18 research outputs found
In vitro and in vivo characterization of therapeutic approaches for solid tumors : natural compounds and novel targets
Cancer represents the second leading cause of death worldwide, and its incidence and mortality are growing. In search for new anti-tumor drugs addressing innovative targets, natural products represent powerful tools in (I) compound-centric, phenotypic and (II) target-centric drug discovery approaches. This study addresses both strategies, aiming to identify novel therapeutic approaches for solid tumors. (I) Compounds from various bacterial sources were characterized, elucidating their effects on different hallmarks of cancer in tumor cells and macrophages as key players of the tumor microenvironment. The compound thioholgamide A stood out for its potent activities against various hallmarks of cancer in tumor cells. Anti-proliferative actions were further confirmed in vivo. These anti-tumor effects were accompanied by a modulation of cell metabolism, i.e., the inhibition of oxidative phosphorylation. Furthermore, the metabolic modulation caused repolarization of tumor-promoting human macrophages into a tumor-antagonizing phenotype. (II) The RNA binding protein IMP2 has been suggested to promote tumorigenesis and tumor progression in several tumor entities. This study revealed a correlation between IMP2 overexpression and tumor progression and poor prognosis in pancreatic cancer. IMP2 was further characterized as a promising anti-cancer target in vitro and in vivo in colorectal cancer, and potential inhibitors of IMP2 demonstrated their anti-proliferative activity in vivo.Weltweit ist Krebs die zweithäufigste Todesursache und seine Inzidenz und Sterblichkeit sind steigend. In der Wirkstoffentwicklung stellen Naturstoffe vielversprechende Werkzeuge, für (I) naturstofffokussierte, phänotypische und (II) targetfokussierte Strategien dar. Diese Arbeit adressiert beide Strategien mit dem Ziel der Identifizierung innovativer Targets in der Tumortherapie. (I) Die Effekte verschiedener bakterieller Naturstoffe wurden in Tumorzellen und Makrophagen als entscheidende Akteure der Tumormikroumgebung charakterisiert. Thioholgamide A stach dabei durch seine potenten Effekte gegen verschiedene Krebsmerkmale hervor. Seine antiproliferativen Eigenschaften wurden in vivo bestätigt. Diese Antitumorwirkungen wurden von einer Hemmung der oxidativen Phosphorylierung begleitet. Darüber hinaus bewirkte diese Modulation des Zellmetabolismus eine Repolarisation tumorfördernder Makrophagen in einen tumorantagonisierenden Phänotyp. (II) Mehrere Studien weisen auf eine Rolle des RNA-bindenden Proteins IMP2 in der Tumorentstehung und dessen Progression in unterschiedlichen Tumorentitäten hin. Diese Arbeit konnte eine Korrelation zwischen einer IMP2 Überexpression und einer verstärkten Tumorprogression und schlechten Prognose für Pankreaskarzinompatienten aufzeigen. IMP2 wurde ferner als vielversprechendes Target in vitro und in vivo gegen Kolonkarzinome charakterisiert und potenzielle IMP2 Inhibitoren demonstrierten antiproliferative Aktivitäten in vivo
The Insulin-Like Growth Factor 2 mRNA Binding Protein IMP2/IGF2BP2 is Overexpressed and Correlates with Poor Survival in Pancreatic Cancer
The insulin-like growth factor 2 (IGF2) mRNA binding protein IMP2 (IGF2BP2) is an
oncogenic protein known to be overexpressed in different tumor types. Pancreatic cancer is a very
lethal cancer that requires early diagnosis and new treatment options. The aim of our study was to
investigate the role of IMP2 in the initiation and progression of pancreatic ductal adenocarcinoma
(PDAC). IMP2 was significantly overexpressed in a human precursor (PanIN) lesions suggesting IMP2
as a marker for early stages of PDAC. In a PDAC cohort of matched normal and tumor samples IMP2
showed overexpression in tumor tissues compared with normal pancreatic tissue. Strict correlation
analysis (threshold R
2 > 0.75) revealed 22 genes highly positively and 9 genes highly negatively
correlating with IMP2. Besides genes involved in the inhibition of apoptosis (Bcl-XL), especially factors
involved in ubiquitination were strongly correlated with IMP2 expression: SMURF1 and FBXO45.
Moreover, protein kinase C (PKC) signaling pathway was distinctly affected: DXS1179E encoding
PKC iota, PKC substrate PLEK2, and inositol triphosphate receptor IP3R3 were positively correlated
with IMP2 expression. Besides tumor initiation, IMP2 also seemed to have an impact on tumor
progression. TGF-β treatment of Panc-1 pancreatic cancer cells to induce epithelial-mesenchymal
transition (EMT) was accompanied by increased IMP2 expression. EMT is important for cancer cells
to gain migratory and invasive potential, which is essential for metastasis. Concordantly, circulating
tumor cells showed higher IMP2 levels as compared with normal tissue from tumor origin and
with normal hematological cells. Accordingly, IMP2 protein levels correlated with poor survival.
