It has been shown that drug resistance is extremely common in hepatocellular carcinoma (HCC) and is one of the major problems in HCC chemotherapy. However, the detailed mechanisms remain largely unknown. We have previously shown that endoplasmic reticulum (ER) stress is involved in the tumorigenesis of HCC. Here, we demonstrated that the unfolded protein response (UPR) inhibits cisplatin-induced HCC cell apoptosis. In HCC cells, cisplatin treatment triggers the UPR, which subsequently inhibits cisplatin-induced apoptosis. Importantly, mild ER stress precondition suppresses the sensitivity of HCC cells to cisplatin-induced apoptosis through autophagy regulation. Furthermore, heat-shock protein 27 (Hsp27) is involved in the cytoprotective role of the UPR in cisplatin-induced apoptosis. We also demonstrated that Hsp27 inhibits cisplatin- induced HCC cell death through autophagy activation. Taken together, our results indicate that the UPR inhibits cisplatin-induced apoptosis in HCC cells, at least in part, by Hsp27-mediated autophagy activation.

• MeSH
• apoptóza fyziologie   účinky léků   MeSH
• autofagie fyziologie   účinky léků   MeSH
• cisplatina farmakologie   metabolismus   MeSH
• dithiothreitol farmakologie   MeSH
• endoplazmatické retikulum fyziologie   účinky léků   MeSH
• hepatocelulární karcinom patologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory jater patologie   MeSH
• proteiny tepelného šoku HSP27 genetika   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• reakce na tepelný šok MeSH
• signální dráha UPR fyziologie   MeSH
• tunikamycin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

• MeSH
• alfa-synuklein * metabolismus   MeSH
• biologické markery metabolismus   MeSH
• chaperon endoplazmatického retikula BiP * metabolismus   MeSH
• demence s Lewyho tělísky * patologie   metabolismus   MeSH
• eukaryotický iniciační faktor 2 * metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mozek metabolismus   patologie   MeSH
• neurotrofní faktory metabolismus   MeSH
• proteiny teplotního šoku * metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• signální dráha UPR * fyziologie   MeSH
• stres endoplazmatického retikula fyziologie   MeSH
• upregulace * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Východiska: Signální dráha UPR (unfolded protein response, tj. odpověď na chybně složené proteiny) pomáhá myelomovým buňkám vyrovnat se se stresovými podmínkami vzniklými v důsledku nadměrné proteosyntézy, a představuje tak pro myelomové buňky prostředek umožňující jejich přežití. Extramedulární onemocnění je agresivnější forma mnohočetného myelomu, při které myelomové buňky ztrácí svoji závislost na mikroprostředí kostní dřeně a mohou infiltrovat jiné tkáně a orgány. Patogeneze vzniku extramedulárního onemocnění není dosud zcela objasněna. Cílem této studie bylo zjistit, zda existuje rozdíl v expresi genů spjatých s UPR mezi plazmatickými buňkami kostní dřeně od pacientů s mnohočetným myelomem a extramedulárním onemocněním. Materiál a metody: Pomocí reverzní transkripce ve spojení s kvantitativní polymerázovou řetězovou reakcí byla analyzována exprese šesti genů spjatých s UPR (ERN1, DDIT3, EIF2AK3, TUSC3, XBP1, HSPA5). Použito bylo celkem 76 vzorků plazmatických buněk kostní dřeně, z toho 44 bylo od pacientů s mnohočetným myelomem a 32 od pacientů s extramedulárním onemocněním. Výsledky: Byl pozorován statisticky významný rozdíl v expresi genů HSPA5, DDIT3, EIF2AK3 a ERN1 mezi skupinou mnohočetného myelomu a extramedulárního onemocnění; exprese byla ve všech případech vyšší u vzorků od pacientů s extramedulárním onemocněním. V případě genů XBP1 a TUSC3 nebyl pozorován statisticky významný rozdíl. Prokázáno bylo také několik statisticky významných korelací mezi hladinou exprese analyzovaných genů a klinickými daty pacientů. Závěr: Výsledky poukazují na možný význam signální dráhy UPR v patogenezi extramedulárního onemocnění. UPR se jeví jako vhodný směr dalšího výzkumu.

