Introduction: The possibility that opioids can influence tumour growth and metastasis is the subject of intense interest. In this study, we set up new methods to quantify the ability of opioids present in biological samples to activate the µ-opioid receptor (MOR) and toll-like receptor-4 (TLR-4). These two receptors can be activated by opioids or their metabolites, and are expressed on cancer cells as well as tumour-associated cells, and they control signalling pathways that play a key role in modulating cancer metastasis. Our objective is to establish the methods for quantifying receptor activation potential in the circulation of mice or patients administered morphine.
Methods: Alphascreen cyclic AMP (cAMP) assay and MOR overexpressing HEK293 cells have been employed to quantify the MOR activation. Cells engineered by co-transfection of the TLR-4 gene and other genes essential for TLR-4 activation (HEK-Blue™ hTLR4) were utilized to measure TLR-4 activity. Both assays were standardised using morphine, its MOR-active metabolite morphine-6 glucuronide (M6G) and its MOR-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G) in the presence/absence of serum or plasma from humans or mice. Specificity was verified using the opioid antagonists naloxone and methylnaltrexone, TLR-4 antagonist LPS-RS and TLR-4 inhibitor amitriptyline.
Results: Plasma is preferred over serum to quantify receptor activation and the optimal amount of plasma used in both assays is 5% (V/V). Morphine and M6G in spiked mouse or human plasma exhibited MOR activation, which M3G lacked. In contrast, M3G showed moderate but consistent activation of the TLR-4.
Conclusions: These assays can be used to measure receptor activation in biological fluids. This will be useful to determine the effect and mechanism of action of administered opioids on the behaviour of cancer and non-cancer cells important in the growth and metastasis of tumours.