Razi Journal of Medical Sciences

Background: 
According to the reports from the International Agency for Research on Cancer, the incidence rate of cancer is rising equal to the growing rate of population. Cancer is a common problem that has been declared as the main cause of mortality among people and included 18.4% of mortality cases in 2018 equal to 9.6 million that has been reported by WHO [1]. Tamoxifen is a non-steroid and anti-estrogen which serves as an option for breast cancer treatment and can reduce the risk of cancer to 50 percent but it has some side effects [2]. Till now, many natural components have been introduced as a remedy for cancer. Thymoquinone is known as a bioactive component of the volatile oil of Nigella sativa seed [3]. Moreover, TQ has an anti-cancer, anti-inflammatory effect, anti-oxidant, anti-bacterial, and modulator of the immune system [5]. Previous studies showed that TQ has different modes of action, in this study we tried to find the cytotoxicity of TQ on breast cancer cells and some of the genes involved in that mechanism. 
Methods: 
To determine the percentage of viable cells following treatment with TQ, the 10-fold dilution of TQ was prepared to evaluate its effect on breast cancer cells, MCF-7. The range of concentrations was between 0–1000 micromolar. TAM 5 micromolar was considered a positive control. The Cell Proliferation assay was used to determine the effect of TQ on cell proliferation. Differences between various concentrations of TQ were evaluated by Tukey multiple comparison test using SPSS V.16. The significant differences between 0, 1, 10, 100, and 1000 micromolar were found by One-way ANOVA test. Annexin V-FITC apoptosis detection kit was used to quantitatively determine the percentage of apoptotic cells in treated and untreated MCF-7 breast cancer cells. Total RNA from each sample of TQ-treated and untreated MCF-7 cell line was isolated using the RNeasyPlus Mini kit. For the quantification of isolated RNAs, the protocol of the NanoDrop 2000C Spectrophotometer was followed. The NanoDrop 2000C Spectrophotometer revealed the sufficient yield and purity of RNA samples at OD230, OD260, and OD280 of wavelength. The ratio of A260 to A230 of samples was more than 2.0 and the ratio of A260 to A280 samples was more than 1.8. After preparing enough amount of RNA, two-step RT-qPCR, Real-time reverse transcription-polymerase chain reaction, was used which involves two reactions. In the first step, the high capacity RNA-to-cDNA Kit was used for reverse transcription of total RNA to a single-stranded cDNA. It was followed by step 2 in which the cDNA was amplified with a thermal cycler. RT-qPCR was used to detect small amounts of mRNA or gene expression levels. The level of gene expression was determined using comparative quantification and its relative expression to the level of internal control that was GAPDH which served to normalize the fluorescence signals relative to the gene of interest or its expression level in samples. The mixture of TaqMan gene expression assay contains a TaqMan probe and primer of the target gene was used to understand the optimal standard curve of each target gene. The Livak method was used to calculate the relative gene expression levels or RQ value in the expression level of the target genes. Therefore, the CT mean value of the target gene to that of the reference gene for both of the treated sample and untreated samples was normalized using the StepOnePlus Real-Time PCR Software v 2.1. In this study, the CT value of the majority of target genes was found to be in the acceptable value of more than 18 but less than 35.
Results:
Initially, Tukey multiple comparison test was selected to determine whether there are significant differences between different concentrations. The results showed there were no significant differences between untreated versus 1 µM and 10 µM concentrations. However, there were significant differences between untreated versus 100 and 1000 micromolar. Similarly, the differences between 1 versus 100 and 1000 micromolar concentrations were found to be significant. These showed concentrations of less than 1000 micromolar would be sufficient for further analysis. The percentage of viable cells at 100 micromolar concentration, mean ± SD = 40 ± 4.2 was almost similar to the percentage of viable cells at 1000 micromolar concentration, mean ± SD = 29 ± 3.8. One-way analysis of variance, ANOVA was done to compare the differences between untreated cells versus TQ treated and TAM treated cells. A great reduction in the number of viable cells was found with 100 micromolar TQ versus untreated cells and TAM treated cells which indicated the highly toxic effect of TQ 100 micromolar concentration. The number of apoptotic cells after 24 hours of treatment of cells with TQ 100 micromolar was about 82 percent, however, it was 17.5 percent with TAM 5 micromolar. UGT1A8 and CARD16 were the genes decreased and increased, respectively obtained by RT-qPCR method. 
Conclusion: 
Up until now, there has been reported much evidence regarding the therapeutic effect of TQ. The results obtained from this experiment showed that TQ is an active compound of black seed that can reduce the side effects of TAM. Trans-4-tamoxifen-glucuronide is an active metabolite of TAM which has a high affinity to the estrogen receptors. If the compound can prevent the accumulation of estrogen inside the body, therefore it can reduce the side effect of estrogen. The results revealed that low expression of the gene UGT1A8 due to TQ can reduce the conversion of trans-4-hydroxy-tamoxifen to trans-tamoxifen-4-o-glucuronide. TQ can activate the glucuronidation process which is one of the processes of xenobiotic and drug metabolism whereby, the trans-tamoxifen would be transferred to glucuronide form and the level of accumulated estrogen would be reduced. UGT1A8, Glucuronosyltransferase Family 1 Member A8, is a gene involved in the family of Cytochrome P450 and active in the metabolism of estrogen. The UGT1A8 protein can maintain the methoxy form of estradiol which the non-cancerous form of TQ [22]. The decrease in the level of this gene in this study suggested the activation and overexpression of 2-methoxy estradiol. This finding highlighted the anti-apoptotic effect of TQ and also, anti-cancer and anti-angiogenesis of this compound. On the other hand, an increase in the expression of CARD16, Caspase Recruitment Domain Family, member 16, about 4-fold suggested the ability of TQ in triggering apoptosis mechanism or cell death through caspase activation, because of the relation between CARD16 with CASP1. CASP1 is involved in inflammation [24]. CASP1 showed an important role in pathological cell death or programmed cell death [25].