β-Caryophyllene Counteracts Chemoresistance Induced by Cigarette Smoke in Triple-Negative Breast Cancer MDA-MB-468 Cells

Abstract

Exposure to cigarette smoke (CS) has been associated with an increased risk of fatal breast cancers and recurrence, along with chemoresistance and chemotherapy impairment. This strengthens the interest in chemopreventive agents to be exploited both in healthy and oncological subjects to prevent or repair CS damage. In the present study, we evaluated the chemopreventive properties of the natural sesquiterpene β-caryophyllene towards the damage induced by cigarette smoke condensate (CSC) in triple negative breast cancer MDA-MB-468 cells. Particularly, we assessed the ability of the sesquiterpene to interfere with the mechanisms exploited by CSC to promote cell survival and chemoresistance, including genomic instability, cell cycle progress, autophagy/apoptosis, cell migration and related pathways. β-Caryophyllene was found to be able to increase the CSC-induced death of MDA-MB-468 cells, likely triggering oxidative stress, cell cycle arrest and apoptosis; moreover, it hindered cell recovery, autophagy activation and cell migration; at last, a marked inhibition of the signal transducer and activator of transcription 3 (STAT3) activation was highlighted: this could represent a key mechanism of the chemoprevention by β-caryophyllene. Although further studies are required to confirm the in vivo efficacy of β-caryophyllene, the present results suggest a novel strategy to reduce the harmful effect of smoke in cancer patients and to improve the survival expectations in breast cancer women.

Keywords: IL-8; STAT3; autophagy; breast cancer; caryophyllene sesquiterpenes; cell migration; chemoresistance; cigarette smoke; environmental pollution; γH2AX.

Figure 1

Chemical structure of β-caryophyllene (ChemSketch, Version 2018.1.1 free software, ACD/Labs, Toronto, ON, Canada).

Figure 2

Scheduled treatment protocols applied to evaluate the chemopreventive properties of β-caryophyllene towards cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells: pre-treatment (A), co-treatment (B) and post-treatment (C).

Figure 3

Scheme of the treatment protocol applied to evaluate the recovery abilities of triple-negative breast cancer MDA-MB-468 cells after a 24 h exposure to β-caryophyllene and cigarette smoke condensate (CSC) alone and in co-treatment.

Figure 4

Cytotoxic effects of β-caryophyllene (A) and cigarette smoke condensate; CSC (B) in triple-negative breast cancer MDA-MB-468 cells after 24 h exposure. Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test).

Figure 5

Cytotoxic effects of β-caryophyllene and cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells under pre-, co- and post-treatment protocols, measured as cell viability (A, C, E) and LDH release (B, D, F). Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference the respect to CSC (Student’s t-test).

Figure 6

Effect of the co-treatment of β-caryophyllene and cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells after 24 h exposure and subsequent 72 h cell recovery. Recovery abilities were expressed as a recovery index, which represents the ratio between the cell number at each time point of recovery and that achieved after 24 h treatment. Cell number was determined by trypan blue exclusion assay at each time point. Data are displayed as mean ± SE of at least two experiments with at least three technical replicates (n = 6). *** p < 0.001, significant lowering of cell viability the respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). ° p < 0.05 and °°° p < 0.001, significant difference the respect to CSC (Student’s t-test).

Figure 7

Intracellular ROS levels induced by β-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (A) Representative images of cells stained by DCF. Original magnification 10×. (B) Bar graph of DCF-fluorescence. Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference with respect to CSC (Student’s t-test). Scale bars = 20 μm.

Figure 8

Bar graphs representing the levels of GSH (reduced glutathione) and GSSG (oxidized glutathione) induced by β-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure as revealed by HPLC analysis. Data are displayed as mean ± SE of at least three independent experiments with at least two technical replicates (n = 6). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference with respect to CSC (Student’s t-test).

