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Effect of Hydroalcoholic Extract of Ephedra major, Momordica charantia, and Resveratrol on Cytotoxicity and Caspase-3 Genes Expression Level in MCF-7 Breast Cancer Cell Line

Background: To increase the therapeutic effect of drugs to combat diseases, combination therapy with current chemical drugs and new medicines derived from medicinal plants is necessary. Objectives: The present work aimed to investigate the effect of hydroalcoholic extract of two medicinal plants, Ephedra major and Momordica cacharantia (Carla), and resveratrol drug on cell viability and expression levels of caspase-3 gene in MCF-7 cell line. Methods: In this experimental study, the hydroalcoholic extraction of tested plants was done with a Soxhlet extractor. The MTT assay and real-time PCR were used to determine cell toxicity and caspase-3 gene expression levels, respectively. Results: The highest and lowest cytotoxic effects of plant extracts and resveratrol were observed at concentrations of 500 and 150 μg/mL, respectively. The highest level of the caspase-3 gene expression was observed after 72 h of incubation by different concentrations of plant extracts and resveratrol. Conclusions: It can be concluded that both plant extracts could influence cell viability in MCF-7 cells via the increase of cell toxicity and expression of caspase-3 gene. Thus, these species could be used in the pharmaceutical industry.


Background
Breast cancer is one of the most common cancers in women, with a high level of mortality over the past decades (1). Surgical side effects and adverse effects after breast cancer surgery and radiotherapy, such as persistent postsurgical and musculoskeletal pain, are major clinical problems after breast cancer surgery and radiotherapy (2). Therefore, numerous researchers are trying to discover and develop plant-based alternatives, particularly for the rational management of cancer control (3). The potential of dietary natural products to influence health has been investigated by scientific research, and it is believed that these compounds could affect the progression of different diseases in various ways (4).
The efficiency and effectiveness of medicinal plants were determined based on traditional experiences and ethnomedical practices (5,6). Ephedra major species belong to the Ephedraceae family and are known to comprise ap-occurring compound, it has been highly studied for the prevention and treatment of many diseases, including cancer disease. The multiple anticancer effects of resveratrol application against both tumor initiation and cancer progression pathways have been extensively reviewed in numerous in-vitro studies (12,13).

Objectives
In this research, we attempted to investigate the effect of ephedra and Carla plant extracts and resveratrol drug on cell viability and expression levels of Caspas-3 gene in a breast cancer cell line (MCF-7) by the MTT assay and realtime PCR method, respectively.

Plant Materials
Ephedra major species were collected at the coordinates 30°03 24 N -54°35 26 E in May 2018. A dry sample of the plant was kept in the Herbarium of Botanic Laboratory of Yazd University, Yazd, Iran (EFYZ-2018). The green fruits of M. charantia were harvested from the northern regions of Iran and subsequently were cleaned and cut into small pieces and dried at room temperature.

Plant Material and Hydroalcoholic Extraction
Shade-dried plant tissues were crushed into powders using a blender. The powdered plant material (20 g) was subjected to hydroalcoholic extraction (80%) at 37°C for 16 h. The mixture was filtered, and the extraction procedure was repeated twice. A rotator evaporator under reduced pressure at 40°C was used to remove the solvent, and it was protected from light (14).

Cell Culture and Treatment
In this study, 105 MCF-7 cell lines were cultured in a cell culture plate containing RPMI-1640 culture medium (Gibco, USA) with 10% Fetal Bovine Serum (FBS, Life Technologies, South Korea), streptomycin (500 µg/mL), and penicillin. The culture plates were transferred to a humidified incubator (Memert, Germany) at 37°C with an atmosphere of 5% CO 2 and 95% air. Cells were passaged when they covered the plate (70 -80% confluence).

Cell Viability Assay
The MTT assay was performed for the determination of the effects of plant extracts and resveratrol drug (15,16) on cell viability. The development of purple color due to the formation of formazan crystals in the presence of viable cells was visualized by the spectrophotometric method. Absorbance was measured at the 570 nm wavelength using an ELISA plate reader (SpectroStar® Nano, Labtech, Germany). All the measurements were done in triplicate and the data were reported as mean ± SD. Cell viability was calculated using the equation: Cell viability = mean OD of treated of cell mean OD of control cells × 100 The IC50 values (µg/mL) were calculated using the graph of cell viability versus plant extract and resveratrol drug concentration for the MCF-7 cell line (17).

