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16 Brain Research, 480 (1989) 16-28 Elsevier

BRE 14144

Opioid antagonist modulation of murine neuroblastoma: a profile of cell proliferation and opioid peptides and receptors

Ian S. Zagon and Patricia J. McLaughlin Department of Anatomy, The M.S. Hershey Medical Center, The Pennsylvania State UniversiOl, Hershey, PA 17033 (U.S.A.)

(Accepted 28 June 1988)

Key words: Neurotumor; Neuroblastoma; Cancer; Opioid receptor; Endogenous opiaid system; Mitosis; Growth; Enkephalin

The endogenous opioids and their receptors are known to play a major role in neoplasia. In the present study, naltrexone (NTX), a potent opioid antagonist, was utilized to explore the interactions of opioids and opioid receptors in mice with transplanted neuroblastoma (S20Y). Tumors from mice subjected to either intermittent (4-6 h/day; 0.1 mg/kg NTX) or complete (24 h/day; 10 mg/kg NTX) opioid receptor blockade exhibited an up-regulation of DADLE and Met-enkephalin binding sites, as well as tissue levels of fl-endorphin and Met-enkephalin. Binding affinity to [D-Ala2,o-LeuS]enkephalin (DADLE) or ethylketoeyclazocine (EKC), the levels of plasma fl-endorphin, and the anatomical location and quantity of Met- and Leu-enkephalin and eytoskeletal components (i.e. tubulin, aetin, brain spectrin(240/235)) were similar in NTX and control tumor-beating animals. Tissue viability of the 0.1 NTX group was increased compared to controls. Both mitotic and labeling indexes were increased during the period of opioid receptor blockade, but decreased in the period subsequent to receptor blockade. NTX treatment produced a 2-fold increase in sensitivity to opioids. Met-enkephalin (10 mg/kg) produced a depression in both mitotic and labeling indexes in tumor-bearing mice that could be reversed by naloxone (10 mg/kg) administration. Thus, the endogenous opioids are trophie agents that inhibit growth by suppressing cell proliferation. The duration of receptor blockade by opioid antagonists modulates these actions, affecting both tumor incidence and survival time. Complete opioid receptor block prevents the interaction of increased levels of putative growth-related peptides with a greater number of opioid receptors, thereby increasing cell proliferation and accelerating tumor growth. With intermittent blockade, an enhanced opioid-receptor interaction occurs during the interval when the opioid antagonist is no Gonger present, producing an exaggerated inhibitory action on cell proliferation and the repression of tumorigenic events.

INTRODUCTION

Carcinogenic events associated with human and animal neoplasias have been shown to be regulated by opioid agonists and antagonists 37. The mechanism underlying this control of tumorigenesis involves endogenous opioid systems (i.e. endogenous opioids _ _ . . 1 ~ • - . a . x l l 12 3 1 - 3 3 3 6 - 3 g mlu upiOiu recepto~j . . . . . . . . Thus, both exogenous and endogenous opioids inhibit neoplasia 11"13'18'20'29-33'36'38, an effect totally blocked by con-

comitant administration of opioid antagonists such as naloxone 29. Opioid antagonists exert dose-depen-

dent, stereospecific inhibitory effects on the growth of cells, particu!arly those in culture; this effect is re-

l 0 12 3 4 versed by naloxone administration , , . The use of opioid antagonists to perturb the endogenous opioid systems has been particularly revealing in regard to carcinogenesis. Both naloxone and naltrexone alter

neoplasia l'tl'3°-33,36.3s stereospecifically 36, although

naltrexone is more than 100 times as potent as naloxone in affecting tumorigenesis. Detailed experiments with opioid antagonists reveal that the duration of receptor blockade is crucial in determining the course of oncogenesis 33. Thus, chronic administration of opioid antagonists at doses (e.g. 0.1 mg naltrexone/ kg) that block receptors for a short period each day (e.g. 4 - 6 h) suppress tumor response and prolong survival time in mice inoculated with a murine neuroblastoma. Blockade of opioid receptors from interaction with endogenous opioids for 24 h/day by either repeated injections of a ' low' dosage (e.g. 0.1 mg/kg given every 6 -8 h/day) of naltrexone or a single injection of a 'high' dosage (e.g. 10 mg/kg; a dosage that is 2% of the LDs0 ) of naltrexone exacerbates tumorigenesis and shortens survival time. These experiments document the delicate balance between

Correspondence: I.S. Zagon, Department of Anatomy, The M.S. Hershey Medical Center, Hershey, PA 17033, U.S.A.

