Stereospecific modulation of tumorigenicity by opioid antagonists

European Journal of Pharmacology, 113 (1985) 115-120

115

Elsevier

STEREOSPECIFIC MODULATION OF TUMORIGENICITY BY OPIOID ANTAGONISTS IAN S. Z A G O N * and PATR1CIA J. M C L A U G H L I N

Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033, U.S.A. Received 27 November 1984, revised MS received 10 April 1985, accepted 1 May 1985

I.S. ZAGON and P.J. McLAUGHLIN, Stereospecific modulation of tumorigenicity by opioid antagonists, European J. Pharmacol. 113 (1985) 115-120. The effects of (+) and ( - ) isomers of naloxone in altering tumor response in A/Jax mice inoculated with S20Y neuroblastoma were determined. Mice given daily subcutaneous injections of 15 mg/kg ( - )naloxone had a 71% tumor incidence, a 33% delay in the time before tumor appearance, and a 28% increase in survival time. Inoculation of neuroblastoma cells in control subjects resulted in 100% tumor incidence within 13 days. Animals given 15 mg/kg (+)naloxone were comparable to control subjects in all aspects of neural neoplasia. These results show that opioid antagonists exert a stereospecific action on neural cancers, and provide further evidence that endogenous opioid systems play an important role in neuro-oncogenic expression. Stereospecificity

Opioidreceptors

Naloxone

1. Introduction

Tumorigenic events have been shown to be modulated by opioid agonists and antagonists (Aylsworth et al., 1979; Plotnikoff and Miller, 1983; Zagon and McLaughlin, 1981a,b; 1984a). The mechanism underlying this regulation of oncogenesis is believed to involve the endogenous opioid systems (Zagon and McLaughlin, 1983a, 1984a). We have found that chronic administration of opioid antagonists at dosages (e.g., 10-20 mg/kg naloxone, 0.1 mg/kg naltrexone) which block opioid receptors for a short period of time (e.g., 4-6 h) each day, inhibit tumor response and extend survival time in mice inoculated with neuroblastoma. Blockade of opioid receptors for 24 h / d a y by injection of opioid antagonists (e.g., 10 mg/kg naltrexone) stimulates tumorigenesis and shortens survival time (Zagon and McLaughlin 1983a, 1984a). The relationship between the pharmacological properties of opioid antagonists and tumor expression was recently clarified (Zagon * To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

Neuroblastoma Endogenousopioids

Tumorigenicity

and McLaughlin, 1984a). A dosage of 0.1 mg/kg naltrexone given repetitively (4 times) each day stimulated neuroblastoma growth, but once daily injections of 0.1 mg/kg naltrexone or 0.4 mg/kg naltrexone (the cumulative dosage of 4 daily injections of 0.1 mg/kg naltrexone) had a remarkable antitumor effect. Thus, tumorigenic events appear to be dictated by the duration of opioid receptor blockade. If tumor response is mediated at the level of the opioid receptor, one would expect that this interaction would be stereospecific. To address the question of the stereospecificity of opioid antagonists in tumorigenesis, the present investigation explored the response of neuroblastoma in mice to the ( - ) and ( + ) isomers of naloxone.

2. Materials and methods 2.1. Animals and tumors

Adult male syngenetic A/Jax mice were obtained from the Jackson Laboratories (Bar

116 Harbour, ME) and groups of 5 or 6 mice were housed under laboratory conditions reported earlier (Zagon and McLaughlin, 1981a, 1984a). Murine tumor cells, S20Y neuroblastoma, cloned from the A / J a x murine C1300 neuroblastoma, were obtained from Dr. M. Nirenberg (NIH, Bethesda, MD) and maintained in culture (Zagon and McLaughlin, 1981a,b). Tumor cells (106 ) were injected subcutaneously (s.c.) in the dorsal surface of the right shoulder; this was considered Day 0. Tumors were measured in 2 dimensions with vernier calipers (accuracy __+0.05 mm); perpendicular dimensions (a,b) were recorded, and tumor size computed as: (a + b) 1/2. Tumor growth was recorded every 3 days after a measurable tumor (5 mm or larger) was observed. All animals were weighed periodically and, at the time of death, mice were autopsied and metastases and lesions noted.

