Aroylhydrazone Diorganotin Complexes Causes DNA Damage and Apoptotic Cell Death: From Chemical Synthesis to Biochemical Effects

Abstract

Under microwave irradiation, eighteen new aroylhydrazone diorganotin complexes (1a-9b) were produced through the reaction of aroylhydrazine, 2-ketobutyric acid, and the corresponding diorganotin. Fourier transform infrared spectroscopy, ¹H, ¹³C, and ¹¹⁹Sn nuclear magnetic resonance spectroscopies, high-resolution mass spectroscopy, X-ray crystallography, and thermogravimetric analysis (TGA) were performed to characterize the complexes. The in vitro anticancer activity for complexes were assessed using a CCK-8 assay on human cancer cells of HepG2, NCI-H460, and MCF-7. Complex 4b revealed more intensive anticancer activity against MCF-7 cells than the other complexes and cisplatin. Flow cytometry analysis and transmission electron microscope observation demonstrated that complex 4b mediated cell apoptosis of MCF-7 cells and arrested cell cycle in S phase. Western blotting analysis showed that 4b induced DNA damage in MCF-7 cells and led to apoptosis by the ATM-CHK2-p53 pathway. The single cell gel electrophoreses assay results showed that 4b induced DNA damage. The DNA binding activity of 4b was studied by UV-Visible absorption spectrometry, fluorescence competitive, viscosity measurements, gel electrophoresis, and molecular docking, and the results show that 4b can be well embedded in the groove and cleave DNA.

Keywords: DNA; apoptosis; crystal structure; diorganotin; synthesis.

Scheme 1

The reactions of 1a–9b.

Figure 1

Molecular structure of 1a–9b.

Figure 2

1D or 2D structures. (a) One-dimensional chain structure of complex 1a, (two phenyl groups on the tin atom have been omitted for clarity). (b) Two-dimensional network structure of complex 3b, (two butyl groups on the tin atom have been omitted for clarity).

Figure 3

Effects of complex 4b on cell cycle and apoptosis of MCF-7 cells. (a) Apoptotic effect of 4b on MCF-7 cell line after treatment for 24 h. Q1, Q2, Q3, and Q4 respectively represent live cells, the earlier apoptotic cells, the late apoptotic cells, and cells damaged during the procedure. (b) Flow cytometric analysis indicated that 4b changed MCF-7 cell cycle distribution at 24 h. (c) The 4b increased apoptotic cell numbers. (d) 4b arrested cell cycle S phase of MCF-7 cells. Error bar show the SD, “*” p < 0.05, “**” p < 0.01, compared with the control cells at 24 h.

Figure 4

Cell morphology of the MCF-7 cell line with 4b after treatment for 24 h. (a,b) Control group, (c,d) treated with 0.4 μM of the 4b for 24 h.

Figure 5

Western blot analysis of complex 4b in MCF-7 cells. Cells were cultured in the presence of different concentrations of 4b for 24 h. Whole-cell lysate was analyzed for Bcl-2, Bax, cleaved caspase-3, Cytochrome c, ATM, p-ATM, Chk2, p-Chk2, and p53 by immunoblotting. A representative anti-β-actin immune blot was shown as the loading control. Error bars show the SD, “**” p < 0.01, compared with the control.

Figure 6

Comet assay of EB-stained MCF-7 cells in the control (a) and treated by 0.4 μM of the complex 4b (b) after 24 h incubation.

Figure 7

The binding of the complex 4b and DNA, (a) UV–Vis absorption spectra of 4b (50 μM) in the absence and presence of CT-DNA (0–80 μM), inset: plot of c_DNA/(ε_A − ε_F) vs. c_DNA, (b) effects of 4b (0–80 μM) on the fluorescent spectra of EB-DNA (30 μM) system, λ_ex = 258 nm, inset: plot of I₀/I vs. c_complex, (c) effect of increasing amounts of the 4b, Bu₂SnO, and ligand, (d) agarose gel electrophoresis of pBR322 treated with different concentrations of 4b (0–80 μM).

Figure 8

Molecular modeling of the interaction between 4b and DNA.