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Keywords:

  • morphine;
  • yohimbine;
  • NG108-15;
  • neurotoxicity;
  • apoptosis

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Short-term incubation with pharmacologically relevant concentrations of morphine has been shown to transiently affect the metabolism and redox status of NG108-15 cells through δ-opioid receptor stimulation, but apparently did not provoke cell death. The present work tries to determine if incubation with morphine at longer time intervals (24 h) provokes apoptosis and/or necrosis, as it has been described in other cell lines. We have also checked the potential modulatory role of yohimbine on these effects, on the basis of the previously described interactions between this drug and opioid receptor ligands. Incubation with morphine 0.1 and 10 μM provoked the appearance of images compatible with apoptosis (bebbling, pyknotic cells with cytoplasmic and nuclear condensation) and necrosis (cells swollen with vacuolated cytoplasm lacking cell processes) that could be observed directly and/or after staining with methylene blue, crystal violet and propidium iodide/4',6-diamidino-2-phenylindole (IP/DAPI). Quantification of apoptosis by activation of caspases 3 and 7 and DNA fragmentation with the Tunel assay revealed a modest but significant increase after incubation with the two concentrations of morphine used. Co-incubation with 10 μM yohimbine prevented all these effects of the opioid. The results extend previous findings of a yohimbine-sensitive, neurotoxic effect of morphine on NG108-15 cells. Copyright © 2012 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

A large number of in vitro studies with neuronal and glial primary cultures as well as with different cell lines have shown that morphine and other related opioids can inhibit cell proliferation and induce cell death, either by apoptosis or necrosis (Dawson et al., 1997; Goswami et al., 1998; He et al., 2011; Hu et al., 2002; Kugawa et al., 1998; Lazarczyk et al., 2010; Oliveira et al., 2002, 2003; Shoae-Hassani et al., 2011; Stiene-Martin and Hauser, 1993; Stiene-Martin et al., 1991; Yin et al., 1999; Zheng et al., 2010). In addition, there is evidence showing opioid-induced autophagy and cell death mediated by beclin-1 and ATG5 (Zhao et al., 2010). These effects are important as they can be related to opioid neurotoxicity in vivo, and they also support a possible utility of opioid drugs as coadjuvants for cancer treatment when neoplasic cells differentially express opioid receptors (Gralow, 2002; Ueda et al., 2003). In spite of these findings, contradictory results have been published regarding the actual effects of morphine on tumor growth and hence some authors have found increased cell proliferation, inhibition of apoptosis and promotion of both angiogenesis and tumor cell migration (see review by Gach et al., 2011).

Most of the studies that previously focused on opioid-induced apoptosis have examined the effects of morphine, which is suggested to result from both increased expression of pro-apoptotic proteins (FasL, Fas, Bax, Bad and caspase 3) and decreased expression of the anti-apoptotic protein Bcl-2 (Boronat et al., 2001; García-Fuster et al., 2003; Mao et al., 2002; Yin et al., 2006). Recent work from our own laboratory has shown that short incubations with pharmacologically relevant concentrations of morphine provoke transient metabolic changes in NG108-15 neuroblastoma × glioma hydrid cells mediated by δ-opioid receptor stimulation, but not substantial caspase activation (Polanco et al., 2009). However, a possible pro-apoptotic effect of the opioid was not carefully examined at longer incubation times, i.e. 24 h. This study is highly recommended as the mechanisms involved in opioid-induced apoptosis have been suggested to be closely related to the development of tolerance (Mao et al., 2002; Tegeder et al., 2003; Wu et al., 1999), and the latter is clearly established for different opioid effects in NG108-15 cells incubated with morphine for 24 h (Polanco et al., 2009; Sharma et al., 1975).

According to these antecedents, the present work has focused on cellular morphology, caspase activation and DNA fragmentation exhibited by NG108-15 cells after 24 h incubation with morphine. One of the reasons for choosing this particular cell line is directly related to our interest in studying pharmacological interactions between opioid and α2-adrenoceptor ligands with potential therapeutic application (see review by Alguacil and Morales, 2004). NG108-15 cells provide a useful model for these studies as they are sensitive to both kind of drugs and exhibit heterologous regulation including cross-adaptations upon prolonged incubation (Lee et al., 1988; Sabol and Nirenberg, 1979; Sharma et al., 1975). In this preliminary work, we have checked if the proapoptotic effects of morphine could be modified by co-incubation with the α2-adrenoceptor antagonist yohimbine, taking into account that this drug was previously shown to prevent morphine-induced inhibition of adenylate cyclase activity and cell metabolism in NG108-15 cells (Polanco et al., 2009, 2011).