2.2. Drugs and drug treatment Beginning 2 days after tumor cell inoculation, animals were divided into 3 groups (N = 17-19 animals/group) and received a single daily s.c. injection of either 15 m g / k g ( - ) n a l o x o n e , 15 m g / k g (+)naloxone, or sterile water. Injections of naloxone or sterile water were continued throughout the experimental period until the death of all tumor bearing animals. The ( - ) isomer of naloxone was provided by Endo Laboratories (Wilmington, DE) and the ( + ) isomer of naloxone was kindly supplied by the National Institute on Drug Abuse. Drug solutions were prepared weekly by dissolution of the powder in sterile water and stored at 4°C.

2.3. Opioid challenge experiments The duration of opioid receptor blockade by naloxone isomers was determined by testing analgesia following a challenge with morphine. Nontumor-bearing mice were given daily s.c. injections of either 15 m g / k g ( - ) n a l o x o n e , 15 m g / k g (+)naloxone, or sterile water for 10 days. Thirty minutes and 2, 4, 6, and 12 h after the last injection of naloxone or sterile water, mice were injected with 2 m g / k g morphine sulfate and tested

30 min later on a hot-plate (55°C; Analgesia Meter, Technilabs). Endpoints for response included licking of forepaws and rapid removal of hindlimbs. Animals were tested only once and they were removed from the hot-plate after 40 s if no response was recorded. Six mice per group were tested at each time point.

2.4. Statistical analyses The proportion of animals developing a tumor (i.e., tumor incidence) was analyzed with the chi square test. Total survival time for all groups of mice inoculated with neuroblastoma (including those animals that did not develop measurable tumors) was analyzed using a two-tailed MannWhitney U-test. Time prior to tumor appearance, the time after tumor appearance, and the total survival time for only tumor-bearing mice were analyzed using analysis of variance. In addition, tumor measurements on the day of death and latency on the hot-plate were analyzed using analysis of variance. Subsequent planned comparisons were made with the Newman-Keuls tests. All analyses were performed using an Apple II Plus computer with Stats-Plus and ANOVA II statistical packages.

3. Results

3.1. General observations By 13 days after tumor cell inoculation, 100% of the mice in the control and ( + )naloxone groups had measurable tumors, but only 47% (8 of 17 mice) in the ( - )naloxone group had tumors at this time. On day 46 after inoculation of tumor cells, when all mice in the control and (+)naioxone groups had died, 29% (5 of 17) of the mice in the ( - ) n a l o x o n e group had not developed a tumor; this proportion was statistically different from both the control and (+)naloxone groups (P < 0.01; table 1). Although the experiment was terminated on day 56, the 5 non-tumor-bearing mice in the ( - ) n a l o x o n e group were observed for 3 additional weeks and no tumors developed. Metastases and lesions were not apparent in

117 TABLE 1 Survival time (days after subcutaneous inoculation of 106 S20Y neuroblastoma cells) of mice injected with 15 mg/kg (+) or ( - ) isomers of naloxone. Controls received daily injections of sterile water. Drug injections began 2 days after tumor cell inoculation. N = proportion of mice with tumors in each group, a Significantlydifferent from control and ( + )naloxone groups at P < 0.01 using a x square test. b Significantlydifferent from control and ( + )naloxone groups at P < 0.01 using analysis of variance. The survival times of only those mice with tumors were analyzed, c Recorded at death. Group

Controls ( + )Naloxone ( - )Naloxone

N

18/18 19/19 12/17 a

Survival (days after tumor cell inoculation)

Tumor size c mean 4- S.E.

Before tumor appearance mean 5=S.E.

Mean _+S.E.

Median

9.8 + 0.3 9.4_+0.4 13.0 + 1.6 b

28.7 + 2.2 27.7 _+1.6 36.7 -+2.5 b

28 28 40

a n y mouse from the control, ( + ) n a l o x o n e , or ( - ) n a l o x o n e groups. Body weights of control a n d naloxone-4-treated a n i m a l s were c o m p a r a b l e t h r o u g h o u t the experiment.

