DNA cleavage mechanism by metal complexes of Cu(II), Zn(II) and VO(IV) with a schiff-base ligand

Metal ions and metal complexes are important components of nucleic acid biochemistry, participating both in regulation of gene expression and as therapeutic agents. Three new transition metal complexes of copper(II), zinc(II) and oxidovanadium(IV) with a ligand derived from o-vanillin and thiophene were previously synthesized and their antitumor properties were studied in our laboratory. To elucidate some molecular mechanisms tending to explain the cytotoxic effects observed over tumor cells, we investigated the interaction of these complexes with DNA by gel electrophoresis, UV–Vis spectroscopy, docking studies and molecular dynamics simulations. Our spectroscopy and computational results have shown that all of them were able to bind to DNA, Cu(II) complex is located in the minor groove while Zn(II) and oxidovanadium(IV) complexes act as major groove binding molecules. Interestingly, only the Cu(II) complex caused double-strand DNA nicks, consistent with its higher cytotoxic activities previously observed in tumor cell lines. We propose that the DNA-complex interaction destabilize the molecule either disrupting the phosphodiester bonds or impairing DNA replication, giving those complexes strong antitumor potential.Fil: Rodríguez, María R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Lavecchia, Martín José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Parajón Costa, Beatriz Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Gonzalez Baro, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Gonzalez Baro, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Cattaneo, Elizabeth Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

<i>GPAT2</i> silencing disrupts membrane integrity.

<p>Cells were grown in a 48-well microplate and LDH activity was determined in the cellular supernatants and in total cell lysates using a commercial colorimetric method. Results are expressed as the percentage of LDH activity in the supernatants for each cell line. Results are the means ± SD of 3 independent experiments. (* p<0.05)</p

mRNA expression of acyltransferases involved in arachidonic acid metabolism.

<p>Total RNA from scr-MDA and sh-MDA cells was extracted, subjected to cDNA synthesis, and amplified by qRT-PCR with primers for human <i>GPAT2</i>, <i>AGPAT11</i>, <i>MBOAT5</i> and <i>MBOAT7</i> genes and normalizing expression levels to that of <i>TATA-Box binding protein (TBP)</i> housekeeping gene (*** p<0.001. ANOVA-Tukey test).</p

Cytoskeleton related genes upregulated or downregulated in sh-MDA cells.

<p>Cytoskeleton related genes upregulated or downregulated in sh-MDA cells.</p

Cells sub-expressing GPAT2 exhibit smoother topography.

<p><b>A)</b> Ra and Rq values of scr-MDA and sh-MDA cells. <b>B)</b> 3D AFM topography images of scr-MDA and sh-MDA cells at 10.0 μm x 10.0 μm showing cell surface details.</p

<i>GPAT2</i> knock-down in MDA-MB-231 cells.

<p>MDA-MB-231 cells were silenced for human <i>GPAT2</i> with HuSH-29 plasmid coding for a shRNA against human <i>GPAT2</i> mRNA (sh-MDA). A non-effective scrambled sequence shRNA plasmid was used to create a negative control (scr-MDA). <b>A)</b> Protein expression was measured by Western blot. Total cell lysate (100 μg protein) from scr-MDA and sh-MDA cells was probed with anti-GPAT2 antibody and with anti-β-actin antibody as a loading control. <b>B)</b> Protein band intensities were quantified with the ImageJ program. Results are representative of three independent experiments (** p< 0.01). <b>C)</b> Fluorescence image showing GFP expression in scr-MDA and sh-MDA at 200x magnification confirm that cells were transfected.</p

Optical microscopy and AFM images of scr-MDA and sh-MDA cells.

<p>AFM images were obtained in tapping topography (A-D for scr-MDA and G-J for sh-MDA) and amplitude modes (E for scr-MDA and K for sh-MDA). Fig 4A presents topographical parameters of a complete scr-MDA cell. Fig 4E presents amplitude image. Fig 4G presents topographical parameters of complete sh-MDA cell, and Fig 4K presents amplitude image. Parameters of the pictures are 50.0 μm x 50.0 μm (Fig 4A, E, G and K); 30.0 μm x 30.0 μm (Fig 4B and H); 15.0 μm x 15.0 μm (Fig 4C and I); 10.0 μm x 10.0 μm (Fig 4D and J). All images have a resolution of 512 x 512 pixels. The height of the cell is expressed in color scale (right bar). White arrows in Figs 4I and J indicate pore-like structures. Optical images (Fig 4F and L) have a magnification of 200x.</p

<i>Gpat2</i> was expressed in mouse primary spermatocytes.

<p>A) Adult mouse testis sections were hybridized in situ with a <i>Gpat2</i> specific antisense probe (left panels) and the corresponding sense probe (right panels). Magnification: 100× (first row) and 600× (second row). A strong signal was detected in primary spermatocytes (black arrow). B) GPAT2 protein was detected in adult rat testis by immunofluorescence in the presence (left panel) or absence (right panel) of a specific GPAT2 antibody (green signal). The GPAT2 signal was detected in spermatocytes as well as in spermatides. Nuclei were stained with propidium iodide (red signal). Magnification: 400×. The highest GPAT2 expression was detected in the spermatocytes. Bar: 50 µm.</p

TAG mass and AA content were maximal in testes from 30-d-old rats.

<p>Total lipids were isolated from testes from 19, 30, 40 and 60-d old rats. A) Lipids were separated by TLC and charred with 5% sulfuric acid in methanol, and the TAG spot was quantified by image processing. B) Fatty acid composition of the TAG fraction was determined by gas liquid chromatography at different stages of sexual development.</p

GPAT2 overexpression increased TAG storage in CHO-K1 cells.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (*<i>p</i><0.05).</p