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cell Culture and Treatment

NG108-15 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and penicillin (100 U ml–1)/streptomycin (100 µg ml–1). Cells were maintained in flasks (75 cm2 surface area; Sarstedt, Barcelona, Spain) at 37 °C under 40% humidity with a 5% CO2 atmosphere. The experiments were conducted in flat-bottom 24-well tissue culture plates (Sarstedt, Barcelona) at a density of 8.5 × 103 cells per cm2 (morphology experiments), in flat-bottom 96-well tissue culture plates of 0.32 cm2 per well (Sarstedt), where cells were plated at 1 × 104/well (caspase assay) or on glass slides into Petri dishes (TUNEL assay). After overnight attachment, cells were incubated during 24 h with the drugs under study dissolved in supplemented DMEM.

Drug incubations were conducted with morphine sulphate (Alcaliber, Madrid, Spain) 0.1 and 10 μM; yohimbine hydrochloride 10 μM (Sigma, Madrid, Spain) and combinations of both drugs. The concentrations of morphine chosen for this study were previously found active to inhibit alamar blue and (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction in NG108-15 cell cultures (Polanco et al., 2009); both of them are pharmacologically relevant as they represent the calculated EC21 and EC95 of morphine to stimulate GTPγS binding in NG108-15 cells, respectively (Polanco et al., 2011). On the other hand, the concentration of yohimbine used was the one that prevented the effects of morphine on the redox status of NG108-15 cells (Polanco et al., 2009) as well as morphine inhibition of adenylate cyclase activity (Polanco et al., 2011).

Morphological Studies

Cell morphology was studied by direct observation and after staining with methylene blue (Hemacolor solution 3; Merck, Barcelona, Spain), 1% crystal violet (Sigma), 0.5–1.0 µg ml–1 propidium iodide (PI; Sigma) and 4',6-diamidino-2-phenylindole (DAPI; Vectashield mounting medium, Vector Labs, Peterborough, UK). Cell photographs were taken with inverted (Nikon TS-100, Izasa, Barcelona, Spain) and fluorescence (Nikon Eclipse 50i, Izasa, Barcelona, Spain) microscopes.

Caspase Assay

Caspase-3 and −7 activities were determined by a luminescent assay (Caspase-Glo 3/7 Assay; Promega, Madison, MI, USA). The kit provides a luminogenic substrate containing the tetrapeptide sequence DEVD in a reagent optimized for caspase activity, luciferase activity and cell lysis. The addition of the reagent to the wells results in cell lysis followed by caspase cleavage of the substrate and generation of a luminescent signal produced by luciferase which is proportional to the amount of caspase activity present. The luminescent signal was measured at 490 nm with a microplate reader (Molecular Devices VERSA Max Tunable, BioNova, Madrid, Spain).

Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick end Labeling (TUNEL) Assay

TUNEL assays were performed according to the TUNEL kit manufacturer's instructions (ApopTag® Fluorescein Direct In Situ Apoptosis Detection Kit; Chemicon -Millipore, Billerica, MA, USA). The DNA fragmentation usually associated with apoptotic process is detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. These new DNA ends that are generated upon DNA fragmentation are typically localized in morphologically identifiable nuclei and apoptotic bodies. In contrast, normal or proliferative nuclei, which have relatively insignificant numbers of DNA 3'-OH ends, usually do not stain with the kit. The kit distinguishes apoptosis from necrosis by specifically detecting DNA cleavage and chromatin condensation associated with apoptosis.

Positive control cells were treated with DNAse I solution in DNAse buffer (0.1 µg ml–1) for 10 min. Cells incubated with drugs for 24 h were then rinsed with phosphate-buffered saline (PBS) (Sigma, Madrid, Spain), fixed in 1% paraformaldehyde in PBS for 10 min and rinsed in PBS. Cells were post-fixed in ethanol-acetic solution at −20 °C for 5 min, rinsed again in PBS, incubated for 10 s in equilibration buffer at room temperature and further incubated for 1 h in Working TdT enzyme Solution in a dark humidified chamber at 37 °C. Cells were put in a coplin jar containing Working Strength Stop/Wash buffer for 10 min at room temperature. The excess of liquid was removed and a mounting medium containing DAPI was applied, using 15 μL for a 22 × 50 mm coverslip with an oil immersion objective. The cell slides were observed in a fluorescence microscope (Nikon Eclipse 50i, Izasa, Barcelona, Spain) using standard fluorescein and DAPI excitation and emission filters.

