Bernd Niggemann

The norepinephrine‐driven metastasis development of PC‐3 human prostate cancer cells in BALB/c nude mice is inhibited by β‐blockers

The development of metastases is a decisive step in the course of a cancer disease. The detection of metastases in cancer patients is correlated with a poor prognosis, and over 90% of all deaths from cancer are not due to the primary tumor, which often can be successfully treated, but are due to the metastases. Tumor cell migration, a prerequisite for metastasis development, is not merely genetically determined, but is distinctly regulated by signal substances of the environment including chemokines and neurotransmitters. We have shown previously that the migration of breast, prostate, and colon carcinoma cells is enhanced by the stress‐related neurotransmitter norepinephrine in vitro, and that this effect can be inhibited by the β‐blocker propranolol. We now provide for the first time evidence for the in vivo relevance of this neurotransmitter‐driven regulation using PC‐3 prostate carcinoma cells. The development of lumbar lymph node metastases in athymic BALB/c nude mice increased with the application of norepinephrine via microosmotic pumps, while propranolol inhibited this effect. However, the growth of the primary tumor was not affected by either treatment. Additionally, experiments using human tissue microarrays showed that 70–90 percent of breast, colon, and prostate carcinoma tissues express the relevant β2‐adrenoceptor. Thus, our work contributes to the understanding of the basic cellular mechanisms of metastasis development, and furthermore delivers a rationale for the chemopreventive use of clinically established β‐blockers for the inhibition of metastases.

Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells

Most patients suffering from breast carcinoma do not die due to the primary tumor but from the development of metastases. Active migration of cancer cells is a prerequisite for development of these metastases. We used time-lapse videomicroscopy and computer-assisted cell tracking of MDA-MB-468 human breast carcinoma cells, which were incorporated into a three-dimensional collagen matrix, in order to analyze the migratory activity of these cells in response to different neurotransmitters. Our results show that met-enkephalin, substance P, bombesin, dopamine, and norepinephrine have a stimulatory effect on the migration of the breast cancer cells; moreover, these cells show positive chemotaxis towards norepinephrine as was analyzed by the directionality and persistence on a single-cell basis. Gamma-aminobutyric acid (GABA) however has an inhibitory effect. Endorphin and leu-enkephalin, as well as histamin and acetylcholine, had no influence on the migratory activity of the cells. In summary, we provide evidence for a strong regulatory involvement of neurotransmitters in the regulation of breast cancer cell migration, which might provide the basis for the use of the pharmacological agonists and antagonists for the chemopreventive inhibition of metastasis development.

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize β1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion

Cell migration may depend on integrin‐mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three‐dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three‐dimensional collagen matrix model with time‐lapse videomicroscopy, computer‐assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co‐clustering of β1, β2, or β3 integrins with F‐actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high‐affinity β1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion‐perturbing anti‐β1, ‐β2, ‐β3, and αv integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, β1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A‐activated cells. Hence, T lymphocytes migrating in three‐dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion‐dependent migration strategies employed by other cells.

Induction of a metastatogenic tumor cell type by neurotransmitters and its pharmacological inhibition by established drugs

The active migration of tumor cells, a crucial requirement for metastasis development and cancer progression, is regulated by signal substances including neurotransmitters. We investigated the migration of tumor cells within a three‐dimensional collagen matrix using time‐lapse videomicroscopy and computer‐assisted analysis of the migration path. Tumor cell migration is induced by norepinephrine, dopamine and substance P. We show that this induced migration, using MDA‐MB‐468 breast and PC‐3 prostate carcinoma cells, can be inhibited by using specific, clinically established receptor antagonists to the β2‐adrenoceptor, the D2 receptor, or the neurokinin‐1 receptor, respectively. All of the investigated neurotransmitters significantly activated the cyclic adenosine‐monophosphate response element binding protein (CREB). Furthermore, microarray analysis revealed changes of gene expression toward a highly motile tumor cell type, including an upregulation of the α2 integrin, which is an essential adhesion receptor for collagen in migration. The gene for the tumor suppressor gelsolin was downregulated. These 2 critical alterations were confirmed on the protein level by flow‐cytometry and immunoblotting, respectively. Neurotransmitters thus induce a metastatogenic tumor cell type by directly regulating gene expression and increased migratory activity, which can be prevented by established neurotransmitter antagonists.

MCP-1 induces migration of adult neural stem cells

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.

