Open Access

Thymoquinone regulates gene expression levels in the estrogen metabolic and interferon pathways in MCF7 breast cancer cells

  • Authors:
    • Marjaneh Motaghed
    • Faisal Muti Al-Hassan
    • Shahrul Sahul Hamid
  • View Affiliations

  • Published online on: November 21, 2013     https://doi.org/10.3892/ijmm.2013.1563
  • Pages: 8-16
  • Copyright: © Motaghed et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

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.

Introduction

The World Health Organization International Agency for Research on Cancer reported that approximately 1.29 million women were diagnosed with breast cancer and >400,000 women succumbed to the disease in 2008 (1). Thymoquinone is an anti-neoplasic agent reported to have medicinal potential in the treatment of breast cancer. The antitumour activity of thymoquinone has been reported in cells derived from ovarian, breast and colon cancers (2). The apoptotic activity of thymoquinone has been reported to induce the total Bax/Bcl-2 ratio in MCF7 (3), HCT-116 (4) and HL-60 cancer cells (5). These findings were based on flow cytometry, western blot analysis and multi-colour fluorescence in situ hybridization (mFISH). Therefore, further investigation is required to to determine its effects on human genome expression using cDNA microarray technology.

Materials and methods

Complete medium preparation

RPMI-1640 (Invitrogen, Gibco, Carlsbad, CA, USA) medium with L-glutamine was used to culture the MCF7 cell line (ATCC® HTB22™). The medium was supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen/Gibco) and 1 unit penicillin/streptomycin (HyClone, Logan, UT, USA).

Thymoquinone solution preparation

The 1 mM stock solution of thymoquinone (Sigma-Aldrich, Saint-Quentin-Fallavier, France) was prepared with DMSO (Sigma-Aldrich). The solution was filtered with a 0.02 μm syringe filter (Hydrophilic Ministart; Sartorius AG, Goettingen, Germany).

Cell culture and treatment

The MCF7 cells were seeded at 3–4×106 cells/well in 96-well plates. They were cultured at 0.5% CO2 in a humidified incubator at 37°C (Thermo Scientific, Waltham, MA, USA) for 24 h. The control cells were treated with 0.05% dimethyl sulfoxide alone (Sigma-Aldrich). Four biological replicates from each sample were prepared in separate culture flasks. The samples were treated with 50 μM thymoquinone for 24 h. EDTA [0.25% (w/v) + Trypsin/0.53% Mm] solution was used to detach the cells from the flask surface. The cells were then centrifuged at 13,000 rpm for 10 min. The supernatant was removed and the cells were washed with PBS solution prior to centrifugation at 13,000 rpm for 5 min.

RNA isolation

RNA was isolated from the MCF7 cells using the RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quantity of RNA was measured using a spectrophotometer (NanoDrop 2000c; Thermo Scientific). Samples with the RNA concentration (A260/A280 ≥1.8 ng/μl) and purity (A230/A260 ≥2.0 ng/μl) were selected. An Agilent 2100 Bioanalyser was used to determine the RNA integrity number (RIN). The degradation level was identified using the RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara, CA, USA). The samples with RIN >9.8 were selected for further analysis.

Two-colour microarray-based gene expression

The Agilent Low Input Quick Amp Labelling kit (USA) was used to generate cRNA with a sample input of 200 ng total RNA in two-color microarray analysis. The RNA Spike-In kit (Agilent Technologies) was used as an external control (positive control). It monitors and calibrates the linearity, sensitivity and accuracy of the microarray workflow. Spike A Mix with cyanine-3 (cye-3) was used to label the samples (thymoqionone-treated and untreated) and Spike B Mix with cyanine-5 (cye-5) was used to label the internal control (Universal Human Reference RNA, Agilent Technologies). T7 RNA polymerase was used to amplify target material.

The array platform used was 8×60K array SurePrint Technology (Slide Human V1 U252800417900-S01; Interscience, Rockland, MA, USA) and gasket slides with SureHyb Technology. The reference design was selected for the study. Four biological replicates from 50 μM thymoquinone-treated MCF7 and four replicates from untreated MCF7 cells were hybridised against Human Universal Reference RNA using a hybridisation kit (Agilent Technologies). The slide chamber was assembled and placed in rotisserie hybridization oven and rotated at 10 rpm in 65°C for 17 h. The array slide was washed and fluorescent imaging system was used to scan the hybridisation signals (DNA Microarray Scanner, Surescan High-Resolution Technology, Agilent Technologies).