3.2. Survival time and tumor growth N o differences in m e a n total survival time (fig. 1), m e d i a n total survival time, survival time after t u m o r appearance, or survival time prior to t u m o r a p p e a r a n c e were detected between control a n d ( + ) n a l o x o n e groups (table 1). F o r t u m o r - b e a r i n g mice in the ( - ) n a l o x o n e group, the m e d i a n length of survival was 43% longer t h a n control mice, whereas m e a n survival time was 28% longer (P < 0.01). The range of life expectancy was 24-56 days for a n i m a l s i n the ( - )naloxone groups in comparison to a range of 19-46 days for control mice (fig. 1). E v a l u a t i o n of latency time from t u m o r inoculation to the a p p e a r a n c e of a m e a s u r a b l e t u m o r (table 1) revealed that mice in the ( - ) n a l o x o n e group had a significant (P < 0.01) delay of 33% in c o m p a r i s o n to control animals. M e a n ( + S . E . ) survival time after t u m o r a p p e a r a n c e (for those mice developing tumors) i n the ( - )naloxone group was 23.7 + 2.3 days; however this value was n o t significantly greater t h a n control levels (18.9 + 2.1 days). T h e p a t t e r n of t u m o r growth appeared to be similar a m o n g all groups. At the time of death (table 1), t u m o r size of n a l o x o n e - t r e a t e d a n d control a n i m a l s was comparable.

23.2 + 1.6 26.6 + 1.3 22.35 -+1.1

3.3. Opioid challenge experiments Prior to challenge with morphine, latency responses on the hot-plate test were similar for all n a l o x o n e a n d control groups (fig. 2). C o n t r o l a n d ( + ) n a l o x o n e a n i m a l s injected with m o r p h i n e a n d e x a m i n e d 1 h after n a l o x o n e a d m i n i s t r a t i o n ex100

80

6O

40

20 --

I

0 ~l. ~ 20

30

40

50

I 60

Days after t u m o r cell inoculation Fig. 1. Effect of dally s.c. injections of (+) or ( - ) isomers of naloxone on survival time (days after tumor cell inoculation) of mice inoculated with 106 S20Y neuroblastoma cells. Survival curves of mice receiving 15 mg/kg (-)naloxone (O), 15 mg/kg (+)naloxone ([3), or sterile water (Q) were analyzed by the Mann-Whitney U test and presented as percentage of survivors.

118 40

30

>~J Z

20

..J 10

Control

F (*}

(-)

Naloxone

Nafoxone

Fig. 2. Latency (s) of response on the hot-plate test for mice receiving 15 m g / k g ( - )naloxone of 15 m g / k g ( + )naloxone or sterile water and examined 1 h after naloxone injections. Open bars = latency time prior to injection of morphine. Stippled bars = latency time 30 rain following injection of 0.2 m g / k g morphine sulfate. ** Significantly (P < 0.01) less than control and ( + )naloxone groups following morphine injection.

hibited analgesia (latency times of 40 s). However, the latency response for ( - ) n a l o x o n e animals following morphine was comparable to pre-injection levels, and differed significantly from analgesic responses of control and (+)naloxone groups. Further investigation of mice given ( - ) n a l o x o n e showed that opioid receptor blockade lasts for 4-6 h. At 4 h, the latency for the ( - ) n a l o x o n e group (mean + S.E. = 22.0 + 0.6 s) was significantly shorter than control (mean _.+S.E. --- 40.0 + 0.0 s) and did not reveal analgesia induced by morphine, but at 6 h, mice in the ( - ) n a l o x o n e group given morphine did not differ from morphine-injected control mice.

