BACKGROUND: Caspase-8 and caspase-9 (encoded by CASP8 and CASP9) are executive caspases of programmed cell death (apoptosis). Dysregulation of apoptosis plays an important role in cancer development, progression, and resistance to anticancer therapy. The goal of this work was to evaluate potential associations between polymorphisms in CASP8 and CASP9, previously linked to breast cancer risk, and the transcript levels of these genes (including their alternative anti-apoptotic variants) in tumor tissues and the clinical characteristics of the patients. MATERIAL AND METHODS: Sanger sequencing, high resolution melting (HRM) analysis, and allelic discrimination were used to identify polymorphisms in DNA samples isolated from tumor tissues and peripheral blood lymphocytes of 60 breast carcinoma patients. Total transcript levels of CASP8 and CASP9, and levels of alternative splicing variants CASP8L and CASP9B, were quantified by real-time PCR in tumor tissues. Clinically interesting associations were validated in DNA from lymphocytes of 615 breast carcinoma patients. RESULTS: A haplotype in CASP9 composed of three polymorphisms rs4645978-rs2020903-rs4646034 was significantly associated with CASP9 expression in tumors, with the expression of the progesterone receptor and ERBB2, and with the TNBC subtype of breast carcinoma in the validation study. The associations between the rs3834129 polymorphism in CASP8 and stage of disease, rs6435074 with grade, expression of estrogen receptor and ERBB2, and rs6723097 with ERBB2 expression have not yet been validated. However, rs6723097 was associated with disease-free survival in patients treated with hormonal therapy. CONCLUSION: This study reveals a previously unknown and presumably functional (in silico) association between a haplotype in CASP9 and molecular and clinical phenotypes of breast carcinoma. The potential clinical utility of this association for prognostication of breast carcinoma should be evaluated by independent studies.Key words: breast carcinoma - caspases - polymorphisms - functional - clinical - importanceThis work was supported by grant of the CU Grant Agency No. 1444313, and grant of the Internal Grant Agency of the Czech Ministry of Health No. 15-25618A.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 3. 2016Accepted: 26. 10. 2016.
- MeSH
- Carcinoma chemistry drug therapy genetics MeSH
- Caspase 8 genetics MeSH
- Caspase 9 genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Breast Neoplasms chemistry drug therapy genetics MeSH
- Polymorphism, Genetic MeSH
- Disease-Free Survival MeSH
- Receptor, ErbB-2 analysis MeSH
- Receptors, Estrogen analysis MeSH
- Receptors, Progesterone analysis MeSH
- Aged MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- ERBB2 protein, human MeSH Browser
- Caspase 8 MeSH
- Caspase 9 MeSH
- Receptor, ErbB-2 MeSH
- Receptors, Estrogen MeSH
- Receptors, Progesterone MeSH
Caspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3-/- TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.
Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.
- Keywords
- ABHD2, Caspase 9, diaPASEF, migration, osteoblasts, proteomics,
- MeSH
- Cell Line MeSH
- Gene Knockout Techniques MeSH
- Caspase 9 * metabolism genetics MeSH
- Mice MeSH
- Osteoblasts * metabolism cytology MeSH
- Cell Movement * MeSH
- Proteomics * methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Casp9 protein, mouse MeSH Browser
- Caspase 9 * MeSH
As an extension of our recently published work (Mlejnek and Kuglík [2000] J. Cell. Biochem. 77:6-17), the role of caspases in N(6)-benzylaminopurine riboside (BAPR)-induced apotosis in HL-60 cells was evaluated in this study. Here, BAPR-induced apoptosis was accompanied by activation of caspase-3 and caspase-9. However, when these caspases were selectively inhibited, the progression of BAPR-induced apoptosis was not markedly affected. Besides that, activation of caspase-3 and caspase-9 was found to be rather late event in apoptotic process. These results suggested that other caspases might be critically implicated. Indeed, pan-specific caspase inhibitor, Z-VAD-FMK, completely prevented DNA cleavage and apoptotic bodies formation. However, Z-VAD-FMK failed to prevent cell death and it was incapable to fully counteract the main apoptotic hallmark-chromatin condensation. Finally, our data indicate that cellular decision between apoptosis and necrosis is made upon the availability of both caspase proteases and intracellular ATP.
- MeSH
- Adenosine analogs & derivatives pharmacology MeSH
- Apoptosis drug effects MeSH
- Cell Membrane drug effects enzymology MeSH
- Cell Nucleus drug effects enzymology metabolism MeSH
- Amino Acid Chloromethyl Ketones pharmacology MeSH
- Chromatin metabolism MeSH
- HL-60 Cells cytology drug effects enzymology MeSH
- Cysteine Proteinase Inhibitors pharmacology MeSH
- Caspase Inhibitors * MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases physiology MeSH
- Humans MeSH
- Oligopeptides pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Adenosine MeSH
- benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone MeSH Browser
- benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone MeSH Browser
- benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone MeSH Browser
- CASP3 protein, human MeSH Browser
- CASP9 protein, human MeSH Browser
- Amino Acid Chloromethyl Ketones MeSH
- Chromatin MeSH
- Cysteine Proteinase Inhibitors MeSH
- Caspase Inhibitors * MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- N(6)-benzyladenosine MeSH Browser
- Oligopeptides MeSH
We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.
