ABSTRACT
The aim of our study was to compare the characteristics and time to initial physician contact in patients with head and neck squamous cell carcinoma (HNSCC) before and during the COVID-19 pandemic at a large Hungarian cancer center. This was a retrospective study of patients 18 years or older presenting at the regional cancer center of Pécs Clinical Center with HNSCC between 1 January 2017, and 15 March 2020 (the pre-COVID-19 period) and between 16 March 2020, and 13 May 2021 (the COVID-19 period). Demographic and clinical data were collected, and the time between initial symptom onset and initial physician contact (TTP) was determined. Descriptive and exploratory statistical analyses were performed. On average, the number of patients diagnosed with HNSCC per month during the pandemic decreased by 12.4% compared with the pre-COVID-19 period. There was a significant increase in stage I and stage II cancers (from 15.9% to 20.3% and from 12.2% to 13.8%, respectively; p < 0.001); a decrease in stage III and IVa,c cancers; and a significant increase in stage IVb cancers (from 6% to 19.9%; p < 0.001) during the pandemic. The median TTP increased during the pandemic from 43 to 61 days (p = 0.032). To our knowledge, this is the first study investigating the effect of COVID-19 on patients with HNSCC in the Central-Eastern European region. We found a bidirectional shift in cancer stages and increased TTP during the pandemic. Our findings highlight the necessity for more nuanced analyses of the effects of COVID-19.
ABSTRACT
BACKGROUND: Rectal cancer constitutes nearly one-third of all colorectal cancer diagnoses, and certain clinical and molecular markers have been studied as potential prognosticators of patient survival. The main objective of our study was to investigate the relationship between the expression intensities of certain proteins, including growth-hormone-releasing hormone receptor (GHRH-R), Hsp90, Hsp16.2, p-Akt and SOUL, in specimens of locally advanced rectal cancer patients, as well as the time to metastasis and 10-year overall survival (OS) rates. We also investigated whether these outcome measures were associated with the presence of other clinical parameters. METHODS: In total, 109 patients were investigated retrospectively. Samples of pretreatment tumors were stained for the proteins GHRH-R, Hsp90, Hsp16.2, p-Akt and SOUL using immunhistochemistry methods. Kaplan-Meier curves were used to show the relationships between the intensity of expression of biomarkers, clinical parameters, the time to metastasis and the 10-year OS rate. RESULTS: High levels of p-Akt, GHRH-R and Hsp90 were associated with a significantly decreased 10-year OS rate (p = 0.001, p = 0.000, p = 0.004, respectively) and high expression levels of p-Akt and GHRH-R were correlated with a significantly shorter time to metastasis. Tumors localized in the lower third of the rectum were linked to both a significantly longer time to metastasis and an improved 10-year OS rate. CONCLUSIONS: Hsp 90, pAkt and GHRH-R as well as the lower-third localization of the tumor were predictive of the 10-year OS rate in locally advanced rectal cancer patients. The GHRH-R and Hsp90 expression levels were independent prognosticators of OS. Our results imply that GHRH-R could play a particularly important role both as a molecular biomarker and as a target for the anticancer treatment of advanced rectal cancer.