The results were extracted with Feature Extraction Project v10.7.3.1 software and analysed using GeneSpring software v12.1. Statistical analysis was carried out using an unpaired t-test and a fold change with cut-off value >2 with a P-value ≤0.05 was considered to indicate a statistically significant difference.

Quantitative reverse transcription PCR (qRT-PCR)

qRT-PCR was carried otu to validate the results from microarray analysis and performed in two steps. The high capacity RNA-to-cDNA kit protocol (Applied Biosystems, Foster City, CA, USA) was followed to transcribe 2 μg total RNA into single-stranded cDNA. This was followed by amplification using a thermal cycler (Eppendrof, Hamburg, Germany). The ΔCT of the thymoquinone-treated cells was subtracted from the ΔCT of the untreated cells to determine the differences (ΔΔCT) and fold change (2−ΔΔCT). The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene was used as an endogenous control to normalize the fluorescence signals in the untreated and treated cells. Five dilution points were prepared in triplicate for each primer to plot the relative standard curve.

Amplification plots and the standard curve of each target gene and endogenous control were created to determine the primer efficiency. Three biological replicates of thymoquinone-treated samples and three biological replicates of untreated samples were examined. The StepOnePlus Real-Time PCR instrument (Applied Biosystems) was used to run this experiment with a 96-well system. The 5′ nuclease assay (TaqMan probe; Life Technologies Corp., Carlsbad, CA, USA) was used to detect the specific PCR product. It was labelled with the reporter fluorescence dye 6-carboxyfluorescein (6-FAM) at the 5′ end and the quencher dye MGBNFQ at the 3′ end. The fast mode was selected to run the experiment for 40 min. The temperature was adjusted to the one recommended in the manufacturer’s instructions (Applied Biosystems,).

The comparative CT (ΔΔCT) method was selected to determine the amount of target nucleic acid sequence in each sample relative to the untreated samples. The normalization of the ΔCT value of the thymoquinone-treated cells to the ΔCT value of the untreated cells was carried out to determine the ΔΔCT value.

Results

Quantification and data filtration

Filtering was carried out to minimise errors and generated 24,971 entities. There were seven entities out of 577 entities with a fold change of >2.0 and and P-value ≤0.05.

Hierarchial clustering

Clustering was carried out to identify entities that were grouped within a cluster which are co-regulated and functionally related. Clustering was carried out on both entities and conditions. This created a two-dimensional dendogram. The untreated samples had a similar expression profile, which linked to form a group. A similar pattern was observed in the thymoquinone-treated samples (Fig. 1).

Gene Ontology (GO) analysis

GO describes gene products in terms of their associated cellular component and molecular function and biological process (Fig. 2). An analysis revealed eight significant GO terms related to biological processes (84%) and molecular functions (16%), as presented in Table I.

Table I

Gene Ontology categories of biological processes and molecular functions.

Table I

Gene Ontology categories of biological processes and molecular functions.

Gene Ontology categoriesTotal genesP-value
Biological processes
 Type I interferon-mediated signaling pathway620.002
 Cellular response to type I interferon620.002
 Response to type I interferon630.002
 Xenobiotic metabolic process1290.021
 Cellular response to xenobiotic stimulus1300.021
 Response to xenobiotic stimulus1310.021
Molecular functions
 Androsterone dehydrogenase activity30.023
 Aldo-keto reductase (NADP) activity180.045
Single experiment analysis (SEA)

SEA identifies matching pathways within the experiment and identifies pathways related to regulated genes. An analysis of the 577 entities with a P≤0.05 and a fold change of >2.0 showed 504 pathways. There were three matched entities in cytochrome p450 (CYP450) (63 entities), phase I and II drug metabolism (49 entities) and interferon type I (54 entities).

Table II presents the downregulated pathways with the highest number of matched entities (16 entities). These were the metapathway biotransformation and interferon signaling pathway (six entities), although these were not significant (P>0.05). The most significant results were observed for the phase I metabolic pathway (P<0.04). The upregulated pathways with the highest number of matched entities were also the metapathway biotransformation (four entities). The most significant results were observed for the GPCR class B secretion pathway (P=0.009).

