Ruthenium(II) complexes of aroylhydrazones: structural, electrochemical and electrostatic interactions with DNA

<p>Four Ru(II) complexes with tridentate ligands <i>viz</i>. (4-hydroxy-N′-(pyridin-2-yl-ethylene) benzohydrazide [Ru(L¹)(PPh₃)₂(Cl)] (<b>1</b>), N′-(pyridin-2-yl-methylene) nicotinohydrazide [Ru(L²)(PPh₃)₂(Cl)] (<b>2</b>), N′-(1H-imidazol-2-yl-methylene)-4-hydroxybenzohydrazide [Ru(L³)(PPh₃)₂(Cl)] (<b>3</b>), and N′-(1H-imidazol-2-yl-methylene) nicotinohydrazide [Ru(L⁴)(PPh₃)₂(Cl)] (<b>4</b>) have been synthesized and characterized. The methoxy-derivative of L³H (abbreviated as <b>L</b>^<b>3</b><b>H*</b>) exists in <i>E</i> configuration with torsional angle of 179.4° around C₇-N₈-N₉-C₁₀ linkage. Single crystal structures of acetonitrile coordinated ruthenium complexes of <b>1</b> and <b>3</b> having compositins as [Ru(L¹)(PPh₃)₂(CH₃CN)]Cl (<b>1a</b>) and [Ru(L³)(PPh₃)₂(CH₃CN)]Cl (<b>3a</b>) revealed coordination of tridentate ligands with significantly distorted octahedral geometry constructed by imine nitrogen, heterocyclic nitrogen, and enolate amide oxygen, forming a <i>cis</i>-planar ring with <i>trans</i>-placement of two PPh₃ groups and a coordinated acetonitrile. Ligands (L¹H-L⁴H) and their ruthenium complexes (<b>1–4</b>) are characterized by ¹H, ¹³C, ³¹P NMR, and IR spectral analysis. Ru(II) complexes have reversible to quasi-reversible redox behavior having Ru(II)/Ru(III) oxidation potentials in the range of 0.40–0.71 V. The DNA binding constants determined by absorption spectral titrations with Herring Sperm DNA (HS-DNA) reveal that <b>L</b>^<b>4</b><b>H</b> and <b>1</b> interact more strongly than other ligands and Ru(II) complexes. Complexes <b>1–3</b> exhibit DNA cleaving activity possibly due to strong electrostatic interactions while <b>4</b> displays intercalation.</p

Kaplan–Meier estimation of cumulative proportion of no tumor recurrence.

<p><b>A:</b> loss of E-cadherin membranous (E-cad^-) expression, <b>B:</b> Loss of β-catenin membranous (β-catM^-) expression, <b>C:</b> loss of E-cadherin and β-catenin membranous (E-cad^−/β-cat M^-) expression, <b>D:</b> ALCAM cytoplasmic positive and E-cadherin membrane loss (E-cad^−/ALCAM C⁺).</p

Association of alterations in ALCAM, E-cadherin and β-catenin expression with evolution of OSCC, clinical parameters and disease prognosis: Multivariate Analysis.

<p>OR, Odd’s ratio; HR, Hazard’s ratio.</p

Analysis of E-cadherin and β-catenin expression in normal oral tissues, hyperplasia, dysplasia and OSCCs: Correlation with clinicopathological parameters.

a<p>TNM stage. OR, Odd’s ratio.</p

Representative oral tissue sections immunostained for E-cadherin and β-catenin.

<p>Histologically normal tissue sections showing membranous immunoreactivity for E-cadherin (A), and β-catenin (G) proteins. Hyperplastic (B), dysplastic (C), OSCC (D) tissue sections depicting loss of membranous staining for E-cadherin. Hyperplastic (H), dysplastic (I), OSCC (J) tissue sections depicting loss of membranous staining for β-catenin. In negative controls, for E-cadherin (E) and β-catenin (F), the primary antibody was replaced by non-immune IgG of the same isotype to ensure specificity. Breast cancer tissue sections used as positive control showed membrane staining for E-cadherin (K) and β-catenin (L) proteins. Original magnification X 200.</p

Alterations in expression of ALCAM, E-cadherin and β-catenin proteins in different stages of oral cancer development and prognosis.

