Biological Evaluation of Triorganotin Derivatives as Potential Anticancer Agents

Abstract

Metal-derived platinum complexes are widely used to treat solid tumors. However, systemic toxicity and tumor resistance to these drugs encourage further research into similarly effective compounds. Among others, organotin compounds have been shown to inhibit cell growth and induce cell death and autophagy. Nevertheless, the impact of the ligand structure and mechanisms involved in the toxicity of organotin compounds have not been clarified. In the present study, the biological activities of commercially available bis(tributyltin) oxide and tributyltin chloride, in comparison to those of specially synthesized tributyltin trifluoroacetate (TBT-OCOCF₃) and of cisplatin, were assessed using cells with different levels of tumorigenicity. The results show that tributyltins were more cytotoxic than cisplatin in all the tested cell lines. NMR revealed that this was not related to the interaction with DNA but to the inhibition of glucose uptake into the cells. Moreover, highly tumorigenic cells were less susceptible than nontumorigenic cells to the nonunique pattern of death induced by TBT-OCOCF₃. Nevertheless, tumorigenic cells became sensitive when cotreated with wortmannin and TBT-OCOCF₃, although no concomitant induction of autophagy by the compound was detected. Thus, TBT-OCOCF₃ might be the prototype of a family of potential anticancer agents.

Keywords: anticancer agents; cell death; cytotoxicity; growth inhibition; organotin derivatives; tin; tumor cells.

Figure 1

¹H-NMR spectra for TBT-O in its dimeric (upper) and monomeric (bottom) forms acquired in phosphate buffer and DMSO, respectively.

Figure 2

Absolute number of viable (a) CAL-27 and (b) MCF-10A cells, evaluated as negative by staining with the trypan blue exclusion test, following 24 h of treatment with the triorganotin compounds or cisplatin at concentrations ranging from 1.25 to 80 μM. * p ≤ 0.03, ** p ≤ 0.002, and *** p < 0.001 versus the control group. Data are expressed as the mean + SD and refer to three experiments performed in triplicate.

Figure 3

(a) ¹H-NMR spectrum region of the T- and G-NH groups (left) and aromatic signals of the 12 bp DNA duplex, the sequence of which is indicated at the top. Signal types were assigned by a combination of chemical shift values and an ¹H-¹H TOCSY experiment. (b) Spectrum obtained after 6 h of incubation with two equivalents of cisplatin. (c) Spectrum obtained after 6 h of incubation with five equivalents of TBT-OCOCF₃.

Figure 4

Glucose uptake assessed by flow cytometry analysis using a deoxy-glucose analog 2-NBDG as a fluorescent indicator in (a) CAL-27 cells and (b) MCF-10A cells treated with the organotin derivatives for 1 h. Data are the means + SD from two experiments in duplicate. * p < 0.03 vs. control; and *** p < 0.001 vs. control.

Figure 5

Fluorescence microscopic analysis of U937 cells treated with TBT-OCOCF₃ (1 µM) alone or with wortmannin (WMN, 0.5 µM) after staining with the fluorescent DNA-binding dye Hoechst. (a) The % of cells showing features of apoptosis after 6 and 18 h of treatment from 6 frames randomly selected from three independent experiments (2 frames each; mean + SD). * p < 0.01; and ** p < 0.001. (b) Representative images of cells subjected to different experimental conditions for 18 h are displayed. In the samples cotreated with WMN plus TBT-OCOCF₃, cells showing typical characteristics of advanced apoptosis with nuclei present as one or more groups of featureless, bright, and spherical beads can be abundantly detected. Objective 63×, scale bar = 10 μm.

Figure 6

Detection of the autophagic flux in U937 cells treated with TBT-OCOCF₃ (1 µM) alone or with wortmannin (WMN, 0.5 µM) after double staining with a fluorescent green dye that selectively labels autophagic vacuoles and with the fluorescent DNA-binding dye DAPI (Autophagy Assay kit, Abcam). As a positive control for autophagy, cells were treated with rapamycin 0.5 µM (Rap) for 18 h. (a) Autophagy fold change with respect to the control cells measured by green fluorescence emission using a fluorescence microplate reader after 6 and 18 h of treatment. The results are from three independent experiments performed in duplicate (mean + SD). * p < 0.01 vs. all groups. (b) Representative images of cells subjected to different experimental conditions after 6 h of treatment. In the samples treated with TBT-OCOCF₃, typical features of apoptosis, especially evident following cotreatment with WMN, can be detected (white arrows). Fluorescent green autophagic vacuoles can be clearly detected only in the cells treated with rapamycin or, occasionally, in the control cells. Objective 40×, scale bar = 10 μm.