ABSTRACT
BACKGROUND: Possible predictive markers of response to neoadjuvant radiochemotherapy (NRCT) of esophageal cancer have been identified. PATIENTS AND METHODS: Patient biopsies were obtained from both tumor and normal tissue before the NRCT of locally advanced esophageal squamous cell carcinoma. Protein solutions were separated and immunoblot analysis was performed with heat shock protein (Hsp)16.2, heme-binding protein 2 (SOUL), BCL2-associated X protein (Bax), B-cell-associated leukemia protein 2 (Bcl-2) and heat shock protein 90 (Hsp90) antibodies. Following NRCT, the patients were restaged according to the Response Evaluation Criteria In Solid Tumors (RECIST). Following resections the pathological down-staging was evaluated. RESULTS: Clinical restaging revealed a response rate of 65%. Pathological examination revealed down-staging in 30% and 25% of the cases for the T and N categories respectively. Compared to the normal esophageal mucosa, a decreased expression of Hsp16.2, Hsp90 and SOUL proteins and an increased Bax/Bcl-2 ratio was found in the responding tumors. CONCLUSION: Hsp16,2, Hsp90 and SOUL expression and Bax/ Bcl-2 ratio correlates to the efficacy of NRCT and predict outcome in patients with locally advanced squamous-cell esophageal cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/therapy , Esophageal Neoplasms/therapy , Neoadjuvant Therapy , Neoplasm Proteins/metabolism , Adult , Aged , Blotting, Western , Carcinoma, Squamous Cell/metabolism , Case-Control Studies , Chemotherapy, Adjuvant , Cisplatin/administration & dosage , Combined Modality Therapy , Esophageal Neoplasms/metabolism , Esophagus/drug effects , Esophagus/metabolism , Esophagus/radiation effects , Female , Fluorouracil/administration & dosage , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins, Small/metabolism , Heme-Binding Proteins , Hemeproteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Male , Middle Aged , Neoplasm Staging , Pregnancy Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Radiotherapy, Adjuvant , Survival Rate , Treatment Outcome , bcl-2-Associated X Protein/metabolismABSTRACT
We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t-TIP47) protected NIH3T3 cells from hydrogen-peroxide-induced cell death, prevented the hydrogen-peroxide-induced mitochondrial depolarization determined by 5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative-stress-induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t-TIP47 increased the mitochondrial membrane potential (Deltapsi), and partially prevented Ca2+ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.
Subject(s)
Apoptosis/drug effects , Apoptosis/genetics , Carrier Proteins/physiology , DNA-Binding Proteins/physiology , Intracellular Signaling Peptides and Proteins/physiology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Pregnancy Proteins/physiology , Animals , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flow Cytometry , HeLa Cells , Humans , Hydrogen Peroxide/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/genetics , Mice , NIH 3T3 Cells , Necrosis/chemically induced , Oxidative Stress/drug effects , Oxidative Stress/genetics , Perilipin-3 , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Vesicular Transport ProteinsABSTRACT
Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.
Subject(s)
Cell Survival/drug effects , Choriocarcinoma/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Placenta/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Choriocarcinoma/pathology , Female , Humans , Immunosuppressive Agents/pharmacology , Methotrexate/pharmacology , Pregnancy , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Radioimmunoassay , Random AllocationABSTRACT
The pituitary adenylate cyclase activating polypeptide (PACAP) has several effects in endocrine and reproductive organs, including the placenta. PACAP is generally known as a survival-promoting peptide acting on divergent signal transduction pathways. However, its effects on the survival and signaling mechanisms of trophoblast cells are not known. In the present study we found that 1-h pretreatment with PACAP38 did not significantly influence the survival of JAR cytotrophoblast cells. However, the survival rate of cells exposed to oxidative stress or CoCl(2)-induced in vitro hypoxia showed a significant further decrease in PACAP-treated cells, implying that PACAP sensitizes the cells to these stressors. This was not observed in the case of lipopolysaccharide or ethanol treatment. Western blot data revealed that, in cells exposed to oxidative stress, PACAP treatment decreased phosphorylation of all extracellular signal-regulated kinase (ERK), phospho-jun N-terminal kinase (JNK), protein kinase B, p38 mitogen-activated protein kinase (MAPK), and phospho-glycogen synthase kinase (GSK) and the expression of bax. The overall effect seems to be a sensitizing effect in almost all examined pathways when oxidative stress was applied, which may explain the enhancing effect of PACAP on cell death in contrast to most other cell types examined so far. Our data show that the signaling mechanism of PACAP may be different in trophoblast cells to that observed in other cell lines.
Subject(s)
Choriocarcinoma/pathology , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Signal Transduction/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Hydrogen Peroxide/pharmacologyABSTRACT
Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.