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Glioblastoma (GBM) is the most common and most lethal type of cancer of the central nervous system in adults. Despite aggressive treatment, which is based on surgical resection, if possible, followed by radiation and chemotherapy, a high recurrence rate and therapy resistance is observed. Thus, additional innovative therapies are urgently needed to improve the poor median survival of only 15 months. Treatment of solid tumours with non-invasive physical plasma (NIPP) represents such a novel and innovative anticancer procedure.In this study, we investigated the effect of NIPP, an ionized argon gas, on the in vitro growth of human GBM cell lines, LN-18 and U-87 MG. Proliferation was measured by live cell count. Subsequently, proliferative factors were analysed at the level of nucleic acids (polymerase chain reaction) and proteins (western blotting).For both GBM lines, a treatment time-dependent decrease in growth was observed compared to controls. Additionally, NIPP treatment resulted in reduced rates of AKT serine/threonine kinase 1 (AKT1) and extracellular-regulated kinase 1/2 ERK1/2 expression, whereas expression of p21, proliferating cell nuclear antigen, and heat-shock proteins 90α and 90β was not affected. In both cell lines, a strong increase in expression of tumour-suppressive microRNA-1 (miR-1) was detected after exposure to NIPP.Our results demonstrated that NIPP is able to efficiently attenuate growth of GBM cells and suggest AKT1, ERK1/2 and miR-1 to be pivotal factors of NIPP-modulated cellular signalling. Translated into the clinical setting, NIPP may represent a promising option for the treatment of GBM.

Purpose:Apoptotic dysregulation, redox adaptive mechanisms, and resilience to hypoxia are major causes of glioblastoma (GBM) resistance to therapy. Commonly known as crucial factors in energy metabolism, OCTN2 (SLC22A5) and its substrate L-carnitine (LC) are increasingly recognized as actors in cytoprotection. This study provides a comprehensive expression and survival analysis of the OCTN2/LC system in GBM and clarifies the system's impact on GBM progression.Experimental Design:OCTN2 expression and LC content were measured in 121 resected human GBM specimens and 10 healthy brain samples and analyzed for prognostic significance. Depending on LC administration, the effects of hypoxic, metabolic, and cytotoxic stress on survival and migration of LN18 GBM cells were further studied in vitro. Finally, an orthotopic mouse model was employed to investigate inhibition of the OCTN2/LC system on in vivo GBM growth.Results:Compared with healthy brain, OCTN2 expression was increased in primary and even more so in recurrent GBM on mRNA and protein level. High OCTN2 expression was associated with a poor overall patient survival; the unadjusted HR for death was 2.7 (95% CI, 1.47–4.91; P < 0.001). LC administration to GBM cells increased their tolerance toward cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to a loss of tumor cell viability. In line herewith, OCTN2/LC inhibition by meldonium resulted in reduced tumor growth in an orthotopic GBM mouse model.Conclusions:Our data indicate a potential role of the OCTN2/LC system in GBM progression and resistance to therapy, and suggests OCTN2 as a prognostic marker in patients with primary GBM.

Expression and survival analyses of CPT1A and CPT1C in GBM specimens

Glioblastoma is the most common and lethal primary brain malignancy that almost inevitably recurs as therapy-refractory cancer. While the success of immune checkpoint blockade (ICB) revealed the immense potential of immune-targeted therapies in several types of cancers outside the central nervous system, it failed to show objective responses in glioblastoma patients as of now. The ability of glioblastoma cells to drive multiple modes of T cell dysfunction while exhibiting low-quality neoepitopes, low-mutational load, and poor antigen priming limits anti-tumor immunity and efficacy of antigen-unspecific immunotherapies such as ICB. An in-depth understanding of the GBM immune landscape is essential to delineate and reprogram such immunosuppressive circuits during disease progression. In this view, the present study aimed to characterize the peripheral and intratumoral immune compartments of 35 glioblastoma patients compared to age- and sex-matched healthy control probands, particularly focusing on exhaustion signatures on myeloid and T cell subsets. Compared to healthy control participants, different immune signatures were already found in the peripheral circulation, partially related to the steroid medication the patients received. Intratumoral CD4+ and CD8+ TEM cells (CD62Llow/CD45ROhigh) revealed a high expression of PD1, which was also increased on intratumoral, pro-tumorigenic macrophages/microglia. Histopathological analysis further identified high PSGL-1 expression levels of the latter, which has recently been linked to increased metastasis in melanoma and colon cancer via P-selectin-mediated platelet activation. Overall, the present study comprises immunophenotyping of a patient cohort to give implications for eligible immunotherapeutic targets in neurooncology in the future.