In conclusion, as IMP2 seems to promote tumor progression of PDAC, it might be an interesting
diagnostic and prognostic marker as well as a novel target for the treatment of PDAC
Induction of Liver Size Reduction in Zebrafish Larvae by the Emerging Synthetic Cannabinoid 4F-MDMB-BINACA and Its Impact on Drug Metabolism
Zebrafish (ZF; Danio rerio) larvae have become a popular in vivo model in drug metabolism
studies. Here, we investigated the metabolism of methyl 2-[1-(4-fluorobutyl)-1H-indazole-3-carboxamido]-
3,3-dimethylbutanoate (4F-MDMB-BINACA) in ZF larvae after direct administration of the cannabinoid
via microinjection, and we visualized the spatial distributions of the parent compound and its
metabolites by mass spectrometry imaging (MSI). Furthermore, using genetically modified ZF larvae,
the role of cannabinoid receptor type 1 (CB1) and type 2 (CB2) on drug metabolism was studied.
Receptor-deficient ZF mutant larvae were created using morpholino oligonucleotides (MOs), and
CB2-deficiency had a critical impact on liver development of ZF larva, leading to a significant
reduction of liver size. A similar phenotype was observed when treating wild-type ZF larvae with
4F-MDMB-BINACA. Thus, we reasoned that the cannabinoid-induced impaired liver development
might also influence its metabolic function. Studying the metabolism of two synthetic cannabinoids,
4F-MDMB-BINACA and methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-
dimethylbutanoate (70N-5F-ADB), revealed important insights into the in vivo metabolism of these
compounds and the role of cannabinoid receptor binding
Characterization of Anti-Cancer Activities of Violacein: Actions on Tumor Cells and the Tumor Microenvironment
Natural products have been shown to serve as promising starting points for novel anti cancer drugs. In this study, the anti-cancer activities of the purple compound violacein,
initially isolated from Chromobacterium violaceum, were investigated. To highlight the
crucial role of the tumor microenvironment on the effectiveness of cancer therapies, this
study includes effects on macrophages as prototypic cells of the microenvironment in
addition to the investigation of tumor-centric activities. Using 2D and 3D cell culture
models, automated live-cell microscopy, and biochemical analyses, violacein was
demonstrated to inhibit tumor cell proliferation and migration. The violacein-triggered
tumor cell death was further associated with caspase 3-like activation and ATP release.
Stimuli released from dead cells resulted in inflammatory activation of macrophages, as
shown by NF-kB reporter cell assays, macrophage morphology, and gene expression
analysis. Moreover, macrophages deficient in the inflammasome component Nlrp3 were
found to be significantly less sensitive towards treatment with violacein and doxorubicin.
Taken together, this study provides new insights into the biological activity of violacein
against cancer. In addition, the in vitro data suggest immunogenic features of induced cell
death, making violacein an interesting candidate for further studies investigating the
compound as an inducer of immunogenic cell death
Targeted delivery of functionalized PLGA nanoparticles to macrophages by complexation with the yeast Saccharomyces cerevisiae
Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly(lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a coculture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency
Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression
Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections
In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections
annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that
can invade and survive within mammalian cells. S. aureus intracellular infections are difficult
to treat because several classes of antibiotics are unable to permeate through the cell wall and
reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver
antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from
the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are
naturally antimicrobial, to target intracellular infections, and investigated how they can affect the
viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether
they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal
laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into
mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus.
We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23
OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000
OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and
IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being
taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation.
This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study,
we provide an important basis for implementing OMVs in the treatment of intracellular infections
Dysregulation of cholesterol homeostasis in human lung cancer tissue and tumour-associated macrophages
Background
Based on reports on elevated cholesterol levels in cancer cells, strategies to lower cholesterol synthesis have been suggested as an antitumour strategy. However, cholesterol depletion has also been shown to induce tumour-promoting actions in tumour-associated macrophages (TAMs).
Methods
We performed lipidomic and transcriptomic analyses of human lung cancer material. To assess whether the TAM phenotype is shaped by secreted factors produced by tumour cells, primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype using tumour cell-conditioned medium.
Findings
Lipidomic analysis of lung adenocarcinoma (n=29) and adjacent non-tumour tissues (n=22) revealed a significant accumulation of free cholesterol and cholesteryl esters within the tumour tissue. In contrast, cholesterol levels were reduced in TAMs isolated from lung adenocarcinoma tissues when compared with alveolar macrophages (AMs) obtained from adjacent non-tumour tissues. Bulk-RNA-Seq revealed that genes involved in cholesterol biosynthesis and metabolism were downregulated in TAMs, while cholesterol efflux transporters were upregulated. In vitro polarized TAM-like macrophages showed an attenuated lipogenic gene expression signature and exhibited lower cholesterol levels compared with non-polarized macrophages. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro TAM-like macrophages. Modulation of intracellular cholesterol levels by either starving, cholesterol depletion, or efflux transporter inhibition indicated that cholesterol distinctly shapes macrophage gene expression.
Interpretation
Our data show an opposite dysregulation of cholesterol homeostasis in tumour tissue vs. TAMs. Polarization of in vitro differentiated macrophages by tumour cell-conditioned medium recapitulates key features of ex vivo TAMs
Comment on: The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ
Recently, first insights into the regulation and the role of the RNA-binding protein IMP2 in macrophage activation have been published by Wang et al. This study addresses differences in the regulation of IMP2 between the human and murine system. While the expression of IMP2 in anti-inflammatory macrophages is synchronous in mice and men, IMP2 expression is regulated differently in inflammatory macrophages