Background: The unfolded protein response (UPR) enables myeloma cells to overcome the stress conditions arising from excessive proteosynthesis and thus provides a survival advantage for myeloma cells. Extramedullary disease is a more aggressive form of multiple myeloma in which myeloma cells lose their dependence on the bone marrow microenvironment and are able to infiltrate other tissues and organs. The pathogenesis of extramedullary disease is not fully elucidated yet. The aim of this study was to determine whether there is a difference in the expression of UPR-related genes between bone marrow plasma cells from multiple myeloma and extramedullary disease patients. Materials and methods: Gene expression of six genes involved in UPR (ERN1, DDIT3, EIF2AK3, TUSC3, XBP1, HSPA5) was analyzed by quantitative reverse transcription polymerase chain reaction. In total, 76 bone marrow plasma cell samples were used, of which 44 were from patients with multiple myeloma and 32 from patients with extramedullary disease. Results: A statistically significant difference was observed between the multiple myeloma and extramedullary disease groups regarding the expression of HSPA5, DDIT3, EIF2AK3, and ERN1 genes. However, in the case of XBP1 and TUSC3 genes, no statistically significant difference in the expression was found. Several statistically significant correlations between the expression levels of the analyzed genes and the clinical data of the patients were observed as well. Conclusion: Our results suggest the importance of UPR in the pathogenesis of extramedullary disease. UPR appears to be a promising avenue for further research.

• MeSH
• exprese genu * genetika   MeSH
• klinická studie jako téma metody   MeSH
• lidé MeSH
• mnohočetný myelom * genetika   patologie   MeSH
• plazmatické buňky patologie   MeSH
• reverzní transkripce MeSH
• signální dráha UPR * genetika   MeSH
• Check Tag
• lidé MeSH

There is an ongoing need for the development of new cancer therapeutics that combine high cytotoxic efficiency with low side effects, and also override resistance to the first-line chemotherapeutics. Copper(ii)-phenanthroline complexes are promising compounds that were shown previously to induce an immediate cytotoxic response over a panel of tumor cell lines in vitro. The molecular mechanism, however, remained unresolved. In this work we performed a thorough study of the copper(ii)-phenanthroline complexes containing different imidazolidine-2-thione ligands in ovarian cancer cells, and revealed that these complexes induce endoplasmic reticulum (ER) stress and subsequently cell death mediated by the unfolded protein response. Alleviation of the ER-stress by tauroursodeoxycholic acid (TUDCA) attenuated the cytotoxic effects. In summary, we have identified a novel, ER-dependent, molecular mechanism mediating cytotoxic effects of copper(ii)-phenanthroline complexes.

• MeSH
• fenantroliny chemie   farmakologie   MeSH
• komplexní sloučeniny chemie   farmakologie   MeSH
• lidé MeSH
• měď chemie   farmakologie   MeSH
• nádorové buněčné linie MeSH
• nádory vaječníků farmakoterapie   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• signální dráha UPR účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

• MeSH
• apoptóza účinky léků   genetika   MeSH
• biologické modely MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kolorektální nádory farmakoterapie   genetika   metabolismus   mortalita   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• MAP kinasový signální systém MeSH
• mutace * MeSH
• nádorové biomarkery MeSH
• nádorové buněčné linie MeSH
• oligopeptidy farmakologie   MeSH
• prognóza MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• proteosyntéza MeSH
• protinádorové látky farmakologie   MeSH
• protoonkogenní proteiny B-raf antagonisté a inhibitory   genetika   metabolismus   MeSH
• pyrimidiny farmakologie   MeSH
• signální dráha UPR účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula účinky léků   genetika   MeSH
• transkripční faktor CHOP genetika   metabolismus   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca(2+) and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na(+), which compromised Na(+)-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.

• MeSH
• aminokyseliny metabolismus   MeSH
• biologický transport MeSH
• blokátory kalciových kanálů farmakologie   MeSH
• buněčná smrt MeSH
• dihydropyridiny farmakologie   MeSH
• draslíkové kanály aktivované vápníkem antagonisté a inhibitory   MeSH
• gliom farmakoterapie   metabolismus   patologie   MeSH
• lidé MeSH
• mykotoxiny farmakologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky patologie   MeSH
• nádory mozku farmakoterapie   metabolismus   patologie   MeSH
• proteomika MeSH
• signální dráha UPR účinky léků   MeSH
• sodík metabolismus   MeSH
• vápníkové kanály - typ T fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Pichia pastoris (Komagataella sp.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli (EcPGA), lipase B from Candida antarctica (CaLB) and xylanase A from Thermomyces lanuginosus (TlXynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing EcPGA, which accumulated in the cells (50-70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing CaLB (20%) and TlXynA (27%). The proportion of cells with up-regulated UPR in CaLB-producing (35%) and TlXynA-producing (30%) strains was lower in comparison to the EcPGA-producing strain, and a smaller proportion of CaLB and TlXynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.

• Publikační typ
• časopisecké články MeSH

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

• Publikační typ
• časopisecké články MeSH

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

• MeSH
• ATM protein genetika   metabolismus   MeSH
• buňky A549 MeSH
• ELISA MeSH
• fosforylace genetika   fyziologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• polyribozomy metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• vnitřně neuspořádané proteiny genetika   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH