Stilbenes are a class of natural compounds with a wide variety of biological effects, such as antitumor activity. The best known stilbene is resveratrol, whose clinical application is limited due to its low bioavailability. Thus, methoxylated derivatives of this stilbene have been synthesized, including cis-trimethoxystilbene (cis-TMS) which has demonstrated not only greater bioavailability than resveratrol, but also more pronounced cytotoxic and antiproliferative effects. In preliminary experiments we obtained the range of cytotoxic concentrations of cis-TMS in breast tumor cells and thus began to investigate the role of this stilbene on death and cell cycle control. Thus, the objective of this study was to evaluate the effect of cis-TMS on the induction of DNA damage, cell cycle kinetics, as well as the effects on mitotic cell progression in MCF-7 (tumor cell line) and its normal counterpart (MCF-10A). DNA damage analysis was performed by the alkaline version of the comet assay, cell cycle kinetics was determined by the analysis of DNA content using propidium iodide by flow cytometry. To determine mitotic progression, the cells were fixed (Carnoy fixative) and stained with Giemsa for mitotic and interphase nucleus counts. In all experiments vehicle and positive controls were dimethyl-sulphoxyde 0.1% (DMSO) and doxorubicin 0.5 µM (DOX), respectively. The results obtained by the comet assay showed that cis-TMS treatment significantly increased the extent of DNA damage in both cell lines at concentrations of 1.25 and 2.5 µM. Cell cycle kinetics were evaluated by flow cytometry and demonstrated G2 cycle phase accumulation of cells for both cell lines (37.2% in the MCF-7 and 30.94% in the MCF-10A), at 2.5 μM. Likewise, the mitotic delay assay, which sought to analyze the percentage of cells in mitosis or interphase, showed that in the MCF-7 cell line, treatment with 2.5 µM of cis-TMS resulted in only 15% mitotic cells whereas in the negative control that frequency was 73.9% of cells in mitosis. Similar effects were observed in the MCF-10A cell line, where only 7.9% of mitotic cells were found after treatment with 2.5 µM of cis-TMS against 69.5% in the negative control. According to the results presented here, it can be concluded that cis-TMS has a genotoxic effect that results in the activation of mechanisms of cell cycle arrest, probably through the activation of the DNA damage response pathway. This effect is not selective between MCF-7 and MCF-10A. The next step of this work will be to analyze the expression of genes related to the DNA damage response pathway, including those of apoptosis and cell cycle control.

Financial Support: FAPESP 2014/12465-0; CAPES.