NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance.

Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug- or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and that Hif-1alpha plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1alpha by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1alpha-dependent manner. As GLUT-1 was induced via Hif-1alpha under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1alpha-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.

Ethionine-dependent inhibition of acute-phase plasma protein synthesis in the rat.

Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins.

Synthesis and secretion of some plasma proteins by tissue slices of Morris hepatoma 7777 and liver from control and turpentine-injected rats.

Slices of Morris hepatoma 7777 or rat liver isolated from control or turpentine-injected rats were incubated for 2 h with 14C-leucine. Radioactivities incorporated into albumin, alpha-fetoprotein, fibrinogen, alpha 1-AP-globulin, haptoglobin and alpha 1-acid glycoprotein were determined after the proteins had been isolated from the incubation medium or tissue homogenate by immunoprecipitation with monospecific antisera. It was found that hepatoma synthesizes fibrinogen, alpha 1-AP-globulin and alpha 1-acid glycoprotein in the amounts comparable to rat liver, whereas formation of albumin and haptoglobin is reduced 5- to 10-fold. Local inflammation elicited by injection of turpentine to tissue donors increased formation of acute-phase protein in liver slices but had no effect on synthesis of these proteins in preparations of Morris hepatoma, although certain ultrastructural changes in the Golgi complex were observed not only in the liver but also in the tumour.

Changes in the blood level and affinity to concanavalin A of rat plasma glycoproteins during acute inflammation and hepatoma growth.

Plasma concentrations of ten individual proteins were measured by electroimmunoassay in young male Buffalo rats following injection of turpentine oil or implantation of Morris hepatoma 7777. The highest relative responses to inflammation and tumour growth were found for alpha 2-macroglobulin, alpha 1-acute-phase globulin and alpha 1-acid glycoprotein. As shown by crossed immuno-affinoelectrophoresis the concanavalin A-reactive fractions of the latter two glycoproteins were predominantly increased in plasma from injured and tumour-bearing rats.