Iotics. Rat normal intestinal epithelial cells (RIEs) were also cultured in the same condition as above. GBL-60 cells (kindly provided by Dr. Sun Ha Paek at Seoul National University Hospital, Seoul, Republic of Korea) isolated from the brain of a patient who suffered from brain-metastasized breast cancer were also cultured in DMEM, which was approved by an Institutional Review Board at the Seoul National University Hospital [31]. 2.2. Cell Viability Assay and Flow Cytometry. Cells were seeded on 96-well plates and treated with different herbal extracts for 24 hours to 72 hours. Cell viability was measured by MTT assays. Absorbance was read at 570 nm on the ELISA reader (Molecular Devices, Palo Alto, CA, USA). Cells were seeded in 6-well plates and treated with each extract for 24 hours. Cells were then harvested and stained with propidium iodide (PI, 50 g/mL) at room temperature in the dark. PI-positive cells were detected using FACSCalibur (BD Biosciences, San Jose, CA, USA). 2.3. Cell Migration, Invasion Assay, and Anchorage-Independent Assay. Cell migration was measured by scratching assays. Cells were seeded in 6-well plates and then scratched. 24 hours after treatments with herbal extracts, migrated cell numbers were counted. For invasion assays, cells were cultured in the upper chambers precoated with matrigels and treated with each extract for 24 hours.Cabergoline After swapping the upper chamber carefully, invaded cell numbers in four fields randomly chosen were counted.Niraparib For anchorage-independent assays, cells were cultured on soft agar plates and treated with extracts every second day.PMID:23376608 At day 15, cells were stained with 0.5 crystal violet to be visualized and colonies were counted with photomicroscope.Mediators of InflammationHerbal compositionAstragalus membranaceus Angelica gigas Trichosanthes Kirilowii MaximowiczAmount used (g) 333 333 333(a)Total amounts0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00 1.00 2.00 3.0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00 2.00 4.Formononetin4.5.6.7.8.9.(AU)SH003 (min)6.00 8.00 10.00 SH003 (min)Decursin(AU)12.14.0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.(AU)10.Nodakenin 20.(b)30.SH003 (min)40.50.Figure 1: HPLC profile of SH003. (a) Composition of SH003. (b) HPLC identification of components in SH003. Formononetin, decursin, and nodakenin were detected in Am and Ag. Three components in SH003 were detected at 3.6 min, 6.1 min, and 11.0 min.5 -GTTGTGTCTTGCCATGCTAAAG-3 , R: 5 -AGAATGAGCCTCAGACATCTCC-3 . ELISAs were performed with human IL-6 ELISA kit (BD Biosciences,San Jose CA, USA) according to the manufacturer’s instructions. 2.7. In Vivo Studies. Animal studies were approved by Kyung Hee University Institutional Animal Care and Use Committee (KHU-IACUC). Six-week-old nude (Nu/Nu) mice were purchased from Oriental Science and injected s.c. with 1 106 MDA-MB-231 cells. When tumor volume reached 50 mm3 , mice were randomly grouped and extracts were p.o. added daily. Body weights and tumor volumes were measured three times a week. At the end of experiments, mice were sacrificed and all organs including tumors were fixed with 4 formaldehyde. Blood was also taken from the heart and subjected to the blood test. Lung metastasis was measured by counting metastatic colony numbers on lungs. Fixed organs were embedded in paraffin and stainedwith hematoxylin and eosin for histological observations. Immunohistochemistry was performed with anti-CD31 antibody (Abcam, Cambridge, UK). 2.8.