Purpose: Apoptotic dysregulation, redox adaptive mechanisms, and resilience to hypoxia are major causes of glioblastoma (GBM) resistance to therapy. Commonly known as crucial factors in energy metabolism, OCTN2 (SLC22A5) and its substrate L-carnitine (LC) are increasingly recognized as actors in cytoprotection. This study provides a comprehensive expression and survival analysis of the OCTN2/LC system in GBM and clarifies the system's impact on GBM progression. Experimental Design: OCTN2 expression and LC content were measured in 121 resected human GBM specimens and 10 healthy brain samples and analyzed for prognostic significance. Depending on LC administration, the effects of hypoxic, metabolic, and cytotoxic stress on survival and migration of LN18 GBM cells were further studied in vitro. Finally, an orthotopic mouse model was employed to investigate inhibition of the OCTN2/LC system on in vivo GBM growth. Results: Compared with healthy brain, OCTN2 expression was increased in primary and even more so in recurrent GBM on mRNA and protein level. High OCTN2 expression was associated with a poor overall patient survival; the unadjusted HR for death was 2.7 (95% CI, 1.47–4.91; P < 0.001). LC administration to GBM cells increased their tolerance toward cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to a loss of tumor cell viability. In line herewith, OCTN2/LC inhibition by meldonium resulted in reduced tumor growth in an orthotopic GBM mouse model. Conclusions: Our data indicate a potential role of the OCTN2/LC system in GBM progression and resistance to therapy, and suggests OCTN2 as a prognostic marker in patients with primary GBM.

Interaction with the dopaminergic system in the central nervous system is either therapeutically intended or it is a side effect. In both cases, dopamine-receptor agonists (DRA) like the ergoline derivative bromocriptine and dopamine-receptor antagonists (DRAn) like metoclopramide have to cross the blood-brain barrier (BBB). The organic anion transporting polypeptides (OATP) 1A2 and 2B1 are cellular uptake carriers for a variety of endogenous and xenobiotic compounds. As both transporters are expressed in endothelial cells of the BBB, the aim of the present study was to determine whether the DRA bromocriptine, cabergoline, and pergolide and the DRAn metoclopramide and domperidone are interacting with OATP1A2 and 2B1 and could therefore be candidate genes modifying wanted and unwanted effects of these drugs. Localization of both transporters in the brain was confirmed using LC-MS/MS and immunofluorescence stainings. For the functional studies, MDCKII cells stably expressing OATP1A2 or 2B1 were used. Initial interaction studies with the well-characterized transporter substrate estrone 3-sulfate revealed that all tested compounds except pergolide inhibit the transport function of both proteins with the most potent effect for bromocriptine (IC50 = 2.2 μM (OATP1A2) and IC50 = 2.5 μM (OATP2B1)). Further studies using the indirect competitive counterflow method identified bromocriptine, cabergoline, and domperidone as substrates of both transporters, whereas metoclopramide was only transported by OATP1A2. These findings were verified for domperidone by direct measurements using its tritium-labeled form as a tracer. Moreover, the transporter-mediated uptake of this compound was sensitive to the OATP1A2 and OATP2B1 inhibitor naringin. In conclusion, this study suggests that OATP1A2 and 2B1 may play a role in the uptake of DR agonists and antagonists into the brain.

Disialoganglioside GD2 is expressed by glioblastoma multiforme (GBM) cells representing a promising target for anti-GD2 immunotherapeutic approaches. The aim of the present study was to investigate anti-tumor efficacy of the chimeric anti-GD2 antibody (Ab) dinutuximab beta against GBM. Expression levels of GD2 and complement regulatory proteins (CRP; CD46, CD55 and CD59) on well-known and newly established primary tumor originated GBM cell lines were analyzed by flow cytometry. Ab-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC) mediated by dinutuximab beta against GBM cells were determined by a non-radioactive calcein-AM-based assay. Analysis of primary GBM cells revealed a heterogeneous GD2 expression that varied between the cell lines analyzed with higher expression levels in the tumor surface compared to the core originated cells. Both GD2-positive and -negative tumor cells were detected in every cell line analyzed. In contrast to CDC, ADCC mediated by dinutuximab beta was observed against the majority of GBM cells. Importantly, CDC-resistant cells showed high expression of the CRP CD46, CD55 and CD59. Our present data show anti-tumor effects mediated by dinutuximab beta against GBM cells providing a rationale for a GD2-directed immunotherapy against GBM. Due to high CRP expression, a combining of GD2-targeting with CRP blockade might be a further treatment option for GBM.

The link between thrombocytosis and malignancy has been well known for many years and its associations with worse outcomes have been reported mainly for solid tumors. Besides measuring platelet count, it has become popular to assess platelet function in the context of malignant diseases during the last decade. Malignant gliomas differ tremendously from malignancies outside the central nervous system because they virtually never form distant metastases. This review summarizes the current understanding of the platelet–immune cell communication and its potential role in glioma resistance and progression. Particularly, we focus on platelet-derived proinflammatory modulators, such as sphingosine-1-phosphate (S1P). The multifaceted interaction with immune cells puts the platelet into an interesting perspective regarding the recent advances in immunotherapeutic approaches in malignant glioma.

A signaling molecule which is involved in proliferation and migration of malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). There are hints for a potential role of S1P signaling in malignant brain tumors such as glioblastoma multiforme (GBM) which is characterized by a poor prognosis. Therefore, a comprehensive expression analysis of S1P receptors (S1P1-S1P5) and S1P metabolizing enzymes in human GBM (n = 117) compared to healthy brain (n = 10) was performed to evaluate their role for patient's survival. Furthermore, influence of S1P receptor inhibition on proliferation and migration were studied in LN18 GBM cells. Compared to control brain, mRNA levels of S1P1, S1P2, S1P3 and S1P generating sphingosine kinase-1 were elevated in GBM. Kaplan-Meier analyses demonstrated an association between S1P1 and S1P2 with patient's survival times. In vitro, an inhibitory effect of the SphK inhibitor SKI-II on viability of LN18 cells was shown. S1P itself had no effect on viability but stimulated LN18 migration which was blocked by inhibition of S1P1 and S1P2. The participation of S1P1 and S1P2 in LN18 migration was further supported by siRNA-mediated silencing of these receptors. Immunoblots and inhibition experiments suggest an involvement of the PI3-kinase/AKT1 pathway in the chemotactic effect of S1P in LN18 cells. In summary, our data argue for a role of S1P signaling in proliferation and migration of GBM cells. Individual components of the S1P pathway represent prognostic factors for patients with GBM. Perspectively, a selective modulation of S1P receptor subtypes could represent a therapeutic approach for GBM patients and requires further evaluation.

Patients with glioblastoma multiforme (GBM) are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs) are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin) as well as differentiation and microglia markers (GFAP, Iba1, and Sparc) in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed.

The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways.

Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.

The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central regulator in the development of several cancer types. In recent years, intriguing information has become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM), the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5) and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq, and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its receptors in GBM. We further highlight the current insights into the signaling pathways considered fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.

The anthracycline-mediated cardiotoxicity is still not completely understood. To examine the impact of cholesterol metabolism and transport in this context, cholesterol and oxysterol levels as well as the expression of the cholesterol transporters ABCA1 and ABCG1 were analyzed in doxorubicin-treated HL-1 murine cardiomyocytes as well as in mouse model for acute doxorubicin-induced cardiotoxicity. Doxorubicin-treated HL-1 cells exhibited enhanced cholesterol (153±20% of control), oxysterol (24S-hydroxycholesterol: 206±29% of control) and cholesterol precursor levels (lathosterol: 122±12% of control; desmosterol: 188±10% of control) indicating enhanced cholesterol synthesis. Moreover, abca1 and abcg1 were upregulated on mRNA, protein and functional level caused by a doxorubicin-mediated activation of the nuclear receptor LXR. In addition, the oxysterols not only induced the abca1 and abcg1 in HL-1 cells but also enhanced the expression of endothelin-1 and transforming growth factor-β, which have already been identified as important factors in doxorubicin-induced cardiotoxicity. These in vitro findings were verified in a murine model for acute doxorubicin-induced cardiotoxicity, demonstrating elevated cardiac (2.1±0.2vs. 3.6±1.0ng/mg) and systemic cholesterol levels (105.0±8.4vs. 130.0±4.3mg/dl), respectively, as well as enhanced oxysterol levels such as cardiac 24S-hydroxycholesterol (2.1±0.2vs. 3.6±1.0ng/mg). In line with these findings cardiac mRNA expression of abca1 (303% of control) and abcg1 (161% of control) was induced. Taken together, our data demonstrate enhanced cholesterol and oxysterol levels by doxorubicin, resulting in a LXR-dependent upregulation of abca1 and abcg1. In this context, the cytotoxic effects of oxysterols and their impact on cardiac gene expression should be considered as an important factor in doxorubicin-induced cardiotoxicity.