Statistical Analysis

Six or more replicates of each drug treatment were performed in the caspase assay and four replicates in the TUNEL assay. The assays were repeated at least three times. The results were expressed as percent of the mean control values and analyzed by one-way anova followed by Newman–Keuls post-hoc tests. A P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

NG108-15 hybrid cell morphology was typically neuronal (Fig. 1). Direct observation of the plates of cells incubated with morphine revealed the presence of cell death images compatible with necrosis and apoptosis; thus, next to cells with normal morphology, others showed cytoplasmic vacuoles and a lack of cell processes (suggestive of necrosis, Fig. 2A), whereas some others exhibited protrusions in the plasma membrane typical of apoptosis (bebbling cells, Fig. 2B). Further processing of morphine-incubated wells with methylene blue, crystal violet and IP/DAPI staining permitted the observation of cells swollen with vacuolated cytoplasm suggestive of necrosis together with pyknotic cells with cytoplasmic and nuclear condensation suggestive of apoptosis (Fig. 3A–C). Cell death images were absent or only marginal after combined incubation with morphine and yohimbine, even when vacuolated cells were still present (Fig. 4).

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Figure 1. Control NG108-15 cells showing the typical neuronal morphology. 40 ×.

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Figure 2. NG108-15 cells incubated with 10 μM morphine. (A) 40 ×. (B) 100 ×.

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Figure 3. NG108-15 cells incubated with 10 μM morphine. (A) (methylene blue staining, 100 ×) and (B) (crystal violet staining, 40 ×) show images of necrosis with structural changes as cytoplasmic vacuolization and nuclear and cellular swelling (thick arrows), and images of apoptosis with chromatin condensation and nuclear and cytoplasmic fragmentation (thin arrows). (C) [propidium iodide/4',6-diamidino-2-phenylindole (IP/DAPI staining, 100 ×)] shows images of apoptotic bodies.

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Figure 4. NG108-15 cells incubated with 10 μM morphine and 10 μM yohimbine. Vacuolated cells are present. Crystal violet staining (40 ×).

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To quantify the pro-apoptotic effects of incubation with morphine, yohimbine or a combination of both drugs, we analyzed the activation of caspases 3 and 7 after 6-, 8-, 10- and 24-h incubations. No effect was observed during the first 10 h, however at 24 h there was a significant increase of around 15% in caspase activity after incubation with morphine 0.1 and 10 μM, an effect that was prevented by co-incubation with yohimbine (Fig. 5). TUNEL assays were performed to confirm that caspase activation was coincident with higher numbers of apoptotic cells labeled with fluorescein isothiocyanate (FITC, green fluorescence) compared with the total number of cells labeled with DAPI (blue fluorescence); these experiments revealed that incubation with morphine 0.1 and 10 μM increased from 2% to 11% the number of apoptotic cells, an effect that disappeared again when yohimbine was added to the incubation medium (Fig. 6). Figure 7 shows some representative images of these experiments.

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Figure 5. The effect of incubation for 24 h with yohimbine (YOH, 10 μM), morphine (MOR, 0.1 and 10 μM) or combinations of these drugs on the activation of caspases 3 and 7. Data are expressed as means ± standard error. *P < 0.05 vs. control. **P < 0.05 vs. morphine.

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Figure 6. The effect of incubation for 24 h with yohimbine (YOH, 10 μM), morphine (MOR, 0.1 and 10 μM) or combinations of these drugs on the percentage of apoptotic cells estimated with the TUNEL assay. Data are expressed as means ± standard error. *P < 0.05 vs. control. **P < 0.05 vs. morphine.

