INTRODUCTION

Japan ranks first in terms of the incidence of gastric cancer within the world and South Korea, Costa Rica, the former Soviet republics, and Japan rank first to fourth in the world in terms of mortality caused by gastric cancer. Its highest prevalence has been observed in the Far East. In Iran, cancer is the third most common cause of death, and more than 30,000 people die each year due to this disease (1,2). Gastric cancer is a common disease in Azerbaijan and Iran. Ardebil has the highest incidence of gastric cancer in Iran after Gilan province. Genetic factors, environmental factors, and food habits are among the causes of gastric cancer in these provinces. Gastric cancer can be divided into completely separate pathologic subtypes with different epidemiological and prognostic features (3). The spread of gastric cancer is a complex and multistage process. It involves genetic and epigenetic changes in cancer-producing genes, tumor suppressor genes, DNA-modifying genes, cell cycle regulators, and signaling molecules. Gastric cancer may be caused by genomic instability, which can be in the form of microsatellite instability or chromosomal instability (4,5).

The main cause of most cancers is the overexpression of anti-apoptotic proteins, such as Bcl-2, and lack of apoptosis. Increasing the expression of these proteins increases resistance to chemotherapy. Thus, inhibiting the expression or function of these proteins in cancer cells can induce apoptosis. The Bax and Bcl-2 genes, other apoptotic genes, and planned deaths are involved in the incidence and development of gastric cancer (6). Chemotherapy is used to control many cancer diseases. However, side effects (e.g., neurological damage and kidney failure) of chemotherapy and the drugs used are sometimes more hazardous than cancer itself. Thus, to reduce these complications and given its resistance to chemotherapy, modern methods are needed to prevent and increase the chance of treating cancer patients (7). Satureja khuzistanica (S. khuzistanica) is a plant with two main effective ingredients, namely thymol and carvacrol, which have anticancer, antioxidant, and antimicrobial effects (8-11). A study on the percentage and ingredients of essential oil of S. khuzistanica showed that the mean essential oil content of S. khuzistanica was 18.3%. Additionally, 43 compounds were identified in the essential oil of S. khuzestanica, which contains 90% of the total carvacrol (11). The objective of this study was to evaluate the antimicrobial, cytotoxic, and anticancer effects of chloroform and water extracts of S. khuzistanica on gastric cancer.

MATERIALS AND METHODS

Antimicrobial Activity

The antimicrobial activities of chloroform and water extracts were screened against pathogenic bacteria [Helicobacter pylori (H. pylori)]. Concentrations of chloroform and water extract of S. khuzistanica (25 and 50 mg/mL) were tested against H. pylori using the disc diffusion method. About 50 μL of each concentration of the extracts was added to each sterile blank disc (Padtan Teb, Iran). From the fresh culture of H. pylori, 1 McFarland solution in brucella broth (Himedia) was prepared, and 100 μL of the solution was taken by sterile swab. Next, it was cultured on a brucella agar medium containing 5% defibrinated sheep blood and 7% inactivated fetal bovine serum. The dried discs of the extracts were placed on the culture at appropriate distances. After placing the discs, the media were kept in a “CO₂” incubator (with 10% “CO₂”) at 37 °C for 72 h. Antibacterial activity was determined by measuring the zone of inhibition around the test discs. The growth inhibition diameter was the average of three different measurements. The result was expressed as the inhibition zone diameter and was compared with the standard antibiotics ampicillin (Amp, 10 μg) and chloramphenicol (C, 30 μg).

Preparation of the Cell Culture Medium

In the present study, AGS and KATO III gastric cancer cell lines and gingival fibroblasts were prepared from Pasteur institute and cultured in RPMI enriched with 10% fetal calf serum, 1% Pen strep, and 5% CO₂ at 37 °C.

