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. 2019 Aug 19;19(1):220.
doi: 10.1186/s12906-019-2628-z.

The cytotoxic effect and glucose uptake modulation of Baeckea frutescens on breast cancer cells

S H Shahruzaman  1 , M F Mustafa  1 , S Ramli  2 , S Maniam  3 , S Fakurazi  1 , S Maniam  4
Affiliations

The cytotoxic effect and glucose uptake modulation of Baeckea frutescens on breast cancer cells

S H Shahruzaman et al. BMC Complement Altern Med. .

Abstract

Background: Baeckea frutescens (B. frutescens) of the family Myrtaceae is a plant that has been used in traditional medicine. It is known to have antibacterial, antipyretic and cytoprotective properties. The objective of this study is to explore the mechanism of B. frutescens leaves extracts in eliminating breast cancer cells.

Method: B. frutescens leaves extracts were prepared using Soxhlet apparatus with solvents of different polarity. The selective cytotoxicity of these extracts at various concentrations (20 to 160 μg/ml) were tested using cell viability assay after 24, 48 and 72 h of treatment. The IC50 value in human breast cancer (MCF-7 and MDA-MB-231) and mammary breast (MCF10A) cell lines were determined. Apoptotic study using AO/PI double staining was performed using fluorescent microscope. The glucose uptake was measured using 2-NBDG, a fluorescent glucose analogue. The phytochemical screening was performed for alkaloids, flavonoids, tannins, triterpenoids, and phenols.

Results: B. frutescens leaves extracts showed IC50 value ranging from 10 -127μg/ml in MCF-7 cells after 72 h of treatment. Hexane extract had the lowest IC50 value (10μg/ml), indicating its potent selective cytotoxic activity. Morphology of MCF-7 cells after treatment with B. frutescens extracts exhibited evidence of apoptosis that included membrane blebbing and chromatin condensation. In the glucose uptake assay, B. frutescens extracts suppressed glucose uptake in cancer cells as early as 24 h upon treatment. The inhibition was significantly lower compared to the positive control WZB117 at their respective IC50 value after 72 h incubation. It was also shown that the glucose inhibition is selective towards cancer cells compared to normal cells. The phytochemical analysis of the extract using hexane as the solvent in particular gave similar quantities of tannin, triterpenoids, flavonoid and phenols. Presumably, these metabolites have a synergistic effect in the in vitro testing, producing the potent IC50 value and subsequently cell death.

Conclusion: This study reports the potent selective cytotoxic effect of B. frutescens leaves hexane extract against MCF-7 cancer cells. B. frutescens extracts selectively suppressed cancer cells glucose uptake and subsequently induced cancer cell death. These findings suggest a new role of B. frutescens in cancer cell metabolism.

Keywords: Apoptosis; Baeckea frutescens; Breast cancer; Glucose uptake; Oxidative phosphorylation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effects of B. frutescens leaves extracts on cell viability. a IC50 values were determined using cell viability assay in MCF-7 and MDA-MB 231 cells treated with either hexane, L90, L70, L50 or water extracts after 24, 48 and 72 h. b Normal mammary epithelial cells (MCF-10A) were treated with either (a) L90, L70 or L50 or (b) water or hexane extract of B. frutescens leaves for 72 h at three different concentrations; lower than IC50 value (low), IC50 value (IC50) and higher than IC50 value (high). Data is expressed as mean ± standard error mean based on three independent experiments with triplicate wells for each concentration
Fig. 2
Fig. 2
Effects of B. frutescens leaves extracts on MCF-7 cell viability. Cytotoxicity was determined using cell viability assay and the IC50 value determined as half maximal percentage of cell viability inhibition is indicated by the red horizontal line. MCF-7 cells were treated with either (a) hexane, b L90, c L70, d L50 or (e) water extract of B. frutescens leaves or (f) etoposide for 48 (left panels) or 72 (right panels) hrs. Etoposide served as the positive control. Data is expressed as mean ± standard error mean based on six independent experiments with triplicate wells for each concentration
Fig. 3
Fig. 3
Morphological observation of B. frutescens leaves extracts treated MCF-7 cells using AO/PI dual staining at X400 magnifications. a Observation of morphological changes in MCF-7 cells. Viable cells are green stained cells with intact nucleus, condensed chromatin marked by intense green stained chromatin, membrane blebbing indicated by the outgrowth of plasma membrane and apoptotic cells are characterised by nuclear disintegration and leakage of plasma membrane. b MCF-7 cells were either treated with DMSO vehicle control (control) or B. frutescens extracts (hexane, L90, L70, L50 and water) for 24, 48 and 72 h at their respective IC50 values
Fig. 4
Fig. 4
Inhibition of glucose uptake of B. frutescens leaves extracts after 24, 48 and 72 h of incubation MCF-7 (left panels) or MCF10A (right panels) cells were either treated with DMSO vehicle control (control) or B. frutescens leaves extracts (a) hexane, b L90, c L70, d L50 or (e) water for 24, 48 and 72 h at three different concentrations; lower than IC50 value (low), IC50 value (IC50) and higher than IC50 value (high). WZB117 served as the positive control. Data is expressed as mean ± standard error mean based on four independent experiments with triplicate wells for each concentration. *p < 0.01, compared with control, # p < 0.01 compared with the positive control (WZB117)
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