- MeSH
- Enzyme Activation MeSH
- Apoptosis drug effects MeSH
- Cell Nucleolus ultrastructure MeSH
- Cisplatin pharmacology MeSH
- Cell Fractionation MeSH
- HeLa Cells MeSH
- Hydrolysis MeSH
- Immunohistochemistry MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases metabolism MeSH
- In Situ Nick-End Labeling MeSH
- Humans MeSH
- Poly(ADP-ribose) Polymerases metabolism MeSH
- Cell Size drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- CASP3 protein, human MeSH Browser
- CASP9 protein, human MeSH Browser
- Cisplatin MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- Poly(ADP-ribose) Polymerases MeSH
The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism, and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K, and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predicts lower regenerative capacity if used as autologous transplants.
- Keywords
- cellular metabolism, endometrial progenitor cells, obesity, self-renewal potential,
- MeSH
- Endometrium metabolism MeSH
- Endothelial Progenitor Cells * metabolism MeSH
- Phenotype MeSH
- Stem Cells metabolism MeSH
- Horses MeSH
- Obesity metabolism MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (Deltapsi(m)) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.
- MeSH
- Enzyme Activation MeSH
- Apoptosis * MeSH
- Cell Line MeSH
- Time Factors MeSH
- Cytochromes c metabolism MeSH
- Cytosol metabolism MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Microscopy, Fluorescence MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases metabolism MeSH
- Microscopy, Confocal MeSH
- Culture Media pharmacology MeSH
- Membrane Potentials MeSH
- Mitochondria pathology MeSH
- Mice MeSH
- Proteins metabolism MeSH
- Flow Cytometry MeSH
- Reactive Oxygen Species MeSH
- Subcellular Fractions MeSH
- Protein Transport MeSH
- Blotting, Western MeSH
- Iron metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Apaf1 protein, mouse MeSH Browser
- Casp3 protein, mouse MeSH Browser
- Casp9 protein, mouse MeSH Browser
- Cytochromes c MeSH
- Apoptotic Protease-Activating Factor 1 MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- Culture Media MeSH
- Proteins MeSH
- Reactive Oxygen Species MeSH
- Iron MeSH
Taxane and platinum-based chemotherapy regimens are standard treatment for advanced ovarian carcinoma. Expression levels of putative markers of taxane resistance in carcinoma tissues and paired peritoneal samples (n=55) and in 16 samples of ovaries without signs of carcinoma were compared with clinical data and the patients' time to progression. KIF14, PRC1, CIT and ABCC1 genes were significantly overexpressed in carcinomas when compared with normal ovarian tissues, while ABCB1 and CASP9 expression was decreased. Associations of protein expression of the proliferation marker Ki-67 with KIF14, PRC1, ABCB1 and CASP2 were found. Lastly, it was discovered that ABCB1 and CASP2 levels associated with FIGO stage and that the CIT level associated with the time to progression of ovarian carcinoma patients (P<0.0001). In conclusion, ABCB1, CASP2, KIF14, PRC1 and CIT genes seem to associate with surrogate markers of ovarian carcinoma progression and CIT gene associates with therapy outcome.
- Keywords
- Clinical course, Cytokinesis, Gene expression, Ovarian carcinoma, Resistance, Taxane,
- MeSH
- ATP-Binding Cassette Transporters genetics MeSH
- Adenocarcinoma diagnosis drug therapy genetics MeSH
- Antineoplastic Agents therapeutic use MeSH
- Drug Resistance, Neoplasm genetics MeSH
- Genetic Association Studies MeSH
- Intracellular Signaling Peptides and Proteins genetics MeSH
- Caspases genetics MeSH
- Kinesins genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Ovarian Neoplasms diagnosis drug therapy genetics MeSH
- Oncogene Proteins genetics MeSH
- Ovary metabolism MeSH
- Peritoneum metabolism MeSH
- Bridged-Ring Compounds therapeutic use MeSH
- Disease Progression MeSH
- Protein Serine-Threonine Kinases genetics MeSH
- Cell Cycle Proteins genetics MeSH
- Taxoids therapeutic use MeSH
- Treatment Outcome MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- ATP-Binding Cassette Transporters MeSH
- Antineoplastic Agents MeSH
- citron-kinase MeSH Browser
- Intracellular Signaling Peptides and Proteins MeSH
- Caspases MeSH
- KIF14 protein, human MeSH Browser
- Kinesins MeSH
- Oncogene Proteins MeSH
- PRC1 protein, human MeSH Browser
- Bridged-Ring Compounds MeSH
- Protein Serine-Threonine Kinases MeSH
- Cell Cycle Proteins MeSH
- taxane MeSH Browser
- Taxoids MeSH