MTT Test

Microculture tetrazolium (MTT) test is a colorimetric method performed based on the reduction and breakdown of tetrazolium yellow crystals with the chemical formula of 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT). The test was conducted using succinate dehydrogenase enzyme and the formation of insoluble blue crystals. In the present study, the MTT test was performed to evaluate the effect of S. khuzistanica on the viability of AGS and KATO III cells and gingival fibroblasts. Accordingly, the cells cultured in a 96-well plate at concentrations of 100, 500, 250, 125, 62.5, and 31.25 μg/mL of the plant extract were treated on normal and cancer cells over 24 h. Then, the medium was replaced with MTT (Sigma Aldrich) solution. After 4 h of incubation in the dark, the MTT solution was replaced with DMSO. The light absorption was then read at 570 nm in an ELISA device. In the next step, the percentage of live cells was calculated using the formula (%) = (ODexp/ODcon) × 100. In this formula, ODexp and ODcon are the light absorption of the exposed and control groups, respectively. All experiments were performed in triplicate.

Generally, to evaluate the gene expression changes, total cell RNA was extracted from the gastric cancer cell line using a solution (Ribospin304-15 Gene All, Korea) according to the manufacturer’s instructions. Spectrophotometer and agarose gel electrophoresis were used for quantitative and qualitative analysis of the concentration and purity of the extracted RNA. In this study, cDNA was synthesized from mRNA using the Sina Colon First Starandn cDNA kit according to the manufacturer’s instructions. To produce cDNAs with similar concentrations, we measured the volume of each sample so that 1000 ng of RNA was available. Eventually, the cDNA samples were produced and stored at -20 °C until the real-time polymerase chain reaction (RT-PCR).

Primers

Each gene fragment was proliferated by selecting a pair of specific primers for each gene, including forward and reverse prime (Table 1).

RT-PCR was performed using a magnetic induction cycler (MIC) device and RealQ Plus 2x Master Mix Green. For this purpose, 12.5 μL of cyber green mixer master, 3 μL of the cDNA sample, 0.5 μL of forward and reverse primers (10 pmol) in water, and 8.5 μL of nuclease-free water were mixed in 25 μL of the reaction mixture. The beta-actin (β-actin) gene was amplified as an internal control, and the fold change in the relative expression of each target mRNA was calculated using the comparative Ct (2-ΔΔCt) method.

Real-time PCR Procedures

In this research, the heat schedule of the RT-PCR device (MIC model) was 95 °C for 15 min in the first stage, 95 °C for 10 s in the second stage, and 49-51-60 °C, respectively, for the Bax, Bcl-2, and β-actin genes C for 15 s, and 72 °C for 20 s in 40 cycles in the last stage. Finally, the data were analyzed by comparing the threshold cycle and the difference in the obtained threshold from the treated cells with the extracts and untreated cells with the target gene ratio (Table 2, 3). 

Melting Curve Analysis

Once PCR is completed, the device can plot the melting curve of each sample. Thus, by analyzing the melting curve, it is possible to identify the presence of non-specific bands and the primer dimer (Figure 1).

Statistical Analysis

Statistical analysis of the data was performed using REST software at a significance level of <0.05.

RESULTS

According to the following equation, the percentage of chloroform and water extracts of the S. khuzistanica plants is 7.5%.

Images taken from the growth of AGS gastric cancer cells before and after treatment with S. khuzistanica extract are illustrated in Figure 2.

Antimicrobial Activity

The antimicrobial activity of the extracts was measured using the disk diffusion method. The antimicrobial activity of the extracts was found within the 25-50 mg/mL concentration range. The inhibition zones of the bacteria were in the range of 18±0.4 mm. According to the disk diffusion method, all the concentrations used were inhibitory for all the reference bacterial strains.

MTT Results

The MTT assay was performed to find a minimum of 50% killing extract in μg/mL, examine the cytotoxic effect of the extract, and determine the IC₅₀ in AGS cells. This test was performed in three replicates. The results of the MTT test at different concentrations of the extract in μg/mL were obtained as a linear chart. In addition, the results showed no significant cytotoxic properties in normal cells. The results for the cancer cells are presented in Figure 3.

