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Chemical composition and antitumor mechanism in triple negative breast cancer cells

Abstract

The genus Cyphostemma (Planch.) Alston (Vitaceae) includes about 150 species distributed in eastern and southern Africa and Madagascar. Some species are used in traditional medicine and their biological activities, including antiproliferative effects against cancer cell lines, have been demonstrated. To date no investigations on Cyphostemma essential oils have been carried out. Essential oils, which play important roles in plant defenses have been demonstrated to be active in the treatment of several human diseases and to enhance bioavability of other drugs. The aim of this paper was to identify the chemical composition of the essential oil of the leaves of Cyphostemma juttae (Dinter & Gilg) Desc. and to verify some biological activities on two triple negative breast cancer cell lines (MDA-MB-231, SUM 149), characterized by the over-expression of the transcription factor NF-κB. In the essential oil, obtained by hydrodistillation and analysed by gas chromatography-mass spectrometry, 39 compounds were detected and with phytol (30%) dominating the chemical composition. C. juttae essential oil reduced cell growth and showed a pro-oxidant activity in both cell lines. Moreover, C. juttae essential oil caused a substantial decrease of NF-κB activation and consequently a significant reduction of some NF-κB target genes. The present study shows for the first time the cytotoxic properties of C. juttae essential oil and highlight its availability to interfere with NF-κB pathway, suggesting a potential therapeutic use in triple negative breast cancers (TNBCs) of this essential oil.

Introduction

The genus Cyphostemma (Planch.) Alston (Vitaceae) includes about 150 species distributed in eastern and southern Africa and Madagascar [1]. Some species from this genus are used in traditional medicine, e.g. roots of C. junceum to treat snake bites [2]. Interestingly, Ochawang’i et al. [3] described the use of C. serpens, alone or in association with other plants, in traditional medicine for the care of breast cancer. Udegbunam et al. [4] used root extracts of C. vogeli to investigate its anti-inflammatory effect on sore throat, cough and pneumonia in mice. The antiproliferative effects of water and methanolic extracts of four Cyphostemma spp. (C. flaviflorums, C. lanigerum, C. natalitium, and one unidentified species) on HepG2 cell line have been studied by Opoku et al. [5], but no chemical analyses of the matrices were performed. Cao et al. [6] found that C. greveana showed antiproliferative activities against the A2780 ovarian cancer cell line and identified one macrolide, lasiodiplodin, three sesquiterpenoids, and a new diterpenoid. Although from a chemical point of view Cyphostemma spp. have been investigated by solvent extractions, to the best of our knowledge no studies have been carried out on hydrodistillated essential oils and their possible biological activities.

The aim of this paper was to identify the chemical composition of the essential oil of the leaves of Cyphostemma juttae Dinter & Gilg (Desc.) obtained by hydrodistillation (HD) and analyzed by gas chromatography-mass spectrometry (GC-MS), and to verify some biological activities on two triple negative breast cancer cell lines (MDA-MB-231, SUM 149). Triple negative breast cancers (TNBCs) are highly aggressive, do not respond to conventional hormonal interventions due to the lack of the respective receptor targets, have chances of early recurrence, metastasize, tend to be more invasive in nature, develop drug resistance and they are also characterized by the over-expression of the transcription factor NF-κB [7]. The relevance of the NF-κB transcription factor in tumor biology is widely known, even in triple negative breast cancer.

Material and methods

Identification of compounds

Compounds were identified by using the mass spectral libraries [9], FFNSC 2, W9N11, and ESSENTIAL OILS (available in MassFinder 3) and the Kovats Retention Indices (KRI) of the compounds based on n-alkane series. We only considered compounds which had a mass spectral similarity more than 70% respected to those present in our digital libraries and which had a calculated Kovats index ± 10 compared to available data bases [9,10] and Nist11.

Cell growth assays

Triple negative breast cancer cell lines (MDA-MB-231, SUM 149) were used. The human breast cancer cell lines MDA-MB-231 (ATCC: HTB-26—Rockville, MD, USA) and SUM149 (SUM149PT—Asterand Bioscience Detroit, MI) were kindly provided by Dr. Elda Tagliabue (Molecular Targeting Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione Institute of Hospitalization and Scientific Care, National Cancer Institute, Milan, Italy) and were authenticated using the short tandem repeat profiling method in their Institute. The non-tumorigenic cell line 1-7HB2 (ECACC 10081201—Cancer Research Technology, London, UK) was kindly provided by Prof. Giulio Ghersi (STEBICEF Department, University of Palermo, Italy).