Tumor-associated macrophages (TAM) are the pre-dominant myeloid cells within malignant glioma and are a poor prognostic factor in patients. TAM in malignant glioma show a tumor-supporting, so-called M2 phenotype. The aim of this study was to characterize phenotype and relevant markers of monocytes/macrophages in the tumor microenvironment following exposure to cold physical plasma Monocytes were enriched from peripheral blood mononuclear cells derived from young healthy volunteers donating blood after informed consent and in accordance with the guidelines of the local ethics committee. Monocytes were co-cultured with a glioma cell line (U87MG). The latter readily induced a M2 phenotype in these monocytes as seen by an increase in CD163 expression. Prior to co-culture, either U87MG cells or monocytes were treated with an atmospheric pressure argon plasma jet (kINPen) for 15 seconds or 5 seconds, respectively. Non-treated co-cultures as well as single monocytes cultures served as controls. FACS immuno-phenotyping of monocytes/macrophages was performed for CD55, CD97, CD162, CD163, CD169, CD204, CD276, HLA-DR, and HLA-ABC after 24h, 48h, and 72h. M2 polarization of monocytes by U87MG was neither attenuated nor reversed by plasma treatment. We next exposed primary glioblastoma patient material to cold physical plasma (kINPen) ex vivo. After treatment, tissues were incubated for 24h, and supernatants were collected. Multiplex cytokine analysis of supernatants. Results of five patients showed an absence of IFNα, IFNγ, IL12p70, IL17α, and IL23, and a presence of IL1β, TNFα, MCP1, IL6, IL8, IL10, IL18, and IL33 in samples. A mixed response was observed. For instance, both of the myeloid-cell interacting proteins MCP1 and IL1β are associated with glioma aggressiveness, and secretion of IL1β decreased after plasma treatment whereas MCP1 was increased. Glioma tissue sections will determine the distribution of myeloid cells within the tumor after plasma treatment compared to controls. Download : Download high-res image (136KB) Download : Download full-size image Figure 1: Fold change of several chemokines/cytokines in kINPen plasma-treated primary human glioblastoma samples compared to untreated glioma tissue.

Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence, treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine interferon-γ (IFNγ). Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability have not been investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly over-represented among genes regulated by doxorubicin exposure, including signal transducer and activator of transcription (STAT) 1 and 2, interferon regulatory factor 9, N-myc and STAT interactor, and caspase 1. Regulation of these genes by doxorubicin was verified with real-time polymerase chain reaction and immunoblotting. An enhanced secretion of IFNγ was observed when HeLa cells were exposed to doxorubicin compared with untreated cells. IFNγ-neutralizing antibodies and inhibition of Janus tyrosine kinase (JAK)-STAT signaling [aurintricarboxylic acid (ATA), (E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490), STAT1 small interfering RNA] significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin-induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFNγ-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin's cytotoxicity in HeLa cells. Immunomodulation is a promising strategy in anticancer treatment, so this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.

