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Rhodiola crenulata induces death and inhibits growth of breast cancer cell lines

Yifan Tu1, Louis Roberts2, Kalidas Shetty1,3, Sallie Smith Schneider1,4,5

1. Program in Molecular and Cellular Biology, 2. Dept of Biochemistry, 3.Dept of Food Science, 4. Dept of Veterinary and Animal Sciences, University of Massachusetts at Amherst, Amherst, MA 01003 USA.5.Pioneer Valley Life Sciences Institute, Springfield, MA 01199 USA.

Running Title: Rhodiola inhibits breast cancer cell growth

Keywords: Rhodiola, phenolic, cell growth inhibition, breast cancer

Corresponding Author and Reprint requests:

Sallie Smith Schneider, Pioneer Valley Life Sciences Institute,

3601 Main Street , Springfield, MA 01199

Tel: 413-794-0941

Fax: 413-794-0857

Abstract:

Diverse compounds from many different chemical classes are currently involved in pre-clinical analyses for their ability to act as both chemopreventative and chemotherapeutic agents. One such class is the plant derived compounds called phenolic phytochemicals. One example is from Rhodiola which is a perennial plant that grows in the Tundra and high elevation regions of Siberia and Tibet, respectively. The phenolic secondary metabolites isolated from Rhodiola are recently being analyzed in a pre-clinical setting for their ability to treat lymphosarcomas and superficial bladder cancers. However, the effects of Rhodiola have yet to be examined for its implications in breast cancer prevention or for its chemotherapeutic abilities. Therefore we sought to investigate the effects of Rhodiola crenulata on breast cancer both in vivo and in vitro. Experiments using aggressive human derived MDA-MB-231 and mouse derived V14 breast cancer cell lines demonstrated that phenolic-enriched R.crenulata extract was capable of inhibiting the proliferation, motility, and invasion of these cells. In addition, the extracts induced autophagic-like vesicles in all cell lines, eventually leading to death of the tumor cell lines, but not the immortal or normal human mammary epithelial cells. Finally, an in vivo experiment showed that phenolic-enriched dietary Rhodiola is effective in prolonging the survival time ofpreventing the initiation of tumor and slowing down the tumor growth in mice bearing tumor grafts, thereby further demonstrating its possible potential for treatment of breast cancer progression and metastasis.

Introduction:

According to the National Cancer Institute, breast cancer is second only to lung cancer in cancer-related deaths for women in the United States (American Cancer Society). Despite a gradual decline in deaths due to breast cancer, which is likely attributable to increased screening, there is a rise in the incidence of newly-diagnosed breast cancer. Thus, protective therapies for breast cancer are a new arena, which are continuing to be of increasing importance. Currently, Tamoxifen, Fenretinide and cyclooxygenase inhibitors have been used in clinical trials and have been shown to have some effect in reducing the risk of breast cancer in women. Pre-clinical trials in animals have suggested that there are a number of additional compounds which might protect or treat breast cancer. This list includes several plant-derived compounds, termed phytochemicals. Many of these phytochemicals have been shown to reduce cyclooxygenase and aromatase activity, increase the cellular antioxidant potential, and shift the proliferation to death ratio of malignant cells.

Rhodiola crenulata is a perennial plant which grows in the high tundra regions of Siberia and highlands of Tibet. Rhodiola crenulata that was used in this study is one genus of Rhodiola. The root of the R. crenulata (Rhodiola) has been used for many years in eastern traditional medicine. Rhodiola has been shown to have antidepressant as well as cardioprotective effects and to enhance mental, physical, and even sexual performance. For these reasons, Rhodiola extracts have been classified as a plant-derived “adaptogen”, defined by Russian researchers as a compound able to maintain a physiological norm upon exposure to stress [1]. Previous studies have also shown that Rhodiola has both anti-bacterial and anti-cancer activities [2]. Salikhova and colleagues found that Rhodiola extracts are anti-mutagens due to their ability to enhance the efficiency of intracellular DNA repair mechanisms [3]. In experiments using rats with transplanted solid Ehrlich adenocarcinoma and metastizing Pliss lymphosarcoma (PLS), it was shown that application of another genus of Rhodiola—Rhodiola rosea extract inhibited the growth of both tumor types, decreased metastases to the liver, and extended survival time [1, 4]. In one human study, oral administration of Rhodiola to patients with superficial bladder carcinoma (T1G1-2) reduced the average frequency of relapses [5]. Taken together with the aforememtioned evidence, Rhodiola has potential as an anticancer agent, and may be useful in conjunction with pharmaceutical antitumor agents.

In this research we have investigated how phenolic-enriched extracts of Rhodiola crenulata are were capable of reducing cell proliferation and death. Further we have investigated the survival time of mice bearing pre-established tumor grafts in response to dietary Rhodiola crenulata and whether there is reduced incidence of tumor formation.