0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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opioids and receptors in regard to cancer, and demonstrate that endogenous opioids exert a tonic and inhibitory influence on neoplasia. In order to elucidate further the role of endogenous opioid systems in cancer, the present report examines the actions of opioid antagonists on a tumor of the nervous system: murine neuroblastoma. This detailed exploration of opioid peptides, receptors, morphology, and cell ki- netics not only characterizes the trophic influence of endogenous opioid systems in neural cancer, but provides the first evidence that opioid peptide-opioid receptor interactions regulate neuro-oncogenic events by controlling cell proliferation.

collected immediately, and the plasma was separated by centrifugation and processed for fl-endorphin levels. Ulcerated and necrotic tissue was dissected from tumor samples, and viable tissues were frozen rapidly in liquid nitrogen and stored at -70 *C until assayed. The tumors of 6-10 animals/group were evaluated. Plasma fl-endorphin levels were estimated with a reagent kit fro:n ImmunoNuclear Corp. (Stillwater, MN). Endogenous opioid peptides in tumor tissue were extracted with cold 0.2 N HCI. Tissue fl-endorphin and Met-enkephalin levels were estimated with reagent kits from ImmunoNuclear Corp. (Stiilwater, MN).

MATERIALS AND METHODS

Animals, tumors, and drug protocol Young adult male syngeneic AJJax mice were ob-

tained from the Jackson Laboratories (Bar Harbor, ME). Groups of mice were housed in stainless steel cages in an environment maintained at 21 + 0.5 °C with a relative humidity of 50 + 10%. Food and water were provided ad libitum. All animals were accli- mated to their surroundings for 5 days prior to the beginning of experimentation.

Murine tumor cells, S20Y neuroblastoma, a cho- linergic cell line cloned from the A/Jax mouse C1300 neuroblastoma (a gift from Dr. M. Nirenberg, NIH, Bethesda, MD) were maintained in culture as described earlier 29'3°. To induce tumor formation, I06 cells were injected subcutaneously (s.c.) into the dor- sal surface of the right shoulder; this was considered Day 0. Tumor size was determined as described previously 33. When tumor diameter measured 12-15 ram, animals were anesthetized (intraperitoneally; i.p.) with 40 mg/kg Nembutal and killed by rapid de- capitation.

Unless otherwise noted, beginning 1 day after tumor cell inoculation, animals received a daily s.c. injection of 0.1 mg/kg or 10 mg/kg naltrexone hydro- chloride (NTX; gift from the National Institute on Drug Abuse, RockviUe, MD) or sterile water in the scapular region. Animals were weighed weekly and appropriate dosage adjustments made.

Radioimmunoassays of fl-endorphin and Met-enkephalin

Animals were decapitated, whole trunk blood was

Receptor binding assays Receptor binding assays were performed accord-

ing to procedures reported elsewhere n2s'4°, and followed those for selective labeling of the/l-, 6, and r- binding sites as described by Gfllan and Kosteditz 4 and Kosterlitz and Paterson s. Briefly, homogenates were incubated for 2 h at 22 °C with tritiated ligand in the presence or absence of specific blocking agents. Selective labeling of the/~-site utilized [3H][D-Ala2- MePheg,Gly-olS]enkephalin (-- [3H]DAGO). For the t~-binding site, [3H][D-Ala2,I)-LeuS]enkephalin (= [3H]DADLE), which binds mainly to the b-site, but also/~-sites, was used in the presence of unlabeled DAGO (10 nM DAGO to each 1 nM [3H]DADLE) to suppress/~ binding. For selective labeling of the r- site, the binding of [3H](+)-ethylketocyclazocine ([3H]EKC) which binds to the/~-, t%, and r-sites, was measured in the presence of unlabeled DAGO (100 nM) and DADLE (100 nM) to suppress/~ and 6 binding, respectively. Non-specific binding for/~-, 6- and r-receptor assays was determined in the presence of DAGO (!00 nM), DADLE (100 nM), and EKC (100 nM for every 1 nM [3H]EKC), respectively. The following radiolabeled ligands were used: [3H]DAGO (47.7 Ci/mmol), [3H]DADLE (36.6 Ci/mmol), and [3H]EKC (27.1 Ci/mmol); all tritiated ligands were purchased from Dupont-New England Nuclear, Bos- ton, MA. The following cold ligands were utilized: DAGO (Peninsula Laboratories, Belmont, CA), DADLE (Sigma, St. Louis, MO) and EKC (Stealing Winthrop, Rensslaer, NY). At least 3 independent assays/ligand were performed on each group, with the tumors of 3-5 mice/group sampled.

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A utoradiography Autoradiographic procedures s were utilized to vis-

ualize opioid (i.e. Met-enkephalin) binding sites in neuroblastoma tissues. In brief, tumor-bearing mice were anesthetized with Nembutal (40 mg/kg, i.p.), decapitated, and tumors excised and frozen in iso- pentane which had been cooled in dry-ice. Frozen sections (20/zm) were obtained on a Slee cryostat and thaw-mounted onto gelatin/alum-coated slides.

