Tualang honey promotes apoptotic cell death induced by tamoxifen in breast cancer cell lines

Abstract

Tualang honey (TH) is rich in flavonoids and phenolic acids and has significant anticancer activity against breast cancer cells comparable to the effect of tamoxifen (TAM), in vitro. The current study evaluated the effects of TH when used in combination with TAM on MCF-7 and MDA-MB-231 cells. We observed that TH promoted the anticancer activity of TAM in both the estrogen receptor-(ER-)responsive and ER-nonresponsive human breast cancer cell lines. Flow cytometric analyses indicated accelerated apoptosis especially in MDA-MB-231 cells and with the involvement of caspase-3/7, -8 and -9 activation as shown by fluorescence microscopy. Depolarization of the mitochondrial membrane was also increased in both cell lines when TH was used in combination with TAM compared to TAM treatment alone. TH may therefore be a potential adjuvant to be used with TAM for reducing the dose of TAM, hence, reducing TAM-induced adverse effects.

Figure 1

Total apoptosis induced by TH, TAM, and TH + TAM in MCF-7 (a) and MDA-MB-231 (b) cells. Cells were treated with TH (1%), TAM (2.5, 5, 10, 15 µM), or their combination for up to 72 h. Apoptosis was detected by flow cytometry using annexin-V-Fluos antibody and propidium iodide. Data are expressed as mean ± standard deviation from three independent experiments. *P < 0.05 significantly different from TH alone. ^σ P < 0.05 significantly different from TAM alone.

Figure 2

Early-and late-stage apoptosis induced by TH, TAM, and TH + TAM in MCF-7 (a) and MDA-MB-231 (b) cells. Cells were treated with TH (1%), TAM (2.5, 5, 10, 15 µM), or their combinations for 24 and 48 h. Apoptosis was detected by flow cytometry using annexin-V-Fluos antibody and propidium iodide. Data are expressed as mean ± standard deviation from three independent experiments. *P < 0.05 significantly different from TH alone. ^σ P < 0.05 significantly different from TAM alone.

Figure 3

Effect of TH, TAM, and TH + TAM on the mitochondrial membrane potential of MCF-7 cells. Cells treated with TH alone or in combination with TH (5 μM) for 24 h were stained with mitochondrial-selective JC-1 dye and analyzed by flow cytometry. (a) The dot plots represent the population of mitochondrial membrane-depolarized cells on the lower right (LR) and mitochondrial membrane-polarized cells on the upper right (UR). (b) Bar graphs represent the percentages of polarized and depolarized cells. Data are expressed as mean ± standard deviation from three independent experiments. ^# P < 0.05 significantly different from untreated control. ^σ P < 0.05 significantly different from TAM alone.

Figure 4

Effect of TH, TAM, and TH + TAM on the mitochondrial membrane potential of MDA-MB-231 cells. Cells treated with TH alone or in combination with TH (5 μM) for 24 h were stained with mitochondrial-selective JC-1 dye and analyzed by flow cytometry. (a) The dot plots represent the population of mitochondrial membrane-depolarized cells on the lower right (LR) and mitochondrial membrane-polarized cells on the upper right (UR). (b) Bar graphs represen the percentages of polarized and depolarized cells. Data are expressed as mean ± standard deviation from three independent experiments. ^# P < 0.05 significantly different from untreated control.

Figure 5

Effect of TH, TAM, and TH + TAM on caspase activation in MCF-7 cells. Treated (6 h) and untreated cells were stained for caspase-3/7 FLICA (FAM-DEVD-FMK), caspase-8 FLICA (FAM-LETD-FMK), and caspase-9 FLICA (FAM-LEHD-FMK) which is indicated by green fluorescence. Cell nuclei are stained blue (Hoechst dye).

Figure 6

Effect of TH, TAM, and TH + TAM on caspase activation in MDA-MB-231 cells. Treated (6 h) and untreated cells were stained for caspase-3/7 FLICA (FAM-DEVD-FMK), caspase-8 FLICA (FAM-LETD-FMK), and caspase-9 FLICA (FAM-LEHD-FMK) which is indicated by green fluorescence. Cell nuclei are stained blue (Hoechst dye).