ABSTRACT
BACKGROUND: Previous studies have shown that patients who underwent renal transplantation were at a greater risk of developing malignancies. Due to advances in effective surgical techniques and immunosuppressive therapies, organ recipients live longer. Yet, there is insufficient information about the recommended type of therapy for colorectal cancer patients following transplantation. We describe the oncological treatment of a patient with renal transplantation, who presented with metastatic colon cancer 5 years after transplantation. CASE PRESENTATION: A 66-year-old Caucasian male patient, with hypertension, type 2 diabetes mellitus, paroxysmal atrial fibrillation, and renal failure underwent successful kidney transplantation in 2013. In April 2018, the adenocarcinoma of the sigmoid colon was found, and surgical resection was performed. The histological diagnosis was low-grade adenocarcinoma. Fluorodeoxyglucose positron emission tomography/computerized tomography scan showed a 2.5-cm metastasis in the VIIth segment of the liver and a metastatic paraaortical lymph node on the left. The clinical diagnosis was, therefore, metastatic (stage IV) sigmoid colon cancer (AJCC TNM system). The ongoing medications of the patient included immunosuppressive drugs and medication for his cardiovascular comorbidities. In July 2018, palliative cetuximab plus folinic acid-fluorouracil-irinotecan chemotherapeutic treatment was initiated, then cetuximab was substituted for panitumumab because of adverse events. In August 2018, the follow-up positron emission tomography/computerized tomography scan revealed stable disease. Because of side effects, the patient was unwilling to continue with the panitumumab plus folinic acid-fluorouracil-irinotecan treatment regimen. Therefore, the patient received 10× 5 Gy stereotactic body irradiation for his liver metastasis and mono-panitumumab therapy. By January 2019, the positron emission tomography/computerized tomography scan showed regression of the liver metastasis but a progression in the paraaortic lymph node. Therefore, 5× 8 Gy stereotactic irradiation was given to the paraaortic lesion. Meanwhile, the patient received altogether 16 cycles of panitumumab until June 2019, when complete remission was attained. In July 2019, the patient suffered a hemorrhagic stroke, probably due to his cardiovascular comorbidities, and died subsequently. CONCLUSIONS: Since information is scarce regarding oncological treatment of patients following organ transplantation, data about their oncological treatment is essential. To our knowledge, this is the first case report to describe the successful chemotherapy and targeted therapy supplemented with stereotactic radiotherapy of a posttransplant patient with metastatic colorectal cancer.
Subject(s)
Colorectal Neoplasms , Diabetes Mellitus, Type 2 , Kidney Transplantation , Rectal Neoplasms , Aged , Antineoplastic Combined Chemotherapy Protocols , Cetuximab , Colorectal Neoplasms/drug therapy , Fluorouracil/therapeutic use , Humans , Male , Panitumumab/therapeutic useABSTRACT
Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)-a metabolite of amiodarone-may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases-ERK and Akt-are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies.
Subject(s)
Amiodarone/analogs & derivatives , Apoptosis/drug effects , Urinary Bladder Neoplasms/pathology , Amiodarone/pharmacology , Cell Line, Tumor , Humans , Membrane Potential, Mitochondrial/drug effectsABSTRACT
With the development of radiation therapy technology, the utilization of more accurate patient fixation, inclusion of PET/CT image fusion into treatment planning, 3D image-guided radiotherapy, and intensity-modulated dynamic arc irradiation, the application of hypofractionated stereotactic radiotherapy can be extended to specified extracranial target volumes, and so even to the treatment of various metastases. Between October 2012 and August 2014 in our institute we performed extracranial, hypofractionated, image-többguided radiotherapy with RapidArc system for six cases, and 3D conformal multifield technique for one patient with Novalis TX system in case of different few-numbered and slow-growing metastases. For the precise definition of the target volumes we employed PET/CT during the treatment planning procedure. Octreotid scan was applied in one carcinoid tumour patient. Considering the localisation of the metastases and the predictable motion of the organs, we applied 5 to 20 mm safety margin during the contouring procedure. The average treatment volume was 312 cm3. With 2.5-3 Gy fraction doses we delivered 39-45 Gy total dose, and the treatment duration was 2.5 to 3 weeks. The image guidance was carried out via ExacTrac, and kV-Cone Beam CT equipment based on an online protocol, therefore localisation differences were corrected before every single treatment. The patients tolerated the treatments well without major (Gr>2) side effects. Total or near total regression of the metastases was observed at subsequent control examinations in all cases (the median follow-up time was 5 months). According to our first experience, extracranial, imageguided hypofractionated radiotherapy is well-tolerated by patients and can be effectively applied in the treatment of slow-growing and few-numbered metastases.