Figure 9

Levels of γH2AX induced by cigarette smoke condensate (CSC) and β-caryophyllene in MDA-MB-468 cancer cells after 24 h exposure. (A) Representative images of cells stained by Alexa Fluor^® 647 anti-gamma H2A.X (phospho S139) antibody and Hoechst 33,258 dye. (B) Data are displayed as mean ± SE of at least three independent experiments with at least two technical replicates (n = 6). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). Scale bars = 20 μm.

Figure 10

Modulation of cell cycle phases induced by β-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (A) Histograms showing the percentages of cells stained by Hoechst 33,258 in different cell cycle phases under the scheduled treatments. (B) Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). * p < 0.05 and *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference with respect to CSC (Student’s t-test).

Figure 11

Apoptosis rate induced by β-caryophyllene, cigarette smoke condensate (CSC) and the positive control doxorubicin in MDA-MB-468 cancer cells after 24 h exposure. (A) Representative images of cells stained by Annexin V and Hoechst 33,258 dye. (B) Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °° p < 0.01, significant difference with respect to CSC (Student’s t-test). Scale bars = 20 μm.

Figure 12

Autophagic cell death induced by β-caryophyllene, cigarette smoke condensate (CSC) and the positive control sorafenib in MDA-MB-468 cancer cells after 24 h exposure. (A) Representative images of cells stained by autophagosome detection reagent. (B) Bar graphs of fluorescence intensity as determined by quantitative analysis using autophagosome detection reagent. Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference with respect to CSC (Student’s t-test). Scale bars = 10 μm.

Figure 13

Immunofluorescence analysis of Beclin-1 and LC3 II expression induced by β-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (A) Representative images of cells stained by a primary anti-Beclin 1 antibody, nucleic acid DAPI dye and secondary red fluorescent antibody. (B) Representative images of cells stained by a primary anti-LC3 II antibody, nucleic acid DAPI dye and secondary green fluorescent antibody. Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). Scale bars = 20 μm.

Figure 14

MDA-MB-468 cells migration rate at 0 h, 4 h and 72 h (n = 3) after co-treatment with β-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells. A. Images of wound healing assay captured at each time point and processed by Gen5™ Microplate Reader and Imager Software (Version 3.11, BioTek, AHSI, Milan, Italy) (4× original magnification). The quantified wound areas are displayed in black. Scale bars = 1000 μm.

Figure 15

Quantification of MDA-MB-468 cells migration rate at 0 h, 4 h and 72 h (n = 3) after treatment with β-caryophyllene and cigarette smoke condensate (CSC). Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). *** p < 0.001, significant difference of each time point with respect to zero time (ANOVA + Dunnett’s multiple comparison post-hoc test). ^§§§ p < 0.001, significant difference with respect to the control at the same time point (Student’s t-test). °°° p < 0.001, significant difference respect to CSC at the same time point (Student’s t-test).

Figure 16

RTPCR analysis of cell migration factors BIRC5 (A), MMP2 (B), TPX2 (C), and IL-8 (D) induced by cigarette smoke condensate (CSC) and β-caryophyllene in MDA-MB-468 cancer cells after 24 h exposure. Data are displayed as mean ± SE of at least three independent experiments with at least three technical replicates (n = 9). * p < 0.05, ** p < 0.01 and *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference respect to CSC (Student’s t-test).

Figure 17

Effect of β-caryophyllene and cigarette smoke condensate (CSC) on the expression levels of phosphorylated STAT3 at tyrosine 705 residue in MDA-MB-468 cancer cells after 24 h exposure. (A) Densitometric bar graph analysis (data are expressed as mean ± standard error obtained from at least two independent experiments) (B) Representative Western blotting membrane, displaying phospho(Tyr705) STAT3, total STAT3 and β -actin, used as protein-loading controls. *** p < 0.001, significant lowering of cell viability with respect to the control (ANOVA + Dunnett’s multiple comparison post-hoc test). °°° p < 0.001, significant difference with respect to CSC (Student’s t-test).