Inhibition of tumor proliferation associated with cell cycle arrest caused by extract and fraction from Casearia sylvestris (Salicaceae)

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. This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo. 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 H1 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 3H-thymidine uptake assay and the trypan blue exclusion method. In vitro, EC50 values found at 24h on MCF-7 cells were 141µg/mL for CAE and 66µg/mL for f-CHCl3. Inhibition on proliferation was recorded at concentrations as low as 4µg/mL in the case of the f-CHCl3 (up to 40%) and up to 50% when CAE was added at 9µg/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 3H-thymidine, thus proliferation was reduced in tumor cells from mice treated with CAE (>30%) or f-CHCl3 (up to 50%) com