4. Discussion

The present results clearly demonstrate that opioid antagonists can significantly change the course of neural neoplasia, supporting conclusions reached in earlier reports (Zagon and McLaughlin, 1981a,b; 1983a,b; 1984a). We now show that the effect of opioid antagonists on cancer is stereospecific. Chronic daily administration of 15 m g / k g ( - ) n a l o x o n e to mice with neuroblastoma had a remarkable antitumor effect that included signifi-

cant reductions in tumor incidence and a marked extension in survival time for tumor-bearing mice. Injections of the enatiomer (+)naloxone, at dosages of 15 mg/kg, resulted in animals which resembled controls in all aspects of tumor response. Naloxone's ability to alter tumorigenicity was made even more prominent when the concentration of neuroblastoma cells used to produce tumors is taken into account. An inoculum of 106 cells utilized in this and other studies (Sheffler et al., 1979; Zagon and McLaughlin, 1981a, 1984a) always yields a 100% tumor incidence in control mice. Thus, in these experiments naloxone's antitumor effects are exhibited under the most strenuous testing conditions. Given lower concentrations of neuroblastoma cells, a more marked lowering of tumor incidence a n d / o r increase in survival time could be envisioned. The dosage (15 mg/kg) of naloxone employed in this study was not high, being well-below the LDso level of 252 m g / k g for mice, as well as below the maximum drug dosage (i.e., 30 mg/kg) that elicits overt effects such as depression or ataxia (Giering et al., 1974). The low general toxicity of the dosages of naloxone utilized also was demonstrated in that tumor-bearing mice receiving naloxone did not lose body weight. Although opioid antagonists such as naloxone have been regarded as 'pure' antagonists to the many biological actions of opioid substances and devoid of significant intrinsic activity (Blumberg and Dayton, 1974; Sawynok et al., 1979), reports indicate that these compounds may have a variety of effects unrelated to opioid receptors (Sawynok et al., 1979). Thus, more rigorous proof (h-at ol~ioid receptors mediate a particular response is essential. Opioid interactions at the receptor level have been shown to be stereospecific (Pert and Snyder, 1973), with isomeric forms showing markedly different affinities for opioid receptors. The ( - ) isomer of naloxone is known to be 3 or 4 orders of magnitude more active than the ( + ) isomer in its ability to bind the opioid receptors in rat brain membrane preparations as well as in antagonizing physiological responses to opioids (Gayton et al., 1978; Iijima et al., 1978; Arndt and Freye, 1979; Faden and Holaday, 1980; Dunwiddie et al., 1982).

119

Indeed, in these reports and others (Sawnyok et al., 1979; Sanger et al., 1981), the demonstration of stereospecificity of opioid antagonist action has been recommended as a criterion for exclusion of pharmacological effects not due to interactions with opioid receptors. The present results showing that the ( - ) isomer of naloxone, but not the ( + ) isomer of naloxone, alters tumor incidence and survival time is consistent with the enantiomeric specificity of opioids. Moreover, it establishes that neuro-oncogenesis is mediated, at least in part, through opioid receptor interactions and provides an important step in demonstrating that endogenous opioid systems are involved in abnormal tissue growth. Data from this investigation provide further evidence which implicates endogenous opioid systems as regulators of neural tumor growth. The S20Y neuroblastoma cell line utilized in this investigation is known to possess opioid receptors (Klee and Nirenberg, 1976). Exogenous opioid agonists such as heroin have been reported to inhibit neoplasia (Zagon and McLaughlin, 1981b), and this action can be completely blocked by concomitant administration of opioid antagonists such as naloxone (Zagon and McLaughlin, 1981b). Results from in vitro studies reveal that opioid agonists exert a growth inhibitory effect on cells in culture that is dose-dependent, stereospecific, and blocked by naloxone (McLaughlin and Zagon, 1984; Zagon and McLaughlin, 1984b). Moreover, at least 2 opioid antagonists, naloxone and naltrexone, have been shown to affect neural neoplasia (Zagon and McLaughlin, 1981a, 1983a). Recent experiments have demonstrated that the duration of opioid receptor blockage is crucial in determining the course of neuro-oncogenesis (Zagon and McLaughlin, 1984a). The present study provides even further support for this line of reasoning by showing that opioid receptors are stereospecifically involved in tumorigenesis.

Acknowledgements This work was supported in part by NIH grants NS-20623 and NS-20500.

