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

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo. MATERIAL AND 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.

Substituted 3­acyl­2­phenylamino­1,4­naphthoquinones intercalate into DNA and cause genotoxicity through the increased generation of reactive oxygen species culminating in cell death.

Naphthoquinones interact with biological systems by generating reactive oxygen species (ROS) that can damage cancer cells. The cytotoxicity and the antitumor activity of 3­acyl­2­phenylamino­1,4­naphthoquinones (DPB1­DPB9) were evaluated in the MCF7 human breast cancer cell line and in male Ehrlich tumor­bearing Balb/c mice. DPB4 was the most cytotoxic derivative against MCF7 cells (EC50 15 µM) and DPB6 was the least cytotoxic one (EC50 56 µM). The 1,4­naphthoquinone derivatives were able to cause DNA damage and promote DNA fragmentation as shown by the plasmid DNA cleavage assay (FII form). In addition, 1,4­naphthoquinone derivatives possibly interacted with DNA as intercalating agents, which was demonstrated by the changes caused in the fluorescence of the DNA­ethidium bromide complexes. Cell death of MCF7 cells induced by 3­acyl­2­phenylamino­1,4­naphthoquinones was mostly due to apoptosis. The DNA fragmentation and subsequent apoptosis may be correlated to the redox potential of the 1,4­naphthoquinone derivatives that, once present in the cell nucleus, led to the increased generation of ROS. Finally, certain 1,4­naphthoquinone derivatives and particularly DPB4 significantly inhibited the growth of Ehrlich ascites tumors in mice (73%).

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis.

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na(3)VO(4)) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na(3)VO(4) was cytotoxic against T24 cells (EC(50)=5.8 µM at 24 h), but in the presence of ascorbate (100 µM) the EC(50) fell to 3.3 µM. Na(3)VO(4) plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na(3)VO(4) did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na(3)VO(4) and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na(3)VO(4) alone, or combined with ascorbate, increased catalase activity, but only Na(3)VO(4) plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na(3)VO(4) plus ascorbate (2-3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na(3)VO(4). The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na(3)VO(4) in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.