Matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS): quantification of [met]enkephalin in cell culture in relation to its growth effects

Abstract

The exact role of endogenous opioids in growth and development of cells is not completely understood. There is good evidence that opioids like [Met]enkephalin, also referred to as opioid growth factor (OGF), have inhibitory effects on growth of certain cell types. Additionally, there is good evidence that enkephalins are rapidly degraded by various specific peptidases in biological systems and this degradation can hinder attempts to determine the true effects of these opioid peptides. Our aims were to monitor opioid production and degradation in SH-SY5Y cell culture with and without the carboxy peptidase inhibitor, kelatorphan, by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), as a novel approach. Regulation of growth of two neuroblastomas (SH-SY5Y and N2A) and a human colon cancer cell line (HT-29) by opioid peptides in the presence and absence of and specific peptidase inhibitors, namely kelatorphan and bacitracin, was also assessed and related to the MALDI results. Neither [Met]enkephalin nor DAMGO (a mu receptor selective agonist) inhibited cell proliferation in any of the cell lines tested. However, naloxone in conjunction with [Met]enkephalin and kelatorphan retarded cell growth of both the N2A and HT-29 cell lines after 72 hours (p<0.05). When looking at pooled data of all three cell lines, [Met]enkephalin with either aminopeptidase inhibitor, kelatorphan or bacitracin, considerably reduced cell numbers after 72 hours. SH-SY5Y cells were shown by MALDI-TOF MS to produce [Met]enkephalin with or without kelatorphan at a rate of 6 to 16 fmoles/min/10,000 cells. The breakdown of [Met]enkephalin in cell culture medium was measured as 40 fmoles/min without kelatorphan. When kelatorphan was added, the rate of degradation remained approximately the same, suggesting that kelatorphan was not able to protect [Met]enkephalin from degradation and therefore, was unable to enhance the growth inhibitory effects of [Met]enkephalin reported in the literature. We conclude from this study that opioid peptides do not have a major inhibitory effect on neuroblastoma or colon cancer cell growth, although growth inhibition could be masked by rapid degradation of exogenously added peptides. MALDI-TOF MS is potentially capable of semi-quantitative measurement of opioid peptides; however, suppression of signal by components found in cell culture medium could be a significant problem.