an Annexin V staining 
assay and a caspase 3/7 activity assay to detect apoptosis events. Our result show in Results High-throughput Screen development and assay performance measurements For optimization of assay performance, the number of cells/well and the length of the incubation period, and the volume of Alamar-Blue reagent were empirically determined. To identify compounds that might have inhibitory effect against HER2 positive cell proliferation, we used a three-step screen experimental approach to assess the activity of 10,000 natural compound library. First step, a one dose HTS was performed using three HER2-positive breast cancer cell liens including BT474, MDA-MB-453, and HCC1569. Compounds that exhibited precipitation were Rutoside excluded from the following screen. We have identified 45 compounds that inhibited cell proliferation by more than 50% at 10 mmol/L after a 72-hour incubation. Second step, a one dose screen was performed using three HER2-negative breast cancer cell lines including MCF-7, SUM190, and MDA-MB-231 using hit compounds from first step. The assay 22576162 steps were summarized in Antitumor activity of hit compounds in vivo To assess the antitumor effect of peonidin-3-glucoside and cyaniding-3-glucoside in a xenograft model of HER2-positive breast cancer, MDA-MB-453 cells were used in female nude mice. After 25 days of peonidin-3-glucoside and cyaniding-3glucoside treatment, animal weight stayed stable during the treatments in each group. Animals treated with drugs did not exhibit signs of organ damage. Tumor growth rates were significantly different from control group. Accordingly, final tumor volumes and weights were suppressed in the peonidin-3-glucoside and cyaniding-3-glucoside groups compared to the control group. This suppression was accompanied by decreased phospo-HER2 levels and decreased proliferation marker Ki67 levels. Anthocyanins Inhibit HER2+ Breast Cancer Cells Step 1 2 3 4 5 6 Event Add reagent Add library compounds or DMSO Incubate Add reagent Incubate Read Parameter 90 mL 10 mL 72 hrs 20 ml 4 hrs Fluorescence Description 1,000 cells/well 10 mmol/L 5% CO2/37uC incubation Alamar-blue reagents 5% CO2/37uC incubation 530 nm Ex/590 nm 22451932 Em, gain = 60. Notes Instrument: Zs-2 plate reader Plates lidded until read. doi:10.1371/journal.pone.0081586.t002 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 HER2negative cell lines. In step 3, we generated the IC50 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. They have been reported to have anti-cancer properties. 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. Catalpol is an irido
