Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

• MeSH
• apoptóza * MeSH
• chondrocyty * metabolismus   MeSH
• kaspasa 9 metabolismus   farmakologie   MeSH
• kaspasy metabolismus   farmakologie   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

• MeSH
• buněčná diferenciace MeSH
• chondrocyty * MeSH
• chondrogeneze * fyziologie   MeSH
• inhibitory kaspas metabolismus   farmakologie   MeSH
• kaspasa 9 genetika   metabolismus   MeSH
• kultivované buňky MeSH
• myši MeSH
• osteogeneze MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.

• MeSH
• antitumorózní látky farmakologie   MeSH
• autofagie účinky léků   MeSH
• buněčná smrt účinky léků   MeSH
• buňky Hep G2 MeSH
• chemorezistence účinky léků   MeSH
• hepatocelulární karcinom metabolismus   patologie   MeSH
• jaterní mitochondrie účinky léků   metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• lidé MeSH
• nádory jater metabolismus   patologie   MeSH
• oxid dusnatý metabolismus   MeSH
• potkani Wistar MeSH
• PPARGC1A metabolismus   MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• sorafenib farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1G93A rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase-like protein, cl-casp8), apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1G93A model of ALS. Stem Cells Translational Medicine 2019;8:535-547.

• MeSH
• amyotrofická laterální skleróza terapie   MeSH
• beclin 1 metabolismus   MeSH
• čtyřhlavý sval stehenní cytologie   metabolismus   MeSH
• dlouhověkost MeSH
• injekce intramuskulární MeSH
• kaspasa 9 metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• mícha cytologie   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• motorické neurony metabolismus   MeSH
• nekroptóza * MeSH
• potkani Sprague-Dawley MeSH
• potkani transgenní MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• spinální injekce MeSH
• superoxid dismutáza 1 genetika   metabolismus   MeSH
• transplantace mezenchymálních kmenových buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. METHODS: Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. RESULTS: CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). CONCLUSIONS: Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

• MeSH
• cílená molekulární terapie * MeSH
• genetická transkripce * MeSH
• genetická variace * genetika   MeSH
• kaspasa 9 genetika   metabolismus   MeSH
• kaspasy * genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory prsu * enzymologie   genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH

SCOPE: The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. METHODS AND RESULTS: Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. CONCLUSION: We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

• MeSH
• antigeny CD95 metabolismus   MeSH
• apoptóza účinky léků   MeSH
• brusnice s jedlými plody chemie   metabolismus   MeSH
• buněčné linie MeSH
• chemorezistence MeSH
• DNA metabolismus   MeSH
• fosfatidylseriny metabolismus   MeSH
• fosforylace účinky léků   MeSH
• fragmentace DNA účinky léků   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 8 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• nádory tračníku metabolismus   patologie   MeSH
• ovoce chemie   metabolismus   MeSH
• proantokyanidiny chemie   izolace a purifikace   toxicita   MeSH
• protein TRAIL toxicita   MeSH
• rostlinné extrakty chemie   MeSH
• TRAIL receptory metabolismus   MeSH
• Vaccinium vitis-idaea chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The intrinsic apoptosis pathway represents an important mechanism of stress-induced death of cancer cells. To gain insight into the functional status of the apoptosome apparatus in non-small cell lung carcinoma (NSCLC), we studied its sensitivity to activation, the assembly of apoptosome complexes and stability of their precursors, and the importance of X-linked inhibitor of apoptosis (XIAP) in the regulation of apoptosome activity, using cell-free cytosols from NSCLC cell lines and NSCLC tumours and lungs from 62 surgically treated patients. Treatment of cytosol samples with cytochrome c (cyt-c) and dATP induced proteolytic processing of procaspase-9 to caspase-9, which was followed by procaspase-3 processing to caspase-3, and by generation of caspase-3-like activity in 5 of 7 studied NSCLC cell lines. Further analysis demonstrated formation of high-Mr Apaf-1 complexes associated with cleaved caspase-9 in the (cyt-c + dATP)-responsive COLO-699 and CALU-1 cells. By contrast, in A549 cells, Apaf-1 and procaspase-9 co-eluted in the high-Mr fractions, indicating formation of an apoptosome complex unable of procaspase-9 processing. Thermal pre-treatment of cell-free cytosols in the absence of exogenous cyt-c and dATP lead to formation of Apaf-1 aggregates, unable to recruit and activate procaspase-9 in the presence of cyt-c and dATP, and to generate caspase‑3‑like activity. Further studies showed that the treatment with cyt-c and dATP induced a substantially higher increase of caspase-3-like activity in cytosol samples from NSCLC tumours compared to matched lungs. Tumour histology, grade and stage had no significant impact on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Upon addition into the cytosol, the XIAP-neutralizing peptides AVPIAQK and ATPFQEG only moderately heightened the (cyt-c + dATP)-induced caspase‑3‑like activity in some NSCLC tumours. Taken together, the present study provides evidence that the apoptosome apparatus is functional in the majority of NSCLCs and that its sensitivity to the (cyt-c + dATP)-mediated activation is often enhanced in NSCLCs compared to lungs. They also indicate that XIAP does not frequently and effectively suppress the activity of apoptosome apparatus in NSCLCs.

