The Study of Anticancer Activity of Satureja Khuzestanica Alcoholic Extracts On Expression of Bcl2 And Bax Genes In The Pc3 Cell Line

Introduction: Prostate cancer is the most common cancer among men after lung cancer. It has grown in Iran in recent years. The use of medicinal plants is one of the most useful ways that causes the least side effects. Due to high levels of antioxidant compounds, Satureja khuzestanica is a good source for drug use to treat and prevent the development and progress of cancers. The aim of the present study was to evaluate the anti-cancer property of Satureja khuzestanica extract on the expression of Bcl2 and Bax genes in prostate cancer cell lines. Methodology: After collecting the plant in spring, the chloroform extract was prepared by rotary device. PC3 cancer cells were incubated at different concentrations of the extract for 24 hours. The inhibitory effect of the extract was evaluated using MTT assay as IC50. To evaluate apoptosis, the level of expression of Bax and BCL-2 genes after RNA extraction and transformation to cDNA were evaluated using Real Time PCR. All data were analyzed using REST software. Results: The results revealed a direct and signicant relationship between the two variables of drug composition and rate of PC3 cell death. This composition increased Bax gene expression and decreased BCL-2 gene expression and induced apoptosis (P <0.05). Discussion and Conclusion: Based on the results, Satureja khuzestanica extract is likely to have anticancer properties and seems to be a new drug for killing prostate cancer cells.


Introduction
Cancer is a fatal and dangerous disease whose treatment is costly and ineffective in most cases. In most countries, chemotherapy is used to control the cancer, but chemotherapy and drugs used to treat cancer have side effects that are sometimes more dangerous than cancer itself, such as liver and kidney failure, nerve damage, and so on. Hence, modern methods to prevent and treat cancer patients are needed to reduce the rate of the side effects (1). In developed countries, prostate cancer is considered as the second most common cancer after skin cancer in men. Prostate cancer indicates the presence of malignancy in men over 50 years. Hereditary history of prostate cancer is an important factor in the development of this cancer. The androgen receptor gene plays a major role in the development and progression of prostate cancer. HSD3B2, HSD3B1, SRD5A2, CYP17 AR genes also play a major role in androgen metabolism and cell proliferation in the prostate. Some polymorphisms in these genes are associated with an increased risk of prostate cancer. Epigenetic changes, especially DNA hyper-methylation in promoter regions, play a major role in the prevention and treatment of prostate cancer. A number of molecular and genetic changes have been observed in prostate cancer. Metastasis inhibitor genes have also been observed in prostate cancer (2). The main cause of non-apoptosis in most cancers is the high expression of antiapoptotic proteins such as Bcl-2. Increased expression of these proteins increases resistance to chemotherapy. Therefore, inhibition of expression or function of these proteins in cancer cells can induce apoptosis (3). There are various treatments for cancer, including surgery, chemotherapy, radiotherapy, and anticancer drugs (4). One of the biggest limitations of anticancer drugs is the resistance of cancer cells to the drug, which can be due to intrinsic resistance to the drug and they might act in a way that resistant cells can be selected among the heterogeneous cells. As a result, with increasing the number of resistant cells, the treatment process will become more di cult. Nowadays, the use of medicinal plants is increasingly considered more than chemical drugs due to their fewer side effects (5). As a result, as the number of resistant cells increases, the healing process becomes more di cult (6). Today, the use of medicinal plants has received less attention than chemical drugs due to its side effects (7).
Based on the studies conducted in this regard, it has been concluded that no study has been conducted on the chloroform extract of the studied plant and the effects of chloroform extract on the prostate cancer cell line have not been investigated, and no similar study has been conducted on the effects of S. khuzestanica species on prostate cancer cell line in Iran. Hence, it was decided to investigate the effect of chloroform extract of this plant in inhibiting the growth and expression of Bax and BCL-2 apoptotic genes in PC3 cancer cell line.