MDA-MB-231 cell line was cultured in RPMI-1640 and the SUM 149 cell line was cultured in DMEM/F-12 supplemented with insulin (5 μg/ml). 1-7HB2 cell line was cultured in DMEM low glucose supplemented with hydrocortisone (5 μg/ml) and insulin (10 μg/ml). All media were supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all reagents were from EuroClone S.p.A., Milan, Italy; GE Healthcare Life Sciences, Logan, UT, USA). The cells were cultured in a humidified atmosphere at 37°C in 5% CO2. After obtaining the cells, the first passage carried out was assigned passage number 1. Cells with a narrow range of passage number (4 ± 6) were routinely tested for Mycoplasma contamination were used for all experiments.

The cells were seeded at 2 × 104 cells/well onto 96-well plates and incubated overnight at 37°C. At time 0, the medium was replaced with fresh complete medium supplemented of essential oil at the indicated concentrations. Following 72 h of treatment, 16 μl of a commercial solution obtained from Promega Corporation (Madison, WI, USA) containing 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and phenazine ethosulfate were added. The plates were incubated in a humidified atmosphere at 37°C in 5% CO2 for 2 h, and the bioreduction of MTS dye was evaluated by measuring the absorbance of each well at 490 nm using a microplate absorbance reader (iMark Microplate Reader; Bio- Rad Laboratories, Inc., Hercules, CA, USA). Cell growth inhibition was expressed as a percentage (mean ± SE) of the absorbance of the control cells.

Anti- and pro-oxidant activity

To evaluate antioxidant activity, DPPH assay was used. The antiradical efficiency of the sample was evaluated by the DPPH stable radical method [11, 12]. 100 μl of sample, the essential oil of C. juttae at different concentrations, was added to aliquots (3.9 mL) of a solution made up with DPPH (4.8 mg) in MeOH (200 ml), and the mixture was incubated for 1 h at room temperature in the dark. Then the absorbance was measured at 517 nm using a UV-VIS spectrophotometer. The initial concentration of DPPH was approximately 60 μM. Lower absorbance values of reaction mixture indicate higher free radical scavenging activity. The results were plotted as the percentage of absorbance disappearance at 517 nm [(1-A/A0) × 100] against the amount of sample divided by the initial concentration of DPPH. Each point was acquired in triplicate. ED50 corresponds to micrograms of fraction able to consume half the amount of free radical divided by micromoles of initial DPPH. The results were expressed as antiradical capacity (ARC), which is the inverse of ED50.

Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) curve was used as the positive control. Pro-oxidant activity was examined by cell counting, adding N-acetyl-L-cysteine (NAC), an antioxidant molecule, 1 h before essential oil. Data were expressed as mean ± standard error (SE) of at least three different experiments performed in duplicate. All the chemicals were supplied by Sigma Aldrich srl, Milan, Italy.

Extraction of cellular RNA and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen Life Technologies). For the evaluation of gene expression, RNA was reverse transcribed using a high capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems Life Technologies). The resulting cDNAs were subjected to real-time RT-PCR using the TaqMan Gene Expression Master Mix kit (Applied Biosystems Life Technologies) in triplicates. The PCR cycling conditions were as follows: Denaturation at 50°C for 2 min, annealing at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and extension at 60°C for 60 min. The running of the samples and data collection were performed on a StepOne AB Real Time PCR system (Applied Biosystems Life Technologies). β-actin was used as an internal standard. The specific primers used were as follows: Survivin Hs00153353, XIAP Hs00236913, Bcl-2 Hs00236329, ABCB1 Hs00184005 (Applied Biosystems Life Technologies).

Relative expression was calculated using the comparative Ct method [ΔCt = Ct(target gene)-Ct(housekeeping gene)]. Where Ct was the fractional cycle number at which the fluorescence of each sample passed the fixed threshold. Fluorescence was measured at 515–518 nm using StepOne AB Real Time PCR System software (Applied Biosystems Life Technologies). The ΔΔCt method was used to determine gene expression levels. ΔΔCt was calculated using the formula: ΔΔCt = ΔCt(each sample)-ΔCt(reference sample). Fold change was calculated using the 2-ΔΔCt equation.

NF-κB activation

The DNA-binding capacity of NF-κB (p65 subunit) was determined in the nuclear extracts of MDA-MB-231 and SUM 149 cells using the TransAM NF-κB and Nuclear Extract kits (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were treated with 46 μg/ml and 64 μg/ml, respectively, of the essential oil for 24 h. Briefly, the determination of binding capacity was based on a 96-well plate, upon which an oligonucleotide containing the NF-κB consensus binding site (5′-GGGACTTTCC-3′) was immobilized. Activated NF-κB contained in the extracts is able to specifically bind to this nucleotide. NF-κB bound to the oligonucleotide may subsequently be detected using an antibody directed against an epitope on p65 (polyclonal rabbit anti-human; cat. no. 40096; 1:1000; Active Motif), accessible only when NF-κB is bound to its target DNA.

