title: Inhibition of tumor proliferation associated with cell cycle arrest caused by extract and fraction from Casearia sylvestris (Salicaceae).
abstract: BACKGROUND
  Casearia sylvestris is a tree found in tropical America. In Brazil it is known mainly as Guaçatonga. Literature reports suggest that the leaves and other plant parts have been used by indigenous populations from South America in preparations, mainly aqueous or hydroethanolic macerations or decoctions, most times taken orally for the primary treatment of several diseases, including cancer.
  
  
  OBJECTIVE
  This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo.
  
  
  METHODS
  Aqueous ethanolic maceration and column chromatography were done to obtain a crude aqueous ethanolic extract (CAE) and a chloroform fraction (f-CHCl3). The human breast cancer cell line MCF-7 was used in culture. In vitro, non-cytotoxic concentrations were determined by MTT assay and the antiproliferative effect was assessed by the colony forming unit assay using non-cytotoxic concentrations. Effects on the cell cycle were observed through flow cytometry using a propidium iodide kit. Casearin C was identified in f-CHCl3 by chromatography and H(1) nuclear magnetic resonance. The effect on some key proteins of DNA damage (phosphorylation on the histone H2AX) and cell cycle control (p53, p16, cdk2) was evaluated through immunoblot. Antiproliferative effects in vivo were measured in tumor tissue from Ehrlich ascites-bearing mice through the (3)H-thymidine uptake assay and the trypan blue exclusion method.
  
  
  RESULTS
  In vitro, EC50 values found at 24 h on MCF-7 cells were 141 µg/mL for CAE and 66 µg/mL for f-CHCl3. Inhibition on proliferation was recorded at concentrations as low as 4 µg/mL in the case of the f-CHCl3 (up to 40%) and up to 50% when CAE was added at 9 µg/mL. The cell cycle arrest was demonstrated by the reduction in terms of number of cells in phases G2/M and S, up to 38.9% and 51.9% when cells were treated with CAE, and 53.9% and 66.2%, respectively, when cells were treated with f-CHCl3. The number of cells in G1 was increased when the cells were treated with CAE (21.4%) or f-CHCl3 (27.8%). Key proteins of cell cycle control were affected. The treatments caused activation of p53, p16 and DNA damage found by the appearance of bands corresponding to γ-H2AX. The treatments caused inhibition of cdk2. CAE and particularly f-CHCl3 caused significant inhibition on tumor growth in mice (40% and 60%, respectively). Uptake of (3)H-thymidine, thus proliferation was reduced in tumor cells from mice treated with CAE (>30%) or f-CHCl3 (up to 50%) compared to cells from control animals. Data from the trypan blue assay indicating a lower number of tumor cells in treated animals. From the overall, data from this study are in line with the traditional claims for the antitumor effect of Casearia sylvestris.
  
  
  CONCLUSIONS
  This investigation suggests that whether the extracts from Casearia sylvestris are cytotoxic at high concentrations, lower concentrations have antiproliferative effect and could be useful to complement conventional cytotoxic chemotherapy, and should be evaluated further.
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Karina Bettega
authors: KB
authors: Felipe
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil; Grupo de Desenvolvimento em Tecnologia Farmacêutica (TECFARMA) da Universidade do Sul de Santa Catarina (UNISUL), Santa Catarina, Brazil.
authors: Maicon Roberto
authors: MR
authors: Kviecinski
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Fabiana Ourique
authors: FO
authors: da Silva
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Nádia Falcão
authors: NF
authors: Bücker
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Mirelle Sinfroni
authors: MS
authors: Farias
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Luiza Sheyla Evenni Porfirio Will
authors: LS
authors: Castro
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Valdelúcia Maria Alves
authors: VM
authors: de Souza Grinevicius
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: Nadia Sandrini
authors: NS
authors: Motta
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors: João Francisco Gomes
authors: JF
authors: Correia
authors: Instituto Biológico de São Paulo, Brazil.
authors: Maria Helena
authors: MH
authors: Rossi
authors: Laboratório de Bioquímica Experimental (LABIOEX), Departamento de Bioquímica da Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil. Electronic address: rozangela.pedrosa@ufsc.br.
authors: Rozangela Curi
authors: RC
authors: Pedrosa
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002286:Carcinoma, Ehrlich Tumor / Q000188:drug therapy; D037821:Casearia; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002725:Chloroform / Q000737:chemistry; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004249:DNA Damage; D006657:Histones / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D008297:Male; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000378:metabolism; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology* / Q000627:therapeutic use; D018515:Plant Leaves / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; C468783:H2AX protein, human; D006657:Histones; D009363:Neoplasm Proteins; D010936:Plant Extracts; D012997:Solvents; D016159:Tumor Suppressor Protein p53; D002725:Chloroform; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
keywords: Antiproliferative; Casearia silvestris; Cell cycle arrest