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New drugs are continuously being developed for the treatment of patients with estrogen receptor-positive breast cancer. Thymoquinone is one of the drugs that exhibits anticancer characteristics based on in vivo and in vitro models. This study further investigates the effects of thymoquinone on human gene expression using cDNA microarray technology. The quantification of RNA samples was carried out using an Agilent 2100 Bioanalyser to determine the RNA integrity number (RIN). The Agilent Low Input Quick Amplification Labelling kit was used to generate cRNA in two-color microarray analysis. Samples with RIN >9.0 were used in this study. The universal human reference RNA was used as the common reference. The samples were labelled with cyanine-3 (cye-3) CTP dye and the universal human reference was labelled with cyanine-5 (cye-5) CTP dye. cRNA was purified with the RNeasy Plus Mini kit and quantified using a NanoDrop 2000c spectrophotometer. The arrays were scanned data analysed using Feature Extraction and GeneSpring software. Two-step qRT-PCR was selected to determine the relative gene expression using the High Capacity RNA-to-cDNA kit. The results from Gene Ontology (GO) analysis, indicated that 8 GO terms were related to biological processes (84%) and molecular functions (16%). A total of 577 entities showed >2-fold change in expression. Of these entities, 45.2% showed an upregulation and 54.7% showed a downregulation in expression. The interpretation of single experiment analysis (SEA) revealed that the cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and UDP glucuronosyltransferase 1 family, polypeptide A8 (UGT1A8) genes in the estrogen metabolic pathway were downregulated significantly by 43- and 11-fold, respectively. The solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11 (SLC7A11) gene in the interferon pathway, reported to be involved in the development of chemoresistance, was downregulated by 15-fold. The interferon-induced protein with tetratricopeptide repeats (IFIT)1, IFIT2, IFIT3, interferon, α-inducible protein (IFI)6 (also known as G1P3), interferon regulatory factor 9 (IRF9, ISGF3), 2′–5′-oligoadenylate synthetase 1, 40/46 kDa (OAS1) and signal transducer and activator of transcription 1 (STAT1) genes all showed changes in expression following treatment with thymoquinone. The caspase 10, apoptosis-related cysteine peptidase (CASP10) gene was activated and the protein tyrosine phosphatase, receptor type, R (PTPRR) and myocyte enhancer factor 2C (MEF2C) genes were upregulated in the classical MAPK and p38 MAPK pathways. These findings indicate that thymquinone targets specific genes in the estrogen metabolic and interferon pathways.

Patients with cancer are prone to several debilitating effects due to chemotherapy or the disease itself. The behavioural disturbances experienced by patients with cancer include fatigue, insomnia, depression, and cognitive disturbances. These symptoms are very common and may persist for months or years even after completion of treatment and may affect patient's quality of life. Functional foods have long been linked to improve human performance and health, and hence are considered beneficial as part of the cancer patients' diet.Beetroot (Beta vulgaris L.) is one of high-nutrient vegetables used for salads and juices, and a valuable source of natural pigments. It belongs to the botanical order Amaranthaceae-Chenopodiaceae, which is cultivated commercially. Beetroot, also known as table or garden beet, is usually grown for its roots. It has a characteristic earthy mushy aroma and flavour, mainly due to the presence of geosmin, a volatile bicyclic alcoholic compound [1]. Beetroot is known for its antioxidant activities and widely used as a remedy for a variety of ailments including cardiovascular-related conditions, anemia, sexual weakness and bladder stones [2]. Some recent clinical studies have also indicated the usefulness of beetroot in managing blood pressure and cardiovascular health [3][4][5][6]. Beetroot has also gained popularity as a supplement to boost energy and improve performance in athletes [7][8][9][10].Interestingly, in the Traditional Persian Medicine practices, beetroot is one of the foods used in the prevention and managing of metastatic progression of cancer [11]. It is also widely used in other medicinal systems including the Arab, traditional Chinese and Ayurvedic medicine. Beetroot, juiced or blended, is a popular functional food among breast, prostate and colorectal cancer patients as reported in Trinidad. A study at two clinical sites on the island revealed that beetroot is being consumed by patients for the purpose of treatment, health improvement and amelioration of side effects associated with chemotherapy treatment [12]. Similarly, beetroot is also the most frequently used alternative dietetic measure among patients with cancer in Germany [13] and among a majority of gastrointestinal cancer patients in Serbia [14].

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