References Arndt, J.O. and E. Freye, 1979, Opiate antagonist reverses the cardiovascular effects of inhalation anaesthesia, Nature 277, 399. Aylsworth, C.F., C.A. Hodson and J. Meites, 1979, Opiate antagonists can inhibit mammary tumor growth in rats, Proc. Soc. Exp. Biol. Med. 161, 18. Blumberg, H. and H.B. Dayton, 1973, Naloxone, naltrexone, and related noroxymorphones, in: Narcotic Antagonists, eds. M.C. Braude, L.S. Harris, E.L. May, J.P. Smith and J.E. Villarreal (Raven Press, New York) p. 13. Dunwiddie, T.V., E. Perez-Reyes, K.C. Rice and M.R. Palmer, 1982, Stereospecificity of opiate antagonists in rat hippocampus and neocortex: responses to ( + ) and ( - ) isomers of naloxone, Neuroscience 7, 1961. Faden, A.I. and J.W. Holaday, 1980, Naloxone treatment of endotoxic shock: stereospecificity of physiologic and pharmacologic effects in the rat, J. Pharmac. Exp. Ther. 212, 441. Gayton, R.J., L.A. Lambert and P.B. Bradley, 1978, Failure of (+)-naloxone to antagonise responses to opioid peptides, Neuropharmacology 17, 549. Giering, J.E., T.A. Davidson, B.V. Shetty and A.P. Truant, 1973, Narcotic antagonist activity of substituted 1,2,4,5-tetrahydro-3H, 3-benzazepines, in: Narcotic Antagonists, eds., M.C. Braude, L.S. Harris, E.L. May, J.P. Smith and J.E. Villarreal (Raven Press, New York) p. 167. Iijima, I., J. Minamikawa, A.E. Jacobson, A. Brossi, and K.C. Rice, 1978, Studies in the (+)-morphinan series. 5.1 Synthesis and biological properties of ( + )-naloxone, J. Med. Chem. 21, 398. Klee, W.A. and M. Nirenberg, 1974, A neuroblastoma x glioma hybrid cell line with morphine receptors, Proc. Natl. Acad. Sci., U.S.A. 71, 3474. McLaughlin, P.J. and I.S. Zagon, 1984, Opioid regulation of neurotumor cell growth in vitro, Soc. Neuroscience, 14th Annual Meeting 10, 1111. Pert, C.B. and S.H. Snyder, 1973, Properties of opiate receptor binding in rat brain, Proc. Natl. Acad. Sci. U.S.A. 70, 2243. Plotnikoff, N.P. and G.C. Miller, 1983, Enkephalins as immunomodulators, Int. J. Immunopharmacol. 5, 437. Sanger, D.J., P.S. McCarthy and G. Metcalf, 1981, The effect of opiate antagonists on food intake are stereospecific, Neuropharmacology 20, 45. Sawynok, J., C. Pinsky and F.S. LaBella, 1979, Minireview on the specificity of naloxone as an opiate antagonists, Life Sci, 25, 1621. Sheffler, B.A., M.A. Repman, and C.-L. Schengrund, 1979, Development of a neonatal and metastatic murine neuroblastoma model, Cancer Res. 39, 711. Zagon, I.S. and P.J. McLaughlin, 1981a, Naloxone prolongs the survival time of mice treated with neuroblastoma, Life Sci. 28, 1095. Zagon, I.S, and P.J. McLaughlin, 1981b, Heroin prolongs survival time and retards tumor growth in mice with neuroblastoma, Brain ges. Bull 7, 25.

120 Zagon, I.S. and P.J. McLaughlin, 1983a. Naltrexone modulates tumor response in mice with neuroblastoma, Science 221, 671. Zagon, I.S. and P.J. McLaughlin, 1983b, Opioid antagonists inhibit the growth of metastatic murine neuroblastoma, Cancer Lett, 21, 89.

Zagon, I.S. and P.J. McLaughlin, 1984a, Duration of opiate receptor blockade determines tumorigenic response in mice with neuroblastoma: a role for endogenous opioid systems in cancer, Life Sci. 35, 409. Zagon, I.S. and P.J. McLaughlin, 1984b, Opiates alter tumor cell growth and differentiation in vitro, NIDA Research Monograph 49, 344.