The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells.

Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo- and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to gamma-radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. gamma-radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by gamma-radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24-48 hr and slightly decreased the viability of the cells. gamma-radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133- cells and the CD133+ cells expressed higher levels of the autophagy-related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to gamma-radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to gamma-radiation.

Cilengitide induces autophagy-mediated cell death in glioma cells.

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

The role of Bcl-x(L) protein in nucleotide excision repair-facilitated cell protection against cisplatin-induced apoptosis.

Many anticancer drugs target the genomic DNA of cancer cells by generating DNA damage and inducing apoptosis. DNA repair protects cells against DNA damage-induced apoptosis. Although the mechanisms of DNA repair and apoptosis have been extensively studied, the mechanism by which DNA repair prevents DNA damage-induced apoptosis is not fully understood. We studied the role of the antiapoptotic Bcl-x(L) protein in nucleotide excision repair (NER)-facilitated cell protection against cisplatin-induced apoptosis. Using both normal human fibroblasts (NF) and NER-defective xeroderma pigmentosum group A (XPA) and group G (XPG) fibroblasts, we demonstrated that a functional NER is required for cisplatin-induced transcription of the bcl-x(l) gene. The results obtained from our Western blots revealed that the cisplatin treatment led to an increase in the level of Bcl-x(L) protein in NF cells, but a decrease in the level of Bcl-x(L) protein in both XPA and XPG cells. The results of our immunofluorescence staining indicated that a functional NER pathway was required for cisplatin-induced translocation of NF-kappaB p65 from cytoplasm into nucleus, indicative of NF-kappaB activation. Given the important function of NF-kappaB in regulating transcription of the bcl-x(l) gene and the Bcl-x(L) protein in preventing apoptosis, these results suggest that NER may protect cells against cisplatin-induced apoptosis by activating NF-kappaB, which further induces transcription of the bcl-x(l) gene, resulting in an accumulation of Bcl-x(L) protein and activation of the cell survival pathway that leads to increased cell survival under cisplatin treatment.

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues induces sustained activation of Erk1/2 via down-regulation of MKP-1: role in the apoptotic effect of etoposide.

The mechanism underlying the important role of protein kinase Cdelta (PKCdelta) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCdelta in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCdelta since the phosphorylation of Erk1/2 was inhibited by a PKCdelta-KD mutant and PKCdelta small interfering RNA. We recently reported that phosphorylation of PKCdelta on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCdeltatyrosine mutants, we found that the phosphorylation of PKCdeltaon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCdelta was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCdelta. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCdelta and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCdeltaon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.