Effect of opioids on growth of human BT474 and SKBR3 mammary carcinoma cells

I. Weingaertner, H. Ammer. Institute of Pharmacology, Toxicology and Pharmacy, Ludwig-Maximilians-University of Munich, Germany

Opioids are potent analgesics and widely used for anaesthetic premedication in cancer surgery and management of cancer pain. Recent data indicate that opioids may also interfere with several aspects of tumor growth, including proliferation, metastasis, vascularization and survival. These effects are mediated via transactivation of receptor tyrosine kinase (RTK) pathways that are critical for tumor growth and, thus, represent promising targets for innovative antibody- and small molecule-based anti-cancer therapies. In a first step to evaluate potential interactions between opioid analgesics and RTK-directed anti-neoplastic strategies, the present study was initiated to identify and characterize opioid receptors in human breast cancer cell lines.

Human epithelial breast cancer BT474 and mammary adenocarcinoma SKBR3 cells were obtained from Cell Lines Service, Eppelheim, Germany, and grown in Dulbecco´s modified Eagle medium (DMEM), containing 10 % fetal calf serum, 4 mM l-glutamine, 100 IU penicillin and 0.1 mg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. RT-PCR studies using opioid receptor type-specific primers revealed that BT474 cells endogenously express mu-opioid receptors, whereas SKBR3 cells contain kappa-opioid receptor transcripts. Protein translation was verified by radioligand binding studies (BT474: 6.6 fmol/mg membrane protein mu-opioid receptors; SKBR3: 14.8 fmol/mg membrane protein kappa-opioid receptors). The functional activity of both receptors was tested in cAMP accumulation assays. Incubation of BT474 cells with the prototypical mu-opioid receptor agonist (-)-morphine (10 µM, 15 min) results in inhibition of forskolin (1 µM)-stimulated cAMP accumulation by about 41%. In contrast, activation of kappa-opioid receptors in SKBR3 cells with the selective agonist U69,539 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide; 1 µM, 15 min) produces an about 3-fold stimulation of cAMP generation. Chronic exposure of BT474 cells for 3 days to 10 µM (-)-morphine enhances cell growth by about 20%, as determined by crystal violet staining. Chronic treatment of SKBR3 cells for 3 days with 1 µM U69,539 produces an about 2-fold increase in cell proliferation. In both cell lines, stimulation of cell growth is blocked by concomitant exposure to the respective mu- and kappa-opioid receptor antagonists (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, CTAP, 1 µM; Norbinaltorphimine, 3 µM), indicating receptor-mediated effects. Further studies revealed that opioids increase cell growth via transactivation of receptor tyrosine kinase pathways. In BT474 cells, incubation with (-)-morphine (10 µM, 5 min) enhances basal and Heregulin (10 ng/ml)-stimulated activation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) and protein kinase B/AKT. In SKBR3 cells, acute stimulation with U69,539 (1 µM, 5 min) only enhances epidermal growth factor (EGF; 10 ng/ml)-stimulated, but not basal ERK1/2 and PKB/ Akt activities. In both cell lines, activation of the protein kinase B/AKT pathway is associated with attenuation of stress-induced apoptosis. The cell protecting opioid effects were assessed by determination of PARP cleavage and Annexin/propidium iodide staining of the cells.

Our results demonstrate that human breast cancer cells may carry endogenous opioid receptors that are functionally coupled to transactivation of RTK pathways. Thus, opioids given therapeutically or as a premedication in tumor surgery may interfere with tumor cell growth. These data also provide the molecular basis for potential interactions between opioids and RTK-targeted antineoplastic strategies.