BACKGROUND: Renal cell carcinoma is difficult to diagnose and unpredictable in disease course and severity. There are no specific biomarkers for diagnosis and prognosis estimation feasible in clinical practice. Long non-coding RNAs (lncRNAs) have emerged as potent regulators of gene expression in recent years. Aside from their cellular role, their expression patterns could be used as a biomarker of ongoing pathology. METHODS: In this work, we used next-generation sequencing for global lncRNA expression profiling in tumor and non-tumor tissue of RCC patients. The four candidate lncRNAs have been further validated on an independent cohort. PVT1, as the most promising lncRNA, has also been studied using functional in vitro tests. RESULTS: Next-generation sequencing showed significant dysregulation of 1163 lncRNAs; among them top 20 dysregulated lncRNAs were AC061975.7, AC124017.1, AP000696.1, AC148477.4, LINC02437, GATA3-AS, LINC01762, LINC01230, LINC01271, LINC01187, LINC00472, AC007849.1, LINC00982, LINC01543, AL031710.1, and AC019197.1 as down-regulated lncRNAs; and SLC16A1-AS1, PVT1, LINC0887, and LUCAT1 as up-regulated lncRNAs. We observed statistically significant dysregulation of PVT1, LUCAT1, and LINC00982. Moreover, we studied the effect of artificial PVT1 decrease in renal cell line 786-0 and observed an effect on cell viability and migration. CONCLUSION: Our results show not only the diagnostic but also the therapeutic potential of PVT1 in renal cell carcinoma.

• MeSH
• karcinom z renálních buněk * genetika   patofyziologie   MeSH
• lidé MeSH
• nádorové biomarkery genetika   fyziologie   MeSH
• nádorové buněčné linie MeSH
• nádory ledvin * genetika   patofyziologie   MeSH
• pohyb buněk genetika   MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u nádorů MeSH
• RNA dlouhá nekódující * genetika   metabolismus   fyziologie   MeSH
• viabilita buněk genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.

• MeSH
• adaptorové proteiny signální transdukční genetika   MeSH
• chemorezistence genetika   MeSH
• down regulace účinky léků   genetika   MeSH
• epiteliální ovariální karcinom farmakoterapie   genetika   MeSH
• karbamoylfosfátsynthasa (amoniak) genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádorové biomarkery genetika   MeSH
• nádorové buněčné linie MeSH
• nádory vaječníků farmakoterapie   genetika   MeSH
• paclitaxel terapeutické užití   MeSH
• proteiny s doménou LIM genetika   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům genetika   MeSH
• taxoidy terapeutické užití   MeSH
• transkripční faktory genetika   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Pancreatic β-cell failure and death contribute significantly to the pathogenesis of type 2 diabetes. One of the main factors responsible for β-cell dysfunction and subsequent cell death is chronic exposure to increased concentrations of FAs (fatty acids). The effect of FAs seems to depend particularly on the degree of their saturation. Saturated FAs induce apoptosis in pancreatic β-cells, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction by saturated FAs in β-cells are not completely elucidated. Saturated FAs induce ER stress, which in turn leads to activation of all ER stress pathways. When ER stress is severe or prolonged, apoptosis is induced. The main mediator seems to be the CHOP transcription factor. Via regulation of expression/activity of pro- and anti-apoptotic Bcl-2 family members, and potentially also through the increase in ROS production, CHOP switches on the mitochondrial pathway of apoptosis induction. ER stress signalling also possibly leads to autophagy signalling, which may activate caspase-8. Saturated FAs activate or inhibit various signalling pathways, i.e., p38 MAPK signalling, ERK signalling, ceramide signalling, Akt signalling and PKCδ signalling. This may lead to the activation of the mitochondrial pathway of apoptosis, as well. Particularly, the inhibition of the pro-survival Akt signalling seems to play an important role. This inhibition may be mediated by multiple pathways (e.g., ER stress signalling, PKCδ and ceramide) and could also consequence in autophagy signalling. Experimental evidence indicates the involvement of certain miRNAs in mechanisms of FA-induced β-cell apoptosis, as well. In the rather rare situations when unsaturated FAs are also shown to be pro-apoptotic, the mechanisms mediating this effect in β-cells seem to be the same as for saturated FAs. To conclude, FA-induced apoptosis rather appears to be preceded by complex cross talks of multiple signalling pathways. Some of these pathways may be regulated by decreased membrane fluidity due to saturated FA incorporation. Few data are available concerning molecular mechanisms mediating the protective effect of unsaturated FAs on the effect of saturated FAs. It seems that the main possible mechanism represents a rather inhibitory intervention into saturated FA-induced pro-apoptotic signalling than activation of some pro-survival signalling pathway(s) or metabolic interference in β-cells. This inhibitory intervention may be due to an increase of membrane fluidity.

