ABSTRACT
D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC(50) value in the nanomolar range. The IC(50) values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 mumol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G(0)-G(1) and G(2)-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036-induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036-mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036-induced DNA damage activated ataxia telangiectasia-mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21(WAF1) in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036-induced cell death was associated with DNA damage-mediated induction of ataxia telangiectasia-mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Organoselenium Compounds/pharmacology , Protein Serine-Threonine Kinases/metabolism , Pyrroles/pharmacology , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins , Carcinoma, Renal Cell/pathology , DNA Adducts/drug effects , Drug Resistance, Neoplasm/drug effects , Enzyme Activation/drug effects , Flow Cytometry , HT29 Cells , HeLa Cells , Humans , Male , Mice , Mice, Nude , Protein Kinase C/metabolism , Protein Kinase C beta , Protein Kinase C-alpha/metabolism , Reactive Oxygen Species/metabolism , S Phase/drug effects , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Mitogen activated protein kinases are of interest as research tools and as therapeutic target for certain physiological disorders. In this study, we found 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone 6 to be a selective inhibitor of MEK1 with an IC(50) of 0.38 microM. An open-chain homologue, 10, showed selective cytotoxicity against renal cancer in the NCI in vitro tumor screening. Structure-activity relationship study of eight compounds showed the cyclic imido-substituted chloro-1,4-naphthoquinone as more potent and selective MEK1 inhibitors than the open chain homologues. The imido-substituted chloro-1,4-naphthoquinones were synthesized in a straightforward fashion by refluxing 2-amino-3-chloro-1,4-naphthoquinone with the appropriate acid chloride or diacyl dichloride.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Naphthoquinones/chemical synthesis , Cell Line, Tumor , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Drug Design , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Humans , Imides/chemistry , MAP Kinase Kinase 1 , Molecular Structure , Naphthoquinones/pharmacology , Protein Kinase C/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
The lymphocyte-specific protein-tyrosine kinase Lck plays a critical role in T cell activation. In response to T cell antigen receptor binding Lck undergoes phosphorylation on serine residues that include serines 59 and 194. Serine 59 is phosphorylated by ERK mitogen-activated protein kinase. Recently, we showed that in mitotic T cells Lck becomes hyper-phosphorylated on serine residues. In this report, using one-dimensional phosphopeptide mapping analysis, we identify serine 59 as a site of in vivo mitotic phosphorylation in Lck. The mitotic phosphorylation of serine 59 did not require either the catalytic activity or functional SH2 or SH3 domains of Lck. In addition, the presence of ZAP-70 also was dispensable for the phosphorylation of serine 59. Although previous studies demonstrated that serine 59 is a substrate for the ERK MAPK pathway, inhibitors of this pathway did not block the mitotic phosphorylation of serine 59. These results identify serine 59 as a site of mitotic phosphorylation in Lck and suggest that a pathway distinct from that induced by antigen receptor signaling is responsible for its phosphorylation. Thus, the phosphorylation of serine 59 is the result of two distinct signaling pathways, differentially activated in response to the physiological state of the T cell.
