To further clarify whether the phosphorylation of AKT and ERK1/2 are the downstream target of growth factor stimulation, we cultured cells in serum-free medium for 24 hours and induced AKT and ERK phosphorylation by addition of 20% FBS for 30 minutes

To further clarify whether the phosphorylation of AKT and ERK1/2 are the downstream target of growth factor stimulation, we cultured cells in serum-free medium for 24 hours and induced AKT and ERK phosphorylation by addition of 20% FBS for 30 minutes. was extended with the inhibition of serum-induced PI3K/AKT and Ras-ERK activation and epidermal growth factor (EGF)-mediated EGFR activation in MDA-MB-231 cells. These results indicate that extracts of could inhibit signal transduction at least involved in EGFR as well as the PI3K/AKT and Ras-ERK pathways, which are crucial players of tumor cell migration and invasion. Our study strongly supports that the extracts of could be a novel botanical drug lead for the development of an antimetastatic agent for the treatment of human malignant breast cancer. is one of genus in the Zingiberaceae. Previous studies have shown that the extracts or phytocompounds from the genus showed anticancer activity by directly modulating cellular signaling pathways.12-14 is a folk plant endemic to SIS3 Taiwan and used as a food-flavoring and traditional Chinese medicine preparation. Previously, we reported that trans-3-methoxy-5-hydroxystilbene isolated from the rhizome of showed antimetastatic effects on human lung carcinoma cells in vitro15; however, the anticancer activity of various extracts of on other cancer models have not been reported. A recent study has shown that galangin, a flavonol isolated from against ER-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines by determining PI3K/AKT and Ras/MAPK pathways. Material and Methods Extracts Preparation was collected in February 2015 from Nantou County, Taiwan, and was identified by Prof Yen-Hsueh Tseng, Department of Forestry, National Chung Hsing University. The voucher specimen (TCF Tseng4568) was deposited in the herbarium of the same university. Air-dried rhizome, stem, and leaves of were extracted with ethanol at ambient temperature and concentrated under vacuum to yield the different parts of extract, namely, ANR, ANS, and ANL as rhizome extract, stem extract, and leaves extract, respectively. Extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare a final concentrations of 2.5, 5, 10, and 20 mg/mL. Chemicals and Reagents MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), the PI3K inhibitor wortmannin, DMSO, hydrocortisone, bovine serum albumin, heparin, and methylcellulose were obtained from Sigma-Aldrich (St Louis, CA). The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech (Rocky Hill, NJ) and reconstituted by the culture medium. Insulin and B-27 supplement were obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies against AKT, EGFR, SIS3 phos-AKTSer473, phos-EGFRTyr1068, and E-cadherin were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against ERK and phos-ERK1/2Thr202/Thyr204 were purchased from Santa-Cruz Biotechnology (Dallas, TX). Anti-vimentin and anti–actin antibodies were procured from Gene Tex (Irvine, CA) and Sigma-Aldrich, respectively. Cell Culture Human breast cancer cell lines (BCCs), MCF-7 and MDA-MB-231, were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in Dulbeccos modified Eagle medium (DMEM) and Roswell Park Memorial Institute Medium-1640 (RPMI-1640), respectively, and supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Carlsbad, CA). Both cell lines were maintained at 37C in a humidified 5% CO2 incubator. Cytotoxicity Assay The MTT stock solution was prepared at a concentration of 5 mg/mL in phosphate-buffered saline (PBS). Cells at a density DLK of 1 1 104 cells/well were seeded in 96-well cell culture plates overnight and then treated with different concentrations (25, 50, 100, and 200 g/mL) of ANR, ANS, and ANL for 48 hours. After treatment, culture media were replaced with fresh media containing MTT (0.5 mg/mL) and incubated for 4 hours at 37C in a humidified 5% CO2 incubator. To determine cell survival, the formazan crystal produced by mitochondrial metabolism was dissolved in DMSO and the intensity measured with a microplate photometer (Multiskan Ascent, MTX Lab Systems, Inc, Bradenton, FL) at 550 nm. Percentage of cell survival was calculated using the following formula: cell survival (% of control) = ODtest/ODcontrol SIS3 100%. Cell Proliferation Assay MCF-7 and MDA-MB-231 cells were seeded in a 96-well cell culture plate at a density of 1 1 103 cells/well. After 24 hours incubation, cells were treated with increasing concentrations (2.5, 5, 10, and 20 g/mL) of ANR, ANS, or ANL for 1 to 4 days. At the end of the indicated time points, the surviving cells were determined by the MTT assay, and the cell growth curve was plotted by the survival of cells in the period of 1 1 to 4 days. Western Blot Analysis The protein extraction and Western blot assay were performed as previously described.17 Cell Migration Assay To determine the capability of cell migration, we used both wound healing and Boyden chamber Transwell assays. For the wound healing assay, a silicon gap insert (Ibidi GmbH, Martinsried, Germany) was placed in a 12-well culture plate to create a cell-free space of 400 m in width between 2-well chambers. MDA-MB-231 cells (2 106 cells/well) were SIS3 seeded into each chamber of the culture insert. After 24 hours of incubation, the silicon insert was.

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