Tributyltin(IV) Butyrate: A Novel Epigenetic Modifier with ER Stress- and Apoptosis-Inducing Properties in Colon Cancer Cells

Abstract

Organotin(IV) compounds are a class of non-platinum metallo-conjugates exhibiting antitumor activity. The effects of different organotin types has been related to several mechanisms, including their ability to modify acetylation protein status and to promote apoptosis. Here, we focus on triorganotin(IV) complexes of butyric acid, a well-known HDAC inhibitor with antitumor properties. The conjugated compounds were synthesized and characterised by FTIR spectroscopy, multi-nuclear (¹H, ¹³C and ¹¹⁹Sn) NMR, and mass spectrometry (ESI-MS). In the triorganotin(IV) complexes, an anionic monodentate butyrate ligand was observed, which coordinated the tin atom on a tetra-coordinated, monomeric environment similar to ester. FTIR and NMR findings confirm this structure both in solid state and solution. The antitumor efficacy of the triorganotin(IV) butyrates was tested in colon cancer cells and, among them, tributyltin(IV) butyrate (BT2) was selected as the most efficacious. BT2 induced G2/M cell cycle arrest, ER stress, and apoptotic cell death. These effects were obtained using low concentrations of BT2 up to 1 μM, whereas butyric acid alone was completely inefficacious, and the parent compound TBT was poorly effective at the same treatment conditions. To assess whether butyrate in the coordinated form maintains its epigenetic effects, histone acetylation was evaluated and a dramatic decrease in acetyl-H3 and -H4 histones was found. In contrast, butyrate alone stimulated histone acetylation at a higher concentration (5 mM). BT2 was also capable of preventing histone acetylation induced by SAHA, another potent HDAC inhibitor, thus suggesting that it may activate HDACs. These results support a potential use of BT2, a novel epigenetic modulator, in colon cancer treatment.

Keywords: ER stress; HDAC inhibitors; apoptosis; colon cancer; histone acetylation; triorganotin(IV) butyrates.

Figure 1

Proposed structure for R₃Sn(IV) butyrates with the numbering scheme referred to the NMR assignments.

Figure 2

Positive full scan spectra of BT1 (a); BT2 (b); BT3 (c); Compounds and zoom scan spectrum of m/z 416 [BT1+SnMe₃]⁺ (d).

Figure 3

Tributyltin(IV) butyrate (BT2) induces cytotoxic effects in colon cancer cells: (a) MTT assay was used to measure cell viability in two colon cancer cell lines (HCT116 and CaCo-2), as reported in Materials and Methods (Section 3). Cells were incubated for 48 h in the presence of the conjugates (BT2 and BT1) and the corresponding parent compounds (TBT and TMT) at the indicated concentrations. The effects of butyric acid (BTA) were also evaluated. The results reported in the histograms are representative of three separate experiments: (*) p-value < 0.05; (***) p-value < 0.001 compared with untreated cells; N.S. = not significant; (b) Morphological analysis of HCT116 cells treated with 0.5 μM BT2 and TBT for 48 h. The cells were visualized under a light microscope at 200× magnification and the pictures were acquired by IM50 Leica Software (Leika Microsystems, Wetzlar, Germany).

Figure 4

Tributyltin(IV) butyrate (BT2) induces cell cycle arrest accompanied by ER stress induction and DNA fragmentation: (a) The cell cycle phase distribution of HCT116 cells was evaluated by flow cytometry analysis. Cells were incubated for 48 h in the presence of 0.5 μM BT2 or TBT. Then, the DNA content was evaluated after incubating the cells in a hypotonic propidium iodide solution, as described in Section 3. Fluorescence was estimated by FacsDiva Software. (b,c) Western blot analysis of p21 and cyclin B1, two proteins differently expressed during the cell cycle phases (b), and Grp78, PERK, Phospho eIF2α, and CHOP markers of endoplasmic reticulum stress (c). Cells were incubated for 48 h in the presence of 0.5 μM BT2 or TBT. The correct protein loading was ascertained by evaluating γ-tubulin levels. Representative blots of three independent experiments and densitometric analysis are shown. (*) p-value < 0.05; (**) p-value < 0.01; (***) p-value < 0.001 compared with untreated cells. N.S. = not significant.

Figure 5

Tributyltin(IV) butyrate (BT2) activates caspase-dependent apoptotic cell death pathway. (a) Cells were incubated for 48 h in the presence of 0.5 μM BT2 or TBT. At the end of incubation, cells were stained with the vital dye Hoechst 333428 that permits to visualize nuclei. Cells were then visualised under fluorescence microscope Leika equipped with a DAPI filter (magnification of ×400). Micrographs are representative of almost two fields from two independent experiments; (b) Western blot analysis of apoptotic markers, pro-caspase-9, pro-caspase-3 and PARP, in cells treated as in (a). The correct protein loading was ascertained by evaluating γ-tubulin levels. Representative blots of three independent experiments and densitometric analysis are shown; (c) AnnexinV positivity confirmed early apoptosis. Cells were treated with the compounds for 24 h and subjected to Annexin V apoptosis detection kit as reported in Materials and methods. Analysis was performed by flow cytometry using FacsCanto BD. The percentage of annexin V positive cells was evaluated by Flowjo BD software. The results are representative of two independent experiments (*) p-value < 0.05; (**) p-value < 0.01; (***) p-value < 0.001 compared with untreated cells. N.S. not significant.

Figure 6

Tributyltin(IV) butyrate (BT2) remarkably reduces histone acetylation. Western blot analysis of acetylated-H3 and H4 histones after treatment for 48 h with 0.5 μM BT2 and TBT (a) or 0.5 μM and 5 mM butyric acid (BTA) (b) are shown. The ratio between acetylated histones and total histone levels was quantified. Representative blots of three independent experiments and densitometric analysis are shown. (***) p-value < 0.001 compared with untreated cells. N.S. = not significant.

Figure 7

Tributyltin(IV) butyrate (BT2) reduces the effect of the HDAC inhibitor SAHA on histone acetylation. The figure shows Western blot analysis of acetylated-H3 and H4 histones after cell treatment for 48 h with 0.5 μM BT2 (lane 2) or 24 h treatment with 10 μM SAHA used alone (lane 4) or after pre-treatment with BT2 (24 h) followed by other 24 h of incubation with the HDAC inhibitor (lane 3). The ratio between acetylated histones and total histone levels was quantified. Representative blots of three independent experiments and densitometric analysis are shown. (*) p-value < 0.05; (***) p-value < 0.001 compared with untreated cells. N.S. = not significant.