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1.  Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells 
International Journal of Oncology  2012;41(6):1967-1976.
Glioblastoma multiforme is the most common primary brain tumor and one of the most aggressive types of cancer in adults. Survival signaling and apoptosis resistance are hallmarks of malignant glioma cells. However, recent studies have shown that other types of cell death such as autophagy can be induced in malignant glioma cells. This suggests that stimulation of this process may be explored in new therapeutic strategies against glioblastoma multiforme. Protein kinase CK2 is a highly conserved and constitutively active enzyme that promotes numerous cellular processes such as survival, proliferation and differentiation. CK2 has been found elevated in several malignancies including brain tumors, and to confer resistance against chemotherapeutic agents and apoptotic stimuli. Recently, we have shown that the siRNA-mediated downregulation of CK2 leads to cell death in DNA-PK-proficient human glioblastoma cells. We show, here, that lack of CK2 results in significant induction of autophagic cell death in two human glioblastoma cell lines, M059K and T98G, as indicated by the positive staining of cells with the acidotropic dye acridine orange, and the specific recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosome membranes. Induction of autophagy is accompanied by CK2-dependent decreased phosphorylation of p70 ribosomal S6 and AKT kinases and significantly reduced expression levels of Raptor. In contrast, phosphorylation and activity levels of ERK1/2 are enhanced suggesting an inhibition of the PI3K/AKT/mTORC1 and activation of the ERK1/2 pathways. Furthermore, siRNA-mediated silencing of CK2 results in increased mitochondrial superoxide production in both glioblastoma cell lines. However, mitochondrial reactive oxygen species release correlates with induction of autophagy only in T98G cells. Taken together, our findings identify CK2 as a novel component of the autophagic machinery and underline the potential of its downregulation to kill glioblastoma cells by overcoming the resistance to multiple anticancer agents.
doi:10.3892/ijo.2012.1635
PMCID: PMC3583692  PMID: 23007634
glioblastoma cells; autophagy; CK2; mammalian target of rapamycin; extracellular signaling-regulated protein kinase 1/2; reactive oxygen species
2.  Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage 
Background
The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks.
Results
In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks.
Conclusions
Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.
doi:10.1186/1471-2199-13-7
PMCID: PMC3316135  PMID: 22404984
3.  2-Triazenoazaindoles: A novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells 
International Journal of Oncology  2011;40(4):914-922.
Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases.
doi:10.3892/ijo.2011.1272
PMCID: PMC3584806  PMID: 22134789
2-triazenoazaindoles; pancreatic cancer; cell death; EGFR; HER-2
4.  Enhancing chemosensitivity to gemcitabine via RNA interference targeting the catalytic subunits of protein kinase CK2 in human pancreatic cancer cells 
BMC Cancer  2010;10:440.
Background
Pancreatic cancer is a complex genetic disorder that is characterized by rapid progression, invasiveness, resistance to treatment and high molecular heterogeneity. Various agents have been used in clinical trials showing only modest improvements with respect to gemcitabine-based chemotherapy, which continues to be the standard first-line treatment for this disease. However, owing to the overwhelming molecular alterations that have been reported in pancreatic cancer, there is increasing focus on targeting molecular pathways and networks, rather than individual genes or gene-products with a combination of novel chemotherapeutic agents.
Methods
Cells were transfected with small interfering RNAs (siRNAs) targeting the individual CK2 subunits. The CK2 protein expression levels were determined and the effect of its down-regulation on chemosensitization of pancreatic cancer cells was investigated.
Results
The present study examined the impact on cell death following depletion of the individual protein kinase CK2 catalytic subunits alone or in combination with gemcitabine and the molecular mechanisms by which this effect is achieved. Depletion of the CK2α or -α' subunits in combination with gemcitabine resulted in marked apoptotic and necrotic cell death in PANC-1 cells. We show that the mechanism of cell death is associated with deregulation of distinct survival signaling pathways. Cellular depletion of CK2α leads to phosphorylation and activation of MKK4/JNK while down-regulation of CK2α' exerts major effects on the PI3K/AKT pathway.
Conclusions
Results reported here show that the two catalytic subunits of CK2 contribute differently to enhance gemcitabine-induced cell death, the reduced level of CK2α' being the most effective and that simultaneous reduction in the expression of CK2 and other survival factors might be an effective therapeutic strategy for enhancing the sensitivity of human pancreatic cancer towards chemotherapeutic agents.
doi:10.1186/1471-2407-10-440
PMCID: PMC2931491  PMID: 20718998
5.  Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells 
The Journal of Experimental Medicine  2008;205(6):1447-1461.
We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand β-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) γ strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by β-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor β, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4+ T cells, IL-1β, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.
doi:10.1084/jem.20071450
PMCID: PMC2413040  PMID: 18490488
6.  Reciprocal Activating Interaction between Natural Killer Cells and Dendritic Cells 
We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-α. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2–activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4+ T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-γ and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.
doi:10.1084/jem.20010938
PMCID: PMC2193595  PMID: 11828007
dendritic cell maturation; cytotoxicity; natural killer cells; cytokines; human

Results 1-6 (6)