Real-time RT-PCR
Treated MCF-7 cells with various concentrations of plant extracts and resveratrol were incubated for different times (24,48, and 72 h) and subjected to RNA extraction using a Cinnagen RNA purification kit (Sina Clon, Iran). The quality and quantity of the extracted RNA were assayed by using agarose gel electrophoresis and NanoDrop TM 1000 (Thermo Scientific, Whatham, MA, USA), respectively. The cDNA synthesis was done with a two-step RT-PCR kit (Sina Clon Bioscience, Iran). The cDNA samples were subjected to quantitative PCR using Eva Green qPCR Mix, ROX (Pishgam, Iran) in a light cycler apparatus (Rotor-Gene 3000, Corbett Robotics, Australia) with 40 cycles consisting of 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s. Melting curve analysis was performed from 60 to 95°C to assess the specificity of PCR products. The caspase-3 gene primers (forward AAGC-GAATCAATGGACTCTGG and reverse CTGTACCAGACCGAGAT-GTC) (18) and GAPDH primers as the reference gene (forward CAGCCTCAAGATCATCAGCA and reverse TGTGGTCAT-GAGTCCTTCCA) were used in this study.

Statistical Analysis
All in vitro experiments were performed at least in triplicate to confirm reproducibility. The differences between means were determined using Student's t test or one-way ANOVA with Dennett's multiple comparison test. Statistical significance was set at *P < 0.05 or **P < 0.01. The 2-∆∆Ct method was used to calculate the data from relative changes in gene expression specified from RT-PCR. 2 Gene Cell Tissue. 2021; 8(3):e110658.

Ephedra Plant Extract
Cytotoxic properties of the two plant extracts and resveratrol drug against MCF7 cells were assessed using a standard MTT assay. The cell inhibition ability of all plant concentrations is shown in Figure 1. The highest cell death occurred in MCF-7 cells incubated for 72 h, and the lowest effect of plant extract was observed at 24 h treatment. The IC50 measured at 72 h time period was found to be better concerning anti-breast cancer activity. The MCF-7 cell line treated with ephedra extract for 24 and 48 h after incubation in the dose-response curve showed modest growth inhibition ( Figure 1).
As shown in Figure 2, cell viability decreased at all concentrations. The increased incubation time and Carla plant extract concentration caused a reduction in cell viability when compared to other incubation times. The comparison of plant extract effects showed that Carla extract was more effective than ephedra extract with IC50 less than 100 µg/mL after 72 h of incubation. Variable IC50 values were observed for different incubation times, from 100 to 500 µg/mL of Carla extract ( Figure 2).

Resveratrol Drug
The results of cell viability performed by the MTT assay showed that the resveratrol drug had a high efficiency for cell viability at low concentrations when compared to the plant extract ( Figure 3). The highest cell inhibitory effect was observed after 72 h of incubation.

Caspase-3 in Breast Cancer Cell Line Treated with Plant Extracts
The expressions of the caspase-3 gene as the target gene and the GAPDH gene as the reference gene were investigated in three groups of 24, 48, and 72 h incubation times with real-time PCR. Gene expression analysis was done on treated and non-treated MCF-7 cells in three time periods.
Data obtained for non-treated cells were used as the negative control. The expression level of the caspase-3 gene increased in the range of 1.85 -2.55 fold change at a concentration of 300 of ephedra extract. The highest increase in gene expression at this concentration was observed at 72 h time period (Figure 4). At a high concentration (400 -500 µg/mL) of the plant extract, the expression level of the caspase-3 gene gradually decreased compared to lower concentrations.
The level of caspase-3 gene expression was constantly stable (> 2 fold change) in 48 h time period. The highest expression level of the caspase-3 gene occurred in 72 h time period (Figure 4). Unlike the ephedra extract, in the Carla experiment, the highest expression level was calculated at 200 µg/mL of the plant extract. The increase in the plant extract concentration did not lead to an increase in the caspase-3 gene. As shown in Figure 5, a time-dependent relationship was observed for gene expression.
The effect of resveratrol as a drug was investigated on the MCF-7 cell line. The use of resveratrol at various concentrations showed a significant change in the expression level of the caspase-3 gene compared to untreated cells. The highest (2.81 fold change) and lowest (0.09 fold change) caspase-3 gene expressions were observed at doses of 25 µg/mL and 100 µg/mL of resveratrol, respectively. At a low resveratrol concentration, the variability of the expression level was not significant in 24 and 48 h time periods, but 72 h after treatment, the expression level of the caspase-3 gene significantly increased ( Figure 6). The melting curve analysis for the caspase-3 gene used in the real-time gene expression analysis showed the high efficiency of specific primers to produce expected products (data not shown).