Doxorubicin is a frequently used anticancer drug, but its use is restricted due to the occurrence of severe side effects, namely strong cardiotoxicity. It is known from cancer cells that doxorubicin enhanced the expression of its efflux pump P-glycoprotein (P-gp), which may modulate local drug concentrations. We therefore studied the cardiac expression of P-gp in doxorubicin-treated mice. Mice were treated with doxorubicin, and P-gp expression was studied after 1, 3, and 5 days. Thereby, we could show a significant upregulation of abcb1a (162 ± 15% of control) and abcb1b (418 ± 110% of control) mRNA transcripts after 3 days. On protein level, western blot analysis and immunofluorescence staining revealed a similar finding 5 days after doxorubicin administration. In addition, these results could be confirmed by in vitro models using primary rat cardiomyocytes and the murine cardiomyocyte-like HL-1 cells. Besides an enhanced mRNA and protein expression, doxorubicin-treated HL-1 cells also demonstrated an enhanced P-gp function as assessed by a daunorubicin accumulation assay. Our in vivo and in vitro results demonstrate a cardiac upregulation of P-gp in doxorubicin-treated mice on expression and functional level. This finding may be relevant for cardiac tissue concentrations of P-gp substrates and may represent a mechanism in cardiac self-protection against xenobiotics. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3951–3958, 2011

Statins are widely used to treat dyslipidemia. Effects of statins in addition to low-density lipoprotein lowering include altered platelet aggregation, requiring drug uptake into platelets. Possible candidates for mediating intraplatelet accumulation of statins include members of the organic anion-transporting polypeptide family such as OATP2B1 (SLCO2B1), a high-affinity uptake transporter for atorvastatin. Therefore, we analyzed OATP expression, localization, and function in human platelets. OATP2B1, but not OATP1B1, was detected in platelets and megakaryocytes on transcript and protein levels. Protein localization was almost exclusively confined to the plasma membrane. Moreover, we could demonstrate significant inhibition of estrone sulfate uptake into platelets by atorvastatin as well as direct transport of atorvastatin into platelets using a liquid chromatography-tandem mass spectrometry method. As a consequence of OATP2B1-mediated uptake of atorvastatin, we observed significant atorvastatin-mediated reduction of thrombin-induced Ca(2+) mobilization in platelets (37.3 +/- 6.7% of control at 15 microM atorvastatin), mechanistically explainable by reduced lipid modification of signal proteins. This effect was reversed by addition of mevalonate. Finally, we demonstrated expression of HMG-CoA reductase, the primary target of atorvastatin, in platelet cytosol. In conclusion, OATP2B1 is an uptake transporter expressed in platelets and is involved in statin-mediated alteration of platelet aggregation.

Doxorubicin is a frequently used anticancer drug, but its therapeutic benefit is limited by acute and chronic cardiotoxicity, often leading to heart failure. The mechanisms underlying doxorubicin-induced cardiotoxicity remain unclear. It was previously shown in men that doxorubicin leads to increased endothelin-1 plasma levels. In addition, cardiac-specific overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling the phenotype following doxorubicin administration. We therefore hypothesized that endothelin-1 is involved in the pathogenesis of doxorubicin cardiotoxicity. In mice (C57Bl/10), we found that doxorubicin (20 mg/kg body weight, i.p.) impaired cardiac function with decreased ejection fraction, diminished cardiac output, and decreased end-systolic pressure points recorded by a microconductance catheter. This impaired function was accompanied by the up-regulation of endothelin-1 expression on mRNA and protein level. In vitro investigations confirmed the regulation of endothelin-1 by doxorubicin and indicated that the doxorubicin-mediated increase of endothelin-1 expression involves epidermal growth factor receptor signaling via the MEK1/2-ERK1/2 cascade, which was further confirmed by immunoblotting studies in the left ventricle of treated animals. Pretreatment of mice with the endothelin receptor antagonist bosentan (100 mg/kg body weight, p.o.) strikingly inhibited doxorubicin-induced cardiotoxicity with preserved indices of contractility. Moreover, bosentan pretreatment resulted in reduced tumor necrosis factor-α content, lipid peroxidation, and Bax expression, as well as increased GATA-4 expression. Thus, endothelin-1 plays a key role in mediating the cardiotoxic effects of doxorubicin and its inhibition may be of therapeutic benefit for patients receiving doxorubicin. [Cancer Res 2007;67(21):10428–35]

