In this study, we examined the role of protein kinase C (PKC)-E in the apoptosis and survival of glioma cells using tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-stimulated cells and silencing of PKCE expression. Treatment of glioma cells with TRAIL induced activation, caspasedependent cleavage, and down-regulation of PKCE within 3 to 5 hours of treatment. Overexpression of PKCE inhibited the apoptosis induced by TRAIL, acting downstream of caspase 8 and upstream of Bid cleavage and cytochrome c release from the mitochondria. A caspase-resistant PKCE mutant (D383A) was more protective than PKCE, suggesting that both the cleavage of PKCE and its down-regulation contributed to the apoptotic effect of TRAIL. To further study the role of PKCE in glioma cell apoptosis, we employed short interfering RNAs directed against the mRNA of PKCE and found that silencing of PKCE expression induced apoptosis of various glioma cell lines and primary glioma cultures. To delineate the molecular mechanisms involved in the apoptosis induced by silencing of PKCE, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that knockdown of PKCE did not affect the expression of Bcl2 and Bax or the phosphorylation and expression of Erk1/2, c-Jun-NH 2 -kinase, p38, or STAT, whereas it selectively reduced the expression of AKT. Similarly, TRAIL reduced the expression of AKT in glioma cells and this decrease was abolished in cells overexpressing PKCE. Our results suggest that the cleavage of PKCE and its down-regulation play important roles in the apoptotic effect of TRAIL. Moreover, PKCE regulates AKT expression and is essential for the survival of glioma cells. (Cancer Res 2005; 65(16): 7301-9)

Protein kinase C␦ (PKC␦) regulates cell apoptosis in a cell-and stimulus-specific manner. Here, we studied the role of PKC␦ in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKC␦ kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKC␦ mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKC␦ decreased it. PKC␦ acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKC␦ within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKC␦ was essential for its protective effect because overexpression of a caspaseresistant mutant (PKC␦D327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKC␦ to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKC␦ on tyrosine 155. Using a PKC␦Y155F mutant, we found that the phosphorylation of PKC␦ on tyrosine 155 was essential for the cleavage of PKC␦ in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKC␦ on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKC␦ protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKC␦ on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKC␦.

Protein kinase CD (PKCD) regulates cell apoptosis and survival in diverse cellular systems. PKCD translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCD constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCD affects its apoptotic and survival functions. PKCD-Cyto, PKCD-Mito, and PKCD-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCD-ER. PKCD-Cyto and PKCD-Mito underwent cleavage, whereas no cleavage was observed in the PKCD-Nuc and PKCD-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCD-Cyto and PKCD-Mito. In contrast to the apoptotic effects of the PKCD-Cyto, PKCD-Mito, and PKCD-Nuc, the PKCD-ER protected the cells from tumor necrosis factor -related apoptosis-inducing ligand -induced and etoposideinduced apoptosis. Moreover, overexpression of a PKCD kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCD and increased tumor necrosis factor -related apoptosis-inducing ligand -induced apoptosis. The localization of PKCD differentially affected the activation of downstream signaling pathways. PKCD-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCD-Nuc increased c-Jun NH 2 -terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCD-Cyto, whereas c-Jun NH 2 -terminal kinase activation mediated the apoptotic effect of PKCD-Nuc. Our results indicate that the subcellular localization of PKCD plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways.

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell-derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell-derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.