Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.
We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.
TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.
A critical problem in the development and implementation of stem cell-based therapy is the lack of reliable, noninvasive means to image and trace the cells post-transplantation and evaluate their biodistribution, final fate, and functionality. In this study, we developed a gold nanoparticle-based CT imaging technique for longitudinal mesenchymal stem cell (MSC) tracking within the brain. We applied this technique for noninvasive monitoring of MSCs transplanted in a rat model for depression. Our research reveals that cell therapy is a potential approach for treating neuropsychiatric disorders. Our results, which demonstrate that cell migration could be detected as early as 24 h and up to one month post-transplantation, revealed that MSCs specifically navigated and homed to distinct depression-related brain regions. We further developed a noninvasive quantitative CT ruler, which can be used to determine the number of cells residing in a specific brain region, without tissue destruction or animal scarification. This technique may have a transformative effect on cellular therapy, both for basic research and clinical applications.
Protein kinase Cδ (PKCδ) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKCδ are involved in these two responses. Transfection of cells with PKCδ mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKCδ wild-type transfectant. Conversely, transfection with PKCδ mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKCδ wild-type transfectant. The tyrosine phosphorylation of PKCδ and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKCδ via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKCδ induced by PDGF. We conclude that the tyrosine phosphorylation of PKCδ and its association with tyrosine kinases may be an important point of divergence in PKC signaling.
Abstract BACKGROUND Recent clinical studies suggest beneficial effects of propofol anesthesia on tumor progression and patient survival in solid tumors but reported benefits are modest. One potential reason is the relatively short, single exposure to propofol, limited to the surgical period. Brain metastases (BM) are the most common brain tumors in adults. Metastatic tumors develop following infiltration of the brain from primary tumors such as lung, breast, melanoma, and colorectal cancers. BM are treated with combination therapies, including surgery, radiotherapy, chemotherapy, and immunotherapy, however the prognosis of most patients with BM remains dismal. In this report we investigated the effects of propofol plus radiation on cancer stem cells derived from human lung cancer brain metastases (BM-CSCs) and their cross-talk with microglia. OBJECTIVES Our hypothesis is that propofol can be repurposed as a treatment of BM in addition to its anesthetic uses. To test this, we first examined the cytotoxic effects of propofol on cancer stem cells established from BM-CSCs alone and with radiation. Also, we studied the effects of propofol on the cross-talk of BM-CSCs and microglia. RESULTS We found that propofol 1) exerted inhibitory effect on BM-CSCs self-renewal, stemness and cell proliferation; 2) increased cell death of cancer cells but not normal neural elements; 3) sensitized BM-CSCs to radiation; 4) inhibited the pro-tumorigenic BM-CSCs/ microglia cross-talk by promoting M1 phenotypes of co-cultured microglia. CONCLUSIONS Propofol exerted anti-tumor effects on BM-CSCs including inhibition of cell renewal, proliferation, and mesenchymal transition. Propofol at sensitized BM-GSCs to radiation and at higher concentrations induced cell death. Propofol exerted anti-tumor cytotoxicity also by inhibiting the pro-tumorigenic CSC-microglia cross-talk via secreted extracellular vesicles (EVs). Propofol effects can be exploited as a general anesthetic of choice during tumor resection and should be examined as an anti-tumor agent in sub-anesthetic doses either alone or in combination with radiation.
Abstract Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here we use an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1,028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA approved drugs led to three promising candidates, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). We show that each of these compounds has the ability to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appear to act on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT. Abstract Figure