• MeSH
• apoptóza * genetika   MeSH
• beta-buňky metabolismus   MeSH
• biologické modely MeSH
• diabetes mellitus 2. typu etiologie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• fyziologický stres MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• metabolismus lipidů MeSH
• mitochondrie genetika   metabolismus   MeSH
• signální transdukce MeSH
• viabilita buněk genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

TP53 gene defects represent the most unfavorable prognostic factor in chronic lymphocytic leukemia (CLL). Although recently introduced small-molecule B-cell receptor signalling inhibitors have revolutionized CLL treatment, data for ibrutinib still point to impaired prognosis for TP53-affected patients. Among cancer-associated TP53 mutations, missense substitutions predominate and typically result in a high mutated-p53 protein level. Therefore, rescuing the p53 tumor suppressor function through specific small molecules restoring p53 wild-type (wt) conformation represents an attractive therapeutic strategy for cancer patients with TP53 missense mutations. We tested the effect of mutated-p53 reactivating molecule PRIMA-1MET in 62 clinical CLL samples characterized for TP53 mutations and p53 protein level. At the subtle PRIMA-1MET concentrations (1-4 μM), most samples manifested concentration-dependent viability decrease and, conversely, apoptosis induction, with the response being similar in both the TP53-mutated and TP53-wt groups, as well as in the TP53-mutated samples with p53 protein stabilization and without it. PRIMA-1MET was able to reduce mutated p53 protein in a proportion of TP53-mutated CLL samples, and this reduction correlated with a significantly stronger viability decrease and apoptosis induction than samples with stable p53 levels. CLL cells are mostly sensitive to PRIMA-1MET apart from those with stable mutated p53.

• MeSH
• apoptóza účinky léků   genetika   MeSH
• chinuklidiny farmakologie   MeSH
• chronická lymfatická leukemie diagnóza   genetika   metabolismus   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• viabilita buněk účinky léků   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular "sensor" and "effector" of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.

• MeSH
• apoptóza účinky záření   MeSH
• buňky Hep G2 MeSH
• fototerapie * MeSH
• laserová terapie s nízkou intenzitou světla * MeSH
• lidé MeSH
• membránový potenciál mitochondrií genetika   účinky záření   MeSH
• mitochondriální membrány metabolismus   účinky záření   MeSH
• mitochondrie genetika   účinky záření   MeSH
• oxidace-redukce účinky záření   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese účinky záření   MeSH
• respirační komplex IV genetika   MeSH
• transport elektronů genetika   účinky záření   MeSH
• viabilita buněk genetika   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. METHODS: Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. RESULTS: Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. CONCLUSIONS: From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.

• MeSH
• androgenní receptory metabolismus   MeSH
• anotace sekvence MeSH
• benzamidy farmakologie   MeSH
• biologické modely MeSH
• biosyntetické dráhy genetika   MeSH
• buněčné sféroidy metabolismus   patologie   MeSH
• buněčný cyklus genetika   MeSH
• chemorezistence účinky léků   genetika   MeSH
• cholesterol biosyntéza   MeSH
• extracelulární matrix metabolismus   MeSH
• fenotyp MeSH
• fenylthiohydantoin farmakologie   MeSH
• fibroblasty asociované s nádorem metabolismus   patologie   MeSH
• kultivační média speciální farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty rezistentní na kastraci genetika   patologie   MeSH
• nádory prostaty genetika   metabolismus   patologie   MeSH
• nitrily farmakologie   MeSH
• progrese nemoci * MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• senioři MeSH
• simvastatin farmakologie   MeSH
• stanovení celkové genové exprese MeSH
• upregulace * MeSH
• viabilita buněk účinky léků   genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzo[a]pyrene (B[a]P) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by B[a]P and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of B[a]P which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.

• MeSH
• buněčné kultury MeSH
• buňky Hep G2 MeSH
• dánio pruhované embryologie   MeSH
• embryonální vývoj účinky léků   genetika   MeSH
• epitelové buňky účinky léků   patologie   MeSH
• kometový test MeSH
• kyslík chemie   MeSH
• lidé MeSH
• monitorování životního prostředí metody   MeSH
• mutageny analýza   toxicita   MeSH
• polycyklické aromatické uhlovodíky analýza   toxicita   MeSH
• poškození DNA * MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.

• MeSH
• buněčná membrána metabolismus   MeSH
• buňky K562 MeSH
• fluorescenční protilátková technika MeSH
• lidé MeSH
• P-glykoproteiny genetika   metabolismus   MeSH
• RNA interference MeSH
• viabilita buněk genetika   fyziologie   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.

• MeSH
• antitumorózní látky hormonální farmakologie   MeSH
• apoptóza účinky léků   genetika   MeSH
• genový knockdown MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• stárnutí buněk účinky léků   MeSH
• tamoxifen farmakologie   MeSH
• transfekce MeSH
• translokátor adeninových nukleotidů 2 genetika   metabolismus   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

• MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• checkpoint kinasa 1 antagonisté a inhibitory   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• HCT116 buňky MeSH
• inhibitor p21 cyklin-dependentní kinasy genetika   metabolismus   MeSH
• kontrolní body fáze S buněčného cyklu účinky léků   MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• nádory farmakoterapie   metabolismus   MeSH
• poškození DNA účinky léků   genetika   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   MeSH
• pyrazoly farmakologie   terapeutické užití   MeSH
• pyrimidinony farmakologie   terapeutické užití   MeSH
• transfekce MeSH
• tyrosinkinasy antagonisté a inhibitory   MeSH
• viabilita buněk účinky léků   genetika   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH