• V-ATPase inhibition increases cancer cell stiffness and blocks membrane related Ras signaling - a new option for HCC therapy.

      Bartel, Karin; Winzi, Maria; Ulrich, Melanie; Koeberle, Andreas; Menche, Dirk; Werz, Oliver; Müller, Rolf; Guck, Jochen; Vollmar, Angelika M; von Schwarzenberg, Karin; et al. (2017-02-07)
      Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide and the third leading cause of cancer-related death. However, therapy options are limited leaving an urgent need to develop new strategies. Currently, targeting cancer cell lipid and cholesterol metabolism is gaining interest especially regarding HCC. High cholesterol levels support proliferation, membrane-related mitogenic signaling and increase cell softness, leading to tumor progression, malignancy and invasive potential. However, effective ways to target cholesterol metabolism for cancer therapy are still missing. The V-ATPase inhibitor archazolid was recently shown to interfere with cholesterol metabolism. In our study, we report a novel therapeutic potential of V-ATPase inhibition in HCC by altering the mechanical phenotype of cancer cells leading to reduced proliferative signaling. Archazolid causes cellular depletion of free cholesterol leading to an increase in cell stiffness and membrane polarity of cancer cells, while hepatocytes remain unaffected. The altered membrane composition decreases membrane fluidity and leads to an inhibition of membrane-related Ras signaling resulting decreased proliferation in vitro and in vivo. V-ATPase inhibition represents a novel link between cell biophysical properties and proliferative signaling selectively in malignant HCC cells, providing the basis for an attractive and innovative strategy against HCC.
    • The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1.

      Wiedmann, Romina M; von Schwarzenberg, Karin; Palamidessi, Andrea; Schreiner, Laura; Kubisch, Rebekka; Liebl, Johanna; Schempp, Christina; Trauner, Dirk; Vereb, Gyorgy; Zahler, Stefan; et al. (2012-11-15)
      The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.
    • The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen.

      Luong, Betty; Schwenk, Rebecca; Bräutigam, Jacqueline; Müller, Rolf; Menche, Dirk; Bischoff, Iris; Fürst, Robert; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      The vacuolar-type H+-ATPase (v-ATPase) is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells (ECs), in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.
    • Vitiosangium cumulatum gen. nov., sp. nov. and Vitiosangium subalbum sp. nov., novel soil myxobacteria from Nepal , and emended descriptions of genus Archangium and Angiococcus, and of Cystobacteraceae family.

      Awal, Ram Prasad; Garcia, Ronald; Gemperlein, Katja; Wink, Joachim; Kunwar, Bikram; Parajuli, Niranjan; Müller, Rolf; Helmholtz-Institue für Pharmazeutische Forschung Saarland (HIPS), Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-01-28)
      Bacterial strains designated MCy10943T and MCy10944T were isolated in 2014 from dried Nepalese soil samples collected in 2013 from Phukot, Kalikot, Western Nepal and Godawari, Lalitpur, Central Nepal. The novel organisms showed typical myxobacterial growth characteristics which include swarming colony and fruiting body formation on solid surfaces, and a predatory ability to lyse microorganisms. The strains were aerobic, mesophilic, chemoheterotrophic and showed resistance to various antibiotics. The major cellular fatty acids common to both organisms were C17:0 2-OH, iso-C15:0, C16:1 and iso-C17:0. The G + C content of the genomic DNA was 72-75 mol %. Phylogenetic analysis showed that the strains belong to the family Cystobacteraceae, suborder Cystobacterineae, order Myxococcales. The 16S rRNA gene sequences of both strains showed 97-98 % similarity to Archangium gephyra DSM 2261T, Cystobacter violaceus DSM 14727T, and 96.7-97 % to Cystobacter fuscus DSM 2262T and Angiococcus disciformis DSM 52716T. Polyphasic taxonomic characterisation suggested that strains MCy10943T and MCy10944T represent two distinct species of a novel genus, for which the names Vitiosangium cumulatum and Vitiosangium subalbum are proposed. The type strain of Vitiosangium cumulatum is MCy10943T (=DSM 102952T =NCCB 100600T) while for Vitiosangium subalbum is MCy10944T (=DSM 102953T =NCCB 100601T). In addition, the genera Archangium and Angiococcus, and the family Cystobacteraceae is herewith emended.
    • Watching DNA replication inhibitors in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies in Mycobacterium .

      Trojanowski, Damian; Kołodziej, Marta; Hołówka, Joanna; Müller, Rolf; Zakrzewska-Czerwińska, Jolanta; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Society of microbiology, 2019-08-05)
      Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria - a causative agents of severe diseases and have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.).