Table II

Regulated pathways identified by single experiment analysis.

Table II

Regulated pathways identified by single experiment analysis.

PathwaysP-valueEntitiesMatched entities
Downregulated pathways
 HS_Metapathway_biotransformation2.3818816
 HS_Benzo(a)pyrene_metabolism1.2595
 HS_Interferon_α_β_signaling2.00266
 HS_AhR_pathway4.32286
 HS_Estrogen_metabolism2.48184
 HS_Oxidative stress7.25304
 HS_Keap1_Nrf2_pathway8.92143
 HS_Tamoxifen_metabolism2.17213
 HS_Glucuronidation0.007263
 HS_Type_II_interferon_signaling (IFNG)1.68374
 HS_Iron_metabolism_in_placenta0.003122
 HS_Tryptophan_metabolism0.026473
 HS_Phase_I_-_functionalization_of_compounds0.004493
 HS_Glutathione_metabolism0.008202
 HS_Spingolipid_metabolism0.034302
 HS_Interferon_type_I_0.040543
 HS_Hypothetical_Network_for_Drug_Addiction0.023322
 HS_Bile_acid_and_bile_salt_metabolism0.041242
 HS_Nicotine_Activity_on_Chromaffin_Cells0.02941
 HS_Cytochrome_P4500.009633
Upregulated pathways
 Hs_One_Carbon_Metabolism0.013272
 HS_GPCRs, _Class_B_secretin_like0.009232
 HS_miRs_in_Muscles_Cell_Differentiation0.015402
 HS_Metapathway_biotransformation0.0241884
 HS_Thyroxine_thyroid_hormone_production0.03251
 HS_Heart_Development0.033472
 HS_Cell_surface_interaction_at_vascular_wall0.020392

The most significantly upregulated genes were large intergenic non-coding RNAs (lincRNAs) and the probe was annotated with its genomic location (Table III). The protein tyrosine phosphatase, receptor type, R (PTPRR) gene was upregulated by 2.2-fold. SEA revealed genes in the estrogen pathway that were downregulated following treatment with thymoquinone (Table IV). The genes that were downregulated were UDP glucuronosyltransferase 1 family, polypeptide A8 (UGT1A8) (by −16.26-fold), cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (by −2.22-fold), cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) (by −2.23-fold) and NAD(P)H dehydrogenase, quinone 1 (NQO1) (by −2.07-fold). Genes in the interferon α and β signaling pathways were also downregulated following treatment with thymoquinone; these genes were the interferon-induced protein with tetratricopeptide repeats (IFIT)1 (by −10.65-fold), IFIT2 (by −4.20-fold), IFIT3 (by −5.27-fold), interferon, α-inducible protein (IFI)6 (also known as G1P3) (-by 7.86-fold) and IFI27 (by −3.19-fold). The genes downregulated in the type II interferon signaling pathway, included interferon regulatory factor 9 (IRF9) (−2.01), IFIT2 (−4.20), 2′-5′-oligoadenylate synthetase 1, 40/46 kDa (OAS1) (−3.5) and IFI6 (−7.86).

Table III

List of genes which were upregulated following with treatment thymoquinone.

Table III

List of genes which were upregulated following with treatment thymoquinone.

Gene symbolDescriptionFold change
XLOC001537BROAD lincRNAs version v26.31
CARD16Homo sapiens caspase recruitment domain family, member 165.25
C2CD4BHomo sapiens C2 calcium-dependent domain containing 4B4.31
UBQLNLHomo sapiens ubiquilin-like4.21
MTR Methyltetrahydrofolate-homocysteine methyltransferase3.91
TMEM100Protein 100, transcript variant 23.70
MOBKL2BBinder kinase activator-like 2B3.67
RNF17(RNF17), transcript variant 13.62
STON1GTF2Homo sapiens ring finger protein 17, transcript variant 13.60
OR52J3Homo sapiens olfactory receptor, family 52, subfamily J, member 33.42
LOC100133130Homo sapiens clone FLB4246 PRO1102 mRNA, complete cds3.30
NCRNA00261Homo sapiens non-protein coding RNA 261, non-coding3.28
FBXW2Homo sapiens F-box and WD repeat domain containing 23.26
LOC100131763Homo sapiens cDNA FL137602 fis, clone BRCOC20093803.19
SCINHomo sapiens scinderin, transcript variant 23.19
PGAP1Homo sapiens post-GPI attachment to protein 13.17
S100BHomo sapiens S100 calcium binding protein B (S100B)3.16
SV2AHomo sapiens synaptic vesicle glycoprotein 2A3.16
C7orf54Homo sapiens chromosome 7 open reading frame 54, non-coding RNA3.15
FAM65BHomo sapiens family with sequence similarity 65, member B, transcript variant 13.15

Table IV

List of genes which were downregulated following treatment with thymoquinone.

