The Warburg effect describes the ability of cancer cells to produce energy via aerobic glycolysis instead of oxidative phosphorylation of pyruvate. This deviation in mitochondrial metabolism inhibits apoptosis, allowing increased proliferation under conditions of reduced oxygen levels. Dichloroacetate (DCA) was successfully used in several human cancer cell lines to reactivate oxidative phosphorylation in mitochondria. The aim of this study was the characterization and response of canine cancer cell lines after DCA exposure. The effect of 10 mM DCA was characterized in vitro on a set of six canine prostate adenocarcinoma and transitional cell carcinoma (TCC) derived cell lines. Cell counts, lactate levels, apoptosis, expression of apoptotic proteins, survival factors and different miRNAs were analyzed. Additionally, metabolic activity, mitochondrial activity and proliferation were investigated. DCA significantly decreased cell number of all but one utilized cell lines and leads to a significant reduction of lactate release. Decreased survivin levels were found in all cell lines, two of which presented a significant reduction in metabolic activity. Increased miR-375 levels were measured in all TCC cell lines. Reactivation of pyruvate dehydrogenase and an elevated mitochondrial activity appear to induce the transition from aerobic glycolysis back to oxidative phosphorylation. Further, these results display that DCA treatment has a suppressant effect on proliferation of canine cancer cells.

The mutation N188T in human connexin46 (hCx46) correlates with a congenital nuclear pulverulent cataract. This mutation is in the second extracellular loop, a domain involved in docking of gap junction hemichannels. To analyze the functional consequences of this mutation, we expressed hCx46N188T and the wild type (hCx46wt) in Xenopus oocytes and HeLa cells. In Xenopus oocytes, hemichannels formed by hCx46wt and hCx46N188T had similar electrical properties. Additionally, a Ca(2+) and La(3+) sensitive current was observed in HeLa cells expressing eGFP-labeled hCx46wt or eGFP-labeled hCx46N188T. These results suggest that the N188T mutation did not alter apparent expression and the membrane targeting of the protein. Cells expressing hCx46wt-eGFP formed gap junction plaques, but plaques formed by hCx46N188T were extremely rare. A reduced plaque formation was also found in cells cotransfected with hCx46N188T-eGFP and mCherry-labeled hCx46wt as well as in cocultured cells expressing hCx46N188T-eGFP and hCx46wt-mCherry. Dye transfer experiments in cells expressing hCx46N188T revealed a lower transfer rate than cells expressing hCx46wt. We postulate that the N188T mutation affects intercellular connexon docking. This hypothesis is supported by molecular modeling of hCx46 using the crystal structure of hCx26 as a template. The model indicated that N188 is important for hemichannel docking through formation of hydrogen bonds with the residues R180, T189 and D191 of the opposing hCx46. The results suggest that the N188T mutation hinders the docking of the connexons to form gap junction channels. Moreover, the finding that a glutamine substitution (hCx46N188Q) could not rescue the docking emphasizes the specific role of N188.

Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.

Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.

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.