Subject(s)
Neoplasms/pathology , Neoplasms/radiotherapy , Radiosurgery , Radiotherapy Planning, Computer-Assisted , Radiotherapy, Conformal/methods , Radiotherapy, Image-Guided , Adenocarcinoma/pathology , Adenocarcinoma/radiotherapy , Adenocarcinoma of Lung , Adrenal Gland Neoplasms/radiotherapy , Adrenal Gland Neoplasms/secondary , Adult , Aged , Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Cancer Care Facilities/trends , Colonic Neoplasms/pathology , Colonic Neoplasms/radiotherapy , Cone-Beam Computed Tomography , Dose Fractionation, Radiation , Female , Humans , Hungary , Lung Neoplasms/pathology , Lung Neoplasms/radiotherapy , Male , Middle Aged , Neoplasm Staging , Radiotherapy Planning, Computer-Assisted/methods , Rectal Neoplasms/pathology , Rectal Neoplasms/radiotherapy , Tumor Burden , UniversitiesABSTRACT
UNLABELLED: Abstract Purpose: Sensitizing cancer cells to irradiation is a major challenge in clinical oncology. We aimed to define the signal transduction pathways involved in poly(ADP-ribose) polymerase (PARP) inhibitor-induced radiosensitization in various mammalian cancer lines. MATERIALS AND METHODS: Clonogenic survival assays and Western blot examinations were performed following telecobalt irradiation of cancer cells in the presence or absence of various combinations of PARP- and selective mitogen-activated protein kinase (MAPK) inhibitors. RESULTS: HO3089 resulted in significant cytotoxicity when combined with irradiation. In human U251 glioblastoma and A549 lung cancer cell lines, Erk1/2 and JNK/SAPK were found to mediate this effect of HO3089 since inhibitors of these kinases ameliorated it. In murine 4T1 breast cancer cell line, p38 MAPK rather than Erk1/2 or JNK/SAPK was identified as the main mediator of HO3089's radiosensitizing effect. Besides the aforementioned changes in kinase signaling, we detected increased p53, unchanged Bax and decreased Bcl-2 expression in the A549 cell line. CONCLUSIONS: HO3089 sensitizes cancer cells to photon irradiation via proapoptotic processes where p53 plays a crucial role. Activation of MAPK pathways is regarded the consequence of irradiation-induced DNA damage, thus their inhibition can counteract the radiosenzitizing effect of the PARP inhibitor.
Subject(s)
Benzimidazoles/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Poly(ADP-ribose) Polymerase Inhibitors , Protein Kinase Inhibitors/pharmacology , Radiation Tolerance/drug effects , Animals , Cell Line, Tumor , Humans , Mice , Signal Transduction/drug effects , Signal Transduction/radiation effectsABSTRACT
The goal of the present study was to compare the efficacy of treatment with irradiation (IR), temozolomide, and quercetin, alone, or in combinations, on 2 glioblastoma cell lines, DBTRG-05 and U-251. Cell viability assay, flow cytometry analysis, colony formation assay, and Western blot analysis were used to compare the effects of treatment on the 2 cell lines. The greatest reduction in cell viability and colony formation was observed when cells were treated with a combination of the agents including quercetin. The treatment of cells with the combination of IR and quercetin was equal to the efficiency of the combination of IR and temozolomide in decreasing cell viability as well as colony formation. Quercetin alone, or in combination with IR, increased the cleavage of caspase-3 and PARP-1 showing an activated apoptosis and significantly reduced the level of phospho-Akt. Moreover, these treatments increased the levels of phospho-ERK, phospho-JNK, phospho-p38, and phospho-RAF1. Our data indicate that the supplementation of standard therapy with quercetin increases efficacy of treatment of experimental glioblastoma through synergism in the induction of apoptosis via the cleavage of caspase-3 and PARP-1 and by the suppression of the actitivation of Akt pathway.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Quercetin/pharmacology , Signal Transduction , Apoptosis/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chemoradiotherapy , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Glioblastoma/drug therapy , Glioblastoma/radiotherapy , Humans , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction/drug effects , Signal Transduction/radiation effects , TemozolomideABSTRACT
We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.
Subject(s)
Cell Membrane Permeability , Hemeproteins/chemistry , Hemeproteins/metabolism , Mitochondrial Membranes/metabolism , Oxidative Stress , Pregnancy Proteins/chemistry , Pregnancy Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/chemistry , Sequence Homology, Amino Acid , Amino Acid Sequence , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cattle , Cell Membrane Permeability/drug effects , Cell Survival , Peptidyl-Prolyl Isomerase F , Cyclophilins/pharmacology , Gene Expression Regulation , HeLa Cells , Heme-Binding Proteins , Hemeproteins/deficiency , Hemeproteins/genetics , Humans , Hydrogen Peroxide/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondrial Membranes/drug effects , Mitochondrial Proteins/metabolism , Molecular Sequence Data , NIH 3T3 Cells , Oxidative Stress/drug effects , Pregnancy Proteins/deficiency , Pregnancy Proteins/genetics , Protein Structure, Tertiary , RNA, Small Interfering/genetics , Rats , Sequence DeletionABSTRACT
Galectin-13 transcripts have been identified in several normal and malignant tissues, but the physiological function of galectin-13 is still poorly understood. Here, we present evidence for its possible role in promoting cell death in the U-937 human macrophage cell line. Transfection of U-937 human macrophages by a galectin-13 cDNA-containing mammalian expression vector increased the galectin-13 level and sensitized the cells to stress stimuli. Galectin-13 overexpression facilitated paclitaxel-induced cell death and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease-G without inducing mitochondrial cytochrome-c release or caspase-3 activation. Immunoblot and immunofluorescence data showed that overexpression of galectin-13 induced long-term activation of c-Jun N-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) pathways, as well as activation of apoptosis signal-regulating kinase-1 (Ask-1) kinase while it suppressed paclitaxel-induced long-term activation of the phosphatidilylositol-3-kinase (PI-3K)-Akt and extracellular signal-regulated kinase (ERK1/2) cytoprotective pathways. In addition, pharmacological inhibition of JNK and p38-MAPK pathways protected the cells from paclitaxel-induced cell death. All this data indicate that galectin-13 overexpression promoted apoptosis presumably by activating the Ask-1 kinase-JNK and p38-MAPK pro-apoptotic pathways and by suppressing the PI-3K-Akt and ERK1/2 cytoprotective pathways.
Subject(s)
Apoptosis/drug effects , Galectins/biosynthesis , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Paclitaxel/pharmacology , Pregnancy Proteins/biosynthesis , Apoptosis/physiology , Cell Culture Techniques , Cell Survival/drug effects , Enzyme Activation , Humans , Immunoblotting , MAP Kinase Kinase 4/metabolism , MAP Kinase Kinase Kinase 5/metabolism , Macrophages/drug effects , Macrophages/enzymology , Proto-Oncogene Proteins c-akt/metabolism , Transfection , U937 Cells , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
UNLABELLED: The aim of this study was to find a possible clinical use of the tail-interacting protein of 47 kDa (TIP47) and further document its expression in smear cytology, different cervical dysplasias, invasive cervical cancer and metastasis. PATIENTS AND METHODS: A new polyclonal anti-TIP47 antibody was developed and used on smears and histological cervix sections of sixty women with different cytological pathologies. Serum TIP47 level of patients with cervical intraepithelial neoplasia (CIN) or carcinoma in stage IIb, IIIa, and IIIb was monitored during treatment. RESULTS: TIP47 was expressed weakly in the dysplasias, stronger in invasive tumors and in lymph node metastasis. In patients with cervical carcinoma, the serum TIP47 level was found to be elevated; it decreased after therapy and elevated again in relapse. CONCLUSION: According to our results, TIP47 could be a good clinical marker for the early detection in blood of the recurrence of cervical carcinoma.