The lysosomal hydrolase beta-glucuronidase (beta-gluc) can be used for the bioactivation of non-toxic glucuronide prodrugs of anticancer agents. The enzyme is present at high levels in many tumours and hence may lead to an enhanced drug targeting by tumour-selective release of the active anticancer drug. Individual expression and regulation of this enzyme is one factor modulating the bioactivation of glucuronide prodrugs. Nevertheless, in contrast to murine beta-gluc, which is inducible by androgens, the human enzyme has been regarded as an unregulated housekeeping gene due to a lacking TATA box and high G+C contents within the putative promotor sequence. Despite these facts, we were able to demonstrate downregulation of human beta-gluc expression by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. However, cis-acting elements responsible for this regulation have not yet been identified. We therefore characterised the 5'-untranslated region of the human beta-gluc gene using transient transfection assays with promotor-luciferase constructs in HepG2 cells and cloned fragments between 3,770 bp and 107 bp. A23187 reduced the beta-gluc promotor activity. This effect disappeared using fragments smaller than 356 bp. Using site-directed in vitro mutagenesis and gel-electrophoretic-mobility shift assays, we found evidence of an involvement of transcription factor activating protein-2 (AP-2) binding sites on the regulation of human beta-glucuronidase by A23187. Our studies provide a basis for the understanding of the transcriptional regulation of the human beta-glucuronidase gene and could be useful for the optimisation of glucuronide prodrug therapy.

Objective Glioblastoma patients (GBM) suffer from an increased incidence of cardiovascular events. Platelets are well known as main player of the primary hemostasis, but have a broad range of additional functions. The formation of heterotypic conjugates between platelets and leukocytes (PLC) represents a pro-inflammatory surrogate marker and is usually increased after platelet activation. The aim of the present study was to evaluate the platelet activation status and the rate of circulating PLC in GBM. Methods Blood samples were drawn of consecutive patients before surgery for a suspected glioblastoma. The formation of PLC and several parameters of platelet activation were determined by flow cytometry before and after stimulation with either ADP or the thrombin receptor-activating peptide (TRAP) in vitro: expression of P-Selectin, CD63, CD40L and fibrinogen-binding to the activated GPIIb/ IIIa. Blood samples from age and gender matched healthy volunteers were used as controls. Statistical analysis was done with the Mann-Whitney-Test. Results Final analysis included 22 patients with histopathological proven virgin glioblastoma (9f, 13m, mean age 67.5 years, range from 55 to 86 years) and their respective controls. Basal platelet activation and in vitro platelet reactivity was increased in GBM. The difference got significant in the basal expression of CD63 (2.8% versus 1.9%, p=0.008), the Fibrinogen-binding after ADP-stimulation (110.3 MFI versus 63.1 MFI, p=0.04) and the CD63 expression after TRAP-stimulation (38.4% versus 33.3%, p=0.04). Furthermore, a reduced number of circulating PLC and in vitro PLC formation was seen in GBM without getting statistically significant. Conclusions In this preliminary report, we show for the first time an increased level of platelet activation and agonist-induced platelet reactivity in GBM. Both could be a reflection of the pro-thrombotic status in these patients. Interestingly, the formation of PLC was not increased, but in tendency decreased. Whether this observation potentially mirrors the intratumoral, anti-inflammatory microenviroment in GBM remains unclear. Citation Format: Sascha Marx, Maximilian Splittstöhser, Heiko Paland, Carolin Seifert, Madlen Juettner, Andreas Boehm, Christoph A. Ritter, Sandra Bien-Moeller, Henry W. Schroeder, Bernhard Rauch. Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2017-1738

Background. The current therapy for glioblastoma multiforme (GBM), the most aggressive and common primary brain tumor of adults, involves surgery and a combined radiochemotherapy that controls tumor progression only for a limited time window. Therefore, the identification of new molecular targets is highly necessary. Inhibition of kinases has become a standard of clinical oncology, and thus the oncogenic kinase Pim1 might represent a promising target for improvement of GBM therapy. Methods. Expression of Pim1 and associated signaling molecules was analyzed in human GBM samples, and the potential role of this kinase in patients’ prognosis was evaluated. Furthermore, we analyzed the in vivo role of Pim1 in GBM cell growth in an orthotopic mouse model and examined the consequences of Pim1 inhibition in vitro to clarify underlying pathways. Results. In comparison with normal brain, a strong upregulation of Pim1 was demonstrated in human GBM samples. Notably, patients with short overall survival showed a significantly higher Pim1 expression compared with GBM patients who lived longer than the median. In vitro experiments with GBM cells and analysis of patients’ GBM samples suggest that Pim1 regulation is dependent on epidermal growth factor receptor. Furthermore, inhibition of Pim1 resulted in reduced cell viability accompanied by decreased cell numbers and increased apoptotic cells, as seen by elevated subG1 cell contents and caspase-3 and -9 activation, as well as modulation of several cell cycle or apoptosis regulatory proteins. Conclusions. Altogether, Pim1 could be a novel therapeutic target, which should be further analyzed to improve the outcome of patients with aggressive GBM.

Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients’ survival.

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR-binding proteins are required to elucidate this network. We affinity-purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high-resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc-1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well-described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin-associated adaptor complex AP-2 showed 4.6-fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP-2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.

Purpose Hepatic veno-occlusive disease (HVOD) is one of the major complications following hematopoietic stem cell transplantation (HSCT). Although high-dose busulfan is associated with the development of HVOD, the underlying molecular mechanisms are still unknown.Methods Transcriptional gene regulation by busulfan was profiled using Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays. Messenger RNA (mRNA) expression of regulated genes was assessed by TaqMan real-time polymerase chain reaction (PCR), and protein expression and secretion was determined by enzyme-linked immunosorbent assay (ELISA)in cell supernatants, lysates, and patient plasma.Results Plasma levels of plasminogen activator inhibitor(PAI)-1 significantly increased 48 h after starting busulfan treatment IV in children preconditioned for HSCT. In vitro,busulfan significantly induced plasminogen activator inhibitor-1 (PAI-1) expression in endothelium-like ECV304 cells in a concentration- and time-dependent manner. Comparative transcriptional profiling of busulfan-treated and control ECV304 cells identified differential expression of genes related to coagulation and fibrinolysis, including tissue factor, tissue factor pathway inhibitor-1, protein S, thrombospondin-1, urokinase receptor, and PAI-1, as well as activin A and transforming growth factor beta 1 (TGF-β1). Ingenuity pathway analysis (IPA) suggested TGF-β1 as a central modulator of gene regulation by busulfan. Consequently, expression of tissue factor, urokinase receptor, and PAI-1 mRNA and PAI-1 protein secretion induced by busulfan were significantly reduced by the activin A/TGF-β1 inhibitor SB 431542 in ECV304 and primary endothelial cells.Conclusions This is the first report that directly relates busulfan exposure to antifibrinolytic activity by PAI-1 and hypercoagulation possibly mediated by members of the TGF-β1 family. This suggests further research to evaluate activin A and TGF-β1 as potential targets for HVOD treatment.

The cysteine protease cathepsin B acts as a key player in apoptosis. Cathepsin B-mediated cell death is induced by various stimuli such as ischemia, bile acids or TNFα. Whether cathepsin B can be influenced by anticancer drugs, however, has not been studied in detail. Here, we describe the modulation of doxorubicin-induced cell death by silencing of cathepsin B expression. Previously, it was shown that doxorubicin, in contrast to other drugs, selectively regulates expression and activity of cathepsin B. Selective silencing of cathepsin B by siRNA or the cathepsin B specific inhibitor CA074Me modified doxorubicin-mediated cell death in Hela tumor cells. Both Caspase 3 activation and PARP cleavage were significantly reduced in cells lacking cathepsin B. Moreover, mitochondrial membrane permeabilization as well as the release of cytochrome C and AIF from mitochondria into cytosol induced by doxorubicin were significantly diminished in cathepsin B suppressed cells. In addition, doxorubicin associated down-regulation of XIAP was not observed in cathepsin B silenced cells. Lack of cathepsin B significantly modified cell cycle regulatory proteins such as cdk1, Wee1 and p21 without significant changes in G(1), S or G(2)M cell cycle phases maybe indicating further cell cycle independent actions of these proteins. Consequently, cell viability following doxorubicin was significantly elevated in cells with cathepsin B silencing. In summary, our data strongly suggest a role of cathepsin B in doxorubicin-induced cell death. Therefore, increased expression of cathepsin B in various types of cancer can modify susceptibility towards doxorubicin.

We have identified the ATP-binding cassette (ABC) transporter ABCC4 as an active constituent of mediator-storing granules in human platelets. In addition to multidrug resistance protein 4, other ABC-type transport proteins may contribute to platelet secretory function as well as determine intended or adverse effects of drugs. Here, we provide a comprehensive expression profiling of ABC transporters in human platelets based on a novel screening approach by combining the TaqMan low-density array RNA screening platform with a recently developed liquid chromatography/mass spectrometry (MS)/MS method for the simultaneous detection of membrane proteins. Transcripts of 25 ABC transporters were detected and showed differential expression compared with megakaryocytic progenitor cells. On the protein level ABCA7, ABCB4, ABCC1, ABCC3 and ABCC4 were identified by liquid chromatography/MS/MS and localized by immunofluorescence microscopy. Their functions may be related to glutathione and lipid homeostasis, secretion of lipid mediators, cell protection as well as drug transport.

