ABSTRACT
Secretory clusterin protein (sCLU) is a general genotoxic stress-induced, pro-survival gene product implicated in aging, obesity, heart disease, and cancer. However, the regulatory signal transduction processes that control sCLU expression remain undefined. Here, we report that induction of sCLU is delayed, peaking 72 h after low doses of ionizing radiation, and is dependent on the up-regulation of insulin-like growth factor-1 as well as phosphorylation-dependent activation of its receptor (IGF-1 and IGF-1R, respectively). Activated IGF-1R then stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately transactivate the early growth response-1 (Egr-1) transcription factor, required for sCLU expression. Thus, ionizing radiation exposure causes stress-induced activation of IGF-1R-Src-Mek-Erk-Egr-1 signaling that regulates the sCLU pro-survival cascade pathway, important for radiation resistance in cancer therapy.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Glycoproteins/metabolism , Immediate-Early Proteins/metabolism , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Molecular Chaperones/metabolism , Receptor, IGF Type 1/metabolism , Transcription Factors/metabolism , src-Family Kinases/metabolism , Cell Line, Tumor , Cell Survival , Clusterin , DNA-Binding Proteins/genetics , Early Growth Response Protein 1 , Enzyme Activation , ErbB Receptors/metabolism , Glycoproteins/genetics , Humans , Immediate-Early Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Molecular Chaperones/genetics , Promoter Regions, Genetic , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Radiation, Ionizing , Receptor, IGF Type 1/genetics , Transcription Factors/genetics , src-Family Kinases/geneticsABSTRACT
PURPOSE: To explore the use of cyclodextrins (CD) to form inclusion complexes with beta-lapachone (beta-lap) to overcome solubility and bioavailability problems previously noted with this drug. METHODS: Inclusion complexes between beta-lap and four cyclodextrins (alpha-, beta-, gamma-, and HPbeta-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and 1H-NMR spectroscopy. Biologic activity and bioavailability of beta-lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). RESULTS: Phase solubility studies showed that beta-lap solubility increased in a linear fashion as a function of alpha-, beta-, or HPbeta-CD concentrations but not gamma-CD. Maximum solubility of beta-lap was achieved at 16.0 mg/ml or 66.0 mM with HPbeta-CD. Fluorescence and 1H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between beta-CD and HPbeta-CD with beta-lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of beta-lap in beta-CD or HPbeta-CD inclusion complexes (TD50 = 2.1 microM). Animal studies in mice showed that the LD50 value of beta-lap in an HPbeta-CD inclusion complex is between 50 and 60 mg/kg. CONCLUSIONS: Complexation of beta-lap with HPbeta-CD offers a major improvement in drug solubility and bioavailability.
Subject(s)
Adjuvants, Pharmaceutic/chemistry , Cyclodextrins/chemistry , Naphthoquinones/pharmacokinetics , alpha-Cyclodextrins , beta-Cyclodextrins , gamma-Cyclodextrins , 2-Hydroxypropyl-beta-cyclodextrin , Adjuvants, Pharmaceutic/pharmacology , Animals , Biological Availability , Cyclodextrins/pharmacology , Humans , Injections, Intraperitoneal , Lethal Dose 50 , Mice , Mice, Inbred C57BL , Naphthoquinones/administration & dosage , Naphthoquinones/pharmacology , Solubility , Tumor Cells, CulturedABSTRACT
The clusterin (CLU) protein has been reported to have both cytoprotective and cytotoxic activities. Previous data from our lab suggest that the secretory form of CLU (sCLU) is cytoprotective and induced after very low, nontoxic doses of ionizing radiation (IR: >0.02 Gy), while a nuclear form is cytotoxic. Cells must presumably suppress sCLU to stimulate cell death, however, factors regulating the stress-inducible expression of sCLU have not been elucidated. Here we demonstrate that p53 can suppress sCLU induction responses. A variety of cytotoxic agents stimulated sCLU expression and DNA damage was sufficient but not necessary for induction. IR-stimulated CLU promoter activity, with concomitant increases in CLU mRNA and protein, showed that CLU induction was delayed with maximal expression observed 48-96 h post-treatment. Expression of the human papillomavirus E6 protein in MCF-7 breast or RKO colon cancer cells enhanced basal CLU levels. Isogenically matched HCT116 colon cancer cell lines that differed only in p53 or p21 status, confirmed a role for p53 in the transcriptional repression of sCLU. Loss of functional p53 in HCT116:p53(-/-) cells augmented CLU de novo synthesis after IR exposure. Repression of sCLU protein levels by p53 may be important for the cascade of p53-mediated events leading to cell death after IR or other cytotoxic agent exposure.