RNA Electrophoresis Results

In order to evaluate the quality of the extracted RNAs, three samples were electrophoresed randomly on Agarose gel. For this purpose, 1.5% gel was made and the congealed samples were loaded onto gel and electrophoresed at V100 for 2 min (Figure 4).

Results of Real-Time PCR

After obtaining the mean, CTs were analyzed using the following formula: in addition, the results were analyzed using REST software, and the following charts were obtained.

Results of relative expression of the Bcl-2 gene and Bax relative to the β-actin gene.

According to the charts of β-actin genes as a reference and Bcl-2 gene and Bax as a target, the expression of Bax and Bcl-2 genes after applying S. khuzistanica extract increased and decreased, respectively. Therefore, after applying a dose of 125 μg/mL, the expression rate of these genes increased and decreased by approximately 1.9 and 0.227 times, respectively, at a significance level of 0.000.

The RT-PCR results show the relative expression of Bax to Bcl-2 (Figure 5).

DISCUSSION

Traditional medicine is effective in many diseases, and its use for treating diseases has been proven. Anxiolytic sources without toxic side effects are a good approach for the treatment and control of diseases such as cancer. Several studies have been conducted on the benefits of S. khuzistanica in medicine. For example, Rezvanfar et al. (12) investigated the benefits and protective effects of S. khuzistanica on cyclophosphamide-induced hemorrhagic cystitis in a rat model. The results showed that the S. khuzistanica extract protects against cyclophosphamide-induced hemorrhagic cystitis in a rat model by reducing radical-induced toxic stress (12).

In another study, Malmir et al. (13) studied new flavonoid monoterpenes in S. khuzistanica. The results showed that S. khuzistanica had a beneficial antidiabetic and antioxidant effect. In addition, Saidi (14) studied S. khuzistanica and its chemical and antioxidant properties and showed that this plant is a rich source of antioxidant compounds. They also examined the effects of S. khuzistanica on inhibiting the expression of iNOS in the macrophage cell line J774A.1. The results showed that S. khuzistanica had anti-inflammatory effects through its effect on iNOS gene expression (15).

Esmaeili-Mahani et al. (16,17) investigated the effect of S. khuzistanica on the tolerance of pain induced by morphine by inhibiting the activation of glial cells. According to their results, S. khuzistanica can have beneficial effects in inhibiting drug tolerance and reducing glial cell activation. Sharafati-Chaleshtori et al. (18) showed that carvacrol of S. khuzistanica reduces the viability rate of cancer cell degeneration of the genome, and genome degradation at concentrations close to IC₅₀ is more significant. Saffari studied the effect of S. khuzistanica on MCF-7 breast cancer cells. These authors showed that the rate of cell apoptosis increased with increasing concentration and time. Furthermore, the alcoholic extract of MCF-7 breast cancer cells showed anticancer activity (19).

Ahmadi et al. (20) evaluated the protective effect of S. khuzistanica essential oil on ovarian failure caused by busulfan. The results showed that busulfan significantly reduced the number and quality of oocytes, fertilization rate, embryo growth before implantation, and embryo quality. S. khuzistanica essential oil significantly reduced the adverse effects of busulfan. According to their results, S. khuzestanica essential oil can protect the fertility of the female sex against damages caused by busulfan (20). Sazgar et al. (2017), combining the hydroalcoholic extract of celeriac and Satureja, showed that it had a toxic effect on HeLa cancer cells, whereas this extract did not have a toxic effect on normal fibroblasts. Shirali and Alizadeh (21) evaluated the antimicrobial properties of the essential oil of S. khuzistanica. The results showed that phenolic compounds such as carocrole, thymol, and gamma-terpinene in essential oil compounds have antimicrobial properties in this plant. Moreover, an increase in phenolic compounds of the essential oil is directly associated with an increase in antimicrobial activity, which prevents lipid oxidation and heart coronary and cancer diseases (21).

