Mitochondria-targeted triphenylphosphonium-based compounds do not affect estrogen receptor α

Background Targeting negatively charged mitochondria is often achieved using triphenylphosphonium (TPP) cations. These cationic vehicles may possess biological activity, and a docking study indicates that TPP-moieties may act as modulators of signaling through the estrogen receptor α (ERα). Moreover, in vivo and in vitro experiments revealed the estrogen-like effects of TPP-based compounds. Here, we tested the hypothesis that TPP-based compounds regulate the activity of ERα. Methods We used ERa-positive and ERα-negative human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231, respectively). Cell proliferation was measured using a resazurin cell growth assay and a real-time cell analyzer assay. Cell cycle progression was analyzed using flow cytometry. Real-time PCR was used to assess mRNA expression of endogenous estrogen-responsive genes. Luciferase activity was measured to evaluate transcription driven by estrogen-responsive promoters in cells transfected with an estrogen response element (ERE)3-luciferase expression vector. Results The TPP-based molecules SkQ1 and C12TPP, as well as the rhodamine-based SkQR1, did not increase the proliferation or alter the cell cycle progression of MCF-7 cells. In contrast, 17β estradiol increased the proliferation of MCF-7 cells and the proportion of cells in the S/G2/M-phases of the cell cycle. TPP-based compounds did not affect the induction of transcription of an ERE-luciferase expression vector in vitro, and SkQ1 did not alter the levels of expression of estrogen-dependent genes encoding GREB1, TFF1, COX6, and IGFBP4. Conclusion TPP-based compounds do not possess properties typical of ERα agonists

Pericentromeric Non-Coding DNA Transcription Is Associated with Niche Impairment in Patients with Ineffective or Partially Effective Multiple Myeloma Treatment

Mesenchymal stromal cells (MSC) ‘educated’ by tumor cells are an essential component of the multiple myeloma (MM) tumor microenvironment (TME) involved in tumor progression. Transcription of tandemly repeated (TR) non-coding DNA is often activated in many tumors and is required for tumor progression and cancer cells genome reorganization. The aim of the work was to study functional properties including the TR DNA transcription profile of MSC from the hematopoietic niche of treated MM patients. Healthy donors (HD) and patients after bortezomib-based treatment (with partial or complete response, PoCR, and non-responders, NR) were enrolled in the study. Their trephine biopsies were examined histologically to evaluate the hematopoietic niche. MSC cultures obtained from the biopsies were used for evaluation of the proliferation rate, osteogenic differentiation, presence of tumor MSC markers, resistance to bortezomib, and pericentromeric TR DNA transcription level. The MSC ‘education’ by multiple myeloma cells was mimicked in co-culture experiments with or without bortezomib. The TR DNA transcription profile was accessed. The histological examination revealed the persistence of the tumor microenvironment (especially of the vasculature) in treated patients. In co-culture experiments, MSC of bortezomib-treated patients were more resistant to bortezomib and protected cancer MM cells of the RPMI8226 cell line more effectively than HD-MSC did. The MSC obtained from PoCR and NR samples differed in their functional properties (proliferation capacity, osteogenic potential, and cancer-associated fibroblasts markers). Transcriptome analysis revealed activation of the TR transcription in cells of non-hematopoietic origin from NR patients’ bone marrow. The pericentromeric TR DNA of HS2/HS3 families was among the most upregulated in stromal MSC but not in cancer cells. The highest level of transcription was observed in NR-MSC. Transcription of HS2/HS3 was not detected in healthy donors MSC unless they were co-cultured with MM cancer cells and acquired cancer-associated phenotype. Treatment with TNFα downregulated HS2/HS3 transcription in MSC and upregulated in MM cells. Our results suggest that the hematopoietic niche retains the cancer-associated phenotype after treatment. Pericentromeric non-coding DNA transcription is associated with the MSC cancer-associated phenotype in patients with ineffective or partially effective multiple myeloma treatment

Efficacy of Mitochondrial Antioxidant Plastoquinonyl-decyl-triphenylphosphonium Bromide (SkQ1) in the Rat Model of Autoimmune Arthritis

Rheumatoid arthritis is one of the most common autoimmune diseases. Many antioxidants have been tested in arthritis, but their efficacy was, at best, marginal. In this study, a novel mitochondria-targeted antioxidant, plastoquinonyl-decyl-triphenylphosphonium bromide (SkQ1), was tested in vivo to prevent and cure experimental autoimmune arthritis. In conventional Wistar rats, SkQ1 completely prevented the development of clinical signs of arthritis if administered with food before induction. Further, SkQ1 significantly reduced the fraction of animals that developed clinical signs of arthritis and severity of pathological lesions if administration began immediately after induction of arthritis or at the onset of first symptoms (day 14 after induction). In specific pathogen-free Wistar rats, SkQ1 administered via gavage after induction of arthritis did not reduce the fraction of animals with arthritis but decreased the severity of lesions upon pathology examination in a dose-dependent manner. Efficacious doses of SkQ1 were in the range of 0.25–1.25 nmol/kg/day (0.13–0.7 μg/kg/day), which is much lower than doses commonly used for conventional antioxidants. SkQ1 promoted apoptosis of neutrophils in vitro, which may be one of the mechanisms underlying its pharmacological activity. Considering its low toxicity and the wide therapeutic window, SkQ1 may be a valuable additional therapy for rheumatoid arthritis

