ABSTRACT
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.
Subject(s)
Maleimides/pharmacology , Mitochondria/metabolism , Protein Multimerization/drug effects , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Apoptosis/drug effects , BH3 Interacting Domain Death Agonist Protein/metabolism , Caspase 8/metabolism , Down-Regulation/drug effects , Enzyme Activation/drug effects , Gene Knockdown Techniques , Humans , Jurkat Cells , bcl-2-Associated X Protein/metabolismABSTRACT
Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.
Subject(s)
Gene Expression Regulation, Neoplastic , Homeodomain Proteins/metabolism , Protein Kinase C-alpha/metabolism , Protein Kinase C-delta/metabolism , Carcinoma , Cell Proliferation , Disease Progression , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Gene Silencing , Hep G2 Cells , Homeodomain Proteins/genetics , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mutagenesis, Site-Directed , Nanog Homeobox Protein , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/enzymology , Nasopharyngeal Neoplasms/pathology , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Promoter Regions, Genetic , Protein Kinase C-alpha/antagonists & inhibitors , Protein Kinase C-alpha/genetics , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , Transcription, GeneticABSTRACT
Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 µM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.
Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Maleimides/pharmacology , Telomerase/antagonists & inhibitors , Cell-Free System , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/toxicity , Humans , Inhibitory Concentration 50 , Jurkat Cells , Leukocytes, Mononuclear/drug effects , Maleimides/chemistry , Maleimides/toxicityABSTRACT
The transcription factor p63 belongs to the p53 protein family and plays an important role in epithelial development. Recent studies showed that p63 is over-expressed in some human squamous cell carcinomas of the head and neck, suggesting a role in carcinogenesis. The p63 gene contains two promoters and alternative promoter usage generates two groups of proteins with (TAp63) or without (ΔNp63) the transactivation domain. Although the roles of TAp63 in epithelial development have been described in numerous recent studies, the regulation of its expression has not been elucidated. In this study, we showed that the transcriptional activity of the TAp63 promoter and TAp63 protein level were both up-regulated by an increased c-jun activity in Hep3B human hepatocellular carcinoma cell. Moreover, the elevated TAp63 expression was coincided with an increased binding of c-jun to the TAp63 promoter. Point mutation of the sp1 binding site within the TAp63 promoter region attenuated the effect of c-jun on TAp63 expression. Knockdown of TAp63 expression by shRNA led to increased proliferation of Hep3B cell compared to that of the mock cell, suggesting a growth suppressive effect of TAp63.