Sections of tumor from the control, 0.1 NTX, and 10 NTX groups, sampled from 3 animals/group, were incubated in a solution of 1 nM pzSI]Met-enkephalin

(2200 Ci/mmol, Dupont-New England Nuciear, Bos- ton, MA) or 1 nM [1251]Met-enkephalin and 1/~M naloxone in 50 mM Tris-HCl (pH 7.4, 22 °C) for 1 h, followed by a series of washing steps and drying un- der a stream of cold air. Sections were placed in X- ray cassettes and exposed to LKB Ultrofilm. Follow- ing 9 days of exposure~ the film was developed in Ko- dak D-19 (5 rain). Quantitative assessment of the an- toradiograms was obtained from densitometric read- ings with a Tobias densitometer (Tobias Associates, Model TCS, Ivyland, PA). At least 20 measure- men~'group were utilized in densitometric analysis.

Histology and morphometry Tumor tissues were excised, immersed in 10%

neutral buffered formalin, embedded in polyester wax, and sectioned at 10gin in the midline parallel to the long axis. The sections were stained with hematoxylin/eosin for general morphology, or protargol silver z8 for neurons and nerve fibers. For morphometry, hematoxylin/eosin-stained sections were pro- jetted (22×) with a Bausch and Lomb projector onto an Apple Graphics Tablet. The outside perimeter, as we!! a~ areas of 'live' (basophi!ic) and 'dead' (acido- philic) tissue were recorded with an Apple II Plus computer. The percentage of viable tissue was calculated as the area of 'live' cells divided by the total area examined. The tumors from at least 6 animals/ group, 2 sections/tumor, were evaluated.

Mitotic and labeling indexes The mitotic index of control and opioid antagonist-

treated animals was determined. At 2 and 10 h following injection of 0.1 or 10 mg/kg naltrexone, o r sterile water, mice were anesthetized with Nembutal (40 mg/kg, i.p.), decapitated, and tumors excised;

tissues were prepared for histology. Using hematoxy- fin- and eosin-stained sections (10 gm), cells located in an area no greater than 100 ;tin from the zurface were counted in an 0.0064 mm 2 grid at 630×. The tumors of at least 3 animals/group were evaluated at the 2 and 10 h time points, and at least 500 cells/section were counted in 7-12 sections/tumor. Cells in metaphase, aaaphase, and telophase were considered mitotic. The mitotic index was calculated as the number of dividing cells relative to the total number of cells counted.

The labeling index of control and opioid antagonist-treated mice was determined. Thirty minutes prior to killing mice at either 2 or 10 h following an acute administration of 0.1 or 10 mg/kg naltrexone, or sterile water, tumor-bearing mice received an in- traperitoneal (i.p.) injection of [3H]thymidine (10 ~Ci/g b. wt.; 6.7 Ci/mmol, Dupont-New England Nu- clear, Boston, MA). The mice were anesthetized with Nembutal, perfused with 10% neutral buffered formalin, and tumors excised and embedded in polyester wax. Tissue sections (10 btm) were coated with NTB-2 nuclear track emulsion (Kodak, Rochester, N-Y), exposed at 4 °C for 2 weeks, developed in D- 19, and stained With hematoxylin/eosin. The labeling index (LI = number of labeled cells/total cells) was determined in randomly selected areas of the tumor, within 100/~m from the surface. Utilizing 2 sections/ tumor, 8-12 tumors/treatment group, a minimum of 3000 cells/treatment group/time point were evaluated at 630x.

Immunocytochemistry Frozen specimens of tumor tissue (6 tumors/group)

were sectioned (20/zm) on a Slee cryostat and the sections collected arid processed for immunocytochemistry on subbed coverslips as described earlier ~9"4°. In brief, sections were treated with 95% ethanol (30 min) and acetone (20 min) at 4 °C. Tis- sues were washed with 50 mM Tris-HCl, pH 7.4, 150 mM NaCI (TBS) for 5 rain at room temperature to re- move endogenous peptides and blocked with 3% fe- tal calf serum (FCS) in TBS for 15 min. The sections were incubated with antisera to either native mouse brain spectrin(240/235) 19 diluted 1:50, rbc spectrin (which recognizes brain spectrin(240/2".SE) TM diluted 1:10, actin (Miles Scientific, Naperville, IL) diluted 1:50, or tubulin (Miles Scientific, Naperville, IL) di.

luted 1:50 for 3 h, or antisera to Met-enkephalin or Leu-enkephalin (lmmunoNuclear Corp., Stillwater, MN), diluted 1:25 as described above for 18-22 h; all antisera were diluted in 1% FCS in TBS with 0.1% Triton X-100. Sections were washed (4 times) with 1% FCS, 0.1% Triton X-100 in TBS and blocked with 3% FCS in TBS for 15 rain, followed by a 3 h in- cubation with 0.3% horseradish peroxidase-conju- gated goat anti*rabbit IgG in 1% FCS, 0.1% Triton X-100 in TBS. Excess peroxidase was removed with 4 changes of TBS over a 30 rain period, and the stained product visualized with 0.06% 4-chloro-l-naphthol, 0.01% hydrogen peroxide in TBS for 15 rain. The sections were washed with TBS and mounted on slides with 9:1 glycerol/TBS.

