Abstract Overexpressed Human epidermal growth factor receptor 2 (HER2) drives the biology of 20% breast cancer and is a prediction of a poor prognosis for patients. HER2-targeted therapies significantly improve outcomes for HER2-positive patients. Traditional Chinese herbs/medicines have been used to treat breast cancer patients including HER2-positive patients in Asia for decades. Although the traditional medicines demonstrate efficacy in clinics for HER2-positive patients, the mechanism is largely unknown. In this article, we screened a 10,000 natural product library in 6 different cell lines representing breast cancer, and assessed the ability of each drug to cause cytotoxicity through a high-throughput screening approach. We have identified eight natural compounds that selectively inhibit the proliferation of HER2-positive cells. Two of the hit compounds, peonidin-3-glucoside and cyaniding-3-glucoside, are both extracts from black rice. They inhibit the phospho-HER2 and phospho-AKT and were confirmed to induce HER2-psotive breast cancer cells apoptosis both in vitro and in vivo. Peonidin-3-glucoside and cyaniding-3-glucoside treatments significantly reduced the tumor size and volume in vivo compared to the control group. There is no significant difference of antitumorgenic effects between peonidin-3-glucoside and cyaniding-3-glucoside treatments.

Citation: Liu W, Xu J, Wu S, Liu Y, Yu X, Chen J, et al. (2013) Selective Anti-Proliferation of HER2-Positive Breast Cancer Cells by Anthocyanins Identified by High-Throughput Screening. PLoS ONE 8(12): e81586. https://doi.org/10.1371/journal.pone.0081586 Editor: Pranela Rameshwar, Rutgers - New Jersey Medical School, United States of America Received: October 4, 2013; Accepted: October 25, 2013; Published: December 3, 2013 Copyright: © 2013 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by Sichuan Province Health Bureau (110465) and by the National Natural Science Foundation of China (81273074). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Breast cancer is a serious and sometimes life-threatening disease. An estimated 232,340 new cases of invasive breast cancer are expected to be diagnosed among women in the US during 2013 according to American Cancer Society. Breast cancer has also become common to Chinese women in recent years possibly due to the change of environments, growth patterns, diet and aging. Based on the model generated by Linos et al., in 2021, the estimated breast cancer incidence rate would be 85.3 to 87.8 per 100,000 woman in China [1]. Evidently, there is a clear need for the development of new therapeutic agents. HER2 overexpression occurs in ∼20% of patients with breast cancer and is associated with aggressive disease and decreased survival. A number of therapeutic approaches have been developed against HER2 worldwide including tyrosine kinase inhibitors, monoclonal antibodies such as Trastuzumab [2]. The mechanism has been largely studied and since then, the disease-free survival and overall survival of patients have all been improved significantly [2]–[5]. In China, there are reports of successful novel therapeutic approaches using traditional medicine for breast cancer patients [6], 7. Traditional Chinese Herbs/Medicines have developed into a mature system for more than three thousand years. Although thousands of Traditional Chinese Medicines have been proved to be effective clinically, the mechanisms of the drug actions are largely unclear. With the modern technology, researchers successfully purified and identified numerous extracts that have not been well defined before. With the rich prior human experiences, we proposed to screen a natural product library which contains 10,000 extracts against representative breast cancer cells and tried to identify compounds that selectively inhibit HER2-positive breast cancer cells.

