Genotoxic Effects of Tributyltin and Triphenyltin Isothiocyanates, Cognate RXR Ligands: Comparison in Human Breast Carcinoma MCF 7 and MDA-MB-231 Cells

Abstract

The cytotoxicity of two recently synthesized triorganotin isothiocyanate derivatives, nuclear retinoid X receptor ligands, was tested and compared in estrogen-receptor-positive MCF 7 and -negative MDA-MB-231 human breast carcinoma cell lines. A 48 h MTT assay indicated that tributyltin isothiocyanate (TBT-ITC) is more cytotoxic than triphenyltin isothiocyanate (TPT-ITC) in MCF 7 cells, and the same trend was observed in the MDA-MB-231 cell line. A comet assay revealed the presence of both crosslinks and increasing DNA damage levels after the 17 h treatment with both derivatives. Differences in cytotoxicity of TBT-ITC and TPT-ITC detected by FDA staining correspond to the MTT data, communicating more pronounced effects in MCF 7 than in the MDA-MB-231 cell line. Both derivatives were found to cause apoptosis, as shown by the mitochondrial membrane potential (MMP) depolarization and caspase-3/7 activation. The onset of caspase activation correlated with MMP dissipation and the total cytotoxicity more than with the amount of active caspases. In conclusion, our data suggest that the DNA damage induced by TBT-ITC and TPT-ITC treatment could underlie their cytotoxicity in the cell lines studied.

Keywords: DNA crosslinks; apoptosis; breast cancer; cytotoxicity; triorganotin isothiocyanates.

Figure 1

Structures of tributyltin isothiocyanate (TBT-ITC) and triphenyltin isothiocyanate (TPT-ITC).

Figure 2

Cytotoxicity of tributyltin and triphenyltin isothiocyanates in MCF 7 and MDA-MB-231 cells. The concentrations of drugs that inhibited cell survival to 50% (IC50) at 48 h measured by MTT assay and determined by Calcusyn software are expressed as the means ± standard deviation (SD) of three to five independent experiments.

Figure 3

DNA damage (alkaline comet assay) caused by treatment with 500 and 1000 nM TBT-ITC and TPT-ITC in human breast cancer MCF 7 (left) and MDA-MB-231 cells (right) expressed either as a mean percentage of tail DNA (DNA-damaging 300 μM hydrogen peroxide, H₂O₂, was used as a positive control) (a,b); or as a percentage of StO-induced DNA migration (crosslinking 20 μM cisplatin, cisPt, was used as a positive control) (c,d). Results are presented as the means of three independent experiments ± standard deviation (SD). Statistically significant differences * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to the negative (untreated) control (C).

Figure 4

Apoptosis and necrosis induction by triorganotin isothiocyanate derivatives in MCF 7 and MDA-MB-231 cells measured by flow cytometry (FDA/PI staining). The proportion of viable (FDA+/PI-), apoptotic (FDA-/PI-), and necrotic (FDA-/PI+) cells is illustrated in histograms after following treatment: (a) control, (b) Taxol 1 µM (positive control), (c) TBT-ITC 500 nM, (d) TBT-ITC 1 µM, (e) TPT-ITC 500 nM, and (f) TPT-ITC 1 µM. The data presented are representative histograms of three independent experiments.

Figure 5

The mitochondrial membrane potential disruption by triorganotin isothiocyanate derivatives in MCF 7 and MDA-MB-231 cells measured by flow cytometry (JC-1 staining). The percentage of cells with depolarized Δψm (JC-1 monomers) is indicated in the right lower quadrant after following treatment: (a) control, (b) Taxol 1 µM (positive control), (c) TBT-ITC 500 nM, (d) TBT-ITC 1 µM, (e) TPT-ITC 500 nM, and (f) TPT-ITC 1 µM. The data presented are representative dot plots of three independent experiments.

Figure 6

Caspase-3/7 activation in human breast cancer cells. Caspase-3/7-positive objects stained by CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent were measured over 24 h in response to increasing concentrations of TBT-ITC and TPT-ITC derivatives. SSP (1 μM) was used as a positive control.