6 ±56dCY —ME (7) 8117 ±3358 149 ±42.1 24.5 ±6.5―

PREVENTION OF MARROW TOXICITY BY MELATONIN

a Control, normal healthy mice; PBS, phosphate-buffered saline; CY, cyclophosphamide; ME, melatonin.bp < o.oi.

Cp < 0.001.

dp < 0.005.

cumulative doses of 40 and 160 mg/kg b.w. or 20 and 40 mg/kg b.w. were used

for cyclophosphamide and etoposide, respectively.In Vitro Induction of Apoptosis. Bone marrow cells were collected from

the long bones, suspended in a-MEM and 5% horse serum, and incubated for

8 h in triplicate and at a concentration of 3 X 106cells/ml in presence of thereported concentrations of etoposide ±melatonin. After incubation at 37°Cand 5% CO2 in 24-well tissue culture plates (Becton Dickinson, Oxnard, CA),cells were harvested by repeated rinsing of the wells. Cell aliquots wereprocessed for the GM-CFU and S-CFU assays and/or fixed in 4% formaldehyde in ethanol overnight, washed three times in water, stained with 10 p@g/mlacridine orange (Fluka AG, Buchs, Switzerland) in saline and examined for

nuclear chromatin morphology in a fluorescence microscope. Apoptotic cellswere clearly identified by their condensed or fragmented chromatin. At least200cellswerecountedfromeachwell,givingreproducibilityto within4%.Incertain experiments, monoclonal rat anti-mouse GM-CSF antibodies wereadded during incubation of bone marrow cells with etoposide + melatonin.The cells were then washed three times and processed for GM-CFU evaluation.

Leukocyte and Platelet Counts. The concentration of leukocytes andplatelets was determined microscopically in a Neubauer chamber in venous

blood obtained from the tail after light ether anesthesia 16 days after tumorinoculation.

GM-CFU and S-CFU. To evaluate the concentration of GM-CFU eitherafter in vivo treatment or after in vitro incubation, i05 viable bone marrow cellswere incubated in 0.3% semisolid agar in a-MEM containing 20% horse serumand 20% lung conditioned medium as source of GM-CSF. Lung conditionedmedium was prepared by mincing the lungs from 2-month-old mice into smallpieces and by incubating the pieces at 37°Cin 5% CO2 in a-MEM and 20%horse serum for 3 days. Cultures were maintained for 7 days at 37°Cin 5%CO2 in humidifiedair and then examined by phase microscopy. Coloniescontaining more than 50 cells were counted as GM-CFU. S-CFU were evaluated by injecting iv. 10@viable bone marrow cells after lethal irradiation (900

cGy; X-ray exposure performed by a linear accelerator; 15 MV energy equivalent) of recipient mice. Spleen colonies were counted 12 days after honemarrow inoculation and 24 h after fixation in Bouin's fluid.

Statistics. Differences were evaluated for significance by analysis ofvariance.

RESULTS

Effect in LLC-bearing Mice. Table 1 reports the effect of theassociation of melatonin with cyclophosphamide or etoposide ontumor growth, blood counts, and number of bone marrow GM-CFU inmice transplanted with LLC. In accordance with previous reports (20),the presence of LLC per se induced an increase of myelopoiesiswhich, in our hands, appeared to be significant only for leukocytecounts (phosphate-buffered saline versus control; Table 1). Such anincrease appeared to be counteracted by melatonin treatment. In theopposite direction, melatonin exerted a significant protection against

bone marrow toxicity induced either by cyclophosphamide or etoposide. Such an effect was apparent and highly significant on leukocytes, platelets, and marrow GM-CFU. On the contrary, melatonin per

se or in association with the antitumor compounds did not influenceeither the size of the primary tumor or the number of lung metastases;also, it did not interfere with the action of the cytotoxic drugs. Toinvestigate whether melatonin acted by preventing bone marrow toxicity or by enhancing the posttreatment recovery of hematopoiesis,experiments were devised in which melatonin was injected only

during cyclophosphamide treatment or only thereafter. Table 2 demonstrates that melatonin was able to antagonize the hematopoietictoxicity of cyclophosphamide when injected together with the drug.Bone marrow protection appeared to be less effective if melatonin wasinjected after cyclophosphamide treatment. These results indicatedthat melatonin is able to protect the bone marrow in the course ofcytotoxic anticancer treatments. However, a similar although lessapparent effect was also observed when melatonin treatment followedcyclophosphamide.

In Vitro Effect of Melatonin on Drug-induced Apoptosis andSurvival of Progenitor Cells We reasoned that the protection ofhematopoiesis exerted by melatonin in vivo might reflect a directaction on bone marrow cells. To challenge this hypothesis, experiments were set up in vitro by incubating normal bone marrow cellswith etoposide in the presence of various concentrations of melatonin.The first question we asked was whether melatonin is able to rescuemarrow cells from apoptosis induced by the anticancer drug. Fig. 1shows that from 10 nr@ito 1 ,.LM,melatonin is able to prevent apoptosisin bone marrow cells incubated with 10 @tMetoposide. Cyclophosphamide was not used because it is well known to be inactive in vitro.However, similar results were obtained with carboplatin, which has a

a PBS, phosphate-buffered saline; CY, cyclophosphamide; ME, melatonin.bp <Cp <dp < 0.001 versus CY.

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. ET(10pM)@ MEDIUM

Table 4 Failure of melatonin to protect bone marrow cells from athymicC57BL/6'°'°'miceBone

marrow cells from C57B116°――,athymic mice were incubated for 8 hourseither in culture medium alone (control) or with the reported concentrations of ET and METhe values represent the mean of 3 experiments ±SD.Incubation

% apoptosis GM-CFU/10@'cellsControl

16.9 ±4.6 132 ±42El―(10 ELM) 49.2 ±5.2 35.7 ±13.5ET (10 @LM)+ ME (1 ELM) 44.5 ±3.4 46.2 ±13.7ME (1 @.LM) 17.2 ±6.3 135 ±38a

ET, etoposide; ME, melatonin.

Table 3 Effect of melatonin on survival of hematopoietic progenitor cellsuponincubationwith etoposide of bone marrow cells from C57BL/6miceBone

marrow cells from C57BL16mice were incubated either in culture mediumalone(control)or with the reported concentrations of etoposide (El) and melatonin (ME).Thevalues

represent the mean of 4 experiments ±SD.Incubation

GM-CFU/10@ ells S-CFU/10@cellsControl

75 ±19 17.6 ±2.6Er(10 p@M) 16.6 ±4.7 6.5 ±3.9

ET(10@M) + ME(1 ELM) 35.7±9.5― 6.8±1.9ME(1 @sM) 72.5 ±16 12.4 ±3.1k?

PREVENTION OF MARROW TOXICITY BY MELATONIN

mechanism of action close to that of cyclophosphamide metabolites(Ref.21 and data not shown). When bone marrow cells, preincubatedwith 1 @LMmelatonin and 10 p@Metoposide were assayed for GM-CFUand S-CFU content, it appeared that melatonin protected GM-CFUprecursors but not the less differentiated, pluripotent S-CFU progenitor cells (Table 3). Thus, the cell types which were rescued fromdrug-induced apoptosis seems to include lineage-committed, myeloidprogenitor cells but not pluripotent S-CFU precursors. When antimouse GM-CSF antibodies were added during the preincubation ofbone marrow cells with etoposide and melatonin, no protection wasevident (Fig. 2). This suggested that the effect of melatonin wasmediated by a factor which is released by bone marrow cells and thatis biologically and immunologically indistinguishable from GM-CSF.

In Vitro Effect of Melatomn on Bone Marrow Cells from Athymic, T-Cell-deflcient Mice. In bone marrow cell suspensions, theprincipal sources of GM-CSF are macrophages and T-cells. Athymicnude mice are T-cell-deflcient and have a poor capacity to mountimmune responses, which is in part balanced by enhanced macrophage functions (22). Therefore, we chose T-cell-deficient, nude miceas donors of bone marrow cells to investigate which cell type couldrelease GM-CSF upon melatonin stimulation. Table 4 shows thatwhen bone marrow cells were obtained from T-cell-deficient mice,melatonin did not exert any protection against etoposide toxicity. Thisindicated that T-lymphocytes are involved in the hematopoietic effectof melatonin and that bone marrow T-cells may be the melatonintarget.

GM-CFU

0 20 40 60 80 100

a-GM-CSF

ET (10pM) + ME ( 1pM) + a-GM-CS}

ET (10pM) + ME ( 1pM

ET (10 pM)

Fig. 2. Monoclonal rat anti-mouse GM-@SFneutralizes the effect of melatonin. Bonemarrow cells from C57B1J6 mice were preincubated for 8 h in culture medium alone orin the presence of etoposide (Efl, melatonin (ME), and/or monoclonal rat anti-mouseGM-CSF (a-GM-CSF). The concentration of a-GM-CSF was 150 pg/ml. The valuesrepresent the mean number ±SD of GM-CFU/10@ bone marrow cells from 3 experiments.a, P < 0.01.

