Thymoquinone Could Increase The Efficacy of Tamoxifen Induced Apoptosis in Human Breast Cancer Cells: An In Vitro Study

Abstract

Objective: Thymoquinone (TQ), as the main component of Nigella Sativa plant, shows anticancer properties. This study was aimed to evaluate the combined effect of TQ and Tamoxifen (TAM) on viability and apoptosis of human breast cancer cell lines.

Materials and methods: In this experimental study, estrogen positive MCF-7 and estrogen negative MDA-MB-231 human breast cancer cell lines were induced by TAM (2 µM) or different doses of TQ (50, 75, 100, 150 µM), individually or in combination. Cell viability and apoptosis were investigated by MTT assay and TdT-mediated deoxy-uracil nick end labeling (TUNEL) assay; Acridine orange (AO)/Ethidium bromide (EB) staining respectively. Data were analyzed by one way ANOVA and P<0.05 was considered significant.

Results: In 24 hours treatment, TAM and all doses of TQ, solely or in combination, significantly reduced cell viability of both cell lines, except in MCF-7 cells treated with 50 µM TQ, and MDA-MB-231 cells treated with 50 or 75 µM TQ (P<0.01). After 48 hours treatment, cell viability of both cell lines was reduced in all treated groups (P<0.05). Remarkable apoptotic index was observed in combination treatment of MCF-7 or MDA-MB-231 cell lines with TAM and TQ (P<0.001).

Conclusion: The synergistic effect of TQ and TAM on human breast cancer cell lines showed cell viability reduction as well as apoptosis induction, independent to estrogen.

Keywords: Apoptosis; Breast Cancer; Necrosis; Tamoxifen; Thymoquinone.

Fig.1

The effects of individual TAM (2 µM) and TQ, or in combination, on viability in MCF-7 and MDA-MB-231 cells. Cells were treated with TAM, TQ and combination of both for A., B. 24 hours, C. and D. 48 hours. Control wells were treated with equivalent amount of agents. Treatment with combined TAM and TQ significantly decreased cell viability compared to individual agents. The results are shown as the mean ± SEM from triplicate experiments. TAM; Tamoxifen, TQ; Thymoquinone, ***; P<0.001, **; P<0.01, *; P<0.05 compared to control, α; P<0.05 compared to TAM and β; P<0.001 in comparison with TAM.

Fig.2

The effects of TAM and TQ alone, or in combination, on morphology of MCF-7. The cells were stained by AO-EB and observed under fluorescence microscope. Images show A. Control group, B. In the presence of 2 µM TAM, C. In the presence of 150 μM TQ, D. In combination of both agents, E. Apoptotic index and F. Necrotic index based on AO-EB stained cells. Arrows show different cell morphologies: Blue; Live, Green; Early apoptotic, Yellow; Late apoptotic and Red; Necrotic cells. Microscopic images were captured with ×200 magnification. TAM; Tamoxifen, TQ; Thymoquinone, AO-EB; Acridine orange–Ethidium bromide, ***; P<0.001, **; P<0.01, *; P<0.05 compared to control, α; P<0.05 compared to TAM, and β; P<0.01 compared to TQ group. The data are presented as mean ± SEM.

Fig.3

The effects of TAM and TQ alone, or in combination, on morphology of MDA-MB-231. The cells were stained by AO-EB and observed under fluorescence microscope. Images show A. Control group, B. In the presence of 2 µM TAM, C. In the presence of 150 µM TQ, D. In combination of both agents, E. Apoptotic index and F. Necrotic index based on AO-EB stained cells. Arrows show different cell morphologies; Blue; Live, Green; Early apoptotic, Yellow; Late apoptotic and Red; Necrotic cells. Microscopic images were captured with ×200 magnification. TAM; Tamoxifen, TQ; Thymoquinone, AO-EB; Acridine orange–Ethidium bromide, ***; P<0.001, **; P<0.01, *; P<0.05 compared to control, α; P<0.05 compared to TAM group and β; P<0.001 compared to TAM and TQ groups. The data are presented as mean ± SEM.

Fig.4

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining assay for apoptosis in MCF-7 cells following treatment with TAM and TQ alone, or in combination. A. Control group, B. In the presence of 2 µM TAM, C. In the presence of 150 µM TQ, D. In combination of both agents and E. Apoptotic index, columns show mean percentage of apoptotic cells from three independent experiments performed in triplicate at images. Microscopic images were captured with ×200 magnitudes. TAM; Tamoxifen, TQ; Thymoquinone, ***; P<0.001 compared to control group, α; P<0.01 compared to TAM and TQ groups. The data are presented as mean ± SEM.

Fig.5

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining assay for apoptosis in MDA-MB-231 cells following treatment with TAM and TQ alone, or in combination. A. Control group, B. In the presence of 2 µM TAM, C. In the presence of 150 µM TQ, D. In combination of both agents and E. Apoptotic index, columns show mean percentage of apoptotic cells from three independent experiments performed in triplicate at images. Microscopic images were captured with ×200 magnitudes. TAM; Tamoxifen, TQ; Thymoquinone, ***; P<0.001 compared to control group, α; P<0.01 compared to TAM and TQ groups. The data are presented as mean ± SEM.