Opioid challenge experiments and nociception Earlier studies 33 have shown that 0.1 NTX blocks

the opioid receptor for 4-6 h and 10 NTX blocks the opioid receptor for the entire 24 h injection period. To secure information as to opioid responsivity of

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mice given 0.1 NTX during the interval when NTX is no longer capable of blocking the receptors, we performed the following experiment. Mice were given daily injections of either 0.1 NTX or sterile water for 1 week. Following procedures described elsewhere 3;, mice were monitored for nociceptive response on a hot-plate (55 °C; Analgesia Meter, Technilabs) 8 b following the last injection of 0.1 NTX or sterile water. Thirty minutes prior to this point, mice were given a s.c. injection of various dosages of ievorphanol tartrate (Hoffmann-LaRoche, Nutley, NJ). End- points for response included licking 6f forepaws and rapid removal of hindlimbs. Five animals/group were tested at each dosage.

Met-enkephalin and cell proliferation In order to examine the action of endogenous opi-

oids on neurotumors, mitotic and labeling indexes were determined.

(a) Mitotic index. At 2 h following injection of either Met-enkephalin (10 mg/kg), Met-enkephalin

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Fig. 1. The concentration of plasma fl-endorphin, and Met-enke )halin and fl-endorphin in neuroblastoma tissues, from mice injected with either 0.1 or 10 mg/kg NTX, or sterile water (CO). Values represent means + S.E.M. Significant difference from controls at P < 0.01 (**).

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(10 mg/kg) plus naloxone (10 mg/kg), or sterile water, mice were anesthetized with Nembutal (40 mg/kg, i.p.), tumors excised, tissues prepared for histology, and mitotic figures evaluated as described earlier. The tissues of at least 4 mice/group were examined, and at least 500 cells/section were counted in 7-12 sections/tumor at 630×.

(b) Labeling index. One and one-half hours following administration of either Met-enkephalin (10 mg/kg), Met-enkephalin (10 mg/kg) plus naloxone (10 mg/kg), or sterile water, tumor-bearing mice received an i.p. injection of [3H]thymidine (10 #Ci/g b. wt.; 6.7 Ci/mmol, DuPont New England Nuclear, Boston, MA). Thirty minutes later, the mice were anesthetized with Nembutal, perfused with 10% neutral buffered formalin, and tumors excised and processed for histology as described earlier. At least 2000 ceils/treatment group were evaluated at 630× utilizing 4-7 sections/tumor and 4 tumors/group.

Statistics Plasma and tissue levels of ~-endorphin and Met-

enkephalin were analyzed using one-factor analysis of variance, with subsequent comparisons made using the Newman-Keuls test. Receptor binding data were analyzed with a Lundon I (Saturation Iso- therm Binding Analysis) computer program (Lundon Software, Cleveland, OH). This analysis utilizes non-linear least-squares data regression. Saturation curves and Scatchard analysis were computer gener- ated. Mean values for binding affinities and capacities from at least 3 experiments were compared using analysis of variance and Newman-Keuls tests. The percentage of 'live' cells was analyzed with a one-factor analysis of variance, and subsequent comparisons made with the Newman-Keuls test. Mitotic indexes of the naltrexone experiments, as well as labeling indexes, were compared with a two-factor analysis of variance, with time (2 and 10 h) and group as factors; subsequent comparisons were conducted with the Newman-Keuls test. Mitotic and labeling indexes of the Met-enkephalin experiments, and data from densitometric analysis, were evaluated by analysis of variance, and subsequent comparisons made with the Newman-Keuls test. Latencies on the hot-plate were examined with the Student's two-tailed t-test. The Stats-Plus and ANOVA programs were used with an Apple IIe computer.

RESULTS

~8-Endorphin and Mez-enkephalin Evaluation of the effect of naltrexone treatment

on opioid peptide levels revealed that levels of plasma ~-endorphin assessed by radioimmunoassay in control and NTX tumor-bearing mice were comparable (Fig. 1). However, levels of/3-endorpbin in tumor tissue from mice given NTX were 1.8-2.3-fold greater than that in control specimens (Fig. 1). Met- enkephalin levels in tumors of NTX mice were also found to be substantially elevated from control values, being 6.3-15.6-fold greater than normal (Fig. 1).

OFioM binding sites In previous studies 27 we discovered binding sites to

DADLE and EKC, but not DAGO, in neuroblastoma tissues and reported that these sites possess the characteristics of opioid binding sites. In the present study, which assessed the effects of NTX on neuroblastoma, chronic exposure to an opioid antagonist resulted in no change in the binding affinity for either [3H]DADLE or [3H]EKC (Fig. 2; Table I). Howev- er, the binding capacity for tumor tissue harvested from animals given 0.1 or 10 NTX was 3.3- and 4.7- fold, respectively, greater than normal for [3H]- DADLE (Fig. 2; Table I). In contrast, the binding capacity for [3H]EKC in 0.1 and 10 NTX mice was decreased 5.0- and 6.8-fold, respectively, from control values (Fig. 2; Table I).

Autoradiography In order to localize opioid binding sites in neuro-

blastoma, and to determine the repercussions of opiold antagonist treatment on the quantity and distribution of binding sites, tissues were incubated in radiolabeled Met-enkephalin and visualized with autoradiographic techniques. Inspection of auto~adio- grams (Fig. 3) revealed a fairly homogenous pattern of [125I]Met-enkephalin labeling in control tumor tissues (Fig. 3A). This binding with a radiolabeled opiold ligand could be blocked by naloxone (Fig. 3B). The autoradiographic images of tumor tissues obtained from animals given either 0.1 or 10 NTX chronically (Fig. 3C,D) were notably more intense than untreated, control specimens (Fig. 3A). Quanti- tative assessment using densitometry supported

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TABLE I

Binding affinity (Kd) and capacity (Bmoz) for [3H]DADLE and [3H]EKC in neuroblas',oma tissues harvested from mice given 0.1 or 10 NTX, or sterile water (control)

Values represent + S.E.M. from at least 3 assays.

[~H]DADLE [3H]EKC

K d B ~ Kd B,~ (nM) (fmol/mg ( r iM) (fmol/mg

protein) protein)

Control 1.0 + 0.3 4.6 + 0.8 0.7 + 0.2 91.9 + 6.5 0.1NTX 1.1+0.3 15.4+3.5" 0.5_+0.1 18.4+2.5"* 10NTX 0.6+_0.1 21.4_+3.4"* 0.4+0.1 13.6_+2.6"*

_eignificant difference from controls at *P < 0,05 or **P < 0 01.

these subjective impressions. Thus, tumor tissues from control mice incubated in [~25I]Met-enkephalin

(optical density = 0.08 +_ 0.02) were significantly denser (P < 0.05) than samples incubated in [12sI]- Met-enkephalin and naloxone (0.04 +_ 0.00). In comparison to controls, [125I]Met-enkephalin binding was 50% greater in tissues from 0.1 NTX mice (0.12 +_ 0.03), and 125% greater in samples from 10 NTX animals (0.18 _+ 0.02); these differences from control levels were statistically significant (P < 0.01).

Histology and morphometry The influence of NTX on the morphology and via-

bility of neuroblastoma was examined. Histology. The cellular elements in neuroblastoma

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tissues consisted of small cells, 9-11 ~m in diameter (Fig. 4A). These cells had a rather large nuclear area, with only a rim of cytoplasm. The nucleus was often oval, and consisted of numerous heterochro- matin aggregations. No differences could be recorded in histological profiles of control and NTX- treated tissues, nor in protargol-stained preparations revealing nerve fibers and neuronal soma.

Morphometry @tissue viability. Tumors harvested from animals in the 0.1 NTX group had a 51.3 + 2.8% viability, in contrast to a 30.3 + 2.9% tissue viability in control specimens; this difference was statistically significant (P < 0.05). The areal percentage of viable tissue in tumors of the 10 NTX group (39.0 _+ 6.1%) did not differ significantly from control values.

Mitotic and labeling indexes The role of opioid antagonists in modulating cell

proliferation in neuroblastoma-bearing mice was evaluated; both mitosis and D N A synthesis were examined.

Mitotic index. Determination of the mitotic index (Fig. 5) revealed 23-33% more dividing cells 2 h following administration of either 0.1 or 10 NTX in comparison to control levels. Ten hours after injection of 10 NTX, tumors in this group had 70% more mitotic figures than control specimens, but tumors in the 0.1 NTX group were markedly subnormal (a de- crease of 30%).

Fig. 3. Autoradiographs of neuroblastoma tissues from control (A,B) animals, or mice given 0.1 (C) or 10 mg/kg NTX (D), and incubated with p25I]Met-enkephalin (A,C,D) or [125I]Met-enkephalin plus naloxone (B). Note the binding in control tumors incubated with [125I]Met-enkephalin, but the absence of activity in tissues incubated with [125I]Met-enkephalin plus naloxone. Tissues from animals chronically treated with naltrexone (B,C) exhibited a marked increase in [t2SI]Met-enkephalin. ×2.2.

