Identification of IRF1 as critical dual regulator of Smac mimetic-induced apoptosis and inflammatory cytokine response.

Smac (second mitochondria-derived activator of caspase) mimetics are considered as promising anticancer therapeutics and used to induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are often abundantly expressed in cancer cells. Here, we identify interferon regulatory factor 1 (IRF1) as a novel critical regulator of Smac mimetic BV6-induced apoptosis and proinflammatory cytokine secretion with impact on the immune response. IRF1 knockdown rescues cells from BV6-induced apoptosis and attenuates BV6-stimulated upregulation of tumor necrosis factor-α (TNFα), indicating that IRF1 mediates BV6-triggered cell death, at least in part, by inducing TNFα. This notion is supported by data showing that exogenous supply of TNFα restores BV6-induced cell death in IRF-knockdown cells. Interestingly, IRF1 selectively controls the induction of nuclear factor-κB (NF-κB) target genes, as IRF1 depletion attenuates BV6-stimulated upregulation of TNFα and interleukin-8 (IL-8) but not p100 and RelB. Concomitant knockdown of IRF1 and p65 cooperate to inhibit BV6-induced cell death, implying a cooperative interaction of IRF1 and NF-κB. In addition, IRF1 silencing hampers TNFα induction by TNFα itself as an another prototypical NF-κB stimulus. Importantly, IRF1 depletion impedes BV6-stimulated secretion of additional proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IL-6 and monocyte chemoattractant protein-1, and migration of primary monocytes to BV6-treated tumor cells. In conclusion, this identification of IRF1 as a dual regulator of BV6-induced apoptosis and inflammatory cytokine secretion provides novel insights into determinants of sensitivity towards Smac mimetic and possible implications of Smac mimetic treatment on tumor microenvironment and immune response.

Smac mimetic promotes glioblastoma cancer stem-like cell differentiation by activating NF-κB.

Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker ß-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis.

Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.

Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells.

As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.

Histone deacetylase inhibitors sensitize glioblastoma cells to TRAIL-induced apoptosis by c-myc-mediated downregulation of cFLIP.

Glioblastoma is the most common primary brain tumor with a very poor prognosis, calling for novel treatment strategies. Here, we provide first evidence that histone deacetylase inhibitors (HDACI) prime glioblastoma cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis at least in part by c-myc-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP). Pretreatment with distinct HDACI (MS275, suberoylanilide hydroxamic acid, valproic acid) significantly enhances TRAIL-induced apoptosis in several glioblastoma cell lines. Monitoring a panel of apoptosis-regulatory proteins revealed that MS275 reduces the expression of cFLIP(L) and cFLIP(S). This leads to decreased recruitment of cFLIP(L) and cFLIP(S) and increased activation of caspase-8 to the TRAIL death-inducing signaling complex, resulting in enhanced cleavage of caspase-8, -9 and -3 and caspase-dependent apoptosis. Also, MS275 promotes TRAIL-triggered processing of Bid, activation of Bax, loss of mitochondrial membrane potential and release of cytochrome c. MS275-mediated downregulation of cFLIP occurs at the mRNA level independent of proteasome- or caspase-mediated degradation, and is preceded by upregulation of nuclear levels of c-myc, a transcriptional repressor of cFLIP. Notably, MS275 causes increased binding of c-myc to the cFLIP promoter and reduces cFLIP promoter activity. Indeed, knockdown of c-myc partially rescues cFLIP(L) from MS275-inferred downregulation and significantly decreases TRAIL- and MS275-induced apoptosis. Also, overexpression of cFLIP(L) or cFLIP(S) significantly reduces MS275- and TRAIL-induced apoptosis. Importantly, MS275 sensitizes primary cultured glioblastoma cells towards TRAIL and cooperates with TRAIL to reduce long-term clonogenic survival of glioblastoma cells and to suppress glioblastoma growth in vivo underscoring the clinical relevance of this approach. Thus, these findings demonstrate that HDACI represent a promising strategy to prime glioblastoma for TRAIL-induced apoptosis by targeting cFLIP.

The cholinergic system in rat testis is of non-neuronal origin.

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

PRIMA-1(MET)/APR-246 targets mutant forms of p53 family members p63 and p73.

The low molecular weight compound PRIMA-1 and the structural analog PRIMA-1(MET), also named APR-246, reactivate mutant p53 through covalent binding to the core domain and induce apoptosis in tumor cells. Here, we asked whether PRIMA-1(MET)/APR-246 can rescue mutant forms of the p53 family members p63 and p73 that share high sequence homology with p53. We found that PRIMA-1(MET)/APR-246 can restore the pro-apoptotic function to mutant TAp63γ and TAp73ß in tumor cells but has less effect on TAp73α. Moreover, PRIMA-1(MET)/APR-246-stimulated DNA binding of mutant TAp63γ and induced expression of the p53/p63/p73 downstream targets p21 and Noxa. The reactivation of mutant p53, p63 and p73 by PRIMA-1(MET)/APR-246 indicates a common mechanism, presumably involving homologous structural elements in the p53 family proteins. Our findings may open avenues for therapeutic intervention in human developmental disorders with mutations in p63.

Disability rating with the IFMSS scales.

The long-term continuous care program of the Göttingen MS Information and Counseling Unit is described. In 120 patients, analysis of environmental situation showed that 1/3 of the patients had endangered family situations, 2/3 were unemployed or pensioned and 2/3 were unable to do housework. 1/3 had unsatisfactory financial situations, 1/3 were in need of better housing, 2/3 required daily personal assistance, and in 1/3 transportation was possible only in special vehicles. An attempt is made to qualify the effort and cost/benefit relation to the unit. A summary of the performance-limiting disturbances showed that there were several severe and frequently occurring disturbances which are difficult to document with the IFMSS systems. To deal with this problem, clinical profiles were developed, examples of which are given.

Intravenous nitroglycerine in refractory unstable angina pectoris.

Sixteen patients with severe coronary artery disease and unstable angina, refractory to standard therapy with nitrates, beta-blockers or calcium antagonists, were given intravenous nitroglycerine (500 micrograms/ml) in an open trial. The infusion was started at 0 . 17 ml/min. The final infusion rate ranged from 0 . 17 ml/min to 2 . 04 ml/min, depending on the symptomatic and haemodynamic response of the individual patient. At the slow infusion rates, the actual dose was probably only 15% of the delivered dose because of the absorption of nitroglycerine to PVC tubing. There was significant pain relief in all patients. In six patients, pain relief was complete; in ten patients, occasional episodes occurred during the nitroglycerine infusion but they were less frequent and less severe and few were associated with ST segment changes. Systolic blood pressure fell by a mean of 100 mmHg at the commencement of therapy but there was no significant change in heart rate. Apart from mild headaches, no other adverse effects were observed. The mean treatment time was 3 . 2 days (range 1-8 days). Eight patients were discharged on oral and/or cutaneous nitrate therapy and eight patients had coronary artery surgery.

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.