Subsequently, the addition of a horseradish peroxidase-conjugated secondary antibody provided a sensitive colorimetric readout that may be quantified by densitometry (iMark Microplate Reader; Bio-Rad Laboratories, Inc.). The specificity of the assay was confirmed by simultaneous incubations in the presence of excess, non-immobilized consensus oligonucleotides, as a competitor, or of a mutated consensus oligonucleotide. The results were expressed as arbitrary units: one unit indicated the DNA binding capacity exerted by 2.5 μg whole cell extract from Jurkat cells (positive control for NF-κB p65 activation; Active Motif) (stimulated with 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore) per microgram of protein from the nuclear extracts.

Statistical analysis

Results of bioassays are given as means ± standard error (SE). Statistical analysis was carried out according to Poma et al. [13] by analysis of variance (one-way ANOVA) followed by Tukey’s test. Statistica ver. 12 (StatSoft Inc. 1984–2014) was used as software for the analyses.

Results

The yield in essential was 0.002%. The plants from which the leaves were collected completed flowering and produced viable seeds. In the year 2018 the plants produced new leaves and the growth was not affected by the previous year removal of leaves.

In vitro anticancer and pro-oxidant activity

We examined the cytotoxic activity of C. juttae essential oil on MDA-MB-231 and SUM 149 cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay. The essential oil of C. juttae reduced cell growth in a dose dependent way, in particular the cell line more sensitive was MDA-MB-231 (Fig 2). The essential oil did not show cytotoxic activity on the non-tumorigenic cell line 1-7HB2. The cell growth inhibition was: 0% at 30 μg/ml; 10 ± 2% at 50 μg/ml; 17.5 ± 1.5% at 70 μg/ml; and 23 ± 2% at 100 μg/ml.

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Fig 2. Cytotoxic activity of C. juttae essential oil on cancer cell lines.

Cell viability was assessed by MTS. Data are expressed as mean ± standard error (SE) of at least three different experiments performed in triplicate. Different letters represent significant differences in cytotoxic activity among the concentrations of each cell line (Tukey test, p < 0.05).


https://doi.org/10.1371/journal.pone.0214594.g002

Since many natural compounds are provided with anti- or pro-oxidant activity, we analyzed if the antitumor activity of the C. juttae oil was due to pro-oxidant activity. The cells were treated with the antioxidant N-acetyl-L-cysteine (NAC) at 2mM 1 h before exposure to the essential oil at the corresponding IC50 values. In both cell lines the addition of NAC reduced cytotoxic activity, in particular the reduction was more evident in MDA-MB-231 cell line (Table 2).

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Table 2. Results of cell counting analysis in the two cell lines following treatment with antioxidant N-acetyl-L-cysteine (NAC) at 2mM before exposure to the essential oil at the corresponding IC50.

Data are expressed as mean ± standard error (SE).


https://doi.org/10.1371/journal.pone.0214594.t002

In addition, the analysis carried out by DPPH (2,2-diphenyl-1-picrylhydrazyl) reduction assay indicated that the essential oil does not possess antioxidant activity, since it was not identified the efficient dose (ED50) of the essential oil of C. juttae (Table 3). These results indicate that the mechanism involves an at least partially pro-oxidant effect.

Inhibition of NF-κB activation

In order to investigate the molecular mechanism underlying the antitumor activity of the essential oil, we analyzed the effects of the essential oil to NF-κB activation by TransAM assay. MDA-MB-231 and SUM 149 cells were treated with the essential oil at the corresponding IC50 values for 24 h: in both cell lines we observed a strong reduction of NF-κB DNA-binding activity (Fig 3).

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Fig 3.

NF‑κB (p65 subunit) DNA binding capacity in nuclear extracts of MDA-MB-231 cells (A) and of SUM 149 cells (B). The cells were treated for 24 h with C. juttae essential oil (EO) (46 and 64 μg/ml, respectively). Results (mean ± standard error of two experiments carried out in duplicate) are expressed as arbitrary units/μg protein of cells nuclear extracts. Statistical differences are *p < 0.05 vs. control.


https://doi.org/10.1371/journal.pone.0214594.g003

In view of this, the treatment with the essential oil also caused a substantial mRNA decrease of some NF-κB target genes, in particular antiapoptotic factors such as Survivin, XIAP, Bcl-2 and the multidrug efflux transporter P-glycoprotein, encoding by ABCB1 gene (Fig 4).

