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.
Background:Nigella sativa or black seed extract has been reported to show various medicinal benefits. Thymoquinone which is an active compound of its seed has been reported to contain anti-cancer properties.Objective:The study addressed the anti-cancer efficiency of long-term in vitro treatment with thymoquinone towards human breast cancer cell lines MCF-7.Materials and Methods:Cell proliferation was determined with CellTiter 96 Aqueous. Non-Radioactive Cell Proliferation Assay Kit. It was followed with trypan blue exclusion test to determine the percentage of viable cells. The study incorporated cell cycle assay to distinguish cell distribution at various cell cycle phases using Cycletest Plus DNA Reagent Kit. The apoptosis detection kit was used to determine the percentage of apoptotic and necrotic cells using flow cytometry.Results:The 50% inhibitory concentration (IC50) value determined using the proliferation assay was 25 μM thymoquinone. Late apoptotic cell percentage increased rapidly when treatment duration was increased to 24 h with 25 and 100 μM thymoquinone. Further analysis using cell cycle assay showed thymoquinone inhibition of breast cancer cell proliferation at minimal dose 25 μM and led to S phase arrest significantly at 72 h treatment (P = 0.009). It was also noted elevation sub-G1 peak following treatment with 25 μM thymoquinone for 12 h. Increase in thymoquinone to 50 μM caused G2 phase arrest at each time-point studied.Conclusion:In general thymoquinone showed sustained inhibition of breast cancer cell proliferation with long-term treatment. Specificity of phase arrest was determined by thymoquinone dose.
About 80% of breast cancers are estrogen-receptor positive. The research carried out herein focused on the effect of Thymoquinone which is an active compound of Nigella sativa seed on estrogen-receptor positive breast cancer MCF7 cell line. The percentage of apoptotic cells was found using Annexin V-FITC apoptosis detection kit. CycleTEST PLUS DNA Reagent was used to distinguish distribution of treated cells between different cell cycle phases. DNA microarray identified the regulated genes, level of expressed genes, gene ontology and pathway networks. Significant arrest of treated cells at G 1 phase suggested cytostatic effect of Thymoquinone 100 µM after 24 hours at p-value < 0.05 which was similar to anti-estrogenic compounds such as Tamoxifen. Cytotoxic effect of Thymoquinone 100 µM was found through highly significant accumulation of cells at sub-G 1 phase after 72 hours at p-value < 0.0001. CYP1A1, CYP1B1, NQO1 and UGT1A8 genes were down regulated after 24 hours treatment with Thymoquinone 50 µM concentration which suggested reduction of catechol estrogens and rising in metoxy forms of estradiol and estrone. Reduction of ER would be predictable due to the down-regulation of CYP1B1 and UGT1A8 genes which reduced affinity of trans-tamoxifen-o-glucuronide to ER. The study proposed the benefits of using Thymoquinone to accelerate Tamoxifen effects in treating breast cancer and reducing its side effects.
Introduction: The dedication of stem cells for dissociation into a specific type of cell requires the over expression of genes related to a particular phenotype and suppression of the other genes. Through imposing corresponding alterations on the genome, the genome modulators such as transcription factors can be regulated by histone-modifying enzymes. Maintenance of the neurogenesis process depend on the function of some of these genes which can regulate shifting of cells from proliferation to differentiation such as Enhancer of zeste homolog 2 (EZH2) known as an evolutionarily conserved gene. Moreover, motor neurons (MN) in spinal cord can be regulated during neuronal differentiation via one of the histone acetyltransferase (P300). Up until now, the mechanism of epigenetic regulation and gene expression underlie transition process of human cord blood mesenchymal stem cells (hCB-MSCs) into MNs has not been clarified very well. Therefore, the aim of this study was to explore the quantitative expression of MN-related genes including ChAT, Islet-1, and Mnx-1 along with two epigenetic regulatory genes P300 and EZH2 involved in neurogenesis during differentiation of hCB-MSCs into MNs, using two morphogens including Sonic hedgehog (Shh) and Retinoic acid (RA) involved in the specification of MNs during the growth of nervous system. Methods: Flow cytometry was done to characterize the cells (hCB-MSCs). The cells were differentiated into MN-like cells according to our previous procedure using RA (0.01mM) and Shh (100ng/ ml) as the inducing morphogens. CB-MSCs with no treatment were assumed as control cells. RT-qPCR and Immunocytochemistry were performed to find the expression of interested genes in this study. Results: The expression of MN-related markers was confirmed at the level of mRNA and protein by induction of differentiation. The results was confirmed by immunocytochemistry showed that a number of cells about 55.33±15.885% and 49.67±13.796% could express Islet-1 and ChAT, respectively. The level of gene expression of Islet-1 and ChAT was significantly increased at the first and second week of exposure, respectively. After two weeks, expression level of P300 and EZH-2 genes was increased remarkably. No significant expression of Mnx-1 was detected when compared with the control sample. Conclusion: In this study MN-related markers, Islet-1 and ChAT, were detected in differentiated cells of hCB-MSCs supporting the potency of cord blood cells in regeneration of MN-related disorders. The over expression of Islet-1, as an early MN marker, in the presence of Shh and RA indicates the supportive effect of these morphogens for the onset of motor neuron generation. We could also detect significant expression of two potent epigenetic regulatory genes involved in neurogenesis, P300 and EZH2 accompanied by ChAT as the mature motor neuron marker at the second week of exposure due to the elimination of Shh and RA at later time of differentiation. To our knowledge, the evaluation of P300 and EZH2 during differentiation of hCB-MSCs into MN-like cells was performed in this study for the first time. However, the assessment of these epigenetic regulatory genes at the level of protein can be suggested to confirm their functional epigenetic modifying effects during motor neuron differentiation.
Abstract:Adenocarcinoma is known as a common type of cancer which includes 85% of breast carcinoma and 95% of colorectal carcinoma. Up until now, the cytotoxic effect of Thymoquinone on different types of tumor cells has been reported. It was hypothesized that Thymoquinone has similar effect on cancer arising from epithelial cells through gene expression analysis in MCF7 breast cancer and HT-29 colon cancer cell lines. The quantity and quality of RNA samples were identified using RNeasyPlus Mini kit and RNA 6000 Nano LabChip kit, respectively. The purified RNA samples of MCF7 cells were used in two-color 8×60K cDNA array platform with SurePrint Agilent technology. The hybridized cRNA/cDNA probes were identified due to labeling with red and green cyanine dyes using GE X hybridization buffer HI-RPM. LOWESS normalization reduced the dye bias on the array slide using feature extraction software. Gene ontology analysis was done after performing different steps of filtering to reduce not satisfied genes using Gene Spring software. Two-step RT-qPCR assay using Taq Man fast advanced master mix analyzed the most up and down regulated genes in MCF7 compared to HT-29 cancer cell lines. The 10 different cancer cell lines in a form of universal reference RNA was used as standard data set comparison and as a positive control RNA in cDNA array and RT-qPCR assay, respectively. The T-test statistical analysis of independent samples showed that there is no significant difference between two types of cell lines due to Thymoquinone treatment with p-value 0.844. Among the selected genes; CARD16, UGT1A8, SLC7A11, IFIT1, IF16 and IFIT3 were expressed significantly (0.009, 0.0001, 0.037, 0.098, 0.0001 and 0.033, respectively) in breast cancer compare to colon cancer cells. The findings indicated similar effect of Thymoquinone on cancer arising from epithelial cells.
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