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Fig. 5. The mitotic index of neuroblastoma tissues at 2 and 10 h following injection of 0.1 or 10 mg/kg NTX, expressed as per- cent difference from control values. Significant difference from controls at P < 0.05 (*) or P < 0.01 (**).

Labeling index. The index of cells labeled with [3H]thymidine (Fig. 6) at 2 h was similar for the control and 0.1 NTX groups, but the 10 NTX group was elevated 27% from control levels. At 10 h, the 0.! NTX group was subnormal, being about 16% less than the index for controls. The 10 NTX group, however, had over a 35% increase in [aH]thymidine-labeled cells in contrast to control values.

Fig. 4. Photomicrographs of murine neuroblastoma tissues stained with hematoxylin and eosin (A), antiserum to Met-cn- kephalin (B), or antiserum to Leu-enkepha!in (C). Neuroblas- toma cells are rather small (9-11 pro), and often have dispro- portionately large nuclei (A). Met-enkephalin and Leu-enkephalin immunoreactivity can be observed in the cytoplasm, but not in cell nuclei (B,C)° Sections of neuroblastoma tissue stained with Met-enkephalin antiserum absorbed with Met-enkephalin (B, inset), or stained with serum from unimmunized animals (C, inset), exhibited no staining, x740.

24

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Immunocytochemical studies of endogenous opioids and the cytoskeleton

A comprehensive exploration of opioid peptides (i.e. Met- and Leu-enkephalin) and cytoskeletal components in neuroblastoma, as well as the effects of opioid antagonist exposure on these features, in tumor-bearing mice was conducted using standard procedures t9"4°. Sections stained with antisera to Met- or Leu-enkephalin revealed cells with immunoreactivity in the cytoplasm, but not the cell nucleus (Fig. 4B,C). Neither the location of the staining reaction nor the intensity of immunoreactivity could be distinguished subjectively among the groups - - control, 0.1 and 10 NTX. Non-immune (Fig. 4C, inset) and preabsorbed (Fig. 4B, inset) treated sections exhibited no staining.

No differences were noted among the groups when sera containing antibodies to actin or to tubulin were used; such antisera stained the cytoplasm, but not the

nucleus of individual neuroblastoma cells (data not shown). Specimens treated with non-immune sera were negative. Brain spectrin(240/235E) IgG did not stain neuroblastoma tissues, whereas brain spectrin(240/235) IgG stained the cytoplasm~ but not the nucleus (data not shown). No apparent differences in staining patterns were noted between experimental and control groups. Sections incubated in non-immune IgO or anti-rbc or anti-brain spectrin antibodies preabsorbed with an excess of rbc spectrin or brain spectrin(240/235), respectively, were not stained.

Opioid challenge experiments and nociception The relationship between opioid antagonist expo-

sure and functional activity was explored using a nociceptive assay. Mice receiving chronic injections of 0.1 N T X and examined 8 h following drug administration were found to be analgesic at dosages of 0.8 mg/kg levorphanol in contrast to a dosage of 1.5 mg/kg levorphanol required for analgesia in control animals (Fig. 7). Thus, the 0.1 NTX group was approximately twice as sensitive to levorphanol than

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Levorphanol, mg/kg Fig. 7. Latency of mice with transplanted neuroblastoma to react to a hot-plate (55 °C). - - - , baseline of sterile water controls, and mice given 0.1 mg/kg NTX. 0 , mice receiving sterile water and challenged with different dosages of levorphanol; O, animals chronically given 0.1 mg/kg NTX and challenged with different dosages of levorphanol. Significant difference from baseline levels at P < 0.05 (*) or P < 0.01 (**).

control subjects. Nociception in animals chronically injected with 0.1 NTX was comparable to that of control mice at all time points prior to administration of levorphanol. In order to examine the efficacy of 0.1 NTX to block the opioid receptor, animals given 0.1 NTX were tested at 2 h for nociceptive response to 1 and 2 mg/kg levorphanol. A dosage of 0.1 NTX was capable of blocking levorphanol-induced analgesia (data not shown), indicating that 0.1 NTX was an ef- fective antagonist 2 h following injection, but not at 8h.

Met-enkephalin and ceU proliferation To explore the effects of opioid peptides on cell

proliferation, mitotic and labeling ~ndexes were determined in tumors from mice injected with Met-enkephalin, Met-enkephalin plus naloxone, or sterile water (Fig. 8). In contrast to controls, the mitotic index of tumors from mice receiving Met-enkephalin was decreased 35%; animals given concomitant injections of Met-enkephalin and naloxone had a mitotic index comparable to controls. The labeling index of tumors from mice injected with Met-enkephalin was markedly subnormal (only 86% of control levels), whereas that of mice receiving both Met-enkephalin plus naloxone were comparable to controls.

