Morphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor kappaB transcriptional activation in neuroblastoma SH-SY5Y cells

Abstract

Morphine is recommended as a first-line opioid analgesic in the pain management of cancer patients. Accumulating evidence shows that morphine has anti-apoptotic activity, but its impact on the therapeutic applications of antineoplastic drugs is not well known. The present study was undertaken to test the hypothesis that morphine might antagonize the pro-apoptotic activity of DOX (doxorubicin), a commonly used antitumour drug for the treatment of neuroblastoma, in cultured SH-SY5Y cells. In the present study we demonstrated that morphine suppressed DOX-induced inhibition of cell proliferation and programmed cell death in a concentration-dependent, and naloxone as well as pertussis toxin-irreversible, manner. Further studies showed that morphine inhibited ROS (reactive oxygen species) generation, and prevented DOX-mediated caspase-3 activation, cytochrome c release and changes of Bax and Bcl-2 protein expression. The antioxidant NAC (N-acetylcysteine) also showed the same effects as morphine on DOX-induced ROS generation, caspase-3 activation and cytochrome c release and changes in Bax (Bcl-2-associated X protein) and Bcl-2 protein expression. Additionally, morphine was found to suppress DOX-induced NF-kappaB (nuclear factor kappaB) transcriptional activation via a reduction of IkappaBalpha (inhibitor of nuclear factor kappaB) degradation. These present findings support the hypothesis that morphine can inhibit DOX-induced neuroblastoma cell apoptosis by the inhibition of ROS generation and mitochondrial cytochrome c release, as well as by blockade of NF-kappaB transcriptional activation, and suggests that morphine might have an impact on the antitumour efficiency of DOX.

Figure 1. Morphine protectes SH-SY5Y cells from DOX-induced cell death

(A) Cells were treated with various concentrations of DOX for 48 h in the absence or presence of an increasing concentration of morphine (Mor), and cell viability was determined using the SRB assay as described in the Materials and methods section. (B) Cells were treated with 4 μM DOX in the presence of increasing concentrations of morphine (Mor) for 48 h, and the apoptotic cells were detected by flow cytometry as described in the Materials and methods section. Values are expressed as a percentage of the untreated control cell samples and represented as means±S.E.M. for at least three independent experiments performed in triplicate. *P<0.05 and **P<0.01 compared with DOX respectively.

Figure 2. Morphine antagonizes DOX-mediated enhancement of intercellular O₂^•− and H₂O₂ levels in SH-SY5Y cells

(A) Cells were treated with 4 μM DOX for the indicated time periods, and the intracellular O₂^•− and H₂O₂ levels were detected by flow cytometry using 5 μM HE and 10 μM DCFH₂-DA as fluorescent probes as described in the Materials and methods section. The Figure is representative of four independent experiments yielding similar results. (B and C) Cells were treated with either 4 μM DOX alone or with 4 μM DOX in combination with 200 μM morphine (Mor) for 24 h, and then the intracellular O₂^•− and H₂O₂ levels were detected. (B) Representative image of five independent experiments yielding similar results. (C) Quantification of O₂^•− and H₂O₂ generation. Values are means±S.E.M. for at least three independent experiments performed in triplicate (^##P<0.01 compared with control; *P<0.05 and **P<0.01 compared with DOX alone).

Figure 3. Morphine inhibites DOX-induced caspase-3 activation, cytochrome c release and changes in Bax and Bcl-2 protein levels in SH-SY5Y cells

Cells were treated with either 4 μM DOX alone or with 4 μM DOX in combination with 200 μM morphine (Mor) for 48 h. Extracts from cells were subjected to SDS/PAGE (12% gels) and immunoblotted with (A) anti-procaspase-3 or anti-(cleaved caspase-3); (B) anti-cytochrome c and (C) anti-Bax or anti-Bcl-2 antibodies. Cytochrome c was isolated from the cytosol and measured by Western blotting as described in the Materials and methods section. Anti-actin antibodies were used as a control for equal loading.

Figure 4. Morphine inhibites DOX-mediated degradation of IκBα in SH-SY5Y cells

(A) Cells were treated with 4 μM DOX for increasing time periods as indicated. Total cell lysates were resolved by SDS/PAGE (12% gels) and then immunoblotted to detect IκBα. (B) SH-SY5Y cells were treated with 4 μM DOX for 12 h in the absence or presence of 200 μM morphine (Mor), and then harvested for Western blot analysis. The image is a representative immunoblot for IκBα from three independent experiments yielding similar results. Anti-actin antibodies were used as a control for equal loading.

Figure 5. Morphine inhibites DOX-induced NF-κB p65 translocation in SH-SY5Y cells

SH-SY5Y cells grown on glass coverslips were pretreated with 200 μM morphine (Mor) for 1 h, and then treated with 4 μM DOX for 16 h. After treatment, cells were immunostained and visualized as described in the Materials and methods section. The images are representative of three independent experiments yielding similar results. Green, NF-κB p65; dark blue, nucleus; white, NF-κB p65 in the nucleus. Values are means±S.E.M. for three independent experiments (^##P<0.01 compared with control; **P<0.01 compared with DOX).

Figure 6. Effects of NAC on DOX-triggered cell death signalling

Cells were treated with 4 μM DOX in the absence or presence of 5 mM NAC for different time periods (24 h for ROS, 48 h for cytochrome c, 12 h for IκBα and 16 h for NF-κB p65 respectively) and then the amount of intracellular ROS, caspase-3, cytochrome c, IκBα and NF-κB p65 were determined as described in the Materials and methods section. (A) NAC inhibited DOX-mediate ROS generation. Values are means±S.E.M. for three independent experiments (^##P<0.01 compared with control; **P<0.01 compared with DOX alone). (B) NAC inhibited DOX-mediated caspase-3 cleavage. (C) NAC inhibited DOX-mediated enhancement of cytosolic cytochrome c levels. (D) NAC failed to inhibit DOX-mediated IκBα degradation. (E) NAC was unable to inhibit DOX-mediated NF-κB p65 translocation. The images are representative of three independent experiments yielding similar results. Green, NF-κB p65; dark blue, nucleus; white, NF-κB p65 in the nucleus.