• Corpus ID: 13460936

Beet Root Juice Promotes Apoptosis in Oncogenic MDA-MB-231 Cells While Protecting Cardiomyocytes Under Doxorubicin Treatment

@inproceedings{Das2013BeetRJ,
  title={Beet Root Juice Promotes Apoptosis in Oncogenic MDA-MB-231 Cells While Protecting Cardiomyocytes Under Doxorubicin Treatment},
  author={Sayantanee Das and Denise S. Williams and Anindita Das and Rakesh C. Kukreja},
  year={2013}
}

• Sayantanee Das, Denise S. Williams, +1 author R. Kukreja
• Published 2013
• Biology

Doxorubicin (DOX) is a well-established chemotherapeutic drug widely used to treat a variety of cancers, though its clinical use is restricted by irreversible cardiotoxicity. Earlier studies show beetroot juice (BRJ) as a potent chemopreventive agent; however its cardioprotective function is yet to be established. The purpose of this experiment is to determine the protective effect of BRJ against DOX-induced cardiotoxicity. It was hypothesized that higher concentrations of BRJ, applied to cardiomyocytes, will provide better cardioprotection against DOX, and simultaneously increase cell death of MDA-MB-231, breast cancer cells, in combination with DOX. Adult rat cardiomyocytes and MDA-MB-231 cells were exposed to different concentrations (0.5, 5, 50, 250, 500 μg/ml) of BRJ with or without DOX, with a control of growth medium. Cell death, measured by trypan blue staining, was significantly reduced in cardiomyocytes but increased in MDA-MB-231 following 24 hours of cotreatment with BRJ and DOX. Cell viability was also significantly reduced after BRJ and DOX co-treatment in MDA-MB-231 cells. Similarly, DOX-induced-apoptosis (programmed cell death), as determined by TUNEL assay, was significantly reduced with BRJ treatment for 48 hours in cardiomyocytes. In contrast, BRJ significantly increased DOX-mediated apoptosis in cancer cells. In conclusion, lower concentrations of BRJ with DOX represented the most effective combination of cardioprotection and chemoprevention. These findings provide insight on the possible cardioprotective ability of BRJ in cancer patients treated with anthracycline chemotherapeutic drugs. Introduction Doxorubicin (DOX), a quinone-containing anthracycline antibiotic, is a well-known chemotherapeutic drug widely used to treat a variety of human carcinomas1 including breast cancer, ovarian cancer, and solid tumors. The clinical use is restricted by its severe side effects, especially cardiotoxicity, leading to cardiomyocyte death2,3. Despite the side effects, DOX still remains a top choice for chemotherapy for its superior chemotherapeutic effectiveness. A mechanism of cardiotoxicity of DOX is its redox-activation of reactive oxygen species (ROS) that ultimately results in myocyte apoptosis4. ROS are chemically-reactive molecules containing oxygens that take electrons from other molecules and become electrically charged, which damages DNA, RNA, proteins, and Beet Root Juice Promotes Apoptosis in Oncogenic MDA-MB-231 Cells While Protecting Cardiomyocytes Under Doxorubicin Treatment Sayantanee Das1*, Denise S. Williams2, Anindita Das3 and Rakesh C. Kukreja3 Student1, Teacher2: Mills E. Godwin High School, 2101 Pump Road, Henrico, Virginia 23238-3500 Mentor3: VCU Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia 23298 *Corresponding author: [e-mail] INTERNSHIP ARTICLE lipids. Tumor formations are associated with inflammation due to oxidative stress and ROS generation5,6. Natural antioxidants play a critical role in human health by averting vital biological molecules from oxidative damage7. Antioxidants are substances found in fruits, nuts, grains and vegetables that inhibit the oxidation process of other molecules and reduce inflammatory properties. Currently, studies show that compounds with antioxidant activity may protect against DOX-induced cardiotoxicity8,9. Few table foods contain betacyanins and other betalains, important anticancer constituents for their high radical scavenging activity10. Beetroot juice (BRJ) contains colorants that can be divided into two categories of betalains: the red betacyanins and the yellow betaxathines. Betalain is important for cardiovascular health. Betanin, which makes up 95% of the total betacyanins, is more stable in the beetroot extract than in pure chemical form11,12. Red beetroot (Beta vulgaris) is a potent chemopreventive agent which reduces cell proliferation, angiogenesis, inflammation and stimulates apoptosis in skin, liver, lung, and esophageal cancer10,13,14. However, due to its well recognized antioxidant properties, BRJ may possess an alleviating effect on DOXinduced cardiotoxicity. Based on this compelling background information, it was hypothesized that the combination of DOX and BRJ can enhance the chemotherapeutic efficacy of DOX while reducing the cardiotoxic side effects. The use of BRJ will be safer and more cost effective and could save cancer patients and improve their quality of life by reducing harsh side effects of DOX such as diarrhea, constipation, hair loss and vomiting. The purpose of this study is to determine the effect of different concentrations and combinations of