DISCUSSION

In this study, we show that melatonin can protect bone marrowfunctions from the toxic effect of cancer chemotherapy compoundswithout interfering with their anticancer action in vivo. This effect isexerted directly on bone marrow T-cells, which may directly releaseor participate in the induction of a GM-CSF-like factor upon melatosun stimulation.

Melatonin per se, did not influence LLC growth and metastasesformation. This is in line with previous results about the effect ofmelatonin on LLC growth (23), but it is in contrast with the widelyreported inhibiting action of melatonin on most tumor histotypes (24).However, melatonin was able to counteract, in part, the tumor-inducedincrease of leukocyte counts. It has been reported that the LLCinduced myelopoiesis is associated with decreased prostaglandin production by tumor-macrophages and immune suppression (25). Theeffect of melatonin on leukocyte counts might thus be related to itsimmunoenhancing properties (3, 5), possibly up-regulating macro

phage activation and prostaglandin production. The cancer chemotherapy compounds decreased the tumor burden and this, in turn,

might have resulted in diminished colony-stimulating activity, activation of macrophages, and prostaglandin production.

The most interesting effect of melatonin was, however, exerted inthe opposite direction, i.e., melatonin protected bone marrow cellsfrom toxicity induced either in vivo or in vitro by the anticancercompounds. Melatonin can rescue myeloid progenitor cells fromdrug-induced apoptosis via a mechanism involving the endogenousproduction of GM-CSF. Consistently, melatonin protected the lineage-committed GM-CFU progenitors but not the less differentiatedCFU-S precursors which are not affected by GM-CSF (26). In linewith previous reports concerning the mechanism of the immunopotentiating action of melatonin (3, 6—8,27), the experiments performedwith bone marrow cells from T-cell-deficient mice suggest that Tcells are involved in the melatonin action. Whether the melatonininduced colony-stimulating factor is directly released by T-cells or

rl)

@ @-

0

@ @,I In@ :@i @—@ @-_@ _[!ñj@ @LJ IJ@ L• Li I U

0 10u1 @o'°i@-@ 10@8 @4 io@

MELATONIN (M)Fig. 1. Prevention of etoposide-induced apoptosis by melatonin. Bone marrow cells

from C57BL/6 mice were incubated with etoposide in the presence of the reportedconcentrations of melatonin. The values are the mean ± SD of 3 experiments. @,percentage of apoptotic cells after 8 h incubation in medium alone.

a@ etoposide; ME, melatonin.

b p < 0.001 ET + ME versus ET.

Cp < 0.005 ME versus control.

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PREVENTION OF MARROW TOXICITY BY MELATONIN

induced by another T-cell cytokine is presently unknown. Recentclinical studies about the mechanism of action of melatonin in cancerpatients are also consonant with a T-cell-mediated effect (28).

Programmed cell death or apoptosis is a normal process by whichcells are eliminated during embryonic development and in adult life.Programmed cell death can be induced in leukemic cells by removalof colony-stimulating factors, by cytotoxic therapeutic agents, or bythe tumor suppressor gene wild-type p53. All these forms of inductionof apoptosis in leukemic cells can be suppressed by the same colonystimulating factors that suppress apoptosis in normal cells (29, 30). Inour case, the rescued GM-CFU precursors are contained in a largercell population which apparently is also protected from the melatonininduced GM-CSF. This might depend on the presence of GM-CSFreceptors on cell types other than GM-CFU precursors (26).

The evidence of a neuroendocrine influence on endogenous colonystimulating factor production has important basic and practical implications. A major problem remains, in fact, the hematological toxicityof cancer chemotherapy compounds.(14). The systemic administration of GM-CSF has significant dose-related toxicity (31). In contrast,

melatonin is well known to have no toxic effects (10—13,28, 32). Amodulation of the production of endogenous CSFs is likely to presentseveral and substantial advantages over systemic administration. Our

finding may thus have straightforward and wide clinical applications.

ACKNOWLEDGMENTS

We thank E. Hertens and P. Galli for excellent technical assistance and Dr.G. Garavaglia,OspedaleS. Giovanni,Bellinzona,Switzerland,for the irradiation facilities.

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1994;54:2429-2432. Cancer Res   Georges J. M. Maestroni, Valeria Covacci and A. Conti  the Pineal Neurohormone Melatonin in Tumor-bearing MiceGranulocyte-Macrophage Colony-stimulating Factor Induced by Hematopoietic Rescue via T-Cell-dependent, Endogenous

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