Fibrin Glue Implants Seeded with Dental Pulp and Periodontal Ligament Stem Cells for the Repair of Periodontal Bone Defects: A Preclinical Study

A technology to create a cell-seeded fibrin-based implant matching the size and shape of bone defect is required to create an anatomical implant. The aim of the study was to develop a technology of cell-seeded fibrin gel implant creation that has the same shape and size as the bone defect at the site of implantation. Using computed tomography (CT) images, molds representing bone defects were created by 3D printing. The form was filled with fibrin glue and human dental pulp stem cells (DPSC). The viability, set of surface markers and osteogenic differentiation of DPSC grown in fibrin gel along with the clot retraction time were evaluated. In mice, an alveolar bone defect was created. The defect was filled with fibrin gel seeded with mouse DPSC. After 28 days, the bone repair was analyzed with cone beam CT and by histological examination. The proliferation rate, set of surface antigens and osteogenic potential of cells grown inside the scaffold and in 2D conditions did not differ. In mice, both cell-free and mouse DPSC-seeded implants increased the bone tissue volume and vascularization. In mice with cell-seeded gel implants, the bone remodeling process was more prominent than in animals with a cell-free implant. The technology of 3D-printed forms for molding implants can be used to prepare implants using components that are not suitable for 3D printing

C₁₂TPP enhances protonophorous effect of 2 nM FCCP (panel A) and 10 µM DNP (panel B) in bilayer lipid membrane (BLM).

<p>Diffusion potential was recorded upon the addition of KOH in one compartment to create ΔpH = 1. Incubation mixture, 10 mM Tris, 10 mM MES, 10 mM KCl, pH 7; C₁₂TPP, 0.1 µM. Control, a record without C₁₂TPP and uncouplers. Plus sign of the potential in the compartment of high pH.</p

Comparison of SkQ1 and C₁₂TPP effects on the uncoupling activity of DNP and FCCP.

<p>Effects of 2 µM SkQ1, 2 µM C₁₂TPP or 200 µM TPP on the dependence of mitochondrial membrane potential on the concentration of DNP (panel A) or FCCP (panel B). The experiments were conducted in a way shown in Fig. 6. Shown are Mean±S.E. of 4–6 experiments.</p

CCCP increases the SkQ-induced efflux of carboxyfluorescein from liposomes.

<p>Carboxyfluorescein (CF) efflux from DPhPC (diphytanoylphosphatidylcholine) liposomes (50 µg/ml), induced by 2.5 µM SkQ1 (panel A) or by 0.5 µM SkQR1 (panel B) was measured with or without CCCP. The efflux was accompanied by an increase in CF fluorescence due to dilution and a relief of CF self-quenching. In panel B, the CF efflux was measured 400 s after the addition of SkQR1. Incubation mixture, 10 mM Tris, 10 mM MES, 100 mM KCl, pH 7.</p

SkQ1 (10-(6-plastoquinonyl)decyl triphenylphosphonium) affects absorption spectra of CCCP in the presence of liposomes.

<p>Incubation mixture: 4 µM CCCP, 1 mM Tris, 1 mM MES, pH = 7.4, containing DPhPC (diphytanoylphosphatidylcholine) liposomes (20 µg/ml) in the absence (curve 1) and in the presence of 1 µM, 2 µM, 4 µM and 9 µM SkQ1 (panel A, curves 2–5, respectively) or in the presence of 1 µM, 2 µM, 4 µM and 9 µM SkQR4 (panel B, curves 2–5, respectively). Insert to panel B shows the dependence of λ_max on the concentration of the SkQ derivatives.</p

C₁₂TPP enlarges the FCCP- and DNP-induced increase in respiration rates of intact yeast cells.

<p>The ordinate represents the ratio of the oxygen consumption rates after (V) and before (V₀) the addition of an uncoupler. (A) FCCP-mediated stimulation of yeast respiration in the absence (solid line) and in the presence (dotted line) of 1 µM C₁₂TPP. (B) DNP stimulation of yeast respiration in the absence (solid line) and in the presence of 0.5 µM (dotted line) or 1 µM (dot and dash line) C₁₂TPP. In the absence of the anionic uncoupler, C₁₂TPP did not increase the rate of oxygen consumption at concentrations below 2 µM (insert). Shown are Mean±S.E. of 4 experiments.</p

C₁₂R4 (rhodamine B dodecyl ester) strongly increases CCCP- (panel A) or DNP-mediated (panel B) electric current through bilayer lipid membrane (BLM).

<p>Where indicated, 0.1 µM CCCP or 1 µM DNP were added to both compartments prior to the measurements; incubation mixture, 0.1 M KCl, 10 mM Tris, 10 mM MES, pH 7.0; at t = 1.5 s, 100 mV voltage was applied to the BLM. Concentration of C₁₂R4 was 0.1 µM.</p