• MeSH
• apoptóza * účinky léků   MeSH
• cytochromy c farmakologie   MeSH
• cytosol metabolismus   MeSH
• deoxyadeninnukleotidy farmakologie   MeSH
• faktor 1 aktivující apoptotickou proteasu metabolismus   MeSH
• inhibitory kaspas farmakologie   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory plic metabolismus   patologie   MeSH
• nemalobuněčný karcinom plic metabolismus   patologie   MeSH
• plíce cytologie   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• senioři MeSH
• X-vázaný inhibitor apoptózy farmakologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic β-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic β-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic β-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.

• MeSH
• apoptóza účinky léků   MeSH
• beta-buňky cytologie   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory   metabolismus   MeSH
• kaspasa 2 chemie   genetika   metabolismus   MeSH
• kaspasa 7 metabolismus   MeSH
• kaspasa 8 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• kyseliny stearové farmakologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• poly(ADP-ribosa)-polymerasy metabolismus   MeSH
• proteiny tepelného šoku metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula účinky léků   MeSH
• transkripční faktor ATF6 metabolismus   MeSH
• transkripční faktor CHOP metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• butyráty farmakologie   MeSH
• dimethylsulfoxid farmakologie   MeSH
• HL-60 buňky MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• kyselina valproová farmakologie   MeSH
• lidé MeSH
• membránový potenciál mitochondrií MeSH
• neutrofily cytologie   MeSH
• tretinoin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: In this study, the effect of novel taxane SB-T-1216 and paclitaxel on sensitive MDA-MB-435 and resistant NCI/ADR-RES human breast cancer cells was compared. MATERIALS AND METHODS: Cell growth and survival were evaluated after 96-hour incubation with tested concentrations of taxanes. The effect on the formation of microtubule bundles was assessed employing fluorescence microscopy and on the cell cycle employing flow cytometric analysis. The activity of caspases was assessed employing commercial colorimetric kits. RESULTS: The IC(50) (concentration resulting in 50% of living cells in comparison with the control) of SB-T-1216 in sensitive cells was 0.6 nM versus 1 nM for paclitaxel. However, the IC(50) of SB-T-1216 in resistant cells was 1.8 nM versus 300 nM for paclitaxel. Both SB-T-1216 and paclitaxel at death-inducing concentrations induced the formation of microtubule bundles in sensitive as well as resistant cells. Cell death induced in sensitive and resistant cells by paclitaxel was associated with the accumulation of cells in the G(2)/M phase. On the contrary, cell death induced by SB-T-1216 took place without the accumulation of cells in the G(2)/M phase but with a decreased number of G(1) cells and the accumulation of hypodiploid cells. Both SB-T-1216 and paclitaxel activated caspase-3, caspase-9, caspase-2 and caspase-8 in sensitive as well as resistant cells. CONCLUSION: Cell death induced by both paclitaxel and novel taxane SB-T-1216 in breast cancer cells is associated with caspase activation and with the formation of interphase microtubule bundles. Novel taxane SB-T-1216, but not paclitaxel, seems to be capable of inducing cell death without the accumulation of cells in the G(2)/M phase.

• MeSH
• antibiotika antitumorózní farmakologie   MeSH
• antitumorózní látky fytogenní farmakologie   MeSH
• apoptóza účinky léků   MeSH
• buněčné dělení účinky léků   MeSH
• chemorezistence MeSH
• doxorubicin farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• G1 fáze účinky léků   MeSH
• G2 fáze účinky léků   MeSH
• kaspasa 2 metabolismus   MeSH
• kaspasa 3 metabolismus   MeSH
• kaspasa 8 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• lidé MeSH
• mikrotubuly účinky léků   MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• paclitaxel farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• taxoidy farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH