Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in neurofibromatosis type 1 (NF1) patients. Current treatment modalities have been largely unsuccessful in improving MPNST patient survival, making the identification of new therapeutic targets urgent. In this study, we found that interference with Usp9X, a deubiquitinating enzyme which is overexpressed in nervous system tumors, or Mcl-1, an anti-apoptotic member of the Bcl-2 family whose degradation is regulated by Usp9X, causes rapid death in human MPNST cell lines. Although both Usp9X and Mcl-1 knockdown elicited some features of apoptosis, broad spectrum caspase inhibition was ineffective in preventing knockdown-induced MPNST cell death suggesting that caspase-independent death pathways were also activated. Ultrastructural examination of MPNST cells following either Usp9X interference or pharmacological inhibition showed extensive cytoplasmic vacuolization and swelling of endoplasmic reticulum (ER) and mitochondria most consistent with paraptotic cell death. Finally, the Usp9X pharmacological inhibitor WP1130 significantly reduced human MPNST growth and induced tumor cell death in an in vivo xenograft model. In total, these findings indicate that Usp9X and Mcl-1 play significant roles in maintaining human MPNST cell viability and that pharmacological inhibition of Usp9X deubiquitinase activity could be a therapeutic target for MPNST treatment.

Simultaneous Interference with HER1/EGFR and RAC1 Signaling Drives Cytostasis and Suppression of Survivin in Human Glioma Cells in Vitro.

We have previously reported that combined inhibition of the epidermal growth factor receptor by erlotinib and of RAC1 by NSC23766 yielded a synergistic antiproliferative effect on established and primary cultured glioblastoma cells. The current study aimed at identifying the molecular mechanism. Staining for annexin V/PI or carboxyfluorescein succinimidyl ester was performed in order to determine the induction of apoptosis, necrosis or cytostasis in established and primary cultured glioblastoma cells. Moreover, expression of Ki-67 was determined by immunofluorescence, and the expression of cell cycle proteins was analysed by Western blot. Our data show that combined treatment with erlotinib and NSC23766 resulted in a reduced number of cell divisions, a significantly decreased Ki-67 expression, increased apoptosis and autophagy when compared to single agent treatments. On the molecular level, concomitant treatment with both agents resulted in a pronounced downregulation of cyclin D1, cyclin-dependent kinases 2, 4 and 6, as well as of survivin when compared to treatments with either agent alone. In conclusion, we demonstrate that combined treatment of human glioma cell lines in vitro with erlotinib and NSC23766 markedly inhibits cell division, induces apoptosis independent of caspase-3 activation and induces autophagy concomitant with suppression of survivin.

The XIAP inhibitor Embelin enhances TRAIL-mediated apoptosis in malignant glioma cells by down-regulation of the short isoform of FLIP.

Embelin has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Embelin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Embelin broadly sensitized malignant glioma cells to TRAIL-mediated apoptosis. Notably, human astrocytes were not significantly affected by the combined treatment consisting of Embelin and TRAIL. Combined treatment with Embelin and TRAIL augmented the activation of initiator caspases-8/-9 and effector caspases-3/-7, respectively. Furthermore, Embelin down-regulated the expression of the long- and short-isoform of c-FLIP. In addition, forced expression of the short isoform of c-FLIP (S) attenuated apoptosis induced by the combination of Embelin and TRAIL. Embelin did not modulate the mRNA levels of c-FLIP (S), suggesting that Embelin modulates the expression of c-FLIP in a posttranscriptional manner. In summary, the short isoform of c-FLIP is a key regulator in TRAIL-Embelin-mediated apoptosis in malignant glioma.

Daidzein overcomes TRAIL-resistance in malignant glioma cells by modulating the expression of the intrinsic apoptotic inhibitor, bcl-2.

The soy isoflavone Daidzein has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Daidzein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Daidzein in combination with TRAIL induces rapid apoptosis in glioma cells. Notably, human astrocytes were not affected by the combined treatment consisting of Daidzein and TRAIL. Combined treatment with Daidzein and TRAIL augmented the activation of caspase-9, suggesting that Daidzein modulated the intrinsic apoptotic pathway. Daidzein did not modulate the expression of death receptors, c-FLIP, XIAP and survivin. However, Daidzein down-regulated bcl-2 and over-expression of bcl-2 attenuated apoptosis induced by the combination of Daidzein and TRAIL. In summary, bcl-2 is a key regulator in TRAIL-Daidzein mediated cell death in malignant glioma.

