Opioids and the apoptotic pathway 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., 1999). 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., 1999), and is targeted to the G0/G1 phase of the cell cycle (Zagon et al., 2000c). OGF has also 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)) and OGF-specific antibodies results in a substantial increase in cell number, cell migration, and cell differentiation compared to control levels (Zagon et al., 2000a; McLaughlin et al., 1999), 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, very little is known about cell survival 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, or in whole, to targeting the ability of cells to survive (i.e., increasing cell death). As a corollary, it may be conjectured that attenuation of the OGF–OGFr axis can lead to an increase in cell number by decreasing cell death. To address this issue, 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: MIA PaCa-2 pancreatic adenocarcinoma, HT-29 colon adenocarcinoma, and CAL-27 squamous cell carcinoma of the head and neck (SCCHN) (McLaughlin et al., 1999; Zagon et al., 1996, Zagon et al., 2000a). Cell death is usually classified into two broad categories: apoptosis and necrosis. Apoptosis occurs under normal physiological conditions and is an active process requiring energy. However, apoptosis can be elicited in a pathological way by toxic injury or during disease progression (Allen et al., 1997; McCarthy and Evan, 1998; Renvoize et al., 1998). Necrosis, in contrast, is a passive, catabolic process, always pathological that represents a cell’s response to extreme accidental or toxic insults. Thus, using quantitative measures and log-phase cells exposed to OGF or NTX at 10−6 M (a concentration known to alter growth), adherent cells were examined for early apoptosis by staining for Annexin V (Allen et al., 1997; Vermes et al., 1995), late apoptosis by utilizing the TUNEL procedure (Allen et al., 1997; Gavrieli et al., 1992; McCarthy and Evan, 1998), and necrosis by the trypan blue exclusion test (McCarthy and Evan, 1998). Cell preparations were examined in a temporal series after drug exposure: 2, 5, and 7 days, in order to understand the full perspective of the cell death process with OGF or NTX modulation. Although previous studies have found that many other opioid peptides – synthetic and natural – at dosages of 10−6 M do not influence the growth of any of the three cell lines used in this study (McLaughlin et al., 1999; Zagon et al., 1996, Zagon et al., 2000a), we included a variety of opioids in our evaluation for comparative purpose. Finally, because dead cells often detach from the substratum and drift into the medium (Clarke et al., 2000), we examined the supernatant for live and dead cells. This provided a comprehensive view of the magnitude of cell death after opioid exposure for both adherent and non-adherent cells.
Section snippets
Cells and cell maintenance
Three human cancer cell lines were studied: MIA PaCa-2 pancreatic adenocarcinoma, HT-29 human colon adenocarcinoma, and CAL-27 SCCHN. All cell lines were purchased from the American Type Culture Collection and maintained within the laboratory; characteristics of the cell lines have been described elsewhere (McLaughlin et al., 1999; Zagon et al., 1996, Zagon et al., 2000a, Zagon et al., 2000c). HT-29 cells were grown in McCoy’s 5A media, and the other two cell lines were maintained in Dulbecco’s
Results
Using the TUNEL technique and MIA PaCa-2 cells growing in culture for either 2, 5, or 7 days following initiation of opioid exposure, at least a 3-fold greater number of apoptotic cells in samples treated with DAMGO, morphine, and etorphine was recorded at all of the time points tested in comparison with control levels (ranging from 0.05% on day 2 to 0.09% on day 7) (Fig. 1). Although no differences were found between these compounds in the magnitude of elevation in TUNEL-positive cells at a
Discussion
This study was designed to address the question of whether a mechanism for the changes in cell number attributed to modulation of the OGF–OGFr axis is related to alterations in cell survival. The results show that stimulation of OGF–OGFr interfacing by a dosage (10−6 M) of exogenous OGF known to result in a decrease in cell number (McLaughlin et al., 1999; Zagon et al., 1996, Zagon et al., 2000a, Zagon et al., 2000c), does not change cell viability – either by necrosis or apoptosis; this would
Acknowledgements
We thank James D. Wylie and Amanda J. Dunkle for technical support.
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This research was supported in part by a grant from the External Research Division of Philip Morris, and a Pennsylvania Settlement Block Grant from The Pennsylvania Department of Health.