Discussion
Medicinal plants are traditionally used to combat diseases because they contain extensive and diverse chemical compositions, some of which have potent activity for cancer treatment. The use of drugs derived either from chemical materials or from natural products is not efficient after cancer development and progression. The use of natural products in the food chain and diet has been recommended in traditional medicine to prevent breast cancer. In this research, based on traditional experiments reviewed in the literature, we selected two plant species to test their efficiency in the MCF-7 cancer cell line.
Ephedra species have been demonstrated to be effective for the treatment of allergies, nasal congestion, and bronchial asthma, but there are a few reports available on the clinical use of ephedra in cancer patients. Carla    some studies on testing the effectiveness of Carla extract in different cancers, but for native Iranian species, there are few studies performed on breast cancer. One-way analysis of variance (ANOVA) used for the IC50 assay indicated that the highest and lowest effects on cell viability (death) occurred 72 h after treatment. The inhibitory effect of the saf-fron extract on cell viability of gastric adenocarcinoma cell line (AGS) showed time and dose-dependent cell toxicity (19), which are in concordance with our results. The limits of crude extracts activity for the 50% inhibition of tumor cells have been defined to be less than 30 mg/mL (20). Anticancer activity of the methanolic extract of micro-algae  showed IC50 values lower than 20 mg/mL (21). Our data showed a low IC50 (200 -300 µg/mL), which was less than the accepted dose (30 mg/mL). This different IC50 level can be due to plant species and plant extraction methods. The high toxicity of ephedra extract could have led to these data. A reduction in cell toxicity with increased concentration and incubation time could be resulted from detoxificaion of plant extract by cell line. The plant extraction method and cancer cell types can affect cell toxicity.
The biological efficiency of plant extract on cell viability depends on multiple factors such as selected plant tissue, solvent application and concentration, the solvent to solid ratio, and extraction time period (22). The  (24), which are approximately in agreement with our results. The chemo-preventive activity of resveratrol as a natural phytoalexin against cancer has been well studied. The effect of resveratrol on cell proliferation is not similar in all breast cancer cell lines (25). The efficiency of resveratrol for 50% growth inhibition of H446 cancer cells was determined at 40 µg/mL resveratrol concentration (26), while in this work, the MCF-7 cell growth inhibition was observed at 75 µg/mL of resveratrol.
The expression levels of the caspase-3 gene have been measured in different cancer cell lines. The treatment of Hone1, AGS, HCT-116, and CL1-0 cell lines with Carla extract led to increased in caspase-3 activity (two-fold changes) (27). An increase in the caspase-3 gene (three-fold changes) was reported in treated cancer stem cells from the human colon with 125 µg/mL of methanolic extract of M. charantia (Carla) when compared to control cells (28), but in this work, the highest expression was recorded at 200 µg/mL of Carla extract after 72 h of treatment, which is in concordance with similar studies.  h with 1.5 mg/mL plant concentrations, showed the timedependent effect of tested plants, which is in agreement with our results (29). In HT29 cancer cells, the activity of Caspase group genes was measured by real-time PCR under treatment of Brucea javanica extract (fruit). The highest expression of these genes was observed at 100 µg/mL concentration after 24 h treatment (30) that is near to that of Carla (200 µg/mL) and resveratrol (75 -100 µg/mL) but is not in agreement with ephedra extract (300 µg/mL). This difference can be due to the plant type (area, tissue, and species) and the method of extraction. Thus, based on these results, the aqueous extract powders of Carla and ephedra as edible vegetables appear to be a safe alternative of treatment for patients with breast cancer.

Conclusion
These results indicate that all the three tested drugs could influence cell viability, but resveratrol was more cytotoxic than the plant extracts. This high effect can be due to drug purity. In this study, we further investigated the characterization of caspase-3 gene expression response in incubated cells during three time periods. The increase in inducers did not show any increase in gene expression, and a moderate concentration was observed to induce gene expression; so, the increase in the drug could have adverse effects.

Footnotes
Authors' Contribution: Seyed Kazem Sabbagh, conception and design of the study and final approval of the version to be published; Mahta Mazaheri, drafting the article; Mohammad Reza Sarafraz, analysis and interpretation of data; Ehsan Ghodrati, Alireza Hajibeiki, and Zahra Shaker, acquisition of data.

Conflict of Interests:
The authors have no conflict of interest to declare.

Ethical Approval: EP/00B/1456262
Funding/Support: The study was supported in part by a grant from the vice chancellorship of Research at the University of Yazd (grant No. 99/P/2728), Iran.