Vasive), and MDA-MB-231 (TNBC, very metastatic) were cultured in DMEM medium
Vasive), and MDA-MB-231 (TNBC, hugely metastatic) had been cultured in DMEM medium with 10 fetal bovine serum and 1 antibiotics. Rat typical intestinal epithelial cells (RIEs) were also cultured in the similar condition as above. GBL-60 cells (kindly supplied 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 have been also cultured in DMEM, which was authorized by an Institutional Review Board in the Seoul National University Hospital [31]. 2.two. Cell Viability Assay and Flow Cytometry. Cells have been 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 around the ELISA reader (Molecular Devices, Palo Alto, CA, USA). Cells were seeded in 6-well plates and treated with every single extract for 24 hours. Cells were then harvested and stained with propidium iodide (PI, 50 gmL) at space temperature within the dark. PI-positive cells have been detected applying FACSCalibur (BD Biosciences, San Jose, CA, USA). two.three. Cell Migration, Invasion Assay, and Coccidia list anchorage-independent Assay. Cell migration was measured by scratching assays. Cells were seeded in 6-well plates and then scratched. 24 hours after treatment options with herbal extracts, migrated cell numbers have been counted. For invasion assays, cells have been cultured inside the upper chambers precoated with matrigels and treated with every single extract for 24 hours. Right after swapping the upper chamber cautiously, invaded cell numbers in 4 fields randomly chosen had been counted. For anchorage-independent assays, cells had been cultured on soft agar plates and treated with extracts every single second day. At day 15, cells have been stained with 0.five crystal violet to become visualized and colonies were counted with photomicroscope.Mediators of InflammationHerbal compositionAstragalus membranaceus Angelica gigas Trichosanthes Kirilowii MaximowiczAmount employed (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 three.0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.ten 0.00 0.00 2.00 four.Formononetin4.five.6.7.eight.9.(AU)SH003 (min)six.00 eight.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)ten.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. CK1 Storage & Stability Formononetin, decursin, and nodakenin were detected in Am and Ag. 3 components in SH003 have been detected at 3.six min, 6.1 min, and 11.0 min.five -GTTGTGTCTTGCCATGCTAAAG-3 , R: five -AGAATGAGCCTCAGACATCTCC-3 . ELISAs have been performed with human IL-6 ELISA kit (BD Biosciences,San Jose CA, USA) according to the manufacturer’s guidelines. 2.7. In Vivo Research. Animal studies were approved by Kyung Hee University Institutional Animal Care and Use Committee (KHU-IACUC). Six-week-old nude (NuNu) mice had been bought from Oriental Science and injected s.c. with 1 106 MDA-MB-231 cells. When tumor volume reached 50 mm3 , mice have been randomly grouped and extracts were p.o. added daily. Body weights and tumor volumes have been measured 3 times a week. In the end of experiments, mice had been sacrificed and all organs which includes tumors had been fixed with four formaldehyde. Blood was also taken in the heart and subjected for the blood test. Lung metastasis was measured by counting metastatic colony numbers on lungs. Fixed organs have been embedded in paraffin and stainedwith hematoxylin and eosin f.