Table IV

List of genes which were downregulated following treatment with thymoquinone.

Gene symbolDescriptionFold change
UGT1A8Homo sapiens UDP glucuronosyltransferase 1 family, polypeptide A8−16.26
SLC7A11Homo sapiens solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11−12.99
IFIT1Homo sapiens interferon-induced protein with tetatricopeptide repeats 1, transcript variant 2−10.65
IF16Homo sapiens interfero, α-inducible protein 6, transcript variant 3−7.86
C17orf64Homo sapiens chromosome 17 open reading frame 64−6.20
ALDH3A1Homo sapiens aldehyde dehydrogenase 3 family, member A1, transcript variant 2−5.85
UGT1A6Homo sapiens UDP glucuronosyltransferase 1 family, polypeptide A6, transcript variant 1−5.68
IFIT2Homo sapiens interferon-induced protein with tetratricopeptide repeats 2−5.32
IFIT3Homo sapiens interferon-induced protein with tetratricopeptide repeats 3, transcript variant 1−5.27
HMOX1Homo sapiens hemeoxygenase (decycling) 1−5.13
DNAH6Homo sapiens dynein, axonemal, heavy chain 6−4.58
TPRG1Homo sapiens tumour protein p63 regulated 1−4.55
PCLOHomo sapiens piccolo (presynaptic cytomatrix protein), transcript variant 1−4.33
CHD5Homo sapiens chromodomain helicase DNA binding protein 5−4.28
CCR1Homo sapiens chemokine (C-C motif) receptor 1−4.26
AKR1C1Homo sapiens aldo-ketoreductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-α (3-α)-hydroxysteroid dehydrogenase)−4.21
GSTA5Homo sapiens glutathione S-transferase α 5−4.21
AKR1B15Homo sapiens aldo-ketoreductase family 1, member B15−4.13
HEATR7B1HEAT repeat containing 7B1−3.98
GPX2Homo sapiens glutathione peroxidase 2 (gastrointestinal)−3.88
Expansion and pathway enrichment analysis

Expansion and pathway enrichment analysis provided information on the biological functions of the entities. Network A (downregulated genes) showed the DAN domain family member 5, BMP antagonist (DAND5) as the focal point among the matched entities (Fig. 3). In addition, CYP1A1 and CYP1B1 were also noted to be highlighted in the interaction. Network B (upregulated genes) highlighted the involvement of regulator of G-protein signaling 4 (RGS4), proteasome (prosome, macropain) 26S subunit, non-ATPase, 9 (PSMD9), ubiquitin specific peptidase 9, Y-linked (USP9Y) and dystrophin (DMD).

Validation of thymoquinone regulation of gene expression by qRT-PCR

The efficiency of each primer was determined using the relative standard curve method. The optimal concentration based on the efficiency of the primers was selected for relative quantification (RQ). The ΔΔCT value of CYP1A1, solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11 (SLC7A11) and UGT1A8 showed a downregulation of 43-, 15-, and 11-fold, respectively. However, the ΔΔCT value of the protein tyrosine phosphatase, receptor type, R (PTPRR) and caveolin 1, caveolae protein, 22 kDa (CAV1) genes showed an upregulation of 2.2- and 1.5-fold, respectively.

Discussion

Almost 75% of breast cancer patients are estrogen receptor-positive (6). Tamoxifen is a selective estrogen-receptor modulator. It has been shown to reduce invasive breast cancer risk by 50% (7,8). Despite advances in treatment, there is a need to investigate the use of new drugs to modulate the expression of the estrogen receptor in estrogen receptor-positive breast cancer cells. Furthermore, recurrence develops after certain durations of treatment. In this study, cDNA microarray technology was used to measure the expression levels of all genes within the human genome. Microarray data analysis provided information on the genes and pathways targeted by 50 μM thymoquinone following 24 h of treatment.