Subject(s)
Biomarkers, Tumor/metabolism , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Pregnancy Proteins/metabolism , Uterine Cervical Dysplasia/metabolism , Uterine Cervical Neoplasms/metabolism , Animals , Biomarkers, Tumor/blood , Blotting, Western , Cell Line, Tumor , Cytoplasm/metabolism , DNA-Binding Proteins/blood , Female , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/blood , Mice , NIH 3T3 Cells , Perilipin-3 , Pregnancy Proteins/blood , Uterine Cervical Dysplasia/blood , Uterine Cervical Dysplasia/pathology , Uterine Cervical Neoplasms/blood , Uterine Cervical Neoplasms/pathology , Vaginal Smears , Vesicular Transport ProteinsABSTRACT
We investigated the response rate and side effects of simultaneous, neoadjuvant radiochemotherapy (RCT) in locally advanced rectal cancer. Between 2005 and 2007, we treated 112 patients in stage II-III rectal carcinoma at the Institute of Oncotherapy, University of Pécs. For staging abdomino-pelvic CT (112) and transrectal US (49) or pelvic MR (10), or PET-CT (1) was performed. Radiation therapy was delivered with 3D CRT-based technique using belly-board with 18 MV photon energy, while patients in prone position. A total dose of 45 Gy (single dose 1.8 Gy) was delivered to the tumor and the pelvic lymph nodes. 5-FU and Ca-folinate was administered concomitantly in the 1st and 5th week of radiotherapy. Four weeks after delivering neoadjuvant RCT the patients' control CT was evaluated according to RECIST criteria. RCT was followed by surgery in 6-9 weeks. We graded the histology using the Mandard regression score system. Side effects were registered using CTCAE v 3.0. Grade 1, 2 or 3 acute gastrointestinal toxicity occurred in 12%, grade 3 hematological toxicity in 9.5% of the patients. The response rate determined by using control CT was 64.85%. According to the Mandard regression score, TRG1 occurred in 15%, TRG2 in 30.4%, TRG3 in 28%, TRG4 in 24% and TRG5 in 2.6% of the cases. Radical surgery was performed in 89 cases, 72 with R0 resection. By assessing the histological samples we found downstaging in 46% of the T and 34.5% of the N stage. We have no information on increased postoperative complications. We followed 86 patients after neoadjuvant therapy. Until March 2009 there was no progression in 48 of our patients. In 13 cases local relapse occurred, and in 25 cases the disease progressed because of distant metastasis, although local control was maintained. 10 patients had local relapse and distant metastases. 17 patients passed away. As a conclusion, neoadjuvant RCT of Stage II-III patients is an effective and well tolerated treatment, allowing for high R0 resection rate and bearing no higher risk for postoperative morbidity.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Neoadjuvant Therapy/methods , Rectal Neoplasms/drug therapy , Rectal Neoplasms/radiotherapy , Adult , Aged , Aged, 80 and over , Chemotherapy, Adjuvant/adverse effects , Disease Progression , Dose Fractionation, Radiation , Female , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/prevention & control , Neoplasm Staging , Positron-Emission Tomography , Radiotherapy, Adjuvant/adverse effects , Rectal Neoplasms/pathology , Rectal Neoplasms/surgery , Tomography, X-Ray Computed , Treatment OutcomeABSTRACT
BACKGROUND: Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. METHODS: Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. RESULTS: Low grade (grades 1-2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. CONCLUSION: Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.
Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cytoplasm/metabolism , Gene Expression Regulation, Neoplastic/physiology , Heat-Shock Proteins, Small/biosynthesis , Heat-Shock Proteins, Small/genetics , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Cytoplasm/genetics , Heat-Shock Proteins, Small/physiology , Humans , Intracellular Signaling Peptides and ProteinsABSTRACT
NCBI database analysis indicated that the human C1orf41 protein (small heat shock-like protein-Hsp16.2) has sequence similarity with small heat shock proteins (sHsps). Since sHsps have chaperone function, and so prevent aggregation of denatured proteins, we determined whether Hsp16.2 could prevent the heat-induced aggregation of denatured proteins. Under our experimental conditions, recombinant Hsp16.2 prevented aggregation of aldolase and glyceraldehyde-3-phosphate dehydrogenase, and protected Escherichia coli cells from heat stress indicating its chaperone function. Hsp16.2 also formed oligomeric complexes in aqueous solution. Hsp16.2 was found to be expressed at different levels in cell lines and tissues, and was mainly localized to the nucleus and the cytosol, but to a smaller extent, it could be also found in mitochondria. Hsp16.2 could be modified covalently by poly(ADP ribosylation) and acetylation. Hsp16.2 over-expression prevented etoposide-induced cell death as well as the release of mitochondrial cytochrome c and caspase activation. These data suggest that Hsp16.2 can prevent the destabilization of mitochondrial membrane systems and could represent a suitable target for modulating cell death pathways.
Subject(s)
Apoptosis , Heat-Shock Proteins, Small/metabolism , Amino Acid Sequence , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cytochromes c/metabolism , Enzyme Activation/drug effects , Etoposide/pharmacology , Gene Expression/drug effects , Genome, Human/drug effects , Genome, Human/genetics , HeLa Cells , Heat-Shock Proteins, Small/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Processing, Post-Translational/drug effects , Protein Structure, Quaternary/drug effects , Protein Transport/drug effects , Sequence Alignment , Sequence Homology , TemperatureABSTRACT
AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase-Akt cytoprotective pathway.
Subject(s)
Apoptosis/drug effects , Apoptosis/physiology , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/pharmacology , Heat-Shock Proteins/physiology , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Amino Acid Sequence , Animals , Cell Line , Enzyme Activation/drug effects , HeLa Cells , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Humans , In Vitro Techniques , Membrane Potential, Mitochondrial/drug effects , Mice , Molecular Sequence Data , NIH 3T3 Cells , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Oxidative Stress/drug effects , RNA, Small Interfering/genetics , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , alpha-Crystallin B Chain/geneticsABSTRACT
We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.
Subject(s)
Calcium/metabolism , Carrier Proteins/pharmacology , Hemeproteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Pregnancy Proteins/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Calcimycin/pharmacology , Carrier Proteins/genetics , Cyclosporine/pharmacology , Etoposide/pharmacology , Heme-Binding Proteins , Hemeproteins/genetics , Humans , Immunosuppressive Agents/pharmacology , Ionophores/pharmacology , Mice , NIH 3T3 Cells , Necrosis/metabolism , Permeability/drug effects , Pregnancy Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
The present article investigated the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on oxidative stress-induced apoptosis in neonatal rat cardiomyocytes. Our results show that PACAP decreased the ratio of apoptotic cells following H2O2 treatment. PACAP also diminished the activity of apoptosis signal-regulating kinase. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis and suggest that its cardioprotective effect is mediated through inhibition of the MAP kinase-dependent apoptotic pathway.
Subject(s)
Apoptosis/drug effects , MAP Kinase Kinase Kinase 5/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Oxidative Stress , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Animals , Cells, Cultured , Enzyme Activation/drug effects , Myocytes, Cardiac/enzymology , Rats , Rats, WistarABSTRACT
Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity was confirmed by 31P-NMR. In this study, recombinant PP13/galectin-13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin-13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin-13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI-TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin-13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin-like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin-13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin-13 may have special haemostatic and immunobiological functions at the lining of the common feto-maternal blood-spaces or developmental role in the placenta.