G Protein-independent G₁ Cell Cycle Block and Apoptosis with Morphine in Adenocarcinoma Cells: Involvement of p53 Phosphorylation¹ Free

Most cancer patients suffer from severe pain. However, physicians often avoid the prescription of potent opioid analgesics, in part for fear of side effects. This attitude is strengthened by a recent study (1) that was published during the review process of the present article. This study (1) shows that morphine promotes the growth of breast cancer xenografts in nude mice by increasing angiogenesis. In addition, tumor-promoting effects of morphine have been found in mice that received an injection with leukemia or sarcoma cells (2). Because morphine caused suppression of concanavalin A-induced proliferation of lymphocytes in this study, the tumor-promoting effects were suggested to be due to an inhibition of the cellular immune response (2). On the other hand, some opioids [methadone, morphine, and buprenorphine (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)] and opioid peptides (14, 15, 16, 17, 18) were found to inhibit tumor cell proliferation and in vivo tumor growth in various models. The reasons for these conflicting results are unclear. It has been suggested that “atypical” opioid binding sites might be involved in tumor suppression because in some studies, the antiproliferative effects were not antagonized with Nx³ (1, 5, 7, 10). However, this was not unequivocally confirmed in other studies (2, 6, 8). Hence, it is not known whether the observed tumor-promoting and/or tumor-suppressing effects of opioids are mediated through the well-characterized opioid-mediated intracellular signaling pathway that involves activation of a PTX-sensitive inhibitory G protein (G_i; Ref. 19), inhibition of adenylyl cyclase, and decrease of cAMP levels and thereby inhibition of PKA. In the present study, we primarily addressed the potential involvement of G_i and p53 for effects of morphine on tumor growth.

MCF-7 [wild-type p53, estrogen receptor positive (20)] and MDA-MB231 [p53 mutation (21), estrogen receptor negative (22)] human breast cancer cells were cultured in DMEM and RPMI 1640, respectively, supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ atmosphere. HT-29 colon cancer cells [dominant negative p53 (20)] were cultured in McCoy’s medium.

DAMGO, Nx, and actinomycin D were from Sigma (Steinheim, Germany). Morphine-sulfate trihydrate was from Merck (Darmstadt, Germany). The drugs were dissolved in water. Forskolin (adenylyl cyclase activator), recombinant human FasL, and Fas-fusion protein were from Alexis (Grünberg, Germany). The caspase 3, 8, and 9 inhibitors Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK, respectively, and 8-bromo-cAMP (PKA activator) were from Calbiochem (Schwalbach, Germany). Antibodies were from Santa Cruz Biotechnology (p21, Fas, FasL, Bax, Erk-2, PARP and secondary antibodies; Heidelberg, Germany), Gramsch Laboratories (μ- and δ-opioid receptor; Schwabhausen, Germany), and Cell Signaling (phospho-p53 and p53; Frankfurt, Germany).

Cells (3 × 10⁵) were treated for 24, 48, and 72 h; harvested by trypsinization; stained with trypan blue; and counted by a blinded observer.

Cells (500 cells/3-cm dish) were treated for 10 days, fixed with 100% methanol, and stained with 0.5% crystal violet, 5% Giemsa solution. Single cell clones were counted using QuantityOne (Bio-Rad, München, Germany). The IC₅₀ was calculated using a standard sigmoidal Emax model.

Cell membranes were prepared as described previously (23). Membranes (100 ng of protein) were incubated for 10 min at room temperature in the presence or absence of morphine or morphine + Nx. The incubation (50 μl, final volume) also contained 50 mm Tris (pH 7.4), 100 μm EDTA, 1 mm DTT, 150 μm MgCl₂, 1 mg/ml BSA, and a protease inhibitor mixture (5 mm benzamidine, 20 μg/ml lima bean trypsin inhibitor, 20 μg/ml leupeptin, and 20 μg/ml aprotinin). To the preincubated reaction mixture, 100 nm [γ-³²P]GTP was added. Hydrolysis of [γ-³²P]GTP was measured as described previously (24). Reactions were stopped after 20 min by transferring the assay mixture into tubes containing 0.75 ml of ice-cold 5% (w/v) Norit A in 50 mm NaH₂PO₄ (pH 8.0). After centrifugation, aliquots (500 μl) of the supernatant were decanted for determination of the [³²P]phosphate content by scintillation counting.

Cells were harvested by scraping them into 200 μl of lysis buffer [135 mm NaCl, 25 mm β-glycerophosphate, 20 mm Tris, 2 mm EDTA, 2 mm Na PP_i, 2 mm DTT, 1 mm Na₃VO₄, 10% glycerol, 1% Triton X-100, 2 μg/ml aprotinin, and 5 μg/ml leupeptin (pH 7.5)]. The protein content was determined using the Bradford method. Proteins were separated electrophoretically by SDS-PAGE and transferred onto nitrocellulose membranes by semidry blotting. Membranes were incubated with primary and secondary antibodies according to standard procedures, and protein-antibody complexes were visualized with enhanced chemiluminescence (Amersham-Pharmacia, Freiburg, Germany).