Steroid hormones play a main role in the maintenance of normal female function and are normally produced by ovaries (9). Estradiol has been known to be the main and most active endogenous estrogen in pre-menopausal women. It is present in the form of estrone in post-menopausal women (10) and in pregnant women it is in the form of estriol (11). It has been reported that elevated circulating estrogen levels cause increased breast cancer risk (12).

CYP450 enzymes are haem-containing enzymes which catalyse phase 1 drug metabolism (13). CYP1A1 is expressed in extrahepatic tissues, such as mammary glands, the prostate, ovaries, uterus, colon, testis, adrenal, thymus and lungs (14). The overexpression of CYP1B1 has been reported in mammary tumours and breast cancer (1517). The enzymes are present in the estrogen metabolic pathway and are known as 17β-estradiol (E2) hydroxylases (18). The conversion of estradiol to 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) is influenced by the CYP1A1 and CYP1B1 genes, respectively (19,20). 4-OHE2 is known as the most carcinogenic metabolite of estrogen (21,22). It has been reported to be a vital factor in hormonal carcinogenesis (23). The CYPIAI gene regulates the metabolism of estradiol into 2-hydroxyestradiol. It is also involved in the metabolism of estrone to 4-hydroxyestrone (4-OHE1). 4-OHE1 is known as one of the most potent carcinogenic estrogen metabolites (20). Our findings revealed a downregulation in the expression of the CYP1A1 and CYP1B1 genes. Further validation with qRT-PCR revealed a significant downregulation of the CYPIAI gene by 43-fold. The downregulation of CYP450 enzymes has been reported to suppress estradiol-2,3-quinone (E2-2,3-Q) and estradiol-3,4-quinone (E2-3,4-Q) activity (22,24).

Previous studies have reported the anticancer and anti-angiogenic effects of 2- and 4-methoxyestradiol (2- and 4-MeOE2) (20,25). Increased levels of the methoxy form of estrone have been shown to have anticancer and anti-angiogenic potential in humans (20). The formation of 2- and 4-MeOE2 occurs through the methylation of 2-OHE2 and 4-OHE2. The oral administration of 2-MeOE2 in phase I and phase II clinical trials has been shown to be well tolerated by patients. Its anti-proliferative effects have been demonstrated in nasopharngeal carcinoma and tumor-derived uterine leiomyoma cell lines (26,27). The phase I enzyme UGT1A8 plays a key role in drug metabolism. In this study, we observed a significant downregulation of the UGT1A8 gene by 16.26-fold. The validation using real-time PCR on relative gene expression revealed a downregulation of 11-fold. The downregulation of this gene would retain 2-MeOE2 levels.

Multidrug resistance is the most important mechanism by which cancer chemotherapeutic drugs fail to take effect and resistance develops against anticancer drugs (28,29). The IFN-related DNA damage resistance signature (IRDS) is associated with resistance to chemotherapy or radiation in various cancer cell lines. Some of the IRDS genes are STAT1, ISG15 and IFIT1. They are associated with the interferon signaling pathway (30). The upregulation of these genes has been reported in different types of cancer. In our study, whole genome microarray data analysis revealed that the IFIT1 gene was downregulated by 10.65-fold. In addition, the IFIT2 and IFIT3 genes were downregulated by 5.32- and 5.27-fold, respectively. The overexpression of the IFIT3 and IFI27 genes has been reported to induced tumour proliferation, angiogenesis and chemoresistance in pancreatic carcinoma cells (32). The findings of our study revealed the downregulation of the IFI27 and IFI6 genes by 3.19- and 7.86-fold, respectively following treatment with thymoquinone. The downregulation of the IFI6 or GIP3 gene has been shown to reduce MCF-7 cell growth (33).

