Abstract 4153: Short term opioid receptor blockade inhibits cancer cell proliferation independent of immune function by upregulation of the OGF-OGFr axis: A tissue culture model of low dose naltrexone (LDN)

Abstract

Abstract The opioid growth factor (OGF) is an autocrine produced and constitutively expressed peptide that interacts with the OGF receptor (OGFr) and subsequently targets the cyclin dependent kinase inhibitory (CKI) pathways to inhibit proliferation at the G1/S phase of the cell cycle. Naltrexone (NTX) is a nonselective opioid receptor antagonist that blocks this interaction. In vivo administration of a low dosage of NTX (LDN, 0.1 mg/kg) blocks opioid receptors for 4-6 h, resulting in a compensatory upregulation of OGF and OGFr. Following clearance of NTX, the elevated levels of OGF and OGFr interact to decrease cell proliferation. To investigate whether LDN inhibits cancer cell proliferation independent of immune function, a tissue culture model using short term NTX in human ovarian cancer cell lines (SKOV-3, OVCAR-3) was established. Cell replication and cell number were determined over 96 h in cultures treated with a single 6 h application of NTX (10−6 M). In SKOV-3 and OVCAR-3 cultures, cell number was decreased by 21-28% and 27-32%, respectively, between 48 and 96 h; DNA synthesis was inhibited ∼ 33% at 72 h. Peptide specificity studies using a variety of classical μ, δ, or κ opioid receptor agonists confirmed that only OGF inhibited cell number. Antibody neutralization of OGF in cultures revealed that endogenous OGF invoked the inhibitory growth effects of short term NTX. To evaluate which opioid receptor mediated the inhibitory action of short term NTX on growth, cultures were treated with siRNAs against the μ, δ, κ, and OGFr receptors; only OGFr was associated with short term NTX treatment. Immunocytochemical studies and semiquantitative analyses documented an upregulation of OGF and OGFr expression following short term NTX exposure in cancer cells; western blot analysis and receptor binding for OGFr revealed 2-fold increases of receptors 48 h after a brief exposure to NTX. NTX's inhibitory action was not related to apoptosis (TUNEL) or necrosis (trypan blue staining). To investigate whether short term NTX's action on cell proliferation was transduced by the p16 and/or p21 CKI pathways, siRNAs for p16 and p21 were utilized in combination with short term NTX. Silencing of p16 and/or p21 blocked the inhibitory action of short term NTX, indicating a mechanism directed toward the CKI pathways. Independent of immune function, short term opioid receptor antagonism has been demonstrated to inhibit cancer cell proliferation by upregulation of the OGF-OGFr axis. Upregulation of the OGF-OGFr axis by a brief opioid receptor blockade may provide a novel target for biotherapy of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4153.