Dichloroacetate affects proliferation but not apoptosis in canine mammary cell lines

Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines. © 2017 Harting et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Inducing myointimal hyperplasia versus atherosclerosis in mice: an introduction of two valid models.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models - both resulting in artery stenosis but each underlying a different pathway of development - are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models – both resulting in artery stenosis but each underlying a different pathway of development – are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked

The potassium channel KCa3.1 as new therapeutic target for the prevention of obliterative airway disease.

BackgroundThe calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD.MethodsTracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28.ResultsSubepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes.ConclusionsOur findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development

Effect of 10 mM DCA on PDH phosphorylation at three different residues after 48 hours treatment.

<p>Data was assessed with Luminex Magnetic Bead technology. PDH-P at Ser²³² decreased significantly in all cell lines. Residue Ser²⁹³ showed no significant response to DCA treatment except in cell line DT14/06T. The third phosphorylation site Ser³⁰⁰ has decreased values in carcinoma cell lines but not in non-neoplastic mammary gland derived cell line MTH53A and benign cell line ZMTH3. Data are shown as mean ± SD, n≥3 and are presented as relative PDH-P values in comparison to untreated control (%). Control was set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p

Effect of 10 mM DCA on PDK-1 expression after 48 hours.

<p>Western Blot analysis of PDK-1 expression in MTH53A, MTH52C, ZMTH3 and DT14/06T. All cell lines showed positivity for PDK-1 and GAPDH but no changes in PDK-1 expression was detectable between untreated and DCA exposed cells in any of the evaluated cell lines. GAPDH was used as loading control.</p

Influence of 10 mM DCA on mammary cell lines after 48 hours.

<p>Statistical significant reduction of cell number was observed in all cell lines. Significant difference in cell number between MTH53A and mammary carcinoma DT14/06T was observed. Data is shown as mean ± standard deviation (SD), n≥3 and are presented as relative cell numbers in comparison to the respective corresponding untreated control (%). Control values were set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p

Influence of DCA on cellular ATP production in mammary cell lines after 48 hours.

<p>In comparison to untreated control significant enhancement of ATP-production was observed in all cell lines. No difference was detectable between non-neoplastic mammary gland derived cell line MTH53A and neoplastic tissue derived cell lines. Data are shown as mean ± SD, n≥3 and are presented as relative ATP-production (mitochondrial respiration) in comparison to untreated control (%). Control was set to 100%. Statistical analysis was performed with two-tailed t-test, *p<0.05, **p<0.01, ***p<0.001.</p