Interferon-stimulated gene factor 3 (ISGF3) is regulated by interferon α. ISGF3 genes consist of STAT1, STAT2 and IRF9. It has been grouped as ISGF3-α and ISGF3-γ(34,35). ISGF3-γ functions through type I interferons (IFN-α and IFN-β) (31). The overexpression of IRF9 has been observed in almost half of breast and uterine tumours. It may be connected to downstream mediators of interferon signaling to drug resistance (31). In this study, thymoquinone induced the downregulation of ISGF3 (IRF9) by 2.01-fold. A previous study using cDNA microarray analysis demonstrated a high expression of the STAT1 and STAT2 genes in MCF-7 cells that overexpressed IRF9(31). The downregulation of IRF9 gene expression may induce the lower expression of interferon-inducible genes (31). This finding further supports the chemopreventive effects of thymoquinone. STAT1 is commonly overexpressed in breast cancer. It is found to be related with increased resistance to radiation and chemotherapy (36). In breast cancer, the increase in estrogen levels leads to a higher expression of STAT1 gene (36).

Glutathione (GSH) is known as a compound that promotes drug-resistance through the removal of free radicals (37). The SLC7A11 gene functions in chemoresistance through the maintenance of intracellular GSH levels (28). In this study, we observed a downregulation of SLC7A11 gene expression by 12.99-fold. Validation using real-time PCR of relative gene expression revealed a downregulation of 15-fold.

The PTPRR gene is known as an inhibitor of MAPK (38). We found that PTPRR was upregulated by 2.20-fold and the myocyte enhancer factor 2C (MEF2C) gene by 2.22-fold, which promotes apoptosis. A previous study demonstrated that the PTPRR gene exerts an inhibitory effect on p44/42 MAPK signaling and transcription factor AP1 expression in cervical cancer (39). In this study, we found that the caspase-10 gene, which is known as one of the initiator caspases, was upregulated by 1.57-fold. Previously, caspase cascade activation has been shown to involve caspase-3, -7 and -9, and not caspase-8 in MCF7/DOX cells (40).

In conclusion, our findings suggest that thymoquinone acts in a synergistic manner involving the estrogen metabolic and interferon pathways as indicated by our microarray findings. The co-adminstration of thymoquinone and tamoxifen requires further investigation in order to fully elucidate the treatment outcomes.

Acknowledgements

This study was funded by Universiti Sains Malaysia short-term grant 304/CIPPT/6311033 and Advanced Medical and Dental Postgraduate Fund. The authors would like to extend their appreciation to the statistician.

References

1 

IARC. World Cancer Report. Geneva: World Health Organization; Lyon: 2008, http://www.iarc.fr/en/publications/pdfs-online/wcr/2008/index.phpurisimplehttp://www.iarc.fr/en/publications/pdfs-online/wcr/2008/index.php.

2 

Banerjee S, Azmi AS, Padhye S, et al: Structure-activity studies on therapeutic potential of Thymoquinone analogs in pancreatic cancer. Pharm Res. 27:1146–1158. 2010. View Article : Google Scholar : PubMed/NCBI

3 

Woo C, Loo SY, Gee V, et al: Anticancer activity of thymoquinone in breast cancer cells: possible involvement of PPAR-gamma pathway. Biochem Pharmacol. 82:464–475. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A and Schneider-Stock R: Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. Int J Oncol. 25:857–866. 2004.PubMed/NCBI

5 

El-Mahdy MA, Zhu Q, Wang QE, Wani G and Wani AA: Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myoloblastic leukemia HL-60 cells. Int J Cancer. 117:409–417. 2005. View Article : Google Scholar : PubMed/NCBI

6 

Rugo HS: The breast cancer continuum in hormone-receptor-positive breast cancer in postmenopausal women: evolving management options focusing on aromatase inhibitors. Ann Oncol. 19:16–27. 2007. View Article : Google Scholar

7 

Fisher B, Costantino JP, Wickerham DL, et al: Tamoifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst. 90:1371–1388. 1998. View Article : Google Scholar : PubMed/NCBI

8 

Jordan VC: Chemoprevention of breast cancer with selective oestrogen-receptor modulators. Nat Rev. 7:46–53. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Duursen MB: Estrogen metabolizing enzymes: biomarkers of exposure, effect and susceptibility for carcinogenesis (Dissertation). Proefschrift Universiteit Utrecht Press; 2005

10 

de Padua Mansur A, Silva TC, Takada JY, et al: Long-term prospective study of the influence of estrone levels on events in postmenopausal women with or at high risk for coronary artery disease. ScientificWorldJournal. 2012:3635952012.PubMed/NCBI

11 

Zhou J and Seidel E: Estrogens induce visfatin expression in 3T3-L1 cells. Peptides. 31:271–274. 2010. View Article : Google Scholar : PubMed/NCBI