<p>β-cat NC: β-catenin nuclear/cytoplasmic accumulation, ALCAM M⁺: ALCAM membrane positive, E-cad M⁻: E-cadherin membrane loss.</p

siRNA knockdown of ALCAM and its effect on E-cadherin and β-catenin.

<p><b>A</b>: RT-PCR analysis of ALCAM (490bp), E-cadherin (160bp) and β-catenin (167bp) amplicons in siRNA transfected (15 nM, lane T) and control (lane U) SCC-4 cells at 48 h post transfection. β-actin (421bp) transcript was used as control for normalization. <b>B</b>: Immunoblot analysis of ALCAM, E-cadherin and β-catenin proteins in SCC-4 cells silenced for ALCAM. SCC-4 cells treated with ALCAM siRNA (15 nM) for 48 h and untreated cells were immunolabelled with respective antibodies and developed using ECL. 7-folds decreased levels of ALCAM (105 kDa) were observed in ALCAM-depleted transfectants (lane T) as compared to untreated SCC-4 cells (lane U); 3-folds increased levels of 110kDa E-cadherin protein (lane T) were observed in ALCAM-depleted transfectants as compared to the untreated SCC-4 cells (lane U); 2-folds increased levels of 92kDa β-catenin protein (lane T) were observed in ALCAM-depleted transfectants as compared to the untreated SCC-4 cells (lane U); α-tubulin (54 kDa) was used as control protein for quantitation. <b>C</b>: Expression of ALCAM, E-cadherin, and β-catenin proteins in ALCAM silenced SCC-4 cells. Cells grown on coverslips were treated with ALCAM siRNA, processed for confocal microscopy, immunolabelled with respective antibodies to ALCAM, E-cadherin and β-catenin proteins followed by FITC conjugated secondary antibody (Green fluorescence), nuclei were counterstained with PI (red fluorescence) (Original Magnification X 400).</p

Immunohistochemical analysis of biotinidase in thyroid tissues.

<p>Paraffin embedded sections of benign thyroid nodules and malignant tumors were stained using anti-biotinidase polyclonal antibody as described in the Methods section: a) Benign tissue section showing nuclear and overall biotinidase immunostaining; b) Papillary non-aggressive thyroid cancer section illustrating reduction in nuclear staining and increase of cytoplasmic biotinidase immunostaining in the tumor cells; c) Papillary aggressive thyroid cancer section showing reduced overall (nuclear and cytoplasmic) biotinidase immunostaining; d) Thyroid cancer section used as a negative control, showing no immunoreactivity in cells (a–d, original magnification x 400).</p

Biotinidase expression in thyroid FNA samples.

<p>FNA specimens from benign (Panel a), non-aggressive papillary thyroid cancer (Panel b), and aggressive papillary thyroid cancer (Panel c) cases were immunostained with 1∶100 α-biotinidase K-17 rabbit polyclonal antibody. FNA specimen used as a negative control shows no immunoreactivity in cells (Panel d). Photomicrographs show a pronounced decrease in nuclear biotinidase expression in more aggressive thyroid cancer cases and are presented at 400× original magnification.</p

(a) Positive Predictive Values as function of time [PPV(t)] and (b) Negative Predictive Values as function of time [NPV(t)] for 32 OSCC patients with E-cadherin loss of membranous expression (E-cad⁻), ALCAM cytoplasmic (ALCAM C⁺) expression, ALCAM C⁺/E-cad⁻, and for all 72 OSCC patients with survival data (overall) when predicting time to cancer relapse in OSCCs.

<p>(a) Positive Predictive Values as function of time [PPV(t)] and (b) Negative Predictive Values as function of time [NPV(t)] for 32 OSCC patients with E-cadherin loss of membranous expression (E-cad⁻), ALCAM cytoplasmic (ALCAM C⁺) expression, ALCAM C⁺/E-cad⁻, and for all 72 OSCC patients with survival data (overall) when predicting time to cancer relapse in OSCCs.</p