Myricetin sensitizes malignant glioma cells to TRAIL-mediated apoptosis by down-regulation of the short isoform of FLIP and bcl-2.

The flavonoid Myricetin has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Myricetin, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Myricetin in combination with TRAIL induces rapid apoptosis in glioma cells. Notably, human astrocytes were not affected by the combined treatment consisting of Myricetin and TRAIL. Combined treatment with Myricetin and TRAIL augmented the activation of initiator caspases-8/-9 and effector caspases-3/-7. Furthermore, Myricetin down regulated the expression of the long and short isoform of c-FLIP and bcl-2 and over-expression of the short isoform of c-FLIP (S) and bcl-2 attenuated apoptosis induced by the combination of Myricetin and TRAIL. Furthermore, Myricetin did not modulate the mRNA levels of c-FLIP, suggesting that Myricetin modulates the expression of c-FLIP in a posttranscriptional manner. In summary, the short isoform of c-FLIP and bcl-2 are key regulators in TRAIL-Myricetin mediated cell death in malignant glioma.

Epigalocatechin-3-gallate (EGCG) downregulates PEA15 and thereby augments TRAIL-mediated apoptosis in malignant glioma.

EGCG is a flavonoid that exhibited therapeutic activity in cancer. In this study three glioblastoma cell lines (U87, A172 and U251) were treated with EGCG, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of EGCG in combination with TRAIL induces rapid apoptosis in TRAIL-resistant glioma cells, suggesting that this combined treatment may offer an attractive strategy for treating gliomas. EGCG treatment down-regulated phosphoprotein-enriched in astrocytes (PEA15) through an Akt (PKB)-dependent mechanism. In addition, over-expression of PEA15 attenuated cytotoxicity induced by the combination of EGCG and TRAIL. In summary, PEA15 is a key regulator in TRAIL-EGCG-mediated cell death in malignant glioma.

TRAIL-mediated apoptosis in malignant glioma cells is augmented by celecoxib through proteasomal degradation of survivin.

Celecoxib is a cyclooxygenase 2-selective nonsteroidal anti-inflammatory drug (NSAID) that exhibited therapeutic activity in cancer. In this study three malignant glioma, U87-MG, U251 and A172, were treated with celecoxib, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Single treatment with celecoxib (25-100muM) for 24h resulted in a concentration-dependant decrease of cellular viability in U87-MG, U251 and A172. Combining subtoxic concentrations of celecoxib with TRAIL strongly increased cell death in human malignant glioma cells. After 8h treatment with celecoxib we found down-regulation of the inhibitor of apoptosis protein survivin that was mediated by proteasomal degradation. In addition, over-expression of survivin not only attenuated celecoxib-induced cytotoxicity but also cytotoxicity induced by the combination of celecoxib and TRAIL. Taken together, in malignant glioma survivin is a key regulator in celecoxib- and TRAIL-celecoxib-mediated cell death.

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion.

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.

Regulation of XIAP and Smac/DIABLO in the rat hippocampus following transient forebrain ischemia.

We investigated the expression of XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac/DIABLO, a newly identified mitochondrial apoptogenig molecule in the hippocampus following transient global ischemia. Transient global ischemia produced by two-vessel occlusion triggers the delayed neuronal death of CA1 neurons in the hippocampus. We demonstrate that CA1 neuronal loss induced by ischemia (10 min) is preceded by a selective and marked elevation of catalytically active caspase-3 in these neurons, indicative of apoptosis. XIAP (X chromosome-linked inhibitor of apoptosis protein) is a member of the inhibitor of apoptosis (IAP) gene family that, in addition to suppressing cell death by inhibition of caspases, is involved in an increasing number of signalling cascades. The present study shows alterations in the levels of XIAP and of Smac/DIABLO (second mitochondrial activator of caspase) after cerebral ischemia. The protein levels of XIAP and the number of XIAP-positive cells were regulated by cerebral ischemia in a strictly time and region dependent manner. The largest change in XIAP-IR was observed in the CA1 sub field, which is the most vulnerable area of hippocampus. The mitochondrial expression level of Smac/DIABLO increased during reperfusion. Smac/DIABLO expression was associated with alteration of the XIAP levels and the appearance of activated form of caspase-3 within the hippocampus during reperfusion in spatial and temporal manners.