Broad Caspase Inhibitor zVAD-fmk Research Articles

Mithramycin A sensitizes cancer cells to TRAIL-mediated apoptosis by down-regulation of XIAP gene promoter through Sp1 sites

Mithramycin A is a DNA-binding antitumor agent, which has been clinically used in the therapies of several types of cancer and Paget's disease. In this study, we investigated the combined effect of mithramycin A and tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) on apoptosis of cancer cells. In Caki renal cancer cells, which are resistant to TRAIL, cotreatment with subtoxic doses of mithramycin A and TRAIL resulted in a marked increase in apoptosis. This combined treatment was also cytotoxic to Caki cells overexpressing Bcl-2 but not to normal mesengial cells. Moreover, apoptosis by the combined treatment with mithramycin A and TRAIL was dramatically induced in various cancer cell types, thus offering an attractive strategy for safely treating malignant tumors. Mithramycin A-stimulated TRAIL-induced apoptosis was blocked by pretreatment with the broad caspase inhibitor zVAD-fmk or Crm-A overexpression, showing its dependence on caspases. We found that mithramycin A selectively down-regulated XIAP protein levels in various cancer cells. Luciferase reporter assay and the chromatin immunoprecipitation assay using the XIAP promoter constructs show that mithramycin A down-regulates the transcription of XIAP gene through inhibition of Sp1 binding to its promoter. Although XIAP overexpression significantly attenuated apoptosis induced by mithramycin A plus TRAIL, suppression of XIAP expression by transfection with its small interfering RNA prominently enhanced TRAIL-induced apoptosis. We present here for the first time that mithramycin A-induced suppression of XIAP transcription plays a critical role in the recovery of TRAIL sensitivity in various cancer cells.

Proteasome inhibition by MG-132 induces apoptotic cell death and mitochondrial dysfunction in cultured rat brain oligodendrocytes but not in astrocytes

Proteasomal dysfunction has been implicated in neurodegenerative disorders and during aging processes. In frontotemporal dementias, corticobasal degeneration, and progressive supranuclear palsy, oligodendrocytes are specifically damaged. Application of proteasomal inhibitors to cultured oligodendrocytes is associated with apoptotic cell death. The present study was undertaken to investigate the death pathway activated in oligodendrocytes by proteasomal inhibition. Our data show that the proteasomal inhibitor MG-132 causes oxidative stress, as indicated by the upregulation of the small heat shock protein heme oxygenase-1 (HO-1) and the appearance of oxidized proteins. Activation of the mitochondrial pathway was involved in the apoptotic process. Mitochondrial membrane potential was disturbed, and cytochrome c was released from the mitochondria. Concomitantly, death-related caspases 3 and 9 were activated and poly(ADP-ribose)-polymerase cleavage occurred. MG-132-induced cell death, DNA-fragmentation, and caspase activation could be prevented by the broad caspase inhibitor zVAD-fmk. In contrast to oligodendrocytes, cultured astrocytes showed resistance to the treatment with proteasomal inhibitors and did not reveal cytotoxic responses. This was also observed in astrocytes differentiated in the presence of dibutyryl cyclic AMP. Hence, individual cells respond differently to proteasomal inhibition and the therapeutic use of proteasomal inhibitors, e.g. for the treatment of cancer or inflammatory diseases, needs to be carefully evaluated.

The anticancer drug mithramycin A sensitises tumour cells to apoptosis induced by tumour necrosis factor (TNF).

In this report we show that mithramycin considerably increases the direct cytotoxic effect of tumour necrosis factor (TNF) on tumour cells in vitro. Sensitisation to TNF-induced apoptosis was prevented by the broad caspase inhibitor zVAD-fmk, whereas overexpression of Bcl-2 had no effect. Mithramycin also potentiated cell death induced by Fas agonistic antibodies. In contrast, mithramycin reduced the percentage of cells undergoing apoptosis due to factor withdrawal. TNF-induced activation of NF-kappaB (NF-κB)-dependent gene expression was not modulated by mithramycin treatment. Concomitantly with the increased sensitivity, the protein level of the short-spliced cFLIP variant was downregulated. These results indicate that mithramycin enhances TNF-induced cell death in an NF-κB-independent manner, and suggest that the Fas-associated death domain protein plays a crucial role in the TNF-sensitising effect of mithramycin.

Mechanisms of viral induced caspase activation in human hepatoma cell lines

For a growing number of liver diseases like viral induced hepatitis or fulminant liver failure, apoptosis induction in hepatocytes plays a key role in the course of the disease. In the effector phase of apoptotic cell death a family of proteases, called caspases, is organized in a hierachial order, thus most members of this cascade are activated by other members being more upstream in the pathway. Therefore, investigation of the very first mechanisms leading to the activation of a so called initiator caspase like caspases-S or -9, are important steps in molecular diagnosis and treatment of various diseases. We have looked for new caspase activation pathways in various cell lines, including human hepatoma lines by infection in our model system with the paramyxovirus Sendai virus (SeV). We could show signs of apoptosis 15 to 20 h after SeV infection by TUNEL assay or FACS analysis of infected cells (e.g. HepG2). A direct influence of caspases on this process could be demonstrated by a complete inhibition of apoptosis by incubation of infected cells with the broad caspase inhibitor z-VADfmk. Subsequently we could not only detect the activation of caspase-3 which is regarded as an effector caspase being downstream of initiating events, but we could also demonstrate caspase-8 and -9 cleavage by immunoblotting in these cell lines. Previous work demonstrated caspase-S as the first caspase in the signal transduction via death receptors, whereas caspase-9 activation is regarded as the first activated caspase mediated by chemotherapeutic agents causing cytochrome c release from the mitochondria. Surprisingly neither the involvement of the death receptors CD95 or TNF-RI (inhibition experiments by decoy proteins), nor the release of cytochrome c (immunoblot, immunofluorescence) could be shown, indicating the existence of an alternative way of caspase-8 and/or caspase-9 activation. To further characterize this pathway we are currently investigating the involvement of other proteases, death receptors or even viral proteins in the activation of initiator caspases.