br Abbreviations A cells Adenocarcinomic human alveolar
Abbreviations: A549 cells, Adenocarcinomic human alveolar basal epithelial cells; AIs, Aromatase inhibitors; ANOVA, One-Way Analyzes of Variance; CCCP, Carbonyl cyanide m-chlorophenylhydrazone; CFBS, Charcoal heat-inactivated fetal bovine serum; CYP19A1 gene, Cytochrome P450 family 19 subfamily A member 1 gene; DCFH2-DA, 2,7-dichlorodihydrofluorescein diacetate; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide probe; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, Dimethyl sulfoxide; DPX, Slide mounting medium (polystyrene – plasticizer – xylene); DVB/CAR/PDMS, Divinylbenzene/carbonex/polydimethylsiloxane; Eos, Essential oils; ER+, Estrogen receptor-positive; FBS, Fetal bovine serum; GC–MS, Gas chromatography-mass spectrometry; HCA, Hierarchical cluster analyzes; HeLa, Human cervical carcinoma cell line; HepG2, Human liver cancer cell line; HFF-1, Human foreskin fibroblast cell line; HO-8910, Ovarian serous cystadeno-carcinoma cell line; HS-SPME, Headspace solid-phase microextraction; LDH, Lactate dehydrogenase; MCF-7, Breast cancer cell line (Michigan Cancer Foundation-7); MCF-7aro, ER+ aromatase-overexpressing human breast cancer cell line; MDA-MB-231, Breast adenocarcinoma cell line; MDA-MB-468, Mammary gland/breast cancer cell line; MEM, Minimum essential medium; MFI, Mean fluorescence intensity; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PA, Polyacrylate; PBS, Phosphate-buﬀered saline; PCA, Principal component analyzes; PDMS, Poly-dimethyl-siloxane; PI, Propidium Iodide; PMA, Phorbol 12-myristate 13-acetate; RI, Retention indices; RLU, Relative luminescence units; ROS, Reactive oxygen species; SERMs, Selective Oxidopamine receptor modulators; SK-OV-3, Ovarian serous cystadenocarcinoma cell line; STS, Staurosporine; T, Testosterone; UACC-257, Melanoma cell line; ψm, Mitochondrial transmembrane potential
Corresponding authors. E-mail addresses: [email protected]ﬀ.up.pt (N. Teixeira), [email protected] (M. Moumni). 1 Both authors contributed equally to this work.
(Augusto, Correia-da-Silva, Rodrigues, Teixeira, & Amaral, 2018; Sobral et al., 2016). Therefore, it is important to find new therapeutic drugs or bioactive molecules that may improve breast cancer treatment. In re-cent decades, modern research in oncology has confirmed the ther-apeutic virtues of natural products, such as essential oils (EOs) from medicinal plants (Elshafie & Camele, 2017; Gautam, Mantha, & Mittal, 2014; Jayaprakasha, Murthy, Uckoo, & Patil, 2013).
J. oxycedrus L. is a widespread tree in the Mediterranean region, including Morocco and Portugal, and is known by the names of “prickly juniper, juniper cade, cada and red-berry juniper” (Taviano et al., 2013). It is a medicinal plant that is a source of several food ingredients and is often used by European gastronomy (Loizzo et al., 2007). The fruits (berries) of J. oxycedrus L. are commonly used as flavoring spices especially for diﬀerent types of meat dishes. Moreover, it is known that berries from this plant are also used in gin due to its distinct flavor (Loizzo et al., 2007). In addition, traditional medicine, in Morocco, has used some extracts of J. oxycedrus L for medicinal purposes. For ex-ample, cade oil, which is an aromatic distilled oil prepared from J. oxycedrus L. plant, is used not only in soaps, creams and lotions, due to its dermatologic properties, but also in folk medicine due to its anti-inflammatory and anti-cancer properties (Loizzo et al., 2007; Skalli, Chebat, Badrane, & Bencheikh, 2013; Zhang, Lin, & Chan, 2015). In fact, this oil has been used by diﬀerent populations as part of anti-cancer therapy (Duke, 2007; Minirth, Krusz, Horewell, & Neal, 2005). Also, some studies showed that certain species of Juniperus can be a potential source of molecules with anti-cancer activity (Kang et al., 2012; Kusari, Zühlke, & Spiteller, 2011; Venditti et al., 2018; Yun, Hyun, Park, Kim, & Kwon, 2012). Widdrol, a sesquiterpene in the EOs of some Juniperus species, was described as one of the potential inducers of apoptosis in cervical and colon cancer cells (Kang et al., 2012; Yun et al., 2012). Despite this, the anti-cancer eﬃcacy of this Moroccan species has not been explored on breast cancer.
Thus, the use and reputation of J. oxycedrus L. extracts that are traditionally prepared to treat diﬀerent illnesses and diseases, like cancer, motivated us to study not only the phytochemical composition of leaves and fruits oils of this plant but also their potential anti-cancer properties. For this purpose, it was used the combination of gas chro-matography-mass spectrometry (GC–MS) analysis with distinct proto-cols of solid-phase microextraction (SPME), to determine the phyto-chemical composition of oils from fruits and leaves of J. oxycedrus L. In addition, the potential anti-cancer activities of these EOs were eval-uated in vitro, using an ER+ aromatase-overexpressing breast cancer cell line (MCF-7aro), which is a good in vitro cell model to study ER+ breast cancer (Amaral et al., 2012; Itoh et al., 2005).