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Fig 4.

Genes mRNA expression levels by quantitative polymerase chain reaction in MDA-MB-231 cells (A) and SUM 149 cells (B). The cells were treated for 24 h with C. juttae essential oil (EO) (46 and 64 μg/ml, respectively). Data are expressed as mean ± standard error (SE) of two different experiments. Different letters (a, b, c and d) in the column of the cell lines and treatments represent significant differences (p < 0.05) among the treatments of each cell line; *differences when treatments are compared to the control (p < 0.05).


https://doi.org/10.1371/journal.pone.0214594.g004

In both cell lines we found a reduction also at protein level of anti-apoptotic factors (Survivin, XIAP and Bcl-2) under essential oil treatment (Fig 5).

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Fig 5. Western blotting analysis in MDA-MB-231 cells and SUM 149 cells.

The cells were treated for 24 h with C. juttae essential oil (EO) (46 and 64 μg/ml, respectively). The data shown are the results of a representative experiment.


https://doi.org/10.1371/journal.pone.0214594.g005

The strong reduction of mRNA level of P-gp under treatment with essential oil in our cell lines is significant but the analysis of the drug resistance implies further experimental evaluations that are in progress. However, it is known that the expression of P-gp is also regulated by post-translational events, such as post-transcriptional glycosylation and membrane localization of P-gp [14].

Discussion

Essential oils and their chemical constituents have potential roles and uses to prevent and treat human disease. The role of these natural products has been discussed with regard to the prevention and treatment of cancer, cardiovascular diseases, and as antibacterial, antiviral, and antioxidant agents, among others [15].

Since C. juttae is a plant species adapted to dry environments with high temperature, high light intensity, and low nutrient and water resources the risk of damage by abiotic and biotic factors can be easy to occur. However, by the biosynthesis of a wide range of secondary metabolites this species, growing in nature in prohibitive habitats, can develop defensive strategies against several biotic and abiotic factors.

Our chemical composition indicates that C. juttae essential oil constituents potentially may play several biological roles. Among others, terpenoids contributing to more than 60% of C. juttae composition are the largest group of plant chemicals which have multiple ecological roles, such as: (1) defense against generalist and specialist herbivores, (2) defense against fungi and bacteria, (3) attraction of pollinators and seed dispersers, (4) allelopathic agents that inhibit seed germination, and (5) protection against abiotic factors (e.g. with reactive troposphere gases) [16, 17]. According to Gershenzon and Dudareva [18] considering the large number of terpenoids produced in nature, very little is known about their mode of action at the molecular level although their highly lipophilic nature suggests that they act on the cell membranes and that their toxicity is caused by loss of chemiosmotic control.

Many plant-derived active principles have gained attention as efficient anticancer agents against TNBCs, with fewer adverse side effects. In this work for the first time we described the strongly inhibitory effect of C. juttae essential oil on NF-κB activation in two TNBC cell lines characterized by a strong expression of the transcriptional factor. Consequently, the inhibition of transcriptional activity due to essential oil leads to a significant decrease of some transcriptional target genes such as Survivin, XIAP, Bcl-2 and P-glycoprotein [19, 20, 21, 22].

Moreover, in the same cancer models, the essential oil showed antitumor capacities in the terms of cytotoxic and pro-oxidant activities and partially pro-apoptotic effects proven by preliminary cytofluorimetric analysis by propidium iodide where only a modest block in preG0-G1 position dose-dependent is observed in all cell lines and also shown in other studies [23]. It is very interesting to note that the essential oil exerts an inhibitory effect on the transcription of the P-glycoprotein the ABCB1 multi-drug efflux pump (MDR). P-glycoprotein is overexpressed in TNBCs and analysis of the MDR reversal activities of essential oil request further analyses [24, 25, 26]. In sight of this plant-derived compounds in combination with classical chemotherapeutic agents could be more efficient in the treatment of TNBCs, possibly with lesser side effects.

Our results on the chemical composition of the C. juttae essential oil shown that Phytol is the main compound (29.6%). Based on these data, it could be hypothesized that the biological activities of essential oil in our TNBC cells, are accountable mainly to phytol which in combination with other terpenoids can act with synergic effects. On the other hand, the antitumor activity of this compound has been already described by Islam et al. [27] in different type of cancer models, such as human gastric adenocarcinoma, lymphoid leukemia, hepatocellular carcinoma and also in one triple negative breast cancer cell line. There are other evidences that showed how the immunomodulatory and antitumor effects of the Phytol can be attributed precisely to the action on NF-κB [27].

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