Opioids and differentiation in human cancer cells☆
Introduction
The endogenous opioid peptide, [Met5]-enkephalin, also termed the opioid growth factor (OGF), is a constitutively expressed native opioid that interacts with the OGF receptor (OGFr) to inhibit growth in neoplasia, development, wound healing, and angiogenesis (Zagon and McLaughlin, 1993, Zagon and McLaughlin, 1999, Zagon et al., 2002). OGF is an autocrine and paracrine produced and secreted peptide that has a direct and rapid action which is stereospecific, reversible, non-cytotoxic, independent of serum, and occurs at physiologically relevant concentrations (Zagon et al., 1996, Zagon et al., 1999, Zagon et al., 2000a, McLaughlin et al., 1999a, McLaughlin et al., 1999b, Zagon, 1988). OGF activity is not cell, tissue, or organ specific (e.g., Zagon et al., 1996, Zagon et al., 1999, Zagon et al., 2000b, McLaughlin et al., 1999a), and is targeted to the G0/G1 phase of the cell cycle (Zagon et al., 2000c). OGF also has been reported to influence cell migration and tissue organization (Zagon et al., 2000d). Interruption of peptide-receptor interaction by sustained opioid receptor antagonism (e.g., the potent and long-acting opioid antagonist, naltrexone (NTX)), OGF-specific antibodies, or antisense constructs for OGFr results in a substantial increase in cell number compared to control levels (Zagon et al., 2002, McLaughlin et al., 1999a, McLaughlin et al., 1999b), suggesting the constitutive nature of OGF-OGFr interfacing.
Of three fundamental processes contributing to the determination of growth (cell number and cell size): cell proliferation, cell survival, and cell differentiation, cell proliferation and cell survival have been studied (Zagon et al., 2000c, Zagon and McLaughlin, 2003). Little is known about cell differentiation as a mechanism of OGF action in inhibiting cell growth. Thus, it could be hypothesized that the repressive effect of OGF on cell number is due, in part, to increasing the ability of cells to undergo differentiation and thereby diminishing the pool of replicating cells. As a corollary, it may be conjectured that attenuation of the OGF-OGFr axis by NTX can lead to an increase in cell number by repressing cell differentiation, thereby expanding the population of cells available for duplication.
To address these issues, the present study uses an in vitro model of three human cancer cell lines that have been documented to be regulated in cell number by the OGF-OGFr axis: SK-N-SH neuroblastoma, SCC-1 squamous cell carcinoma of the head and neck (SCCHN) and CAL-27 SCCHN (McLaughlin et al., 1999a, McLaughlin et al., 1999b, Zagon et al., 2000c). Differentiation was monitored by assessment of neurite formation (Schubert and Jacob, 1970, Monard et al., 1973), process length (Schubert and Jacob, 1970, Monard et al., 1973), and the presence of βIII-tubulin (Katsetos et al., 2003, Ohucihi et al., 2002) for SK-N-SH cells, and involucrin immunoreactivity for SCC-1 and CAL-27 cells (Watt, 1983, Chou et al., 2004). Thus, using quantitative measures and log-phase cells exposed to OGF or NTX at 10−6 M (a concentration known to alter growth – McLaughlin et al., 1999a, McLaughlin et al., 1999b), adherent cells were examined for differentiation. Cell preparations were employed in a temporal series extending up to 6 days after drug exposure in order to understand the full perspective of the differentiation process with OGF or NTX modulation. To examine whether other opioid peptides – synthetic and natural – can modulate differentiation of any of the three cell lines used in this study, we included a variety of opioids in our evaluation as well. Finally, the effects of chronic exposure to OGF and NTX on cell morphology (cell and nuclear areas) and the cytoskeleton (F-actin, α-tubulin) of SK-N-SH cells were investigated as a structural correlate to differentiation. These studies provide a comprehensive view of the role of the OGF-OGFr axis on the processes of differentiation in various cancer cells of human origin.
Section snippets
Cell lines and cell maintenance
Human cancer cell lines utilized in this study included: SK-N-SH neuroblastoma (Biedler et al., 1973), and UM-SCC-1 (SCC-1) (Krause et al., 1981) and CAL-27 (Gioanni et al., 1988) SCCHN. SK-N-SH and CAL-27 cell lines were purchased from the American Type Culture Collection (Manassas, VA), and SCC-1 cells were obtained from the Cancer Research Laboratory at the University of Michigan (Dr. Thomas E. Carey, Director). SK-N-SH cells were grown in MEM media, whereas SCC-1 and CAL-27 cell lines were
Process differentiation
SK-N-SH cells grown in culture for either 3 or 6 days following initiation of opioid exposure were assessed for properties of process differentiation (Fig. 1). The percentage of control cells with a process of 40 μm or greater was 8% and 21% at 3 and 6 days, respectively, an increase of 2.5-fold over a 3-day period. Mean process length for controls was 51.5 ± 3.1 and 66.9 ± 5.9 μm at 3 and 6 days, respectively. Exposure to OGF, OGF blockade with naloxone, naloxone alone, or NTX did not alter either the
Discussion
This study addressed the question of whether a mechanism for the decrease in cell number attributed to OGF exposure is due to a stimulation of cell differentiation which would have the net effect of reducing the population of dividing cells. In addition, this investigation examined whether a mechanism for the increase in cell number related to treatment with NTX is the result of an inhibition in cell differentiation, thereby expanding the pool of cells capable of duplication. The results show
Acknowledgements
We thank Jody Hankins, Caitlin Groff, and Bethany Rommel for technical assistance.
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Grant Support: This research was support in part by Philip Morris USA Inc. and Philip Morris International.