Elsevier

Neuroscience Research

Volume 29, Issue 2, October 1997, Pages 121-127
Neuroscience Research

Etorphine inhibits cell growth and induces apoptosis in SK-N-SH cells: involvement of pertussis toxin-sensitive G proteins

https://doi.org/10.1016/S0168-0102(97)00080-1Get rights and content

Abstract

Opiates have been used extensively in the treatment of pain but with the severe side effect of addiction, which is believed to be related to opiates' direct (primary) or indirect (secondary) neurotoxicity. In this study, the effects of opioids on cell growth and apoptosis have been examined in human neuroblastoma cell line SK-N-SH. Etorphine, a wide-spectrum and potent agonist of opioid receptors, was found to significantly inhibit cell growth and to induce apoptosis. The inhibitory and apoptotic activities of etorphine followed a dose- and time-dependent manner. The more specific agonists of opioid receptors such as morphine, [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO), [d-Pen2, d-Pen5]-enkephalin (DPDPE), dynorphin A and nociceptin/orphanin FQ did not show similar toxic activities under the same conditions. In addition, the effects of etorphine could not be blocked by the opioid receptor antagonist naloxone, suggesting that the effects of etorphine might not be mediated by a classical opioid receptor. However, pretreatment of SK-N-SH cells with pertussis toxin (PTX) blocked the inhibition of cell growth and apoptosis induced by etorphine, indicating the involvement of PTX-sensitive G proteins in the processes. It was also shown that etorphine-induced apoptosis was prevented by actinomycin D (AD) and interleukin-1β converting enzyme inhibitor I. Interestingly, etorphine was similarly potent to inhibit growth of pheochromocytoma (PC12) cells but less effective in SH-SY5Y neuroblastoma cells and C6 glioma cells. We propose that inhibition of cell growth and induction of apoptosis may be one mechanism of opioid neurotoxicity.

Introduction

Chronic use of opiates leads to addiction, which is characterized by the phenomena of tolerance, sensitization, dependence and withdrawal (Nestler et al., 1993). Though cellular and molecular mechanisms underlying opiate addiction are far from clear, opiate addiction has been shown to be related to adaptive changes of neuronal functions in many regions of the central nervous system (Nestler, 1996, Wise, 1996). As typified in the ventral tegmental area (VTA) of the brain, chronic opiate exposure could induce major structural adaptations with gross impairment of dopaminergic neurons, perhaps even neural injury (Nestler, 1996).

It has been reported recently that opioids exert their immunosuppressing effects through altering many aspects of immunocyte functions: inhibition of proliferation and trafficking of T lymphocytes (Flores et al., 1995, Shahabi and Sharp, 1995); suppression of natural killer cell activity (Freier and Fuchs, 1994) and induction of apoptosis in thymocytes (Fuchs and Pruett, 1993). Opioids have been also shown to induce apoptosis in human lung cancer cells, which express both opioid peptides and receptors (Maneckjee and Minna, 1994). However, little is known about the direct effects of opioids on the growth of neuronal cells.

SK-N-SH cell line was derived from a human neuroblastoma and its growth, biochemical and cytogenetic properties confirmed its neuronal nature (Biedler et al., 1973). SK-N-SH was used as a target cell line in cell-mediated cytotoxicity assays (Bluestein, 1978). The expression of both delta and mu opioid receptors in SK-N-SH cells (Hochhaus et al., 1986) makes it an ideal cellular system to investigate the potential neurotoxic effects of opiates. Data from the present study mainly using SK-N-SH cells revealed that etorphine, a wide-spectrum and potent agonist of opioid receptors (Paterson et al., 1983, Wang et al., 1995) significantly inhibits cell growth and induces apoptosis. The data also show that pertussis toxin-sensitive G proteins are required for etorphine-induced neurotoxicity.

Section snippets

Opioids and related materials

Etorphine-HCl was provided by Professor Bo-Yi Qin (Academy of Military Medical Sciences, Beijing, China) and by Professor Cui-Bai Qiu (Shanghai Medical University, Shanghai, China) respectively. The drug from both sources gave the same result. Morphine-HCl was kindly provided by Professor De-He Zhou (Shanghai Institute of Materia Medica, Shanghai, China). [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO), [d-Pen2, d-Pen5]-enkephalin (DPDPE), naloxone, pertussis toxin (PTX) and actinomycin D (AD)

Etorphine significantly inhibited growth of SK-N-SH cells

As measured by [3H]thymidine incorporation assay, etorphine (10–100 μM) significantly inhibited growth of SK-N-SH cells after 24 h treatment (Fig. 1A). However, morphine, a less potent opioid receptor agonist and naloxone, a non-specific antagonist of opioid receptors, did not have significant effect on the cell growth under the same conditions (Fig. 1A). The inhibitory effect of etorphine was dose- and time-dependent, and the [3H]thymidine incorporation in SK-N-SH cells was reduced to about

Discussion

Recent progress has revealed that opiate addiction may lead to major structural adaptations and a gross impairment, even neuron injury, in the ventral tegmental area (VTA) of the brain and these changes could in turn contribute to motivational dependence and an aversive state during opiate withdrawal (Nestler, 1996). The molecular and cellular mechanisms underlying such neurotoxicities of opiates, however, are not clear. Although our laddering may not be considered the perfect one, it has been

Acknowledgements

This study was supported by research grants from the National Natural Science Foundation of China (No. 39600157), Chinese Academy of Sciences, Shanghai Education Development Foundation and the German Max-Planck Society. The authors thank Y.Q. Wu, W. Hu and Y.P. Wang for skilled technical assistance.

References (30)

  • H.G. Bluestein

    Neurocytotoxic antibodies in serum of patients with systemic lupus erythematosus

    Proc. Natl. Acad. Sci. USA

    (1978)
  • S.-Z. Bu et al.

    Progesterone induces apoptosis and up-regulation of P53 expression in human ovarian carcinoma cell lines

    Cancer

    (1997)
  • L.R. Flores et al.

    Mechanisms of morphine-induced immunosuppression: effect of acute morphine administration on lymphocyte trafficking

    J. Pharmacol. Exp. Ther.

    (1995)
  • D.O. Freier et al.

    A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression on natural killer cells activity

    J. Pharmacol. Exp. Ther.

    (1994)
  • B.A. Fuchs et al.

    Morphine induces apoptosis in murine thymocytes in vivo but not in vitro: involvement of both opiate and glucocorticoid receptors

    J. Pharmacol. Exp. Ther.

    (1993)
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