Anandamide is an endogenous inhibitor for the migration of tumor cells and T lymphocytes

Cell migration is of paramount importance in physiological processes such as immune surveillance, but also in the pathological processes of tumor cell migration and metastasis development. The factors that regulate this tumor cell migration, most prominently neurotransmitters, have thus been the focus of intense investigation. While the majority of neurotransmitters have a stimulatory effect on cell migration, we herein report the inhibitory effect of the endogenous substance anandamide on both tumor cell and lymphocyte migration. Using a collagen-based three-dimensional migration assay and time-lapse videomicroscopy, we have observed that the anandamide-mediated signals for CD8+ T lymphocytes and SW 480 colon carcinoma cells are each mediated by distinct cannabinoid receptors (CB-Rs). Using the specific agonist docosatetraenoylethanolamide (DEA), we have observed that the norepinephrine-induced migration of colon carcinoma cells is inhibited by the CB1-R. The SDF-1–induced migration of CD8+ T lymphocytes was, however, inhibited via the CB2-R, as shown by using the specific agonist JWH 133. Therefore, specific inhibition of tumor cell migration via CB1-R engagement might be a selective tool to prevent metastasis formation without depreciatory effects on the immune system of cancer patients.

Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)‐induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC) ‐γ1 due to c‐erbB‐2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long‐term PLC‐γ1 tyrosine phosphorylation and sustained levels of inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c‐erbB‐2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short‐term PLC‐γ1 tyrosine phosphorylation and short‐term IP3 and DAG turnover. A third cell line expressing a point‐mutated c‐erbB‐2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC‐γ1 activation was attributed to this structure. Neither PLC‐γ1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC‐γ1 activation time course by c‐erbB‐2 signaling. Induction of cell migration was solely observable in the c‐erbB‐2‐positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D‐ collagen lattice. In summary, c‐erbB‐2 up‐regulation switches on the cell migration program by modulating the time course of PLC‐γ1 activation.

Boswellic acid acetate induces differentiation and apoptosis in highly metastatic melanoma and fibrosarcoma cells

The aim of the study was to investigate the antitumor and/or preventive effect of BC-4, an isomeric compound isolated from the plant Boswellia carteri Birdw. containing alpha- and beta-boswellic acid acetate in 1:1, MW 498.3. We used the MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) assay to study the growth inhibition activity of BC-4. Tumor cells migration within a three-dimensional collagen matrix was recorded by time-lapse videomicroscopy and computer-assisted cell tracking. Topoisomerase II was isolated from mouse melanoma B16F10 cells and its activity was determined by its ability to cut plasmid pBR322 DNA. The secretion and activity of matrix metalloproteinases (MMPs) from human fibrosarcoma HT-1080 cells were determined by gelatin zymography. BC-4 was a cytostatic compound and could induce the differentiation of B16F10 mouse melanoma cells, blocked the cell population in G1 phase and inhibited topoisomerase II activity. The G1 phase population of B16F10 cells was increased from 57.4 to 87.7%, while S phase population was reduced from 33.3 to 5.9% after treatment with BC-4 at 25 microM concentration for 48 h. BC-4 also inhibited the migration activity of B16F10. BC-4 could induce apoptosis of HT-1080 cells, as proved by acridine orange fluorescence staining, Wright-Giemsa staining, electromicroscopy, DNA fragmentation and flow cytometry. BC-4 inhibited the secretion of MMPs from HT-1080 cells, too. In conclusion, if it turns out that BC-4 is a well tolerated substance, exhibiting no significant toxicity or side effects, being evaluated currently in China, BC-4 is a good candidate for the prevention of primary tumor, invasion and metastasis.

Role of the β1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell–cell and cell–matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the β1-integrin subunit using the specific β1-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the β1-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the β1-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the β1-integrin subunit in forming the cell–cell and cell–matrix bonds necessary for adhesion, extravasation and migration.

Signal processing in migrating T24 human bladder carcinoma cells: Role of the autocrine interleukin‐8 loop

T24 human bladder carcinoma cells reveal a high locomotor activity (70% locomoting cells) within a 3‐dimensional collagen matrix. This high migratory activity is induced by an autocrine engagement of the interleukin‐8 receptor A, as was shown by antibodies neutralizing the secreted interleukin‐8. Treatment of the cells with these specific antibodies reduced the locomotor activity by half. The intracellular signal transduction underlying the interleukin‐8‐induced T24 locomotion involves the activity of protein tyrosine kinases (PTKs), the phospholipase Cγ (PLCγ) and the protein kinase C (PKC), as proven by the use of specific enzyme inhibitors. These results suggest the following model for the regulatory signal transduction of interleukin‐8‐induced human T24 bladder carcinoma cell migration: The engagement of the interleukin‐8‐receptor, a receptor of the serpentine family, leads to the β‐arrestin‐mediated activation of PTKs. These kinases phosphorylate the PLCγ, which generates the second messengers diacylglycerol (DAG) and inositol‐1,4,5‐trisphosphate (IP3). DAG activates the PKC, whereas IP3 mediates the release of calcium from the endoplasmatic reticulum. By means of confocal laser microscopy, we observed an oscillation of the cytosolic calcium concentration in migrating T24 cells, which were loaded with the calcium‐dye fluo‐3/AM. Here, we report on a new autocrine function of secreted interleukin‐8 and the intracellular signal transduction leading to the regulation of cytosolic calcium and to a migratory tumor cell phenotype.