BACKGROUND: Autophagy is a crucial factor contributing to radioresistance during radiotherapy. Although Lys05 has proven its ability to improve the results of radiotherapy through the inhibition of autophagy, molecular mechanisms of this inhibition remain elusive. We aimed to describe the molecular mechanisms involved in Lys05-induced inhibition of autophagy. MATERIALS AND METHODS: Radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) and methods of quantitative phosphoproteomics were employed to define the molecular mechanisms involved in Lys05-induced inhibition of autophagy. RESULTS: We confirmed that at an early stage after irradiation, autophagy was induced, whereas at a later stage after irradiation, it was inhibited. The early-stage induction of autophagy was characterized mainly by the activation of biosynthetic and metabolic processes through up- or down-regulation of the critical autophagic regulatory proteins Sequestosome-1 (SQSTM1) and proline-rich AKT1 substrate 1 (AKT1S1). The late-stage inhibition of autophagy was attributed mainly to down-regulation of Unc-51 like autophagy-activating kinase 1 (ULK1) through phosphorylation at Ser638. CONCLUSION: This work contributes to emerging phosphoproteomic insights into autophagy-mediated global signaling in lung cancer cells, which might consequently facilitate the development of precision medicine therapeutics.

• MeSH
• aminochinoliny farmakologie   MeSH
• autofagie * MeSH
• fosfoproteiny analýza   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory plic farmakoterapie   metabolismus   patologie   radioterapie   MeSH
• nemalobuněčný karcinom plic farmakoterapie   metabolismus   patologie   radioterapie   MeSH
• polyaminy farmakologie   MeSH
• proteom analýza   metabolismus   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Background. Extracts from plant and/or animal tissues are frequently used in alternative medicine as drugs or food supplements. Such extracts may contain a complex of pharmacologically or physiologically active factors but frequently there exists no experimental confirmation as to precise mechanisms of action. This work aimed to verify if a long used bovine tissue extract Imuregen registered as a food supplement has desirable effect on tumor cells. Methods. Two independent methodological approaches were used. Viability of cell cultures was evaluated using WST-1-based cell cytotoxicity assay. Cell growth was monitored in real time using xCELLigence cell analysis. Normal human adherent lung fibroblasts (NHLF) were used to represent non - tumor lung cells. A human non-small cell lung carcinoma cell line H1299 was used as a model of tumor cells. Results. Our study demonstrated a direct influence on viability of the H1299 tumor cell line (p < 0.005) and a cytostatic/cytotoxic effect of the bovine tissue extract after 72h. of cultivation while leaving non-tumor NHLF cell line unaffected. The extract (0.1 μg/ml and 1 μg/ml, resp.) also significantly affected the viability of irradiated H1299 tumor cell line (p < 0.005, Co, 4Gy) compared to non-tumor irradiated counterparts. In addition to the cytotoxic effect, the extract slightly modified the generation time of the cells and substantial differences between the effects on tumor and non-tumor cell lines were observed. Conclusion. The data presented here might suggest the extract intervenes into the proliferative cell cycle and subsequently influences the generation time of cells. Further analyses should be oriented toward the effects of animal tissue extracts on cellular systems defending against tumors and/or infections and intercellular communications that lead to influencing the fate of individual cell types.

• Klíčová slova
• Imuregen,
• MeSH
• nádorové buněčné linie imunologie   účinky léků   MeSH
• nemalobuněčný karcinom plic genetika   imunologie   MeSH
• oncogene addiction MeSH
• proliferace buněk genetika   účinky léků   účinky záření   MeSH
• protinádorové látky imunologicky aktivní * MeSH
• techniky in vitro MeSH
• tkáňové extrakty imunologie   terapeutické užití   MeSH
• Publikační typ
• práce podpořená grantem MeSH

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

• MeSH
• apoptóza účinky záření   MeSH
• fluorescenční mikroskopie MeSH
• ionizující záření * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory plic metabolismus   MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 μM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 μM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.

• MeSH
• aminokyselinové motivy MeSH
• ATM protein antagonisté a inhibitory   MeSH
• biologické markery MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• genová ontologie MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• lidé MeSH
• metabolom * MeSH
• metabolomika metody   MeSH
• nádorové buněčné linie MeSH
• proteom * MeSH
• proteomika metody   MeSH
• pyraziny farmakologie   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• signální transdukce MeSH
• sulfony farmakologie   MeSH
• tolerance záření účinky léků   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• výpočetní biologie metody   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to compare the effects of DNA repair inhibitors in the context of radio-sensitization of human lung cells. The radio-sensitizing effects of NU7441 (1 mM), an inhibitor of DNA-dependent protein kinase (DNA-PK); KU55933 (10 μM), an inhibitor of ataxia-telangiectasia mutated kinase (ATM); and VE-821 (10 μM), an inhibitor of ATM-related kinase (ATR) were tested by the xCELLigence system for monitoring proliferation, fluorescence microscopy for DNA damage detection, flow-cytometry for cell cycle and apoptosis analysis and western blotting and ELISA for determination of DNA repair proteins. We employed normal human lung fibroblasts (NHLF, p53-wild-type) and non-small cell lung cancer cells (H1299, p53-negative). DNA-PK inhibition (by NU7441) in combination with ionizing radiation (IR) increased the number of double strand breaks (DSB), which persisted 72 h after irradiation in both cell lines. Additionally, NU7441 and KU55933 in combination with IR caused G2-arrest. ATR inhibitor (VE-821) together with IR markedly inhibited proliferation and induced G2/M arrest accompanied by apoptosis in H1299, but not in NHLF cells, and thus diminished DNA-repair of tumour cells but not normal lung fibroblasts. Our findings indicate that ATR inhibition could be a promising therapeutic strategy in p53-deficient lung tumours.

• MeSH
• enzymy opravy DNA antagonisté a inhibitory   genetika   MeSH
• fibroblasty účinky záření   MeSH
• kultivované buňky účinky záření   MeSH
• nádorové buňky kultivované účinky léků   účinky záření   MeSH
• nemalobuněčný karcinom plic genetika   radioterapie   MeSH
• oprava DNA účinky léků   MeSH
• proliferace buněk účinky záření   MeSH
• radiosenzibilizující látky farmakologie   izolace a purifikace   terapeutické užití   MeSH
• techniky in vitro MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

We examined the effect of epidermal growth factor (EGF) treatment in mice that received bone marrow transplantation (BMT) after 11 Gy whole-body irradiation. C57Bl/6 mice were divided into three treatment groups: 0 Gy; 11 Gy ((60)Co, single dose, 0.51 Gy/min) with BMT (5 × 10(6) bone marrow cells isolated from green fluorescent protein syngeneic mice, 3-4 h postirradiation); and 11 Gy with BMT and EGF (2 mg/kg applied subcutaneously 1, 3 and 5 days postirradiation). Survival data were collected. Bone marrow, peripheral blood count and cytokines, gastrointestine and liver parameters and migration of green fluorescent protein-positive cells were evaluated at 63 days postirradiation. Epidermal growth factor increased survival of irradiated animals that received BMT from 10.7 to 85.7% at 180 days postirradiation. In the BMT group, we found changes in differential bone marrow and blood count, plasma cytokine levels, gastrointestinal tissues and liver at 63 days postirradiation. These alterations were completely or in some parameters at least partially restored by epidermal growth factor. These findings indicate that epidermal growth factor, administered 1, 3 and 5 days postirradiation in combination with bone marrow transplantation, significantly improves long-term prognosis.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• bezpečnost MeSH
• časové faktory MeSH
• celotělové ozáření škodlivé účinky   MeSH
• cytokiny krev   MeSH
• epidermální růstové faktory farmakologie   MeSH
• kostní dřeň účinky léků   imunologie   účinky záření   MeSH
• mitóza účinky léků   účinky záření   MeSH
• myši MeSH
• počet buněk MeSH
• radiační poranění krev   farmakoterapie   patologie   terapie   MeSH
• slezina účinky léků   patologie   účinky záření   MeSH
• střeva účinky léků   patologie   účinky záření   MeSH
• transplantace kostní dřeně * MeSH
• velikost orgánu účinky léků   účinky záření   MeSH
• vztah dávky záření a odpovědi MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Radiation-induced autophagy is believed to represent a radioprotective mechanism of cancer cells. Thus, its inhibition should support radiation treatment and increase its efficacy. On the other hand, there is evidence that radiation alone or in combination with various chemical agents can induce autophagy that results into increased cell death, especially within transformed apoptosis-resistant cells. In this paper, besides description of autophagic process and its relation to cancer and radiotherapy, we compared two contradictory radiosensitization approaches that employ inhibition and induction of autophagy. In spite of the classical concept based on cytoprotective model, there is a plethora of recently developed inducers of autophagy, which indicates the future trend in radiosensitization via modulation of autophagy. Because contemporary literature is conflicting and inconsistent in this respect, we reviewed the recent studies focused on enhancement of sensitivity of cancer cells toward radiation in regard to autophagy, revealing some striking discrepancies. The deeper the knowledge, the more complex this situation is. To interpret results of various studies correctly one has to take into account the methodology of autophagy assessment and also the fact that radiosensitization might be mediated by other than intrinsic mechanisms related to autophagy. Notwithstanding, targeting autophagy remains an attractive anti-tumor strategy.

• MeSH
• ATM protein fyziologie   MeSH
• autofagie účinky záření   MeSH
• chlorochin farmakologie   MeSH
• fosfatidylinositol-3-kinasy fyziologie   MeSH
• hypoxie buňky MeSH
• lidé MeSH
• membránové proteiny fyziologie   MeSH
• nádory radioterapie   MeSH
• protoonkogenní proteiny fyziologie   MeSH
• tolerance záření * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH