Morphine, a potential antagonist of cisplatin cytotoxicity, inhibits cisplatin-induced apoptosis and suppression of tumor growth in nasopharyngeal carcinoma xenografts

Abstract

Morphine is an opioid analgesic drug often used for pain relief in cancer patients. However, there is growing evidence that morphine may modulate tumor growth, progression and metastasis. In this study, we evaluated whether morphine modulates cisplatin-induced apoptosis in human nasopharyngeal carcinoma CNE-2 cells and whether morphine affects the antitumor activity of cisplatin on tumor growth in human nasopharyngeal carcinoma CNE-2 xenografts in nude mice. We showed that a pretreatment with morphine (1 μg/ml) inhibited the sensitivity of CNE-2 cells to cisplatin by inhibiting cisplatin-induced CNE-2 cell apoptosis, decreasing caspase-3 activity and increasing the Bcl-2/Bax ratio. However, a high dose of morphine (1000 μg/ml) had the opposite effect. We also showed that at a low dose, morphine enhances chemoresistance in an in vivo nasopharyngeal carcinoma (NPC) model by inhibiting cisplatin-induced apoptosis and decreasing neovascularization. Taken together, our results indicate that a low dose of morphine may lead to chemoresistance of cisplatin in NPC models in vitro and in vivo by inhibiting cisplatin-induced apoptosis and decreasing neovascularization.

Figure 1. Effect of different concentrations of morphine on cisplatin-inhibited proliferation of CNE-2 cells.

(A) Cells were treated with cisplatin (CDDP) 4 μg/ml for 24 h, 48 h, and 72 h. (B) Cells were treated without or with morphine (Mor) (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml or 1000 μg/ml) for 24 h, 48 h, and 72 h. (C) Cells were treated with cisplatin (CDDP) 4 μg/ml for 24 h in the presence of an increasing concentration of morphine (Mor). (D) Cells were treated with cisplatin (CDDP) 4 μg/ml for 24 h in the presence of an increasing concentration of morphine (Mor). (E) Cells were treated with cisplatin (CDDP) 4 μg/ml for 24 h in the presence of an increasing concentration of morphine (Mor). Cell viability was determined using the MTT assay as described in the Materials and Methods section. *P < 0.05, **P < 0.01. Error bars represent the mean ± SD of triplicates.

Figure 2. Effect of the different concentrations of morphine on cisplatin-induced apoptosis in the CNE-2 cells.

Cells were treated with cisplatin (CDDP) 4 μg/ml for 72 h in the absence or presence of an increasing concentration of morphine (Mor). Thereafter, the cells were stained with Annexin V/PI and detected by fluorescence microscopy. (A) The apoptosis rate of the cells was determined as described in the Materials and Methods section. (B) The survival rate of the cells was determined as described in the Materials and Methods section. *P < 0.05, ***P < 0.001. Error bars represent the mean ± SD of triplicates.

Figure 3. Effect of different concentrations of morphine on cisplatin-induced caspase-3 activation and the changes in the Bax and Bcl-2 protein levels in the CNE-2 cells.

Cells were treated with either 4 μg/ml cisplatin (CDDP) alone or with 4 μg/ml cisplatin (CDDP) in combination with 1 μg/ml morphine (Mor) or 1000 μg/ml morphine (Mor) for 72 h. Extracts from cells were subjected to SDS/PAGE (12% gels) and immunoblotted with (A) anti-procaspase-3 or anti-(cleaved caspase-3). (B,C) The percent of relative intensity obtained from the corresponding western blots. (D) Anti-Bax or anti-Bcl-2 antibodies. (E,F) Percentage of relative intensity obtained from the corresponding western blots. Anti-β-actin antibodies were used as a control for equal loading. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent the mean ± SD of triplicates.

Figure 4. Morphine inhibited the antitumor effect of cisplatin on tumor growth of human CNE-2 xenografts in nude mice.

(A) The effects of Mor, cisplatin (CDDP) or their combination on tumor growth in CNE-2 xenograft-bearing nude mice. The saline solution treatment served as a control. The tumor length diameter (A) and width diameter (B) were measured every 2 days. The tumor volumes were calculated as (A × B²)/2. (B) Comparison of the tumor weight of four groups. (C) TUNEL staining, cleaved-caspase-3 immunohistology and CD34 immunohistology of the CNE-2 xenograft tumor sections after the treatment. (D) The number of TUNEL-positive cells per × 40 high-power field (HPF) is indicated. Numbers indicate the average of 10 mice per group. (E) The number of cleaved-caspase-3-positive cells per × 40 high-power field (HPF) is indicated. Numbers indicate the average of 10 mice per group. (F) The number of CD34-positive cells per × 40 high-power field (HPF) is indicated. Numbers indicate the average of 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent the mean ± SD of 10 mice per group.