Panaxadiol (PD) is a purified sapogenin of ginseng saponins, which exhibits

Panaxadiol (PD) is a purified sapogenin of ginseng saponins, which exhibits anticancer activity. two different sites of the annexin V protein. Data from this study suggested that apoptosis might play an important role in the EGCG enhanced antiproliferative effects of PD on human colorectal cancer cells. Keywords: Panaxadiol, Epigallocatechin gallate, Antiproliferation, Cell cycle, Apoptosis, Docking analysis, Human colorectal cancer INTRODUCTION Colorectal cancer is the third most common malignancy in the United States and the second most frequent cause of cancer-related death (HawkLevin, 2005; Jemal et al., 2010). Although curative surgical resection is possible in 70C80% of patients after diagnosis, almost half of them will die from recurrence of cancer (Yao et al., 2008). However, natural products have been a valuable source of new candidate compounds for adjuvant therapy (Cragg et al., 2009; Gullett et al., 2010). We recently observed that a ginsenoside aglycon protopanaxadiol (PPD) showed antiproliferative effects on human colorectal cancer cells (Du et al., 2011). Panaxadiol (PD), a pseudoaglycone of diol-type triterpenoid with a dammarane skeleton (Fig. 1), is an active anticancer compound in steamed American ginseng (Qi et al., 2010). We also showed that steamed American ginseng extract increased reactive oxygen species (ROS) levels and induced mitochondrial damage in colorectal cancer cells (Li et al., 2010; Wang et al., 2009). We hypothesize that antioxidants may decrease the level of ROS and enhance PD-induced apoptosis in colorectal cancer cells, and this hypothesis was proven in part by our recent observation that the tumoricidal effect of PD was enhanced by a natural phenol compound epicatechin (Rodriguez et al., 2010). Fig. 1 Chemical structures of panaxadiol (PD) and epigallocatechin gallate (EGCG). Epigallocatechin gallate (EGCG), a well-known Posaconazole antioxidant, is believed to be the major biologically active compound in green Posaconazole tea (Kawai et al., 2005; Kim et al., 2009; Nakazato et al., 2005). A highly popular beverage globally, green tea contains a number of polyphenol compounds referred to as catechins, which have been demonstrated to possess strong ROS scavenging activity. Of these, EGCG showed the most potent antioxidant potential (LambertElias, 2010). In this study, we investigated the possible synergistic antiproliferative effects of PD and EGCG in two human colorectal cancer cell lines, HCT-116 and SW-480. The effects of these compounds on cell cycle and apoptosis were also evaluated, and the docking model of these compounds to annexin V, a protein related to apoptosis, was simulated. MATERIALS AND METHODS Reagents and materials All cell culture plasticware were obtained from Falcon Labware (Franklin Lakes, NJ) and Techno Plastic Products (Trasadingen, Switzerland). Trypsin, McCoys 5A and Leibovitzs L-15 media, and phosphate buffered saline were obtained from Mediatech, Inc. (Herndon, VA). Penicillin G/streptomycin and EGCG was obtained from Sigma-Aldrich (St. Louis, MO). An MTS assay kit, CellTiter 96 Aqueous One Solution Cell Proliferation Assay, was obtained from Promega (Madison, WI). An annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (Rockville, MD). PI/RNase Rabbit Polyclonal to TCF2 staining buffer was obtained from BD Biosciences Pharmingen (San Diego, CA). PD was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Cell culture The human colorectal cancer cell lines HCT-116 and SW-480 were obtained from American Type Culture Collection (ATCC). Cells were routinely grown in McCoys 5A medium (for HCT-116) or Leibovitzs L-15 medium (for SW-480) supplemented with 10% fetal bovine serum Posaconazole (FBS) and penicillin/streptomycin (50 units/ml). Cells were maintained in a tissue culture flask and kept in a humidified incubator (5% CO2 in air at 37C). The medium was changed every 2C3 days. When the cells reached 70%C80% confluence, they were trypsinized, harvested, and seeded into a new tissue culture flask. Cell proliferation assay The effect of PD and EGCG on the proliferation of HCT-116 and SW-480 cell lines was determined using a modified trichrome stain (MTS) assay. Cancer cells were plated into a 96-well plate at a density of 1104 cells/well. After seeding for 24 h, the cells were treated with PD, EGCG or both at various concentrations. All experiments were performed in triplicate. At the end of the.

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