Continuing the study of the physicochemical and biological properties of ruthenium-quinolone adducts, four novel complexes with the general formula [Ru([9]aneS3)(dmso-κS)(quinolonato-κ(2)O,O)](PF6), containing the quinolones levofloxacin (1), nalidixic acid (2), oxolinic acid (3), and cinoxacin (4), were prepared and characterized in solid state as well as in solution. Contrary to their organoruthenium analogues, these complexes are generally relatively stable in aqueous solution as substitution of the dimethylsulfoxide (dmso) ligand is slow and not quantitative, and a minor release of the quinolonato ligand is observed only in the case of 4. The complexes bind to serum proteins displaying relatively high binding constants. DNA binding was studied using UV-vis spectroscopy, cyclic voltammetry, and performing viscosity measurements of CT DNA solutions in the presence of complexes 1-4. These experiments show that the ruthenium complexes interact with DNA via intercalation. Possible electrostatic interactions occur in the case of compound 4, which also shows the most pronounced rate of hydrolysis. Compounds 2 and 4 also exhibit a weak inhibition of cathepsins B and S, which are involved in the progression of a number of diseases, including cancer. Furthermore, complex 2 displayed moderate cytotoxicity when tested on the HeLa cell line.

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models.Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.

Background:The lack of cysteine cathepsin inhibitor, stefin B (cystatin B), results in progressive myoclonus epilepsy, type 1. Results: Stefin B deficiency in macrophages resulted in increased inflammasome activation. Conclusion: Stefin B-deficient mice are significantly more sensitive to e LPS-induced sepsis due to increased caspase-11 expression and mitochondrial damage. Significance: Stefin B has an important role in limiting the inflammatory response during LPS-induced sepsis.

BackgroundIn vivo imaging of orally administered lactic acid bacteria (LAB) and commensal bacteria in mice is shown to provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract. The bacteria can be detected and monitored using bioluminescence or near-infrared fluorescence.ResultsFluorescence imaging of bacteria was established by expressing the infrared fluorescent protein IRFP713 in Lactococcus lactis, Lactobacillus plantarum and Escherichia coli. All three bacterial species were monitored in live mice and no major differences in transit time were observed. Bacteria passed through the stomach and small intestine in 1 h and the majority were secreted from the large intestine after 6–8 h. Intestinal localization of bacteria was confirmed by imaging the isolated intestines and culturing the intestinal content. The use of fluorescence tomography for spatial localization of fluorescent bacteria has been established. The expression of an additional infrared fluorescent protein IRFP682 enabled concomitant detection of two bacterial populations in live mice.ConclusionsThe present work provides a methodological basis for future studies of probiotic and theranostic actions of LAB in mouse disease models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0376-4) contains supplementary material, which is available to authorized users.

Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.

Endoglin (CD105), a transforming growth factor (TGF)-β coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro, indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

L‐leucyl‐leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft‐vs‐host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U‐937 and THP‐1 promonocytes as models for monocytic cells, U‐87‐MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C‐dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C—the most upstream molecule in the pathway—in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.