Material and Methods:

Rhodiola crenulata:

Rhodiola crenulata root extract was obtained from and quality controlled by Barrington Nutritionals (Harrison, NY). Extracts arrived in powder form, were dissolved in 10% ethanol and filter distilled water. The concentration used in the in vitro experiments ranged from 25 µg/ml to 100 µg/ml, (180-720 µM). The total soluble phenolic content of extracts was an average range of 125 mg/ gram dry weight and it was optimized for 1% Salidroside.

Cell lines:

The V14 cell line is a murine breast cancer cell line that was derived from a p53 heterozygous BALB/c mouse. They are an estrogen and progesterone receptor negative (ER-, PR-), Her2/Neu receptor positive cell line. Cells were cultured in DMEM/F12 media buffered with HEPES and NaHCO3 and with the following components: 2% FBS (Invitrogen, Carlsbad, CA), gentamycin (10 µg/mL; Life Technologies, Grand Island, NY). 76Ntert cells are an immortalized non-malignant human mammary epithelial cell line which were derived from a human reduction mammoplasty that was immortalized with TERT overexpression [6]. 76N TERT cells were cultured in DMEM/F12 buffered with HEPES and NaHCO3 (Invitrogen) and the following components from Invitrogen: 1% FBS, 1X Antibiotic-Antimycotic liquid, and 20 µg/mL Gentamycin. The following components from Sigma (Sigma-Aldrich, St. Louis, MO) were also used: 10 µg/mL L(+)-Ascorbic acid sodium salt, 1 ng/ml Cholera Toxin Vibrio, 12.5 ng/ml Epidermal Growth Factor, 2 nM β-Estradiol, 0.1mM Ethanolamine, 1µg/ml Hydrocortisone, 1 µg/ml Insulin, 100 µM O-Phosphorylethanolamine, 35 µg/ml Pituitary Extract bovine, 2.5 ng/ml Sodium selenite, 10µg/ml Apo-transferrin human, and 10nM 3,3',5-Triiodo-L-thyronine. Cells were maintained in 95% humidified air plus 5% CO2 and subcultured weekly. MDA-MB-231 cells are a highly invasive, p53 mutant, ER and PR negative human breast cancer cell line and were cultured in 10% FBS and 20 µg/ml gentamycin.

Human Mammary Epithelial Cells (HMECs) were isolated from human mammary tissue which was obtained from reduction mammoplasties. Approximately 1 g of tissue was minced and dissociated in DMEM/F12 medium (Invitrogen, Place) supplemented with 2% BSA [7], 5 µg/mL insulin [7], 0.5 µg/mL hydrocortisone [7], 100 µg/mL cholera toxin (Invitrogen, Carlsbad, CA), 300 U/mL collagenase type I (Invitrogen, Carlsbad, CA), 100 U/mL hyaluronidase type I-S [7], 100 U/mL penicillin [7], 100 µg/mL streptomycin [7], and 0.25 µg amphotericin [7], and incubated at 37°C for 18 hours. The digested solution was passed through sterile gauze and divided into equal volumes in two 50 ml tubes. The epithelial pellet was collected by centrifuging at 80 g for 10 minutes. The pellet was washed with cold Hanks Balanced Salt Solution (Invitrogen, Carlsbad, CA) with 2% FBS (HF) and spun at 100 g for 5 minutes. The epithelial pellet was then treated with 2 ml trypsin/EDTA (Invitrogen,Carlsbad, CA) for 5 minutes and 10 ml HF was added to stop the digestion and the cells were spun at 100 g for 5 minutes. The cell pellet was subsequently treated with 2 ml dispase (BD Bioscience, Texarkana, Texas) plus 10 µl DNaseI (Pierce Biotechnology, Rockford, IL) for 5 minutes. Ten milliliter cold HF was added to stop the digestion and the cell suspension was sieved sequentially through a 100 µm and 40 µm cell strainer, respectively. The cells were then centrifuged at 100 g for 5 minutes and resuspended in 1 ml mammary epithelial growth media (MEGM), (Cambrex, Walkersville MD) with the following components from Sigma: 5 µg/ml transferrin, 1 x 10-5 M isoproterenol and 70 µg/ml bovine pituitary extract (BD Bioscience, Texarkana, TX).

Animals, Treatments, and Tissue Procedure:

All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Eight week old virgin BALB/c female mice were housed in plastic cages under a 12-hour dark/light cycle, and were permitted free access to food and water. Each mouse was injected with 1x106 V14 cells in 100µl Hanks’ Balanced Salt Solution (HBSS) (Cambrex Bio Science Inc. Walkersville, MD) subcutaneously in the right hind flank. Rhodiola crenulata was dissolved in the drinking water of two different treatment groups at 20 or 0.5 mg/kg/day. The mice were euthanized when their tumors reached 1cm3 and the tumors were removed for analysis. Half of the tumor was fixed and paraffin embedded for immunohistochemical staining and TUNEL assays and the other half was frozen for protein analysis.