A one-tube RT-PCR system (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer’s instructions. PCR conditions were as follows: 30 min at 50°C; 35 cycles of 94°C for 1 min, 57°C (Fas) or 52°C (β-actin) for 1 min, and 72°C for 1.5 min; and 7 min at 72°C. Amplification products were separated by 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining. Amplification of Fas mRNA was confirmed by DNA sequencing.

Primers were as follows: Fas, 5′-CTGTATGTGAACACTGTGACCC-3′ (forward) and 5′-AATTTATTGCCACTGTTTCAGG-3′ (reverse); and β-actin, 5′-CCGGATCCTCTTTGCTACTGAGACAGG-3′ (forward) and 5′-CCGAATTCGGGATCTGAATGCAATGTT-3′ (reverse).

Cells (5 × 10⁵) were starved for 72 h and then treated in full medium for 24 or 48 h. Cells were harvested by trypsinization, fixed with ice-cold 80% ethanol, and kept at −20°C for 24 h. Cells were stained with PI, and the DNA content was determined using flow cytometry (Becton Dickinson). The cell cycle distribution was assessed with WinMDI 2.8.

MCF-7 cells (1 × 10⁶) were treated for 24 h and harvested by trypsinization. Cells were stained with phycoerythrin-labeled annexin V and 7-AAD, and fluorescence was measured by flow cytometry. Ten thousand cells were counted.

Tumor cells (5 × 10⁶ MCF-7 cells, 1 × 10⁷ MDA-MB231 cells, and 1 × 10⁷ HT-29 cells in 150 μl of medium per mouse) were injected s.c. at the right dorsal flank of NMRI-nu/nu female mice (20–25 g; Harlan Winkelmann GmbH, Borchen, Germany). In case of MCF-7 cells, mice received 10 mg/kg β-estradiolvalerate i.m. at day 1, 7, and 14. Mice were treated with morphine, Nx, and morphine + Nx or vehicle (5% glucose/Ringer lactate) starting right after tumor cell implantation (8–15 mice/group). Because of a potential desensitization of opioid receptors, the dose of morphine and Nx was increased stepwise (10, 20, and 30 mg/kg i.p. for the first, second, and third week). For the drug combination, the Nx dose was one-tenth of the morphine dose because this ratio is generally considered to result in a complete antagonism of antinociceptive effects of morphine (25). The tumor volume was assessed three times a week [tumor volume = (long diameter × short diameter²)/2] for 2 or 3 weeks. The experiments were approved by the local ethics committee for animal research. Morphine plasma concentrations were analyzed by a liquid chromatography tandem mass spectrometry method as described previously (26).

Data are presented as mean ± SE. For statistical comparisons, univariate ANOVA was performed. Subsequently, treatment groups were mutually compared using t tests with a Bonferroni α correction for multiple comparisons (α at 0.05). For time courses, the area under the curve was calculated, and areas under the curve were then submitted to one-way ANOVA.

Cell proliferation was significantly reduced at ≥10 μm morphine in MCF-7 cells (Fig. 1 A). Nx (250 μm) significantly reduced the proliferation in MCF-7 cells but had no significant effect in MDA-MB231 and HT-29 cells. The combination of morphine (250 μm) and Nx (250 μm) was as effective as either agent alone in MDA-MB231 cells and was more potent than either agent alone in MCF-7 and HT-29 cells. The effects of the selective μ-agonist DAMGO were indistinguishable from those of morphine.

In the colony forming assay (Fig. 1 B), the IC₅₀ for morphine was 144.0 ± 23.3, 104.6 ± 18.7, and 182.9 ± 7.3 μm in MCF-7, MDA-MB231, and HT-29 cells, respectively. When morphine was combined 1:1 with Nx, the concentration effect curve was shifted to the left, indicating increased potency. The IC₅₀ for the mixture of each drag of morphine and Nx was 30.7 ± 1.2, 10.6 ± 0.9, and 32.3 ± 10.6 μm in MCF-7, MDA-MB231, and HT-29 cells, respectively.

Flow cytometry (Fig. 2 A) revealed a dose-dependent relative increase of the number of cells in the G₁ phase after morphine treatment. This indicates that morphine inhibited the progression from G₁ to S phase and caused a G₁ block. In MDA-MB231 cells, morphine also increased the number of sub-G₁ cells, indicating apoptosis. Nx caused a G₁ block in MCF-7 and HT-29 cells but had no obvious effect in MDA-MB231 cells. Again, morphine + Nx was more effective than either agent alone. The mixture caused a considerable increase of sub-G₁ cells in all three cell lines. Nx was added 10 min before morphine in all combination experiments.

PARP cleavage was determined by Western blot analysis to further assess induction of apoptosis (Fig. 2 B). Both morphine and Nx caused a PARP cleavage at 2 or 3 mm for 24 h in MDA-MB231 and MCF-7 cells, respectively. In HT-29 cells, PARP cleavage occurred at 3 mm for 48 h. Again, effects of the mixture were somewhat stronger than those of either agent alone.

In MCF-7 cells, we additionally assessed the binding of annexin V to phosphatidylserine of the plasma membrane and 7-AAD DNA binding by means of flow cytometry (Fig. 2 C). Both morphine and Nx caused an increase of late apoptotic cells (stained with 7-AAD and annexin V) and necrotic cells (stained with 7-AAD) at concentrations of 3 mm. Effects of morphine were not antagonized by Nx or vice versa.

Inhibition of the effector caspase 3 caused a near complete inhibition of morphine-induced apoptosis in MDA-MB231 and HT-29 cells, as indicated by a disappearance of sub-G₁ cells in cell cycle measurements (Fig. 2 D). Because MCF-7 cells lack caspase 3, inhibition of this effector caspase had no effect in these cells. Inhibition of caspase 8, which is primarily activated upon death receptor stimulation, caused a 50–70% inhibition of apoptosis in all three cell lines. Inhibition of caspase 9, which is activated in the course of the mitochondrial apoptosis cascade, caused a reduction of morphine-induced apoptosis by 30–50% in all three cell lines.

In mice treated with morphine, growth of MCF-7 and MDA-MB231 tumors was significantly reduced as compared with that seen in control mice (P = 0.001 for both cell lines; Fig. 3 A). Tumor growth was also inhibited by the combination of morphine + Nx in MCF-7 (P < 0.001) and MDA-MB231 tumors (P < 0.05), whereas Nx itself had no significant effect. HT-29 tumors were resistant to all treatments.

Plasma morphine concentrations were determined after administration of the last dose (Fig. 3 B). Peak morphine plasma concentrations ranged from 50–60 μm at 10–25 min after i.p. morphine injection. The elimination half-life was approximately 18 min. Concentrations were in the range of 0.9–3.4 μm between 1 and 2 h. These concentrations are similar to those found in cancer patients chronically receiving oral morphine for pain management (27, 28).

Morphine stimulated the steady-state GTPase activity in MDA-MB231 cell membranes with increasing concentrations (Fig. 4,A) and reached a maximum at 0.1–10 μm. Similar results were obtained in MCF-7 cells (data not shown). There was no increase of GTPase activity with HT-29 membranes (data not shown), although all three cell lines show immunoreactivity for at least one type of opioid receptor (Fig. 4 B). In MDA-MB231 cells pretreated with PTX (200 ng/ml for 24 h), the morphine-dependent increase in GTPase activity was significantly reduced. Addition of equimolar concentrations of Nx abolished morphine-stimulated steady-state GTPase activity.

To evaluate whether the typical opioid receptor-coupled signaling cascade was responsible for the growth-inhibitory effects of morphine, various antagonists were evaluated by flow cytometry. Neither PTX (200 ng/ml for 24 h), forskolin (5 μm for 24 h), nor 8-bromo-cAMP (10 μm for 24 h) was able to antagonize the morphine-induced cell cycle arrest (Fig. 4 C). The GTPase data suggest that morphine could signal through activation of a PTX-sensitive G protein in MDA-MB231 and MCF-7 cells as described for other cells expressing opioid receptors. However, because PTX and Nx abolished the morphine-induced G_i activation but not the morphine-induced growth inhibition, cell cycle arrest and apoptosis are obviously G_i independent.

Differences between MCF-7 (wild-type p53), MDA-MB231 (p53 mutation), and HT-29 (dominant negative p53) cells suggested that the p53 status might be important for the effects of morphine. Therefore, we assessed p53 and phospho-p53 (Ser¹⁵, Ser⁹) levels by means of Western blot analysis. Morphine and DAMGO caused an NH₂-terminal phosphorylation of p53 at Ser¹⁵ in MCF-7 cells and at Ser⁹ in MDA-MB231 cells (Fig. 5,A, left panel and right panel, respectively). In MCF-7 cells, p53 phosphorylation was associated with an increase of total p53 protein levels (Fig. 5,B). Because p53 mRNA remained constant (data not shown), this was probably caused by a stabilization of p53 (29). The combination of morphine and Nx also caused a p53 stabilization, whereas Nx alone had no effect (Fig. 5 B, right panel). In MDA-MB231 cells, total p53 protein remained constant (data not shown).

Morphine-induced p53 phosphorylation was associated with an increase of the p53-dependent proteins p21 (cell cycle inhibitor) and Bax (proapoptotic mitochondrial protein; Fig. 5 C). Similar effects were observed with morphine + Nx. Nx alone had no effect.

Because inhibition of caspase 8 was able to reduce morphine-induced apoptosis (Fig. 2 D), death receptors were likely to be involved. p53 regulates the transcription of Fas (30), DR5 [tumor necrosis factor-related apoptosis-inducing ligand receptor (31, 32)], the tumor necrosis factor α receptor (33), and Pidd, a new death domain-containing protein (34). Of these targets, we primarily addressed Fas because previous studies with lymphocytes and neurons have suggested that morphine might induce Fas expression (35, 36, 37).

CD95/Fas protein expression was found in untreated MDA-MB231 and HT-29 cells but was minimal in untreated MCF-7 cells (Western blot analysis; Fig. 6,A), suggesting that MCF-7 cells in particular down-regulate Fas, which is a common feature of tumor cells (38, 39). Therefore, we used this cell line to assess the effects of opioid treatment. DAMGO (500 μm) caused a rapid increase of Fas mRNA (RT-PCR; Fig. 6,B). Its effects were stronger than those of 1 μg/ml actinomycin D (positive control) and were not antagonized by Nx. Fas protein levels were increased at 6 h after DAMGO treatment (Fig. 6 C).

If cells were treated with morphine together with Fas-fusion protein (200 ng/ml), which inhibits the activation of the Fas receptor, morphine-induced apoptosis was partly antagonized (Fig. 6,D). However, Fas-fusion protein was less antagonistic than the caspase 8 inhibitor (Fig. 2,D). On the other hand, treatment with recombinant human FasL (60 ng/ml) caused apoptosis only in the presence of morphine at morphine concentrations that per se did not cause apoptosis (Fig. 6 D). However, the sensitivity of the tumor cells toward FasL remained low even in the presence of morphine.

Previously, morphine was found to cause a delay of normal cell death in neurons (40), to protect astrocytes from apoptosis-promoting agents (41), and to increase the proliferation of endothelial and tumor cells (1, 2, 42, 43). On the other hand, opioids were also found to promote cell death in lymphocytes (44, 45, 46), tumor cells (3, 4, 5, 6, 8, 10, 47, 48, 49, 50), and neurons (51, 52, 53). Effects of Nx were highly controversial [antagonism (8, 42, 43) or no antagonism (1, 5, 7)]. The protective effects of morphine have been linked to a Gβ/γ-mediated activation of phosphatidylinositol 3′-kinase (54), PKB/Akt (54), and Erks [Erk-1 and Erk-2 (1)]. On the other hand, the proapoptotic effects of morphine have been suggested to involve activation of p38 mitogen-activated protein kinase (37), nuclear factor κB inhibition (3), or up-regulation of death receptors (35, 36). In the present study, antiproliferative effects of morphine were associated with p53 activation and up-regulation of p53-dependent genes, including CD95/Fas. Although morphine facilitated FasL-evoked apoptosis, the sensitivity toward this death receptor ligand remained low even in the presence of morphine, suggesting that up-regulation of the Fas receptor might contribute in part to the growth-inhibitory effects of morphine but probably does not represent the primary mechanism. This is probably due to the variety of adaptations that allow tumor cells to escape from Fas-induced killing, of which down-regulation of the receptor is only one mechanism (39). In addition to the role of p53, the lack of in vivo effects of morphine toward HT-29 tumors in mice that did not respond with an increase of GTPase activity upon morphine exposure suggests that the antitumoral effects of morphine also depend on the abundance of opioid receptors. At first sight, this appears illogical because the growth-inhibitory effects of morphine occurred primarily at concentrations where G_i was no longer active. This suggests that possibly the uncoupling of G_i, rather than its activation, may be the initiating event that ultimately leads to p53 phosphorylation. This idea is supported by the finding that neuronal apoptosis induced by morphine was associated with morphine tolerance (52), which is known to be caused by opioid receptor desensitization and uncoupling of the G_i protein (55, 56, 57). On the other hand, increase of tumor growth with opioids occurred at very low doses (1, 43) and/or rapidly discontinued administration (1–3 doses; Ref. 42), i.e., with a dosing regimen avoiding tolerance. Because treatment of cancer pain usually requires long-term morphine use with increasing doses, opioid tolerance generally develops, possibly along with proapoptotic effects of morphine. However, morphine-induced apoptosis is not restricted to tumor cells. In particular, effects on immunocytes may have a negative impact on the antitumoral immune response (2), which may result in a faster progression of tumor growth or metastasis despite direct proapoptotic effects of morphine on tumor cells. In the present study, the growth-inhibitory effects of morphine were increased by Nx, suggesting that the combination of morphine and Nx might be useful to supplement cancer therapy, provided that tumors express opioid receptors and have no loss of function mutation of p53.