BRJ and DOX on adult rat cardiomyocytes and a human breast cancer cell line (MDA-MB-231) to demonstrate the possible cardioprotective role of BRJ against DOX, as well as chemotherapeutic ability. Based on numerous studies discussing the antioxidant potency and chemotherapeutic effects of BRJ, in this study it is hypothesized that BRJ will protect cardiomyocytes against DOX-toxicity; as well as, BRJ will enhance DOX-induced cancer cell death. A null hypothesis states that BRJ will have no significant effect on DOXinduced cardiotoxicity or cancer cell death. Materials and Methods Adult male Wistar rats (300 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Ventricular cardiomyocytes were isolated using an enzymatic technique as Sayantanee Das, Denise S. Williams, Anindita Das and Rakesh C. Kukreja Page 2 of 6 Results The effects of BRJ on DOX-induced cardiotoxicity were studied and the results are displayed in Figures 1-7 and Supplementary Data Tables 1-7. The mean was determined for each level of the independent variable. The variance, standard deviation, and ANOVA test at a 0.001 level of significance were calculated and analyzed. In cardiomyocytes, DOX (2 μM) significantly increased cell death (35.390±2.998%) compared to the control (7.893±1.522%) after 24 hour treatment (n=8, p<0.001; Figure 1, Supplementary Data Table 1). Remarkably, most BRJ treatment with DOX significantly reduced DOX toxicity to cardiomyocytes (n=8, p<0.001), except 500 μg/mL of BRJ+DOX (38.950±3.756%). However, from Figure 1, it can be noted that with increasing concentrations of BRJ alone, cell death increased compared to the control (n=8, p<0.001); though it is lower compared to DOX alone (n=8, p<0.001). Cardiomyocyte apoptosis was significantly increased with DOX (23.190±4.068%) when compared to the control (1.969±0.753%) after 48 hour treatment (n=4, p<0.001; Figure 2, 3; Supplementary Data Table 2). BRJ treatment with DOX significantly reduced cell apoptosis compared to DOX alone (n=4, p<0.001), except for 500 μg/mL of BRJ+DOX (17.22±1.422%; p<0.01). Though the apoptosis of BRJ+DOX treatment was significantly lower than DOX alone, it was significantly higher than the control (n=4, p<0.001). BRJ alone Figure 1. Percentage of rat cardiomyocyte cell death after 24 hour treatment with DOX (2μM) and/or BRJ (0.5, 5, 50, 250, 500 μL). Significance α to all p<0.001, to DOX p<0.001. previously reported15,16. In brief, the mouse was anesthetized with pentobarbital sodium (100 mg/kg intraperitoneally), and the heart was quickly removed from the chest. Within 3 min, the aortic opening was cannulated onto a Langendorff perfusion system, and the heart was retrogradely perfused (37 °C) at a constant pressure of 55 mm Hg for ~5 min with a Ca2+-free bicarbonate-based buffer containing 120 mm NaCl, 5.4 mm KCl, 1.2 mm MgSO4, 1.2 mm NaH2PO4, 5.6 mm glucose, 20 mm NaHCO3, 10 mm 2,3-butanedione monoxime, and 5 mm taurine (Sigma-Aldrich Co., St. Louis, MO) that was continuously gassed with 95% O2 + 5% CO2. The enzymatic digestion was commenced by adding collagenase type II (Worthington, Lakewood, NJ; 0.5 mg/ml each) and protease type XIV (0.02 mg/ml) to the perfusion buffer and continued for ~15 min. 50 μm Ca2+ was then added in to the enzyme solution for perfusing the heart for another 10– 15 min. The digested ventricular tissue was cut into chunks and gently aspirated with a transfer pipette for facilitating the cell dissociation. The cell pellet was resuspended for a three-step Ca2+ restoration procedure (i.e. 125, 250, and 500 μm Ca2+). The freshly isolated cardiomyocytes were plated on 4-chamber slides (for cell death and apoptosis assays) with Medium 199 (Invitrogen Corp., Grand Island, NY) containing 2 mm l-carnitine, 5 mm creatine, 5 mm taurine, 5 mm glucose, 0.1 μm insulin (Sigma-Aldrich Co., St. Louis, MO), 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) (Invitrogen Corp., Grand Island, NY). Cells were placed in a 37oC incubator with 5% carbon dioxide. BRJ extract, bought from a local vitamin store, was dissolved in growth medium to prepare a stock solution of 10 mg/mL. Ten milliliters of 0, 0.5, 5, 50, 250, and 500 μg/mL BRJ were prepared by adding 0, 0.5, 5, 50, 250, and 500 μL BRJ (10 mg/ mL) in 10 mL medium. DOX (molecular weight 579.98, Sigma-Aldrich Co., St. Louis, MO) was dissolved in water to prepare 1 mg/mL solution. Ten milliliters of 2 μM DOX was prepared by adding 11.6 μL of DOX (1 mg/mL) to 10 mL of medium. An hour after plating, the cells were treated with 2 μM DOX with/without the different concentrations of BRJ. After 24 hours, the cell death was measured by adding 15 μL Trypan blue solution (0.4% solution, Sigma-Aldrich Co., St. Louis, MO). The dead cells, stained blue, were counted under a microscope. The percentage of cell death was determined and recorded. For apoptosis, cells were fixed with 4% paraformaldehyde solution after 48 hours of the treatments and stained using a Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) kit (BD Bioscience, San Jose, CA). The apoptotic cells (fluorescent green) and total cells were counted under a fluorescence micros