[3H]Thymidine Incorporation Assay:

MDA-MB-231, V14, 76Ntert and HMECs cells were seeded into 24-well plate at a density of 1x104 cells per well (Becton Dickinson Labware. NJ) and incubated at 37°C in 5% CO2 overnight. Cells were then treated in triplicate in 1ml of medium supplemented with doses of Rhodiola crenulata extracts according to the requirement of each experiment. Cells in the control group were treated in triplicate in 1ml of medium supplemented with 10% ethanol which was used to dissolve Rhodiola crenulata. At the same time, cells were labeled with 1µCi/ml of [3H]thymidine (Amersham, Arlington Heights, IL). Cells were harvested by washing with PBS and 10% TCA, mixed with 0.1% SDS and 0.1 N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer (Perkin Elmer, Wellesley, MA). Differences in the quantity of [3H]thymidine incorporated by cells with or without Rhodiola crenulata were determined using a two-sample t-test with a 95% confidence interval.

MTS assay:

The CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of living cells in proliferation assays. Cells were seeded in 96-well plate at a concentration of 1x103cells/100ul/well. 24 hours later the cells were treated with Rhodiola extracts at different concentrations in triplicate. The cells were assayed 48 hours later. Assays were performed by removing the medium containing the Rhodiola extracts, as it affected the colorimetric staining, and replacing it with the media with fresh growth media prior to the addition of 20 µl CellTiter 96® AQueous One Solution, The cells were incubated 1-4 hours and the absorbance was measured at 490 nm using 96-well plate reader. The production of formazan is supposed to be proportional to the number of living cells.

Motility/invasion assays:

Cell motility was assessed using a modified wounding assay [8]. A nearly confluent monolayer of cells was grown overnight in reduced serum medium (1% FBS). The monolayer of cells was pre-treated with Rhodiola crenulata extracts at different concentrations for 30 minutes and then wounded with a pipette tip. Cells in the control group was treated with same amount of 10% Ethanol as Rhodiola crenulata in the media. The percentage of cells that migrated into the cleared, wounded area was determined for each sample by counting under a light microscope after five hours. The number of migrated cells in each group was divided by that of the control group to get a “% control motile cells”.

The Matrigel invasion assay was used to determine the invasive potential of breast cell lines in response to treatment with Rhodiola crenulata. 50 µL of 1 mg/mL Matrigel (BD Biosciences, San Jose, CA) was loaded onto the Transwell (Corning) filter inserts (6.5 mm) with 8.0 µm pore size in 24 well tissue culture plates (as described in [9]), to form a gel layer after one hour at 37ºC. In experimental groups, cCells with Rhodiola crenulata at defined concentrations in medium with 2% fetal bovine serum were added to the upper chamber of Transwells transwells (100 µL at 2x106 cells/mL), and cultured for 18 hours. 600 μl cell culture media with 2% fetal bovine serum was added to the lower chamber of transwells. In the control groups, same amount of 10% Ethanol was added to the media in the upper chamber. All groups of cells were cultured for 18 hours. Matrigel and noninvasive cells were then removed from the upper surface of the Transwells with a cotton swab. The membranes were rinsed with PBS and then fixed in 10% formalin for 45 minutes. Cells were stained for 45 minutes with hematoxylin. The membranes were mounted onto a microscope slide for observation, counting, and photography using an Olympus bright field microscope equipped with a Pixera digital camera. 10 microscope fields were counted for the invaded cells of each membrane. The invaded cell number in each group was divided by the invaded cell numbers in the control group to get a “% control invasion”.

Cell migration and invasion assay were repeated twice. The differences of the cell numbers migrated or invaded between Rhodiola crenulata treated groups and control group were determined using a two-sample t-test with a 95% confidence interval.

Death Assay:

Cells in the experiment groups were treated with 75 μg/ml or 100 μg/ml Rhodiola extracts in the media depending on the cell lines when they reached 70-80% confluence in 100mm dishes. Cells in the control groups were treated with same amount of 10% Ethanol in the media. Floating and adherent cells were lifted off the plates by trypsinization and collected as a pellet by centrifugation. The pellet was resuspended in 5 ml ice-cold phosphate-buffered salt solution. 1 ml of the solution was transferred into a round bottom 12x75 mm plastic culture tube (VWR international, NJ, USA) and incubated with 1µg propidium iodine (1.0 µg/µl, Invitrogen, Oregon, USA) in dark for 15 minutes at room temperature to stain the dead cells. The percentage of dead cells was determined by flow cytometry (BD FACS Calibur, BD Bioscience, CA, USA). Each experiment was repeated three times. The percentage of the dead cells in the experimental groups was compared with the percentage of dead cells in the respective control groups using a two-sample t-test with 95% confidence interval to see whether the induction of death by Rhodiola crenulata was statistically significant.

Results

Rhodiola crenulata extract inhibits the proliferation and mitochondrial activity of breast cancer cells.