ABSTRACT
The biological functions and reaction pathways of lipocalins in mammalian system were sought. Mouse uterine 24p3 protein is a secreted lipocalin from mouse uterus. To evaluate the effect of uterine 24p3 protein on the reproductive system, endometrial carcinoma cell line (RL95-2) was an experimental target for achieving the in vitro study. The cells were treated with 0.75 microM dexamethasone (DEX) or under serum-free medium to mimic the stress environment for various time periods, then employing Western blot to measure the 24p3 protein secretion. It showed the time-dependent induction effect on 24p3 protein and suggested the level of protein secretion correlating to environmental stress. Furthermore, the supplementation of 24p3 protein to the medium accompanied the reduction of cell viability. It showed that the 24p3 protein may be a death factor under conditional media via PI and annexinV-FITC assay. Based on the autocrine hypothesis, we investigated the effect of 24p3 protein on cultured RL95-2 cells upon the 24p3 protein interaction. We have demonstrated significant increase in intracellular reactive oxygen species upon 24p3 protein interaction. While the changes of mitochondrial membrane potential and cytochrome c release from mitochondria occurred, the activities of caspase-8, -9 and -3 were found to have increased. The condensation of DNA was occurred suggesting that 24p3 protein induced irreparable DNA damage, which in turn triggered the process of apoptosis. It shows evidence for the direct effect of this protein on endometrial cells. These findings suggest that 24p3 protein creates an intracellular oxidative environment that induces apoptosis in RL95-2 cells.
Subject(s)
Acute-Phase Proteins/toxicity , Apoptosis/drug effects , Carcinoma/pathology , Endometrial Neoplasms/pathology , Proto-Oncogene Proteins/toxicity , Uterus/metabolism , Blotting, Western , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Coloring Agents , Culture Media , Cytochromes c/metabolism , DNA, Neoplasm/genetics , Dexamethasone/pharmacology , Female , Flow Cytometry , Fluorescein-5-isothiocyanate , HeLa Cells , Humans , Lipocalin-2 , Lipocalins , Membrane Potentials/drug effects , Microscopy, Fluorescence , Mitochondria/drug effects , Reactive Oxygen Species/metabolism , Trypan Blue , Uterus/chemistryABSTRACT
Serum amyloid P component (AP) and C-reactive protein (CRP) are normal serum components which belong to the pentraxin family of proteins. These proteins have been previously localized by immunohistochemical method to the brain lesions of Alzheimer's disease (AD). AP is a constant constituent of amyloid deposits including those found in AD. Both AP and CRP have been localized to AD neurofibrillary tangles. An indirect role for these proteins has been previously suggested in the etiology of AD. We studied the effects of serum AP and CRP on a human-derived neuronal cell line (hNT). In treated cell cultures, AP and CRP were detected immunohistochemically within hNT neurons, indicating cellular uptake of these proteins. Serum AP at the lowest serum physiological concentration (8 microgram/ml) showed a marked toxicity to hNT neurons. CRP also displayed toxicity to the hNT neurons but at a level compatible with inflammatory states (50 microgram/ml). These results suggest a more direct role for serum AP and CRP in the pathogenesis of AD.
Subject(s)
Alzheimer Disease/metabolism , C-Reactive Protein/toxicity , Neurons/enzymology , Serum Amyloid P-Component/toxicity , Acute-Phase Proteins/toxicity , Antibodies , Blood Proteins/pharmacology , Blotting, Western , C-Reactive Protein/analysis , C-Reactive Protein/immunology , Fluorescent Antibody Technique , Humans , L-Lactate Dehydrogenase/metabolism , Neurofilament Proteins/analysis , Neurofilament Proteins/immunology , Neurons/chemistry , Neurons/drug effects , Serum Amyloid P-Component/analysis , Serum Amyloid P-Component/immunology , Teratocarcinoma , Tumor Cells, Cultured/metabolismABSTRACT
Vascular leakage in the middle ear cavity was studied after i.v. administration of various substances in rats and determined by the Evans blue technique. Bradykinin, histamine, serotonin, acetylcholine, substance P (SP) and vasoactive intestinal polypeptide (VIP) resulted in extravasation of Evans blue. In the case of bradykinin and histamine, the leakage was dose dependent. Calcitonin gene-related peptide (CGRP) did not affect vessel permeability. In other experiments the effect of histamine antagonists was tested on production of middle ear effusion, caused by blowing air at 14 degrees C into the external auditory canal (EAC). The increase in vessel permeability in this otitis media model was inhibited by the H2-receptor antagonist cimetidine, at doses 0.1 and 1.0 mg/ml. Diphenhydramine, an H1-receptor antagonist, arrested only partly middle ear fluid accumulation. Our study demonstrated that various inflammatory mediators and neuropeptides are capable of inducing vascular leakage in the middle ear cavity. It was also concluded that H2-receptors are involved in the regulation of middle ear vascular permeability.