12 

Abrahamsson A, Morad V, Saarinen N and Dabrosin C: Estradiol, tamoxifen, and flaxseed alter IL-1beta and IL-1Ra levels in normal human breast tissue in vivo. J Clin Endocrinol Metab. 97:E2044–E2054. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Androutsopoulos VP, Tsatsakis AM and Spandidos DA: Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention. BMC Cancer. 9:1872009. View Article : Google Scholar : PubMed/NCBI

14 

Furukawa M, Nishimura M, Ogino D, et al: Cytochrome p450 gene expression levels in peripheral blood mononuclear cells in comparison with the liver. Cancer Sci. 95:520–529. 2004. View Article : Google Scholar : PubMed/NCBI

15 

Fu J, Weise A, Falany J, et al: Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression. Breast Cancer Res Treat. 120:35–45. 2010. View Article : Google Scholar : PubMed/NCBI

16 

Zhao YN, Zhang W, Chen YC, Fang F and Liu XQ: Relative imbalances in the expression of catechol-O-methyltransferase and cytochrome P450 in breast cancer tissue and their association with breast carcinoma. Maturitas. 72:139–145. 2012.PubMed/NCBI

17 

Gajjar KL, Martin-hirsch PL and Martin FL: CYP1B1 and hormone-induced cancer. Cancer Lett. 324:13–30. 2012. View Article : Google Scholar : PubMed/NCBI

18 

Mikstack R, Rimando AM, Dutkiewicz Z, Stefanski T and Sobiak S: Design, synthesis and evaluation of the inhibitory selectivity of novel transresveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1. Bioorg Med Chem. 20:5117–6126. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Ashley-Martin J, Vanleeuwen J, Cribb A, Andreou P and Guernsey JR: Breast cancer risk, fungicide exposure and CYP1A1*2A gene-environment interactions in a province-wide case control study in Prince Edward Island, Canada. Int J Environ Res Publ Health. 9:1846–1858. 2012.PubMed/NCBI

20 

Williams-Brown MY, Salih SM and Xu X: The effect of tamoxifen and raloxifene on estrogen metabolism and endometrial cancer risk. J Steroid Biochem Mol Biol. 126:78–86. 2012. View Article : Google Scholar : PubMed/NCBI

21 

Scornaienchi ML, Thornton C, Willett KL and Wilson JY: Cytochrome P450-mediated 17b-estradiol metabolism in zebrafish (Danio rerio). J Endocrinol. 206:317–325. 2010. View Article : Google Scholar : PubMed/NCBI

22 

Han EH, Kim HG, Hwang YP, Song GY and Jeong HG: Prostaglandin E2 induces CYP1B1 expression via ligand-independent activation of the ERalpha pathway in human breast cancer cells. Toxicol Sci. 114:204–216. 2010. View Article : Google Scholar : PubMed/NCBI

23 

Guengerich PF, Chun Y, Kim D, Gillam E and Shimada T: Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies. Mutat Res. 523–524:173–182. 2003.PubMed/NCBI

24 

Zahid M, Kohli E, Saeed M, Rogan E and Cavalieri E: The greater reactivity of estradiol-3,4-quinone vs estradiol-2,3-quinone with DNA in the formation of depurinating adducts: implications for tumor-initiating activity. Chem Res Toxicol. 19:164–172. 2006. View Article : Google Scholar : PubMed/NCBI

25 

Lakhani NJ, Sarkar MA, Venitz J and Figg WD: 2-Methoxyestradiol, a promising anticancer agent. Pharmacotherapy. 23:165–172. 2003. View Article : Google Scholar : PubMed/NCBI

26 

Newman SP, Foster PA, Stengel C, et al: STX140 is efficacious in vitro and in vivo in taxane-resistant breast carcinoma cells. Clin Cancer Res. 14:597–606. 2008. View Article : Google Scholar : PubMed/NCBI

27 

Verenich S and Gerk PM: Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges. Mol Pharm. 7:2030–2039. 2010. View Article : Google Scholar : PubMed/NCBI

28 

Huang Y, Dai Z, Barbacioru C and Sadee W: Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance. Cancer Res. 65:7446–7454. 2005. View Article : Google Scholar : PubMed/NCBI

29 

Krishna R and Mayer LD: Multidrug resistance (MDR) in cancer: mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci. 11:265–283. 2000. View Article : Google Scholar : PubMed/NCBI

30 

Weichselbaum RR, Ishwaran HYT, Nuyten DSA, et al: An interferon-related gene signature for DNA damage resistance is a predictive marker for chemotherapy and radiation for breast cancer. Proc Natl Acad Sci USA. 105:18490–18495. 2008. View Article : Google Scholar : PubMed/NCBI

31 

Luker KE, Pica CM, Schreiber RD and Piwnica-Worms SD: Overexpression of IRF9 confers resistance to antimicrotubule agents in breast cancer cells. Cancer Res. 61:6540–6547. 2001.PubMed/NCBI

32 

Camaj P, Ischenko I, Seeliger H, Arnold G, Jauch KW and Bruns CJ: Overexpression of the gene IFIT3 enhances tumor growth, angiogenesis, metastasing and chemoresistance of the pancreas carcinoma cells. In: Chirurgisches Forum und DGAV Forum; 2009; 38. Springer; Berlin Heidelberg: pp. 17–18. 2009

33 

Cheriyath V, Kuhns MA, Jacobs BS, et al: G1P3, an interferon- and estrogen-induced survival protein contributes to hyperplasia, tamoxifen resistance and poor outcomes in breast cancer. Oncogene. 31:2222–2236. 2012. View Article : Google Scholar : PubMed/NCBI

34 

Veals SA, Schindler C, Leonard D, Fu XY, Aebersold R, Darnell JE Jr and Levy DE: Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins. Mol Cell Biol. 12:3315–3324. 1992.

35 

Wong LH, Hatzinisiriou I, Devenish RJ and Ralph SJ: IFN-γ priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs. J Immunol. 160:5475–5484. 1998.

36 

Englert NA, Spink BC and Spink DC: Persistent and non-persistent changes in gene expression result from long-term estrogen exposure of MCF-7 breast cancer cells. J Steroid Biochem Mol Biol. 123:140–150. 2011. View Article : Google Scholar : PubMed/NCBI

37 

Pham AN, Bower PE, Alvardo O, Ravula R, Gout PW and Huang Y: Pharmacogenomic approach reveals a role for the x(c)- cystine/glutamate antiporter in growth and celastrol resistance of glioma cell lines. J Pharmacol Exp Ther. 323:949–958. 2010. View Article : Google Scholar : PubMed/NCBI

38 

Lin G, Aranda V, Muthuswamy SK and Tonks NK: Identification of PTPN23 as a novel regulator of cell invasion in mammary epithelial cells from a loss-of-function screen of the ‘PTP-ome’. Genes Dev. 25:1412–1425. 2011.PubMed/NCBI

39 

Su PH, Lin YW, Huang RL, et al: Epigenetic silencing of PTPRR activates MAPK signaling, promotes metastasis and serves as a biomarker of invasive cervical cancer. Oncogene. 32:15–26. 2012.PubMed/NCBI

40 

Arafa el-SA, Zhu Q, Shah ZI, Wani G, Barakat BM, Racoma I, El-Mahdy MA and Wani AA: Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells. Mutat Res. 706:28–35. 2011. View Article : Google Scholar : PubMed/NCBI

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December 2013
Volume 33 Issue 1

Print ISSN: 1107-3756
Online ISSN:1791-244X

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APA
Motaghed, M., Al-Hassan, F.M., & Hamid, S.S. (2013). Thymoquinone regulates gene expression levels in the estrogen metabolic and interferon pathways in MCF7 breast cancer cells. International Journal of Molecular Medicine, 33, 8-16. https://doi.org/10.3892/ijmm.2013.1563
MLA
Motaghed, M., Al-Hassan, F. M., Hamid, S. S."Thymoquinone regulates gene expression levels in the estrogen metabolic and interferon pathways in MCF7 breast cancer cells". International Journal of Molecular Medicine 33.1 (2013): 8-16.
Chicago
Motaghed, M., Al-Hassan, F. M., Hamid, S. S."Thymoquinone regulates gene expression levels in the estrogen metabolic and interferon pathways in MCF7 breast cancer cells". International Journal of Molecular Medicine 33, no. 1 (2013): 8-16. https://doi.org/10.3892/ijmm.2013.1563