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Radiotherapy, Curcumin, Ginsenoside Rg3, What&,rsquo s Known Ginsenoside 20(S)-Rg3 and curcumin have been shown to inhibit tumor growth and induce apoptosis in cancer cells. What&,rsquo s New For the first time, the effect of co-treatment with ginsenoside 20(S)-Rg3 and curcumin together with radiotherapy was evaluated. Co-treatment with these herbal drugs and radiotherapy had a synergistic and complementary effect on breast cancer cell apoptosis. IntroductionBreast cancer is the second most common cancer in women and becoming more prevalent among those younger than 40 years old. 1, The standard treatment modalities are surgery, chemotherapy, radiotherapy, and endocrine therapy, out of which, radiotherapy is one of the main treatments. 2, However, in many cases, it becomes less effective due to cellular resistance against radiation. 3, An adverse effect of irradiation is that, in addition to tumor cells, normal healthy cells are also destroyed. For many years, researchers have studied synthetic and herbal drugs to increase the radiosensitivity of cancer cells while protecting normal healthy cells. 4, The main challenge in cancer treatment is the use of therapeutic agents with the least toxic potential, e.g. herbal plants. 5, One such plant is ginseng, as it contains ginsenoside in its root, known for its significant therapeutic effects in cancer therapy. 5, , 6, It has been reported that ginsenoside 20(S)-Rg3 has anti-tumor activity in gastric carcinoma, melanoma, leukemia, breast, liver, ovarian, and colon cancer. 7, Ginsenoside Rg3 has been shown to induce apoptosis in cancer cells by activating the caspase-3 pathway as well as inhibiting cellular metabolism and tumor growth. 8, - 10, Sin and colleagues showed that ginsenoside Rg3 is an active tumor suppressor. 11, Based on the proteomic analysis, Lee and colleagues reported that ginsenoside 20(S)-Rg3 and curcumin have anti-metastatic properties. 12, Curcumin is a natural component of the rhizome of Curcuma longa and one of the most powerful chemopreventive and anticancer agents. 13, A review study reported that curcumin contains biological and pharmacological properties and exhibits anti-oxidant, anti-inflammatory, immunomodulatory, anti-microbial, anti-ischemic, anti-carcinogenic, hepatoprotective, nephroprotective, hypoglycemic, and anti-rheumatic activities. 14, Considering the above, it is important to look for suitable natural compounds that can be used as sensitizers in combination with ionizing radiation. Hence, the present study assessed the anti-cancer effect of co-treatment with ginsenoside 20(S)-Rg3 and curcumin on MDA-MB-231 breast cancer cell line with and without radiation therapy.Materials and MethodsThe present experimental study was conducted at Tehran University of Medical Sciences (Tehran, Iran) in 2019. The study was approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1396.2151). Preparation of Ginsenoside 20(S)-Rg3 and CurcuminGinsenoside 20(S)-Rg3 and curcumin were purchased from Sigma-Aldrich (UK). Solutions of ginsenoside 20(S)-Rg3 and curcumin were prepared by dissolving the compounds in powder form in dimethyl sulfoxide (DMSO). The solutions were diluted using a cell culture medium (RPMI-1640 medium). Cell CultureThe MDA-MB-231 cell line was obtained from the Pasture Institute (Tehran, Iran) and cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The cell line was then incubated in a humidified CO2 incubator at 37 &,deg C. 15, All reagents were purchased from Merck (Germany).IrradiationIrradiation was performed at a dose of 4 Gy using a 6MV X-ray beam from a medical linear accelerator with 25&,times 25 cm2 field size and 100 cm source-cell distance at 180 degrees gantry angle.Cell Viability Assay3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to determine the effect of the treatment on cell viability. MDA-MB-231 cell lines were cultured in 96-well plates. When the cells became adherent after 24 hours, the cell culture medium in the experimental group was replaced with 100 &,mu L medium containing different concentrations of ginsenoside 20(S)-Rg3 (0, 10, 80, 150 &,micro mol/L) and curcumin (0, 10, 30, 50, 90 &,micro g/mL). Cells in the control group were only treated with the same