Mechanism of cancer evading metronomic chemotherapy and action of Archazolid as an anti-metastatic drug

In the present study the mechanisms leading to acquired chemoresistance, as well as new treatment strategies implying the prevention evading of tumor cells were addressed. Resistance formation is one of the major hurdles in cancer therapy. Metronomic antiangiogenic treatment of xenografted prostate cancer tumors in mice with cyclophosphamide (CPA) results in the appearance of resistant tumors. To investigate the complex molecular changes occurring during resistance formation, a comprehensive gene expression analysis of the resistant tumors in vivo was performed. A multitude of differentially expressed genes, e.g. PAS domain containing protein 1 (PASD1), annexin A3 (ANXA3), neurotensin (NTS) or plasminogen activator tissue (PLAT), were observed, when comparing resistant to in vivo passaged tumor samples. Moreover, tumor cells from in vivo and in vitro conditions showed a significant difference in target gene expression. For clarification of the mechanisms leading to the survival of tumor cells during maintained anti-angiogenic CPA therapy the differentially expressed genes were assigned to functional pathways like: axon guidance, steroid biosynthesis and complement and coagulation cascades. As blood flow might play a crucial role during maintained anti-angiogenic therapy, further analysis was focused on the genes grouped in complement and coagulation cascades. pregulation of anti-coagulatory ANXA3 and PLAT and downregulation of SERPIN A1 and other SERPIN-family members was shown by qPCR analysis. In contrast coagulation factor F3 was upregulated, accompanied by the expression of an altered gene product. Taken together, a potential role of anticoagulation as a resistance mechanism for anti-angiogenic CPA therapy could be described. Furthermore, the role of archazolid, a novel myxobacterial V-ATPase inhibitor in cancer treatment and in particular its action on the secreted cellular proteome was evaluated. As extracellular protein secretion may have an impact on invasive properties of tumor cells, the changes of the secretome profile of highly migratory urinary bladder carcinoma cells upon archazolid treatment were analyzed. An induced secretion of prometastatic lysosomal proteins such as the cathepsin family was observed. Interestingly, intracellular cathepsin B activity however strongly decreases and mature cathepsin B protein diminishes. It could be shown that archazolid inhibits the mannose-6-phosphate receptor mediated trafficking of procathepsin B from the trans-Golgi network to prelysosomal compartments, leading to an impaired cathepsin B maturation process. This results in an unnatural secretion of the inactive proenzyme and a dramatic decrease in intracellular cathepsin B activity. Importantly, also in vivo an archazolid induced reduction of cathepsin B activity was proven and archazolid treatment resulted in a reduced formation of distant metastases in the lungs. In summary these results indicate that archazolid in addition to its known anti-migratory properties might exert an anti-metastatic effect by reducing the activity of pro metastatic proteases like cathepsin B

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background: A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent. Methods: In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis. Results: In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction. Conclusions: Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion

Cell-Based Sensor System Using L6 Cells for Broad Band Continuous Pollutant Monitoring in Aquatic Environments

Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni2+ and Cu2+) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity

Pretubulysin : a new option for the treatment of metastatic cancer

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF(Fbw7) E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer

The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1.

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 V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1

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Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy

Abstract Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin

Chondramide affects activation of the RhoGTPase Rho.

<p>(A) A Rac1 pull down was performed for untreated and Chondramide treated cells (24 h) upon EGF-stimulation (5 min). (B) After same treatment a Rho pull down was conducted. (C) Myosin light chain 2 (MLC2) was analyzed on its activation state via Western blot analysis upon EGF stimulation (5 min). A,B,C: Left panel: one representative Western blot is shown. Right panel: Densitometric analysis of Western blots. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3.</p

Treatment with Chondramide reduces breast cancer cell migration, invasion and adhesion.

<p>(A) Chondramide treated and untreated MDA-MB-231 cells were allowed to migrate in a Boyden chamber for 16 h. (B) Chondramide inhibits invasion of MDA-MB-231 cells through matrigel in Boyden chamber (48 h). A,B: For positive control (PC) lower compartment was filled with medium plus 10% FCS, for negative control (NC) only medium without FCS was added. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (C) Pretreated MDA-MB-231 cells were seeded freshly on indicated surfaces and counted after fixation. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (D) Cells from C were stained for F-actin, nuclei and vinculin. Bar represents 50 µm.</p