Glutamate (Kainate) Receptors

XY, ML, LC, XP, ZJQ and HMA performed the experiments

XY, ML, LC, XP, ZJQ and HMA performed the experiments. RKO cells. -Solanine induced apoptosis of RKO cells, as indicated by morphological changes and positive Annexin-FITC/propidium iodide staining. Additionally, -solanine activated caspase-3, ?8 and ?9 in RKO cells, which contributed to -solanine-induced apoptosis. -Solanine also increased the generation of reactive oxygen species, which contributed to caspase activation and induction of apoptosis. -Solanine inhibited the migration, invasion and adhesion of DMP 696 RKO cells, as well as the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. In addition, -solanine inhibited cell proliferation, activated caspase-3, ?8 and ?9, induced apoptosis, and inhibited the migration and invasion of HCT-116 cells. Furthermore, -solanine inhibited tumor growth and induced apoptosis L. (Longkui) is one of the most commonly used anticancer Chinese herbs in clinical practice, and it has been confirmed to have important anticancer properties (10). In colorectal cancer, L. was found ti inhibit cell proliferation, induce autophagy, enhance the therapeutic effect of chemotherapy, and inhibit cell adhesion, migration and invasion (11,12). -Solanine is one of the main components of L, and it has been shown to exert anticancer effects on various types of cancer cells: It can inhibit proliferation, induce apoptosis and inhibit angiogenesis in breast cancer (13); it also inhibited the proliferation, migration and invasion of pancreatic cancer cells and suppressed tumor growth (14); in addition, -solanine was shown to inhibit the invasion of prostate cancer cells DMP 696 by inhibiting epithelial-to-mesenchymal transition and lowering the expression levels of matrix metalloproteinases (MMPs) (15); furthermore, it promoted apoptosis of hepatocellular carcinoma cells by enhancing the generation of reactive oxygen species (ROS), accompanied by an increase in the expression levels of apoptosis signal-regulating kinase 1 and thioredoxin-binding protein-2 and a decrease in histone deacetylase 1 expression (16). The aim of the present study was to investigate the effects of -solanine on human colorectal cancer cells. Materials and methods Chemicals and reagents RPMI-1640 medium, fetal bovine serum (FBS) and trypsin were purchased from Gibco; Thermo Fisher Scientific, Inc. The Cell Counting Kit-8 (CCK-8) was provided by Dojindo Co., Ltd. The Hoechst 33258 staining kit, caspase activity assay kit, ROS assay kit, Z-VAD-FMK, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and N-acetyl-L-cysteine AGAP1 (NAC), were purchased from Beyotime Institute of Biotechnology. Antibodies against cyclin D1 (CCND1; cat no. DMP 696 BS1741), cyclin-dependent kinase (CDK)4 (cat no. MB0027), MMP-2 (cat no. BS1236), MMP-9 (cat. no. bs1241) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat no. AP0063) were purchased from Bioworld Technology, Inc. The Cell cycle and FITC Annexin V Apoptosis Detection kits were obtained from BD Pharmingen; BD Biosciences. The CytoSelect? 48-Well Cell Adhesion Assay and CytoSelect? 24-Well Cell Invasion Assay kits were manufactured by Cell Biolabs, Inc. -Solanine was purchased from Santa Cruz Biotechnology, Inc. (CAS no. 20562-02-1, cat no. sc-252340, lot no. E1619) and its purity (as assessed by high-performance liquid chromatography) was >95%. The chemical structure of -solanine is shown in Fig. 1. -Solanine (10 mM) was dissolved in dimethyl sulfoxide (DMSO) and the same volume of DMSO was used as control. Open in a separate window Figure 1. Chemical structure of -solanine. Cell culture Human colorectal cancer RKO and HCT-116 cells were obtained from and authenticated with short-tandem repeat analysis by The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium with 10% FBS and 1% streptomycin–penicillin, and maintained at 37C in a humidified atmosphere containing 5% CO2. Cell proliferation assay In this study, 1104 colorectal cancer cells were inoculated into 96-well plates, and different concentrations of -solanine with the same volume of DMSO were added after 24 h; an equal volume of DMSO was used as control. At different time points during treatment, 10 l CCK-8 solution was added to the wells and incubated at 37C for 2 h, and the optical density (OD) was measured by a plate reader (SpectraMax M5; Molecular Devices LLC) at the read mode of absorbance (450 nm). The cell survival rate was calculated as follows: Cell survival rate (%)=(experimental OD value/control OD value) 100%. The IC50 of -solanine was calculated using IBM? SPSS? Statistics 25 (IBM Corp.). Cell cycle detection RKO cells (3105) were inoculated in 6-well plates. At 24 h after inoculation, the cells were treated with 0C25 M -solanine (final concentration) with the same volume of DMSO. After 48 h of treatment, the cells DMP 696 were collected, in with 75% ethanol, washed with phosphate-buffered saline (PBS), stained with 400 l propidium iodide (PI) solution in the dark at room temperature for 15 min, and the cell cycle distribution was detected by flow.