HepG-2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2-D gel-based and gel-free methods. The analysis of crude HepG2 cell extracts by 2-D DIGE provided data on 1835 protein spots which was then complemented by MS-centered analysis of stable isotope labeling by amino acids in cell culture-labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin-induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin-associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.

Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatin-treated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, −29%; dp/dtmax, −45%; cardiac output, −68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor α and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and anti-inflammatory effects. [Cancer Res 2009;69(2):695–9]

Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity. Methods and results: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure–volume loops were measured in wild-type and TLR4-deficient mice (TLR4−/−) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-α expression and enhanced content of TUNEL-positive cells. In contrast, TLR4−/−Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression. Conclusions: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy.

Functional analysis of intracellular structures requires isolation and purification of these cellular compartments. With regard to platelet function both delta and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited and restricted to density gradient centrifugation. Because this method is time-consuming and the resulting products are often of limited purity, we designed a new purification method based on immunolabeling followed by magnetic sorting. We directly compared this new method with the conventional method of ultracentrifugation. We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIbβ, for plasma membrane) could be detected by Western blotting. The method describes purification of membranal structures of human platelets such as the plasma membrane and both types of granules. Therefore, studies requiring highly purified material (e.g. identification of specific transporters and receptors) will benefit from these results.

High-dose busulfan is an important component in many conditioning protocols for hematopoietic stem cell or bone marrow transplantation. Treatment with busulfan results in the inhibition of cell cycle progression and apoptosis of tumor cells. As Rho GTPases are involved in cell cycle regulation, we investigated the influence of modified Rho guanine nucleotide dissociation inhibitor-alpha (GDI), a physiological inhibitor of Rho GTPases, on busulfan activity in cancer cells. RhoGDIalpha has been shown to be overexpressed in multiple types of tumors such as ovarian and breast cancer. To investigate the role of RhoGDIalpha, we established a RhoGDIalpha knockdown by the transient transfection of HEK293 cells with specific small interfering RNA resulting in strongly reduced RhoGDIalpha mRNA and protein expression. Other members of the RhoGDI family such as RhoGDIbeta and RhoGDIgamma were not affected. In RhoGDIalpha knockdown cells, cell cycle regulation was not altered by the downregulation of RhoGDIalpha; however, the rate of apoptotic cells increased when compared with the control small interfering RNA-transfected cells. In addition, treatment of cells with busulfan resulted in a further increased apoptotic rate, as determined by fluorescence-activated cell sorter analysis and caspase-3 activation. Such a sensitization of RhoGDIalpha small interfering RNA transfected cells was also found upon treatment with doxorubicin and taxol. In summary, we could demonstrate that the expression of RhoGDIalpha influences the sensitivity of cells toward busulfan-induced cytotoxicity.

Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin. The enzyme cathepsin B catalyzes the activation of these prodrugs, and hence, the regulation of cathepsin B by antitumor agents could influence the efficacy of peptide prodrugs using this protease. In the present investigation, the effects of doxorubicin on cathepsin B expression in the human cervix carcinoma cell line HeLa were examined. Exposure to doxorubicin induced a time- and dose-dependent up-regulation of cathepsin B expression on mRNA, protein, and activity levels. In the cathepsin B gene promoter region, a potential nuclear factor kappaB (NF-kappaB) binding site could be identified. Pretreatment of HeLa cells with specific NF-kappaB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IkappaB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicin-induced cathepsin B expression. Finally, binding of NF-kappaB subunits p50 and p65 to the NF-kappaB binding site in the cathepsin B gene promoter region could be demonstrated by electrophoretic mobility shift assay. In summary, our data clearly indicate that doxorubicin induces cathepsin B expression and activity via NF-kappaB. These findings contribute to a better understanding of tumor targeting with peptide prodrugs and help to define a possible mechanism of doxorubicin toxicity in tumor cells.

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.