medium. One hour after the treatment, the cells were radiated and then incubated at 37 &,deg C. After 24, 48, and 72 hours of exposure to ginsenoside 20(S)-Rg3, curcumin, and radiation. The MTT solution (0.5 mg/ml final concentration) was added and incubated for an additional four hours at 37 &,deg C. The liquid was removed, and 100 &,mu L of the MTT solvent was added, and then it was shaken for 10 min. Finally, spectrophotometric absorbance was measured at 570 nm using ELISA reader, and the optical density (OD) for each well was determined.Apoptosis Assay by Flow CytometryCell lines were seeded in 6-well plates, added with 2 mL medium/well, and cultured overnight. Afterwards, they were treated with 30 &,micro g/mL of curcumin and 80 &,micro mol/L ginsenoside 20(S)-Rg3 for one hour and exposed to 4 Gy X-rays. After 48 hours, flow cytometry was performed with Annexin V-FITC/PI kit (BioLegend, USD), according to the manufacturer&,rsquo s instructions. The treated cells were rinsed twice with PBS and trypsinized for three min to obtain a single-cell suspension. Trypsinization was stopped by adding the medium. Cells were rinsed twice in pre-cooled PBS and then resuspended in Annexin V Binding Buffer at a concentration of 1&,times 106 cells/mL. Initially, 100 &,micro L of cell suspension was transferred in a 5 mL test tube, and then 5 &,micro L of FITC Annexin V and 10 &,micro L of PI solution were added. Cells were vortexed and incubated for 15 min at 25 &,deg C in the dark. Next, 400 &,micro L of Annexin V binding buffer was added to each tube. Finally, the samples were analyzed with BD FACSCaliburTM (Biosciences, San Jose, CA, USA) using flow cytometry. The experiment was repeated three times. 16, Statistical AnalysisData were analyzed using IBM SPSS software version 19.0 with the ANOVA and Kruskal-Wallis test. P values&,lt 0.05 were considered statistically significant. Graphpad prism 8.3.1 Mac (GraphPad Software Inc., USA) was used to plot the charts.ResultsThe inhibitory effect of ginsenoside 20(S)-Rg3 and curcumin combined with radiation on cell viability of MDA-MB-231 cells was examined using MTT assay. The results showed that the growth of MDA-MB-231 cells decreased in all groups, however, the decrease in the treated groups was relatively higher (figures 1A,, 1B,, 1C,). The combination of ginsenoside 20(S)-Rg3 and curcumin at concentrations of 80 &,micro mol/L and 30 &,micro g/mL, respectively, resulted in increased cell death (figure 1D,). In comparison with the control group, the effect was significant (P&,lt 0.05) except for the concentration of 10 &,micro g/mL curcumin without radiation at 24 hours and 48 hours and the concentration of 10 &,micro mol/lLginsenoside 20(S)-Rg3 without radiation at 24 hours (table 1,).Figure 1. The results of MTT assay showed that various concentrations of ginsenoside 20(S)-Rg3 and curcumin inhibited cell viability of MDA-MB-231 cells in a concentration-dependent manner, with and without 4 Gy radiation, at 24, 48, 72 hour. Effect of ginsenoside 20(S)-Rg3 and curcumin plus radiation on cell death(A), after 24 hour, (B), 48 hour, (C), 72 hour, and (D), combined effect Statistically significant differences were determined using Kruskal-Wallis test. The data were significant with respect to the control group (P&,lt 0.05) except for 10 &,micro g/mL curcumin (P=0.520) and 10 &,micro mol/L ginsenoside 20(S)-Rg3 (P=0.513) concentrations. W, Non-irradiated samples, R, Irradiated samplesTreatment durationConcentration of ginsenoside Rg3 (&,micro mol/L)P valueTreatment durationConcentration of ginsenoside Rg3 (&,micro mol/L)P valueWith radiationWithout radiation24 hour100.02124 hour100.513800.020800.0301500.0211500.04048 hour100.03448 hour100.038800.021800.0181500.0211500.01872 hour100.02072 hour100.021800.020800.0211500.0101500.020Treatment durationConcentration of curcumin (&,micro g/mL)P valueTreatment durationConcentration of curcumin (&,micro g/mL)P valueWith radiationWithout radiation24 hour100.02124 hour100.520300.020300.040500.020500.030900.020900.03048 hour100.02048 hour100.237300.021300.017500.020500.018900.030900.01772 hour100.01972 hour100.020300.019300.030500.021500.030900.010900.010All the comparisons were made with the control group Kruskal-Wallis test was done The significance level was 0.05 |