Methodology
This study was an experimental study whose all steps were performed in cell culture laboratory of Maragheh University of Medical Sciences. All of its steps were brie y as follows: Satureja khuzestanica was identi ed and approved by the herbalists of Khorraman Herbal Plants Company in mid-spring of 2018. After direct sending of dried leaves of Satureja khuzestanica, they were powdered by mill. Next, 100 g of Satureja khuzestanica powder was mixed with one liter of chloroform, and after 2 weeks, the mixture of Satureja khuzestanica with chloroform solvent was passed through lter paper and extraction was performed through a vacuum distillation method using a rotary device. The concentrated extract at value of 9.53 g was kept at refrigerator away from heat and light until cell culture (8).
Gas chromatography/ mass spectrometry Fid-GC was carried out using a Hewlett-Packard 6890 with HP-5 capillary column (phenyl methyl siloxane. 25 m, 0.25 mm i.e., ratio, 1:25, and ame ionization detector. Temperature programmer: 60 ºC (2 min) rising to 240 ºC at 4 ºC/min: injector temperature 250 ºC, detector temperature, 260 ºC. GC-MS was performed using a Hewlett-Packard 6859 with a quadruple detector, on a HP-5 column, operating at 70 eV ionization energy, using the same temperature programmer and carrier gas as above. Retention indices were calculated by using retention times of n-alkanes that were injected after the oil chromatographic.
Cell culture: PC3 cancer cells were used as cell lines in the current study. Prostate cell lines were obtained from the cell bank of Pasteur Institute of Iran and cultured in RPMI1640 with 10% fetal bovine serum (FBS) along with 100 μl of penicillin streptomycin antibody in an incubator at 37 ° C with su cient humidity and 5% carbon dioxide. The cell culture medium was replaced every two days on average, when needed, and it was passaged when reached to 85% con uency. To perform different tests and when the cells reached at least 80% cell growth, they were removed from the bottom of the ask by trypsin / EDTA enzyme and centrifuged at 1000 rpm for 7 min. Cell deposition was prepared in suspension in 1 ml culture medium and percentage of cells viability in cell suspension was determined by mixing equal ratio of trypan blue using a hemocytometer slide and by examining the light microscope. MTT assay was used to investigate the cytotoxic effect of Satureja khuzestanica chloroform extract on PC3 cell line. The MTT assay is a metabolic test of mitochondrial competition and based on the breakdown of tetrazolium salt by the mitochondrial succinate dehydrogenase enzyme, living cells is transformed into purple waterinsoluble crystals in formazan water (9).
Then, cell suspension was prepared at a concentration of 1 10 4 cells / ml and incubated in 96-well microplates for 24 hours. Then, concentrations (1000, 500, 250, 125, 62.5 and 31.25 µg / ml) of the extract were added. The cell group of cell suspension was considered as having no extract. The micro-plates were incubated for 24 h under the same conditions. Then concentrations of 10 µl (5 µg / ml) of MTT solution were added to each well and incubated for four hours. The supernatant was replaced with 100 µl DMSO (dimethyl sulfoxide) and dissolved the formazan crystals. Then, it was measured by ELISA (Statfex. 2100) at 560 nm.
Cell viability for each concentration was calculated using the following formula: After obtaining IC50, cells treated with that concentration were used for RNA extraction and Bax and BCL-2 genes` examination. After extraction of RNA from PC3 cancer cells treated with chloroform extract and untreated cells, cDNA synthesis was performed and then, it was used along with the preparation of Bax and BCL-2 and actine-β primers in reaction with 25 μl Real Time PCR to understand the mechanisms involved in the induction of cell death. After 24 hours, their RNA was extracted according to common guidelines (9). Then, the steps of RNA transformation to cDNA were performed. C-DNA synthesis kit (Sinagen Company) was used to transform the RNA into cDNA. After cDNA synthesis, Bax and BCL-2 genes were selected to evaluate the expression of genes involved in apoptosis. To validate the test, actine -control gene (expressed in all cells) was used. For this purpose, primers shown in Table 2 were used in RT-PCR test Based on the guidelines, after preparing the substances in a 0.2 ml microtube, along with the control and treatment group, they were transferred to Thermo Cycler device. After completion of the Thermo cycler work, RT-PCR product was applied to 1% agarose gel. Then, Real time PCR was used to evaluate the expression of the desired genes. In this method, the primers of Table 2 with program Table 4 were used and the contents of Master mix-Real Time PCR were added according to Table 3.

Statistical analysis of data
The results of this study were analyzed using REST software and ANOVA tests.

Identi cation of components
The linear retention indices for all the compounds were determined by conjection of the sample with a solution containing the homologous series of C8-C22 n-alkanes. The individual constituents were identi ed by their identical retention indices, referring to known compounds from the literature and also by comparing their mass spectra with either the known compounds or with the Wiley mass spectral database Table 1.

Analysis of MTT results
Results of MTT assay showed that chloroform extract of the studied plant at different concentrations inhibited the proliferation of cancer cells. Figure 1 shows the cytotoxic effect of chloroform extract of Satureja khuzestanica on PC3 cell line. Cell viability was determined by MTT assay. The absorbance was measured by ELISA device at 560 nm. The viability rate of the control group was considered 100 and the level of signi cance was considered at p <0.05. Comparison of cell growth percentages at different doses showed that rosemary extract at 125 mg / ml of Satureja khuzestanica extract had more effect in 24 hours and reduced the growth of cancer cells.

Real Time PCR test results
The expression levels of BCL-2 and BAX genes are shown in the following diagrams. As seen, the expression of BAX gene as a promoter of apoptosis was higher at 24 hours after treatment with chloroform extract at 125 mg / ml than the other groups, which was in accordance with MTT assay. At the same dose, the expression level of BCL-2 gene as an apoptotic inhibitor gene was signi cantly lower Figure 2 and Table 5.

RNA electrophoresis results
To evaluate the accuracy of the quality of the extracted RNA used in Real Time, 4 samples were randomly electrophoresed in 1% agarose gel whose results were in the form of 5s and 28s bands. According to this Figure3, the extracted RNAs had almost good quality.
In order to con rm the results of PCR Real Time, the sample was electrophoresed on 1% Electrophoresis gel Figure 4. The presence of the desired band indicated that all bands have been proliferated and the results were correct.

