Inhibitor of apoptosis (IAP) proteins are frequently expressed at high levels in cancer cells and represent attractive therapeutic targets. We previously reported that the Smac (second ...mitochondria-derived activator of caspases) mimetic BV6, which antagonizes IAP proteins, sensitizes glioblastoma cells to temozolomide (TMZ)-induced cell death in a nuclear factor-κB (NF-κB)-dependent manner. However, BV6-induced NF-κB target genes responsible for this synergistic interaction have remained elusive. Using whole-genome gene expression profiling, we here identify BV6-stimulated, NF-κB-dependent transcriptional upregulation of interferon-β (IFNβ) and IFN-mediated proapoptotic signaling as critical events that mediate BV6/TMZ-induced apoptosis. Knockdown of IFNβ significantly rescues cells from BV6/TMZ-induced cell death. Similarly, silencing of the corresponding receptor IFNα/β receptor (IFNAR) confers a significant protection against apoptosis, demonstrating that IFNβ and IFN signaling are required for BV6/TMZ-mediated cell death. Moreover, BV6 and TMZ cooperate to transcriptionally upregulate the proapoptotic B-cell lymphoma 2 family proteins Bax (Bcl-2-associated X protein) or Puma (p53-upregulated modulator of apoptosis). Knockdown of Bax or Puma significantly decreases BV6/TMZ-induced apoptosis, showing that both proteins are necessary for apoptosis. By identifying IFNβ as a key mediator of BV6/TMZ-induced apoptosis, our study provides novel insights into the underlying molecular mechanisms of Smac mimetic-mediated chemosensitization with important implications for the development of novel treatment strategies for glioblastoma.
Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the ...second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index <0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-κB-inducing kinase and NF-κB activation. BV6-stimulated NF-κB activation is critically required for sensitization toward TMZ, as inhibition of NF-κB by overexpression of the mutant IκBα super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6-mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFα) production. Also, TNFα, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6- and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-κB- and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ.
Highly porous in situ hardening systems mechanically stable and elastic.
Goal of the present study was to develop and to characterize in situ-hardening, porous PLGA-based systems for their future ...application as bone grafting materials. Therefore, we investigated the precipitation behavior of formulations containing PLGA and a water-miscible solvent, DMSO, PEG 400, and NMP. To increase porosity, a pore forming agent (NaCMC) was added and to enhance mechanical properties of the system, an inorganic filler (α-TCP) was incorporated. The behavior upon contact with water and the influence of the prior addition of aqueous media on the morphology of the corresponding hardened implants were investigated. We proved cell-compatibility by live/dead assays for the hardened porous polymer/ceramic-composite scaffolds. The IsHS formulations can therefore be used to manufacture hardened scaffolds ex vivo by using molds with the desired shape and size. Cells were further successfully incorporated into the IsHS by precultivating the cells on the α-TCP-powder prior to their admixing to the formulation. However, cell viability could not be maintained due to toxicity of the tested solvents. But, the results demonstrate that in vivo cells should well penetrate, adhere, and proliferate in the hardened scaffolds. Consequently, we consider the in situ hardening system being an excellent candidate as a filling material for non-weight-bearing orthopedic indications, as the resulting properties of the hardened implant fulfill indication-specific needs like mechanical stability, elasticity, and porosity.
Inhibitor of apoptosis (IAP) proteins are frequently expressed at high levels in cancer cells and represent attractive therapeutic targets. We previously reported that the Smac (second ...mitochondria-derived activator of caspases) mimetic BV6, which antagonizes IAP proteins, sensitizes glioblastoma cells to temozolomide (TMZ)-induced cell death in a nuclear factor-κB (NF-κB)-dependent manner. However, BV6-induced NF-κB target genes responsible for this synergistic interaction have remained elusive. Using whole-genome gene expression profiling, we here identify BV6-stimulated, NF-κB-dependent transcriptional upregulation of interferon-β (IFNβ) and IFN-mediated proapoptotic signaling as critical events that mediate BV6/TMZ-induced apoptosis. Knockdown of IFNβ significantly rescues cells from BV6/TMZ-induced cell death. Similarly, silencing of the corresponding receptor IFNα/β receptor (IFNAR) confers a significant protection against apoptosis, demonstrating that IFNβ and IFN signaling are required for BV6/TMZ-mediated cell death. Moreover, BV6 and TMZ cooperate to transcriptionally upregulate the proapoptotic B-cell lymphoma 2 family proteins Bax (Bcl-2-associated X protein) or Puma (p53-upregulated modulator of apoptosis). Knockdown of Bax or Puma significantly decreases BV6/TMZ-induced apoptosis, showing that both proteins are necessary for apoptosis. By identifying IFNβ as a key mediator of BV6/TMZ-induced apoptosis, our study provides novel insights into the underlying molecular mechanisms of Smac mimetic-mediated chemosensitization with important implications for the development of novel treatment strategies for glioblastoma.
Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the ...second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index <0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-κB-inducing kinase and NF-κB activation. BV6-stimulated NF-κB activation is critically required for sensitization toward TMZ, as inhibition of NF-κB by overexpression of the mutant IκBα super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6-mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFα) production. Also, TNFα, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6- and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-κB- and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ. PUBLICATION ABSTRACT
Fra-1: a novel target for retinoid action Kaiser, Astrid; Brembeck, Felix H.; v. Marschall, Zofia ...
FEBS letters,
April 02, 1999, Letnik:
448, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Using a cDNA gene expression array system, we identified fra-1 as a novel target molecule for retinoid action in the human pancreatic carcinoma cell line Dan-G. Retinoid treatment resulted in a ...significant and time-dependent induction of the fra-1 expression on the post-transcriptional level. Supershift assays revealed that fra-1 participates in the activator protein 1 complex together with c-fos, c-jun and junB. Transient transfection experiments using a reporter plasmid containing an activator protein site upstream of the CAT reporter gene revealed that the phorbolester-induced CAT activity was suppressed by retinoids. Since fra-1 lacks a transactivation function, we therefore suggest that the retinoid-mediated induction of fra-1 might function as a negative regulator of the activator protein 1 activity in human pancreatic carcinoma cells.
We have investigated the role of protein kinase C (PKC) isoenzymes in the differential growth regulation of human pancreatic carcinoma cell lines by all-trans retinoic acid (RA). RA treatment results ...in dose-dependent stimulation of anchorage-independent growth in AsPc1 cells and growth inhibition in Capan 2 cells. Both cell lines express an identical pattern of nuclear RA and retinoid X receptors as determined by RT-PCR. Western blotting using monospecific antibodies revealed that both cell lines express PKC isoenzymes alpha and zeta, whereas beta, gamma, delta, and epsilon were not detected. Incubation with RA in the growth-stimulated AsPc1 cell line resulted in induction of PKC alpha expression, whereas PKC alpha expression was decreased by RA in the growth-inhibited Capan 2 cell line. In contrast, PKC zeta expression was not affected by RA in either cell line. Incubation of AsPc1 cells with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate resulted in a time- and dose-dependent selective down-regulation of PKC alpha but not zeta. The dose-dependent decrease of intracellular PKC alpha concentration correlated well with the anchorage-independent growth rate of AsPc1 cells. Furthermore, selective down-regulation of PKC alpha blocks subsequent growth stimulation by RA in AsPc1 cells. When PKC alpha concentration was decreased by stably transfecting AsPc1 cells with a PKC alpha complementary DNA antisense construct, RA-stimulated growth could also be partially blocked. These data, therefore, suggest that differential regulation of PKC alpha expression plays a central role in determining the bidirectional effects of RA on growth in pancreatic carcinoma cells.
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