DISCUSSION

The present study, which utilizes opioid antago-

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Fig. 8. The labeling and mitotic indexes of neuroblastoma tissues at 2 h following injection of sterile water (C), 10 mg/kg Met-enkephalin (M), or 10 mg/kg methionine enkephalin and 10 mg/kg naloxone (MN). Pulse time for [3H]thymidine, utilized in the labeling index study, was 30 min. Significant difference from controls at P < 0.05 (*). Bars = S.E.M.

25

nists to perturb endogenous opioid-opiold receptor interactions, serves to elucidate the role of opioid peptides in regulating neoplasia. Examination of neuroblastoma tissue from mice exposed to naltrexone revealed an up-regulation of DADLE binding sites using receptor binding assays and of Met-enkephalin binding sites by autoradiographic methodolo- gy, as well as an up-regulation of tissue levels offl-endorphin and Met-enkephalin utilizing radioimmunoassays. However, binding sites to EKC were down- regulated by opioid antagonist administration. No changes were observed in binding affinity to either [3H]DADLE or [3H]EKC, plasma fl-endorphin levels, nor in the distribution or quantity of opioid peptides (i.e. Met- and Leu-enkephalin) or cytoskeletal components (i.e. tubulin, actin, brain spectrin(240/ 235)) as recorded from inspection and subjective impressions of immunocytochemical preparations. Ad- ministration of opioid antagonists produced a supersensitivity to opioids in nociceptive tests. Finally, the morphological and cell kinetic features of neuroblastoma transplanted into mice were influenced by opioid antagonists. Daily intermittent (i.e. 0.1 NTX), but not complete (i.e. 10 NTX), opioid receptor blockade produced a greater viability of tumor tissue relative to control tumors, suggesting that necrotic processes had not progressed as rapidly in these 0.1 NTX-treated animals. Cell replication in neurotumors appeared to be modulated tightly by endogenous opioid-opioid receptor interactions. During opioid receptor blockade (i.e. 0.1 NTX at 2 h and 10 NTX at 2 and 10 h) 33 an increase in the mitotic index, and the labeling index (with the exception of the 0.1 NTX group at 2 h), could be recorded. Mitotic and labeling indexes were found to be subnormal subsequent to receptor blockade (i.e. 0.1 NTX at 10 h) 33. Finally, our studies demonstrate for the first time, that both DNA synthesis and mitosis are governed by endogenous opioid peptides. Met-enkephafin de- pressed incorporation of [3H]thymidine and mitosis in an opioid antagonist-reversible fashion.

Our finding that opioid antagonist administration to tumor-bearing animals does not alter the binding affinity of opioid receptors is consistent with previous observations 2,11A4'2L23,25,41,42 in normal and abnormal neural tissues. Moreover, the increase in the binding of a putative 6-receptor ligand (i.e. [3H]DADLE) is consonant with previous observations in neural tis-

26

sues n.14,~,42. However, the effects of opioid antagonists on putative u-receptors is unclear, with up-regulation and/or no change in the binding capacity of u- related ligands (e.g. [3H]EKC) previously recorded in neural tissues (including human neuroblastoma) n34,23,2s'42. We have found a reduction of [3H]EKC binding sites in murine neuroblastoma tissues frcm mice given nalt:exone. Thus, the nature of the EKC binding site and/or its reaction to opioid antagonist exposure in normal neural tissue and human neuroblastoma, and murine neuroblastoma, may differ. However, our data for neurotumor tissues sup- ports the suggestion by other investigators studying neural tissues 14,2s,42 that opioid binding sites in neural cancers may be differentially regulated in regard to opioid antagonist action. The effects of opioid an: tagonist administration on plasma and/or tissue endorphin/enkephalin levels are inconclusive, with increases 7"9'1u7'24~6, decreases ~6, or no change 6'u,26 reported. Our study with tumor-bearing animals found little alteration in plasma/~-endorphin levels, but substantial increases in tissue concentrations of ~-endorphins and Met-enkephalin. Whether this increase in tissue-related neuropeptides represents a sequestering by tumor tissues and/or an increased production by tumor cells, requires further investigation. Finally, and presumably in conjunction with an increase in opioid receptors_, a supersensitivity to opioids has been reported in animals chronically subjected to opioid antagonists 2,21,22,25. The present find- ings are consistent with this observation, with almost a 2-fold increase in sensitivity to ievorphanol recorded in naltrexone-treated mice.

