Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase

Abstract

Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F₀F₁ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC₅₀: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit ^PLUS) into culture media at eugenol concentration >EC₅₀. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential.

Figure 1

(a) The effect of spices on viability of MCF-7 cell line. The seven different spices: “Clove, 7-spices, Black Pepper, Curry, Ginger, Turmeric and Nutmeg” were purchased from Spices and Herbs Store in Lebanon. Each spice was soaked, for 30 minutes, in 50 ml double distilled boiled water, then filtered using a filter paper to prepare a specific % stock solutions that were distributed in small aliquots and stored at −20 °C. All values were tested for normal distribution using Shapiro-Wilkis Test: (Clove: p = 0.30; 7-spices: p = 0.32; Black Pepper: p = 0.192; Curry: p = 0.587; Ginger: p = 0.10; Turmeric: p = 0.735; Nutmeg: p = 0.790). Each value represents mean ± SEM of nine determinations from three different experiments compared to control of vehicle (water) treated cells; *P value < 0.05 was considered significant. (b) The effect of clove extract on different cell lines. The effect of aqueous clove extract on cultured HepG2, Hek293, Caco2, and MCF-7 cells were compared. |Cells were treated for 24 hours with varying clove extract concentrations (0.05–1%) following which viability was determined using MTT assay. All values were tested for normal distribution using the Shapiro-Wilkis Test (HepG2: p = 0.33; Hek293: p = 0.172; Caco2: p = 0.117; MCF-7: p = 0.12). Each value represents mean ± SEM of nine determinations from three different experiments compared to control of vehicle (water) treated cells; *P value < 0.05 was considered significant.

Figure 2. Effect of eugenol on viability of MCF-7 cells, MDA-MB-231 cells and Bcl-2 over expressing MCF-7 cells.

All values were tested for normal distribution using the Shapiro-Wilkis Test (p = 0.612). Each value represents mean ± SEM of nine determinations from three different experiments. *P value < 0.05 was considered significant compared to a control.

Figure 3

Effect of eugenol on mitochondrial membrane potential of: (a) MCF-7 and (b) Bcl-2 overexpressing MCF-7 treated cells: (i) control; and eugenol at (ii) EC₅₀ (0.9 mM); (iii) 5 mM.

Figure 4

(a) Variation in ATP level in eugenol-treated MCF-7 cells. (b) Effect of eugenol on LDH release from MCF-7 and MDA-MB-231 treated cells. All values were tested for normal distribution using Shapiro-Wilkis Test: (ATP: p = 0.321; LDH: p = 0.112). Each value represent mean ± SEM of nine determinations from three different experiments; *P value < 0.05 was considered significant compared to control.

Figure 5

(a) Effect of eugenol on NBT reduction and H₂O₂ production in MCF-7 cells. All values were tested for normal distribution using the Shapiro-Wilkis Test (NBT reduction: p = 0.553; H₂O₂ production: p = 0.205); (b) Effect of antioxidants on viability and NBT reduction in eugenol-treated MCF-7 cells. All values were tested for normal distribution using the Shapiro-Wilkis Test (NBT reduction: p = 0.200; viability: p = 0.161). Each value represent mean ± SEM of nine determinations from three different experiments; *P value < 0.05 was considered significant compared to control and eugenol treated cells.

Figure 6

(a) Effect of eugenol on expression of cyt-c in MCF-7 treated cells (b) Eugenol induces release of cyt-c in culture media; and (c) expression of anti- and pro-apoptotic proteins in eugenol-treated MCF-7 cells. Results of densitometry of bands in cropped gels are expressed as mean expression ratio relative to control and are normalized relative to the internal control expression of GAPDH. Each value represent mean ± SEM of nine determinations from three different experiments; *P value < 0.05 was considered significant.

Figure 7

Effect of eugenol on MCF-7 cells using RTCA: (a) proliferation (b) migration and (c) invasion. Each value represent mean ± SEM of three different experiments; *P value < 0.05 was considered significant.

Figure 8

(a) Representative dot plots of cell apoptosis for control (vehicle treated: ethanol) and eugenol (0.9 mM) treated MCF-7 cell after Annexin V/PI dual staining. The proportion of dead cells (Annexin V−/PI+), live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−) and late apoptotic/necrotic cells (Annexin V+/PI+) were measured and compared. (b) Percentage of early and late apoptotic MCF-7 cells. Each value represents the mean ± SEM of two different experiments; *P value < 0.05 was considered significant compared to a control cells. (c) Representative histogram on the effect of eugenol on MCF-7 cell cycle distribution as measured by PI flow cytometry, compared to control. Each value represents the mean ± SEM of two different experiments; *P value < 0.05 was considered significant.