Materials and Methods Compound Library The natural product library contains 10,000 natural products with a minimum of 98% purity confirmed by NMR and HPLC (Pharmanic, Chengdu, Sichuan, China). Briefly, compounds were extracted by supercritical CO 2 extraction (SFE-CO 2 ) and the residues after SFE-CO 2 extraction were then refluxed with 80% ethanol and the ethanol extracts were spray-dried to obtain the extracts. Then the extracts samples were compared to the reference chemical standards purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) by Pharmanic. The conditions of the solvent gradient elution were 8-20% (A) in 0–20 min, 20–40% (A) in 25–30 min, 40–70% (A) in 30–45 min, 70–90% (A) in 55–60 min at a flow-rate of 1.0 ml/min. Detection was conducted with different wavelengths of 230, 240, 270, 262, and 420 nm with the reference wavelength of 550 nm at room temperature. Compounds were present at 10 mmol/L in DMSO. Afatinib (BIBW2992) was gifted from the Pharmacology department of Chengdu Medical College with a >98% purity. Cell Culture All cell lines were obtained from the American Type Culture Collection (ATCC) except SUM190 (Table 1). SUM190 cells were gifted from Chengdu Medical College bio-core facility [8]. MCF-7, MDA-MB-453 and MDA-MB-231 cells were maintained in DMEM supplemented with 2 mmol/L L-glutamine, 10% fetal bovine serum and 1% penicillin/streptomycin. BT474 cells were maintained in DMEM: Ham's F12 medium (1∶1 mixture) supplemented with 2 mmol/L L-glutamine, 5 µg/ml insulin, 10% fetal bovine serum and 1% penicillin/streptomycin. HCC1569 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were maintained in a 5% CO 2 atmosphere at 37°C. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Breast cancer cell lines used in HTS and their molecular classifications [38]. Breast cancer cell lines used in HTS and their molecular classifications [4] https://doi.org/10.1371/journal.pone.0081586.t001 High-throughput Screening Natural Compounds for Activity in Breast Cancer Cells To identify compounds that might have inhibitory effect on breast cancer cell proliferation, we used a high-throughput drug screen experimental approach to assess the activity of 10,000 natural compounds. Prior to the screen, the cell viability assay was miniaturized to a 96-well, low-volume, black, flat-bottom polystyrene microplates (Corning, Shanghai, China) format to accelerate assay throughput. Cells were grown to 80% confluence, harvested and aliquoted into 96-well plates at concentrations of 1,000 cells per well in a total volume of 90 µL/well. The outer wells were inoculated with medium to minimize evaporation from the sample wells. Cells were allowed to attach overnight and 10 µl culture medium that containing either vehicle or drug at a 100 µmol/L concentration were added to give a final concentration of 10 µmol/L. Cell proliferation was evaluated using 20 µL Alamar-Blue reagent per well according to manufacturer's instructions after 72-h incubation. The plates were then incubated at 37°C for an additional 4 h and the fluorescent signal was measured. Fluorescence was measured with an excitation at 530 nm and emission at 590 nm on ZS-2 plate reader. After background subtraction, cell viability values were normalized to vehicle controls and expressed as percentage of the mean of the relative vehicle controls. Compounds which showed a percentage of inhibition against cell proliferation more than 50% are defined as “hit compounds” for each cell line. Vehicle only wells served as intra-plate controls on each plate were uniformly distributed throughout the screen. Background plates containing vehicle only served as inter-plate controls were inserted throughout the screen. The controls were used to calculate the background levels/noises of the assay and to evaluate assay response as well as performance. Performance of the assay was assessed using Z′ factors. Quantitative Compounds Screen Assay Cells were grown to 80% confluence, harvested and aliquoted into 96 well plates at concentrations of 1,000 cells per well in a total volume of 90 µL/well. Hit compounds or vehicle (DMSO) were then loaded to each well to give a final doses range from 50 µmol/L to 0.03 µmol/L (serial four-fold dilutions) in 100 µL total volume. Cells were cultured for 72 hours at 37°C in a 5% CO 2 atmosphere. Aliquots of 20 µL Alamar-Blue reagent were added directly to each well, the plates were incubated at 37°C for 3 h and the fluorescent signal was measured with an excitation at 530 nm and emission at 590 nm on ZS-2 plate reader. Data were normalized as percentage inhibition relative to vehicle control. IC 50 values were estimated using a four parameter logistic curve model by SigmaPlot 12.0 (Systat Software, INC). Western Blotting Cells were grown to 70–80% confluence, harvested and aliquoted into 100 mm dishes. Media was removed and replaced by media supplied with different doses of compounds the next day. Dished were incubated for an additional 6 h. Cells were washed with PBS and cell lysates were prepared by scraping cells into RIPA buffer with protease inhibitor and phosphatase inhibitor. The homogenates were centrifuged. The supernatants were transferred to fresh tubes and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.) per manufacturer's instructions. Equal amounts of total protein (25 µg) were subjected to electrophoresis (10% tris-HCl, 1.0 mm gels) and transferred to PVDF membranes. Membranes were blocked in TBS containing 0.05% Tween 20 (TBST) and 5% nonfat milk or 5% BSA for 1 hour at room temperature. Primary antibodies were diluted per manufacturer's instructions and incubated in TBST/5% nonfat milk or TBST/5% BSA for 2 h at room temperature or overnight at 4°C. Secondary antibodies were diluted (1∶10,000) and incubated with PVDF membranes for 1 h at room temperature. Proteins were detected by ECL plus western blotting detection system (GE healthcare). The following antibodies were used: anti-phospho-HER2 (Tyr1248), anti-HER2, anti-phospho-AKT (Thr308 or Ser473), anti-AKT, anti-phospho-p42/44MAPK, anti-p42/44MAPK, and anti-β-actin antibodies (Rui Biological Ltd., Shanghai, China). Annexin V-FITC assay Cells were treated with hit compounds for 48 h at 37°C, pooled and washed with PBS for three times, and re-suspended in binding buffer. Cells were then stained with annexin-V-FITC antibody supplied by the Chengdu Medical College Flow-cytometry core facility. Propidium iodide (PI) (final concentration 1 g/ml) was added immediately prior to analysis. Bivariant analysis of FITC fluorescence and PI fluorescence gave different cell populations, where FITC (−) and PI (−) were designated as viable cells, FITC (+) and PI (−) as apoptotic cells, and FITC (+) and PI (+) as late apoptotic or necrotic cells. Caspase 3/7 activity assay Cells were grown to 70–80% confluence, harvested and aliquoted into 96-well plates. Different doses of compounds were added to the plates the next day. Plates were incubated for an additional 48 h at 37°C. Aliquots of Alamar-Blue reagent were added directly to each well, the plates were incubated at 37°C for 3 h and the fluorescent signal was measured with an excitation at 530 nm and emission at 590 nm on ZS-2 plate reader. Then equal volume of caspase 3/7 activity assay reagent (Promega, China) was added to each well and the luminescence signal was measured on ZS-2 plate reader. Data were normalized as luminescence relative to fluorescence. In vivo efficacy in xenograft models In vivo experiments were carried out under pathogen-free conditions at the animal facility in accordance with the institutional guidelines of the Chengdu Medical College Institutional Animal Care and Use Committee (IACUC). All protocols were reviewed and approved by IACUC and HER2-positive breast cancer cell line MDA-MB-453 cells were resuspended to 2×106 cells/100 µl in PBS and implanted subcutaneously into the flank region of 6–7-week-old female nude mice weighing 18 to 22 gram. When tumors reached 50 to 60 mm3 in volume, animals were randomly assigned to 3 groups, receiving either saline (control) or peonidin-3-glucoside (6 mg/kg) and cyaniding-3-glucoside (6 mg/kg) as oral gavage 7 times a week for a total of 25 days. Tumors were measured every 5 days with a caliper and tumor volume (in mm3) was calculated using the following formula: V = 4/3π[(w+l)/4]3, where V = volume (mm3), w = width (mm), l = length (mm). Once the control tumors reached 1000 mm3, the animals were euthanized due to ethical requirements. After 25 days treatment, all animals were euthanized using overdosed CO 2 , and the tumor tissues were extracted for immunostaining and weighing. All values are expressed as the mean ± SEM. Statistical analysis In vitro data were reported as mean ± SD, each treatment performed in duplicate or triplicate. Data were log-transformed to stabilize variances for proliferation assays. In vivo data were reported as mean ± SEM. Values were analyzed using the Student's t test or with one-way ANOVA when three groups were present. Statistical significance was considered as p≤0.05.