Loizzo et al. (22) investigated the anticancer effects of the essential oil of Satureja. They studied the cellular cytotoxicity of this essential oil on various cancer cells, including breast cancer. They reported that Satureja essential oils could inhibit breast cancer cells, and this ability increases with increasing concentration (22). This study showed that the treatment of cells with S. khuzistanica leads to an increase and reduction in the expression of apoptotic Bax and Bcl-2 genes. In addition, they reported that the extract of this plant had significantly increased anti-apoptotic properties against the AGS cell line. Therefore, the expression of Bax and Bcl-2 genes changes in gastric cancer cells. A significant reduction in Bax gene expression as a pro-apoptotic regulator of apoptosis and an increase in Bcl-2 gene expression as an anti-apoptotic regulator demonstrate the high capacity of S. khuzistanica to reduce the incidence of cell apoptosis. S. khuzestanica significantly increases apoptosis by reducing the expression of Bcl-2 and increasing the expression of Bax (23). In addition, this study provided significant information on the correlation between busulfan and apoptosis due to oxidative stress in sperm and testicular tissue in adult rats. An imbalance between the production of ROS and the reduction of antioxidant mechanisms resulted in the toxicity induced by oxidative stress in adult male rats after treatment with busulfan. However, a 1-week treatment with S. khuzistanica in rats treated with busulfan-S. khuzistanica reduced the apoptosis induced by oxidative stress, cytotoxicity, and genotoxicity in sperm [Nasimi et al. (23), 2018]. Natural substances in S. khuzistanica extract can increase and decrease the relative expression of Bax and Bcl-2 genes, respectively. This study showed that the expression of Bax and Bcl-2 genes after application of S. khuzistanica extract (125 μg/mL) increased and decreased, respectively. These results suggest that the expression of Bax and Bcl-2 genes can be changed by the application of S. khuzistanica extract. Data from a study show that S. khuzistanica has anticancer effects on MCF-7 cancer cells and shows synergistic effects in combination with vincristine (17).

In a previous study, the methanol extract of S. khuzistanica induced cytotoxic effects in a dose-dependent manner. In this regard, almost half of the cells were destroyed during 24 h with 125 μg/mL, and MCF-7 was detected as the most sensitive cell line (24,25).

CONCLUSION

In our study, the results of antimicrobial activity showed that among the extracts, chloroform extract had a more powerful antimicrobial effect than water extracts against H. pylori bacteria. The antimicrobial effect of thymol and carvacrol is due to the damage to membrane integrity induced by a change in pH hemostasis.

In the cytotoxic assay, chloroform and water extracts exhibited cytotoxic effects on the gastric cancer cell lines (AGS and KATO III). Chloroform extract had a more powerful cytotoxic effect than water extract on gastric cancer cell lines.

Dysregulation of the mitochondrial apoptotic pathway is one of the most important events during carcinogenesis. The Bcl-2 protein family, including anti-apoptotic (Bcl-2 and Bcl-xl) and pro-apoptotic (Bax and Bak) members, plays an essential role in regulating this pathway. According to our results, the Bax/Bcl-2 ratio was significantly (p<0.05) correlated with the gastric cancer cell line.

This is the first report demonstrating the potential pro-apoptotic effects of S. khuzistanica extracts on gastric cancer cell lines (AGS and KATO III). However, further investigations of the extracts in animal models with gastric cancer are needed for additional understanding of their in vivo activity.

Acknowledgement: We thereby appreciate the Khoraman Company for their cooperation in providing the S. khuzistanica plant.

Ethics

Ethics Committee Approval: This study was approved by the Bonab Islamic Azad University (approval number: 1394.625).

Informed Consent: It wasn’t obtained.

Author Contributions

Concept: B.B., Design: L.A., H.S., Data Collection or Processing: B.B., H.S., Analysis or Interpretation: A.T., Literature Search: F.A.R., G.S., S.K., Writing: B.B., H.S.

Conflict of Interest: No conflict of interest is declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.