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Figure 7. TUNEL images representative of the effects of different incubations on NG108-15 cells. (A) Cells labeled with 4',6-diamidino-2-phenylindole (DAPI), 20 ×. (B) Cells labeled with fluorescein isothiocyanate (FITC), 20 ×. (C) Cells labeled with DAPI + FITC, 20 ×. (D) Cells labeled with DAPI + FITC, 100 ×.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The results obtained clearly show that 24 h of incubation with morphine 0.1 and 10 μM increases to a similar extent the activity of caspases 3/7, which is coincident with DNA fragmentation as shown by dUTP-FITC labeling and thus demonstrate morphine-induced apoptosis in NG108-15 cells. These findings were corroborated by morphological observations currently considered the main tool to define apoptosis (Kroemer et al., 2009). This morphological evaluation also revealed an apparent increase of necrosis images after morphine incubation. In any case, cell death induced by morphine was quantitatively limited, a reason that can explain why it was not previously described in NG108-15 cells. The possibility exists that apoptosis and/or necrosis could affect a subpopulation of cells particularly sensitive to morphine toxicity, an idea compatible with other studies that described up to four different types of cultured neuroblastoma cells regarding their sensitivity to opioid-induced calcium dyshomeostasis (Perez-Alvarez et al., 2010). In any case, our results replicate previous findings of morphine-induced increases of apoptotic figures including nuclear alterations such as chromatin condensation, cells showing the typical bebbling and cell fragmentation without inflammation or discharge, leaving the cell debris as apoptotic bodies with a typical ‘hourglass’ image. This neurotoxic effect of morphine was more delayed than the one previously described affecting the redox status of the cell but shared a lack of concentration dependency (Polanco et al., 2009); the reason for this lack of dose effect is presently unknown, but it may hint at the time of exposure being too long.

The increase in cell death by morphine found in our experiments is apparently contradictory with the results obtained by Heiss et al. (2009) in NG108-15 cells. These authors reported that incubation with DPDPE 1 μM or etorphine at the same concentration is accompanied by a cytoprotective effect mediated by δ receptors and activation of the PI3K/Akt cascade. There are, however, significant differences between conditions in both studies to be taken into account. Thus, Heiss et al. observed an anti-apoptotic effect of the opioids only when cells were deprived of serum during incubation, which increases five times the presence of the active form of caspase-3 and decreases cell viability more than 60%. As our pro-apoptotic effect occurs in normal cell growth, it appears that the effects of opioids may vary depending on the state of the cells, these drugs being neuroprotective only in very adverse situations. This hypothetical duality of effects according to specific environmental conditions could explain at least part of the apparent contradictions in the literature showing both apoptotic and anti-apoptotic effects of opioids (Boronat et al., 2001; Hassanzadeh et al., 2011; Mao et al., 2002; Tramullas et al., 2007; Xie et al., 2010). Some limitations of our study must be highlighted before predicting a significant contribution of the effects found with morphine to opioid neurotoxic symptoms in vivo (i.e. impairments of memory and cognitive skills). First, the NG108-15 cell line is a mixture of rat astrocytoma and mouse neuroblastoma particularly susceptible to drug neurotoxicity. Second, the concentrations used clearly exceeded the minimum effective analgesic concentration of morphine for non-tolerant patients (6–26 nM), although the lowest of them approached the plasmatic levels reported in tolerant patients under treatment for cancer pain (Peterson et al., 1990).

Concerning the modulatory role of yohimibine on morphine-induced cell death, we found an interaction qualitatively similar to that described at shorter time intervals concerning cell metabolism. Thus, co-incubation with yohimbine for 24 h prevented morphine-induced caspase activation, dUTP-FITC cell labeling and images consistent with cell death, as co-incubation with yohimbine for up to 16 h was previously found to prevent morphine inhibition of alamar blue and MTT cellular reduction (Polanco et al., 2009). The mechanism of this new interaction has not been explored yet, but in any case the results are consistent with findings from various in vivo studies suggesting that α2-adrenergic antagonists may have neuroprotective properties, both in the absence and presence of opioids. Thus, dexefaroxan increases cell survival in the olfactory bulb and dentate gyrus of the hippocampus of rats (Bauer et al., 2003; Rizk et al., 2006), and yohimbine itself was found to prevent reactive astrogliosis provoked by chronic morphine treatment in the rat brain (Alonso et al., 2007; Garrido et al., 2005). The present study extends the number of interactions already described between opioid and α2-adrenoceptor ligands and further supports the idea that opioid neurotoxicity can be negatively modulated by concomitant exposure to α2-adrenoceptor antagonists, as previously described with analgesia and addiction. In spite of these findings, it is clear that much more work is needed to fully understand the effects of morphine in these cells as well as their modulation by different opioid and adrenergic ligands, both agonists and antagonists.

In summary, the incubation of NG108-15 cells with pharmacologically relevant concentrations of morphine for 24 h leads to a limited but significant cell death that can be prevented when yohimbine is present in the incubation medium. This effect was more delayed than the transient, yohimbine-sensitive modification of the redox status of NG108-15 cells previously shown to be induced by similar concentrations of morphine. Therefore, the study of a possible causative relationship between both phenomena seems highly recommended.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (PI05/2503), Spain.

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  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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