BACKGROUND: The aim of the study was to contribute to our understanding of the mechanisms responsible for the resistance of breast cancer cells to taxanes. MATERIALS AND METHODS: Cell cycle characteristics, DNA fragmentation, p53 and p21(WAF1/CIP1) expression, caspase-3 and caspase-9 activity, cytochrome c release from mitochondria during cell death induction by the taxanes paclitaxel and docetaxel in highly-sensitive MDA-MB-435 and highly-resistant NCI-ADR-RES human breast cancer cells were compared. RESULTS: Approximately 300-fold higher concentrations of the taxanes were required to induce death in resistant NCI-ADR-RES cells than in sensitive MDA-MB-435 cells. Cell death induced by the taxanes in both sensitive and resistant cells was preceded by the accumulation of cells in the G2/M-phase. Neither cell type produced any DNA fragmentation (DNA ladder) typical of regular apoptosis. The p53 and the p21(WAF1/CIP1) levels did not change in sensitive or in resistant cells during cell death induction by the taxanes. The activity of the executioner caspase-3 increased significantly (2 to 2.5-fold) and, similarly, the activity of caspase-9 increased significantly (2 to 3.5-fold) in both cell types. However, cytochrome c was found to be released from mitochondria into the cytosol only in the resistant NCI-ADR-RES cells, but not in the sensitive MDA-MB-435 cells. CONCLUSION: The death induced by the taxanes in the studied breast cancer cells can be characterized as an apoptosis-like death, including caspase-3 and caspase-9 activation but not oligonucleosomal DNA fragmentation. However, the mechanisms of death induction by the taxanes in sensitive MDA-MB-435 cells and resistant NCI-ADR-RES cells differ. Cytochrome c is released from the mitochondria in resistant but not in sensitive cells.
- MeSH
- Antineoplastic Agents, Phytogenic pharmacology MeSH
- Cell Death drug effects physiology MeSH
- Cell Growth Processes drug effects MeSH
- Drug Resistance, Neoplasm MeSH
- Cytochromes c metabolism MeSH
- Docetaxel MeSH
- DNA Fragmentation drug effects MeSH
- Cyclin-Dependent Kinase Inhibitor p21 biosynthesis MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases metabolism MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 biosynthesis MeSH
- Breast Neoplasms drug therapy metabolism pathology MeSH
- Paclitaxel pharmacology MeSH
- Taxoids pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Antineoplastic Agents, Phytogenic MeSH
- CASP3 protein, human MeSH Browser
- CASP9 protein, human MeSH Browser
- CDKN1A protein, human MeSH Browser
- Cytochromes c MeSH
- Docetaxel MeSH
- Cyclin-Dependent Kinase Inhibitor p21 MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases MeSH
- Tumor Suppressor Protein p53 MeSH
- Paclitaxel MeSH
- Taxoids MeSH
- TP53 protein, human MeSH Browser
BACKGROUND: Recently, infliximab dependency has been described. AIM: To assess frequency of ID in 82 consecutive Crohn's disease children treated with infliximab 2000-2006 and to describe clinical and genetic predictors of long-term infliximab response. METHODS: A phenotype model of infliximab dependency was used to assess treatment response: 'immediate outcome' (30 days after infliximab start)--complete/partial/no response. 'Long-term outcome': (i) prolonged response: maintenance of complete/partial response; (ii) infliximab dependency: relapse < or = 90 days after intended infliximab cessation requiring repeated infusions to regain complete/partial response or need of infliximab >12 months to sustain response. Polymorphisms TNF-308 A>G, TNF-857 C>T, Casp9 93 C>T, FasL-844 C>T, LTA 252 C>T and CARD15 (R702W, G908R, 1007fs) were analysed. RESULTS: Ninety-four per cent of children obtained complete/partial response. In long-term outcome, 22% maintained prolonged response, 12% had no response, while 66% became infliximab dependent. Perianal disease and no previous surgery were associated with infliximab dependency (OR 5.34, 95% CI: 1.24-22.55; OR 6.7, 95% CI: 1.67-26.61). No association was found with studied polymorphisms. The cumulative probability of surgery 50 months after starting infliximab was 10% in infliximab dependency, 30% in prolonged responders and 70% in nonresponders (P = 0.0002). CONCLUSIONS: Sixty-six per cent of children became infliximab dependent. Perianal disease and no surgery prior to infliximab were associated with infliximab dependency phenotype.
- MeSH
- Time Factors MeSH
- Crohn Disease complications drug therapy genetics MeSH
- Child MeSH
- Phenotype MeSH
- Gastrointestinal Agents administration & dosage adverse effects MeSH
- Remission Induction MeSH
- Infliximab MeSH
- Humans MeSH
- Adolescent MeSH
- Antibodies, Monoclonal administration & dosage adverse effects MeSH
- Substance-Related Disorders * genetics MeSH
- Retrospective Studies MeSH
- Treatment Outcome MeSH
- Dose-Response Relationship, Drug MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Gastrointestinal Agents MeSH
- Infliximab MeSH
- Antibodies, Monoclonal MeSH