Discussion Identification and characterization of new pharmacological activities from existing Chinese Traditional Medicines represents an effective way to accelerate the translation of discoveries at the bench to clinical applications partially due to the known clinical effects of the drugs. To date, a number of interesting hits have been identified. In the present study, we performed a three-step screen for searching anti-HER2 positive breast cancer agents. In step 1, we screened the entire 10,000 Chinese Traditional Medicine extracts library against three HER2-positive cell lines. In step 2, we screened the hit compounds from step 1 against three HER2-negative cell lines. In step 3, we generated the IC 50 values for the hit compounds which selectively inhibit HER2-positive cell proliferation. Peonidin-3-glucoside and cyaniding-3-glucoside are anthocyanin pigments in colored rice cultivars [10]. They have been reported to have anti-cancer properties [11]-[16]. Anthocyanins have been used as medicine or supplements for many years and it has been reported that depending on the nutrition habits, the daily intake of anthocyanins in humans could be up to 150 mg/day [14]. Catalpol is an iridoid glucoside and exhibits anti-inflammatory and anti-diabetes activities [17]–[19]. Quercetin is a natural flavonoid widely distributed in plants that acts as a neuroprotective, anti-cancer, anti-ROS formation agent [6], [20]–[23]. Methyl protodioscin is one of the main bioactive extracts from the Traditional Chinese Medicine Dioscorea collettii var hypoglauca. Studies show that methyl protodioscin has anti-proliferative effects on cancer cells due to the induction of cell cycle arrest and/or apoptosis [13], [24]–[29]. Leonurine exerted cardioprotective, antioxidant, and anti-inflammation properties [30]–[33]. Digoxin is a purified cardiac glycoside extracted from Digitalis lanata. Digoxin is widely used in the treatment of various heart conditions including atrial fibrillation, atrial flutter and heart failure that cannot be controlled by other medication [34]–[36]. Digoxin is a very potent Na+/K+ ATPase pump inhibitor with a very narrow therapeutic index. Therefore extra cautious is needed when use this drug [35], [37]. Europinidin is a water soluble, bluish red plant dye derived from Plumbago zeylanica. There is no citable reference for this compound regarding its activities. We prioritized to study peonidin-3-glucoside and cyaniding-3-glucoside and showed significant anti-tumor activity in vitro. To further evaluate the efficacy of these two compounds, we conducted in vivo anti-tumor experiments using immune-compromised mice model. It has been reported that depending on the nutrition habits, the daily intake of anthocyanins in humans could be up to 150 mg/day [14]. We conservatively choose the safe human dose as 30 mg/day to convert the dosage to the mouse equivalent does using the recommended conversion of animal doses to human equivalent does based on body surface area by US Food and Drug Administration (2005). We calculated the equivalent doses of human (30 mg/day) is equal to 6 mg/kg/day to mouse assuming the human body weight is 60 kg and the mouse body weight is 0.020 kg. The formula listed below has been used: Animal equivalent dose = (30 mg/60 kg) x 12.3 Both peonidin-3-glucoside and cyaniding-3-glucoside showed significant anti-tumor activity in vivo, however, there is no significant difference between the two treatments.

Conclusions In this study, we screened a 10,000 Chinese Medicine extracts library against three representative HER2-positive breast cancer cell lines for anti-proliferation drugs and identified 45 hits in step 1. In step 2, the hits identified from step 1 were profiled against three representative HER2-negative breast cancer cell lines. We identified eight compounds as selective anti-proliferation agents of HER2-positive breast cancer cells. In step 3, those eight compounds have been validated to generate IC 50 values. Very interestingly, there are two compounds, peonidin-3-glucoside and cyaniding-3-glucoside were extracted from the same source (Black rice). To initiate the validation, we also tested the ability of these drugs to induce the early apoptosis and further confirmed the anti-proliferation abilities. Studies using nude mice bearing MDA-MB-453 cells confirmed the antitumorgenic effects of peonidin-3-glucoside and cyaniding-3-glucoside treatments in vivo. In conclusion, the discovery of HER2 selective anti-proliferation of breast cancer cells by Chinese Traditional Medicines have important implications in the development of these drugs as new anti-breast cancer agents.

Acknowledgments We would like to thank the Chengdu Medical College flow-cytometry core facility and the animal core facility for helping conducting the in vitro and in vivo studies.

Author Contributions Conceived and designed the experiments: WL JX SW YL XY JC XT ZW XZ XL. Performed the experiments: WL JX SW YL XY JC XT ZW XZ. Analyzed the data: WL JX SW YL XY JC XT ZW XZ XL. Contributed reagents/materials/analysis tools: WL JX SW YL XY JC XT ZW XZ XL. Wrote the paper: WL JX XL.