Discussion
Environmental factors such as air pollution, stress, lifestyle and diet are among the causes associated with the rising incidence of cancer. Eating foods that have antioxidant properties have been shown to be effective in preventing and reducing the incidence of cancer (10)(11).
Despite the use of treatment strategies such as surgery, chemotherapy and radiotherapy, the mortality rate in cancer patients is still high, which indicates the ineffectiveness of these treatment strategies. In addition, the detrimental effect of chemotherapy and radiation therapy on dividing normal cells is another disadvantage associated with these therapeutic processes (12). showed that the antioxidant activity of Satureja hortensis extract increases with increasing concentration and is able to prevent oxidation processes (17).

S.khuzestanica is
By examining the antifungal activity of the oil and the methanolic extract of Satureja khuzestanica against Aspergillus phallus, Dikbas N et al. (2008) concluded that the essential oil of Satureja khuzestanica has high antifungal activity against the mentioned pathogen (18). Studies conducted at (2015) have shown that carvacrol is a poly-phenolic compound that has very powerful antioxidant ability such as vitamin E and ascorbic acid. Carvacrol and many of its antioxidants have a signi cant role in preventing diseases such as cancer. Through genome degradation, they reduce the viability of cancer cells and genome degradation is much more pronounced at concentrations close to IC50 (19). Based on the studies conducted by Shirali, 43 compounds were identi ed in the essential oil of Satureja khuzestanica, which make up 99.96% of all essential oils. Then, to determine its antimicrobial effects, they used disk diffusion method against two microorganisms of Escherichia coli and Candida albicans. They concluded that the presence of phenolic compounds such as carvacrol, thymol and Gammaterpinene in the essential oils also caused antimicrobial activity in this plant. Increasing phenolic compounds of essential oils is also directly associated with increased antimicrobial activity. This property prevents the oxidation of lipids and coronary heart disease and cancer (20).
In a study conducted by Sazghar et al. (2017) to investigate the anti-cancer properties of hydroalcoholic extract of Celeriac and savory on Hela cancer cells, it was found that BAX mRNA expression level increased signi cantly. However, no change was seen in BCL-2 expression. BAX protein expression level was also increased signi cantly in Real Time PCR, while it had no effect on BCL-2 expression. In this study, the cytotoxic effect of this extract on Hela cancer cells was con rmed, but this extract had no toxic effect on broblast cell line. In the present study, the same results (signi cant expression of BAX gene) were obtained using chloroform extract of Satureja khuzestanica. In the current study, the highest expression of BAX gene was observed at 125 mg / ml after 24 hours, which was in accordance with MTT assay results (21).

Declarations Funding Info
This article was adapted from the MS.c. project(14430513951001) of Saman Kazemi, where Hossein Soltanzadeh supervised, and Asghar Tanomand advised this project.
This study was supported in a MS.C program by bonab Islamic Azad University

Con icts of interest/Competing interests
We con rm that this manuscript has not been published elsewhere and is not under consideration by another journal. All authors have approved the manuscript and agree with submission to Molecular Biology Reports journal. The study was supported in a Master program by Bonab Islamic Azad University. The authors have no con icts of interest to declare.
Ethics approval 1) This material is the authors' own original work, which has not been previously published elsewhere.
2) The paper is not currently being considered for publication elsewhere.
3) The paper re ects the authors' own research and analysis in a truthful and complete manner.
4) The paper properly credits the meaningful contributions of co-authors and co-researchers.

5)
The results are appropriately placed in the context of prior and existing research. 6) All sources used are properly disclosed (correct citation). Literally copying of text must be indicated as such by using quotation marks and giving proper reference. 7) All authors have been personally and actively involved in substantial work leading to the paper, and will take public responsibility for its content.
Ethical principles in writing the article have been Compliance according to the instructions of the National Ethics Committee and the COPE regulations. And due to we used the cancer cell line and didn't have a human sample, according to the National Ethics Committee, there is no need for a code of ethics.  Tables   Table 1. Identification of components The linear retention indices for all the compounds were determined by conjection of the sample with a solution containing the homologous series of C8-C22 n-alkanes. The individual constituents were identified by their identical retention indices, referring to known compounds from the literature and also by comparing their mass spectra with either the known compounds or with the Wiley mass spectral database.  Bax is UP-regulated in sample group (in comparison to control group) by a mean factor of 4/733 (S.E range is 3/934-6/130) Bax sample group is different to control group P(H1)=0/000

Authors' contributions
Bcl2 is DOWN-regulated in sample group (in comparison to control group) by a mean factor of 0/889 (S.E range is 0/879-0/900) Bcl2 sample group is different to control group P(H1)=0/024    Total RNA Electrophoresis results of β-actine, Bax, and BCL-2 genes to ensure correct operation of Real time PCR      Diagram 6. Relative expression to BCL-2