The results of this investigation are consonant with previous reports 27,40 demonstrating opioid receptors in murine neuroblastoma transplanted into A/Jax mice. Thus, specific and saturable binding to DA- DLE and EKC (but not DAGO) have been recorded, with a general similarity noted between studies in regard to binding affinities and capacities for each ligand. Both ~-endorphin and Met-enkephalin were also detected in murine S20Y neuroblastoma tissues in this report using tumors of 12-15 mm and in an earlier study utilizing tumors of various sizes 4°. The concentration of Met-enkephalin was quite similar in both studies (approximately 1 ng/g tissue), but nearly 3 times more/~-endorphin was detected in tumors studied previously; presumably, this could have

been due to the variable (and generally larger) diameter tumors (and hence, increased blood contamina- tion) employed in this earlier work.

The present results provide a basis for understand- ing the mechanism by which opioid antagonists alter neural neoplasia as shown in previous reports n, 30-33.36,3s. In vivo and in vitro experi~nentation with a variety of opioid peptides has shown that one of the putative opioids playing a major role in regulating neurocarcinogenic events is Met-enkephalin n'13,15. Thus, opioid antagonists such as naltrexone appear to up-regulate the levels of opioid peptides such as Met-enkephalin, as well as related binding s~tes (e.g. DADLE and Met-enkephalin), and to enhance the physiological response to opioids. In the case of complete opioid receptor blockade" by the use of a single daily injection of a relatively high dose of NTX (e.g. 10 NTX) 33, or injections of a relatively low dose of NTX given at repeated intervals each day (e.g. 0.1 NTX given 4 times daily) 33, opioids do not have any opportunity to interact with receptors. The removal of opioid control results in accelerated tumor growth through an increase in cell division. When animals in-

. oculated with tumors are given an intermittent receptor blockade each day, by administration of a single daily injection of a low dose of NTX 33 or a high dose of the short-acting opioid antagonist - - naloxone 3°, neurocarcinogenesis appears to be either totally suppressed or delayed. Thus, during the daily interval when the opioid antagonist is no longer present, the increase in neuropeptides and receptors can interact. This interfacing produces an exaggerated action on cell proliferation, resulting in decreased cell replication and the repression of tumorigenic events. The present studies showing that an endogenous opioid, Met-enkephalin, can depress mitotic and labeling indexes in a naloxone-reversible manner provides validation to the notion that one (or more) endogenous opioid(s) function in an inhibitory fashion on tumor growth; and do so by influencing cell replication by way of the opioid receptor.

Collectively, these data and those of previous studies now permit the formulation of a number of prin- ciples about endogenous opioid systems and neoplasia. First, opioids serve as trophic agents in regulating cancer, exerting their influence by inhibitory mechanisms. This is evident from tumor transplantation studies in which exogenous (i.e. heroin) or en-

27

dogenous (e.g. Met-enkephalin) opioids inhibit tumor growth 13,~5,ts'2°'29, from investigations demon-

strating that endogenous opioid systems control brain development 39, and from tissue culture studies t°'n'24 in which both endogenous and exogenous opioids suppressed the growth of neurotumor cells. Finally, complete disruption of endogenous op io id - opioid receptor interaction in t 'amor transplantation experiments using opioid antagonists produces an ac- celeration in tumorigenic events 3m3,36. Second, opioid action in regard to tumor biology is mediated by opioid receptors. Tumor transplantation studies show that the effects of exogenous and endogenous opioids on cancer can be reversed by concomitant administration of opioid antagonists 29, and the effects of opioid antagonists are stereospecific, indicating that the site of action is located at the opioid receptor 36. Moreover, tissue culture studies with neurotumor cells have demonstrated a dose-dependent, stereospecific, and opioid antagonist-reversible effect of opioids on growth t°'m24, indicating a direct action that is not mediated nor dependent upon immunolog- ical and hormonal factors. Third, opioids exert their

control on growth by regulating cell proliferation. This has been clearly demonstrated by in vivo and in vitro assessment of mitotic and labeling indexes in this and other studies t°,12.34. Fourth, the endogenous opioids and opioid receptors are in a tonic and deft- cate equilibrium that strictly governs growth. In the present study, within a few hours of disruption of the endogenous opioid systems, both mitotic and labeling indexes were markedly altered. Moreover, previous tumor transplantation studies 11,3°-33,35,36,3s in-

dicated that blockade of endogenous opioid-opioid receptor interactions profoundly influenced the course of tumorigenesis.

ACKNOWLEDGEMENTS

This work was supported by NIH Grants NS-20623 and NS-20500. We are grateful to Laura Nagy and Susan Ditty for technical assistance, Therese Segneri for manuscript preparation and Drs. Steven R. Goodman and Beat M. Riederer for the antisera to the spectEn isoforms.

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