Antitumorigenic Effect of Tramadol and Synergistic Effect With Doxorubicin in Human Breast Cancer Cells

Abstract

Background: Breast cancer in women is one of the leading causes of cancer mortality worldwide, and curative therapy is the main focus of clinical treatment. Anesthetic-analgesic techniques might alter stress responses and immunity and thereby influence outcomes in cancer patients. This study investigated the effect of tramadol on breast cancer progression and metastasis.

Methods: The effects of tramadol on two different subtypes of human breast adenocarcinoma cell lines, MDA-MB-231 and MCF-7, were studied with regard to cell growth, migration, colony formation and invasion and normoxic or hypoxic microenvironment for the expression of hypoxia-inducible factor-1α, reactive oxygen species, epithelial-mesenchymal transition related and cyclin-related proteins. The co-administration of tramadol and doxorubicin was studied to determine whether the effective doxorubicin dose might be reduced in combination with tramadol.

Results: The results showed that tramadol inhibited cell growth at concentrations more than 0.5 and more than 1.0 mg/mL in MDA-MB-231 and MCF-7 cells, respectively. Additionally, cell migration, colony formation and invasion were inhibited in a dose-dependent manner by tramadol in both cell lines. The combination of tramadol and doxorubicin induced synergistic effects in MDA-MD-231 cells and, with specific dosage combinations in MCF-7 cells.

Conclusions: Tramadol may regulate epithelial-mesenchymal transition and possess cytotoxic effects in breast cancer cells. Tramadol inhibits the progression of breast cancer cells and might be a candidate for combination therapy, especially for triple-negative breast cancer, and is a promising treatment strategy for breast cancer.

Keywords: HIF-1α; breast cancer; doxorubicin; epithelial–mesenchymal transition; tramadol.

Figure 1

The effects of tramadol on cell viability and the cell-cycle profiles of human breast cancer cells. (A–D) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.1, 0.5, 1.0, 2.5 and 5 mg/mL) for 24 h. (A, B) Cell viability was measured according to the MTT method. (C, D) For cell-cycle profiles, the cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Bars depict the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t-tests).

Figure 2

Analysis of the effect of tramadol on the cell migration of human breast cancer cells in a wound-healing assay. (A, B) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.01, 0.05, 0.1, 0.25 and 0.5 mg/mL) for 24 h. Quantification of the migration area of ​​untreated and tramadol-treated cells within 24 h using Image J Bars depict the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t-tests).

Figure 3

Analysis of the effect of tramadol on the colony-formation ability of human breast cancer cells. (A, B) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.05, 0.1, 0.2, 0.3 and 0.4 mg/mL) for 14 days. Bars depict the mean ± SD of three independent experiments. * p <0.05, ** p <0.01 and *** p <0.001 (Student’s t-tests). Bars depict the mean ± SD of three independent experiments.

Figure 4

Analysis of the effect of tramadol on the invasiveness of human breast cancer cells. (A, B) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.1, 0.5 and 1 mg/mL) for 24 h. Bars depict the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t-tests). Bars depict the mean ± SD of three independent experiments.

Figure 5

The effects of tramadol on protein expression in human breast cancer cells. (A, B) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.05, 0.1, 0.5, 1 and 1.5 mg/mL) for 4 h. β-actin (lower panel) served as the loading control.

Figure 6

The combination index (CI) of combination treatment of tramadol and doxorubicin in human breast cancer cells. (A, B) MDA-MB-231 and MCF-7 cells were treated with tramadol (0, 0.0625, 0.125, 0.25, 0.5, 1, 2 and 4 mg/mL) and doxorubicin (0, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM) for 24 h. The experimental points below the line correspond to CI <1, indicating a synergistic effect.