Ingrid A. M. van Roosmalen

Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor

BackgroundMacrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.MethodsDifferentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 μg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.ResultsHIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.ConclusionsExpression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1α protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.

Two death-inducing human TRAIL receptors to target in cancer: similar or distinct regulation and function?

The emergence during evolution of two tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, receptor-1/DR4 and -2/DR5, able to induce apoptosis has raised the question whether they differ in function and regulation, which is of key importance for selecting either DR4 or DR5 selective pro-apoptotic agents for cancer treatment. In this review we found practically no information regarding possible differences in DR4 and DR5 function based on structural differences. On the other hand, a panel of different DR4 or DR5 selective pro-apoptotic agonists have been developed that were explored for efficacy in different tumour types in a large number of studies. Leukemic cells appear mainly sensitive for DR4-induced apoptosis, contrasting the situation in other tumour types that show heterogeneity in receptor preference and, in some cases, a slight overall preference for DR5. Both receptors were found to mediate intracellular stress-induced apoptosis, although this is most frequently reported for DR5. Interestingly, DR5 was also found to transmit non-apoptotic signalling in resistant tumour cells and recently nuclear localization and a role in microRNA maturation has been described. DR4 expression is most heavily regulated by promoter methylation, intracellular trafficking and post-translational modifications. DR5 expression is predominantly regulated at the transcriptional level, which may reflect its ability to respond to cellular stressors. It will be important to further increase our understanding of the mechanisms determining TRAIL receptor preference in order to select the appropriate TRAIL receptor selective agonists for therapy, and to develop novel strategies to enhance apoptosis activation in tumours.

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.

Correction: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

Erratum to: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

Correction: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma (vol 3, 495, 2014)

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

Apoptosis activation by TRAIL receptor selective variants in glioblastoma (stem) cells

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumour in humans. The 5-year survival rate of patients is Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) induces apoptosis in tumour cells, without causing harm to healthy cells. TRAIL can bind to five different receptors; two death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), and three decoy receptors, DcR1, DcR2 and OPG. To overcome this promiscuous behaviour, TRAIL variants have been designed that have a higher selectivity to either DR4 or DR5. In this study we investigated the sensitivity of various glioblastoma (stem) cell lines for TRAIL using the TRAIL variants alone and in combination with the proteasome inhibitor bortezomib. Recombinant human (rh)TRAIL and the generated TRAIL variants, two DR4-specific (4C7 and 4C9) and one DR5-specific (D269H/E195R) variants, were produced and purified. A panel of GBM cells was used: GSC-23 (with CSC characteristics, provided by H. Colman (Houston, Tx), U87, A172 and normal human astrocytes. First, the basal level of DR4, DR5, DcR1 and DcR2 expression was determined using flow cytometry. All cell lines displayed high DR5 and DcR2 expression, whereas DR4 and DcR1 levels were not detectable. Cell viability assays using resazurin conversion indicated that GSC-23 cells when maintained as neurospheres are resistant to TRAIL and variants, also when combined with bortezomib. TRAIL treated monolayer A172 and U87 cells were stained with AnnexinV and analysed by flow cytometry. A172 cells were sensitive for TRAIL-induced apoptosis, whereas U87 are resistant. Combined treatment with bortezomib effectively sensitized both cell lines for TRAIL-induced apoptosis. Next, a retroviral vector was generated containing the DR4 encoding sequence to examine the effect of enforced DR4 expression in GBM cells. GSC-23-DR4 neurospheres remained resistant to TRAIL and the variants, however, when combined with bortezomib sensitization towards both 4C7, 4C9 and D269H/195ER was seen. In conclusion, our preliminary results show predominantly DR5 expression in GBM suggesting benefit from DR5 selective TRAIL variants. Combined treatment with bortezomib sensitized for TRAIL-induced apoptosis in GSC-23-DR4 neurospheres, whereas GSC-23 cells remained resistant. In ongoing experiments the apoptosis inducing properties of TRAIL and TRAIL variants in GBM stem cells and differentiated derivatives is further examined. Mechanisms of resistance in relation to the effect of bortezomib are further explored to achieve optimal TRAIL-induced apoptosis in GBM (stem) cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2011-3399