Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.
- MeSH
- androgenní receptory genetika metabolismus MeSH
- antitumorózní látky farmakologie MeSH
- buňky PC-3 MeSH
- CD antigeny genetika metabolismus MeSH
- cyklopentany farmakologie MeSH
- docetaxel farmakologie MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- inhibitor p27 cyklin-dependentní kinasy genetika metabolismus MeSH
- kadheriny genetika metabolismus MeSH
- lidé MeSH
- lymfatické metastázy MeSH
- malá interferující RNA genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory prostaty genetika metabolismus patologie MeSH
- posttranslační úpravy proteinů * MeSH
- prostata metabolismus patologie MeSH
- protein NEDD8 genetika metabolismus MeSH
- proteiny asociované s kinázou S-fáze antagonisté a inhibitory genetika metabolismus MeSH
- pyrimidiny farmakologie MeSH
- regulace genové exprese u nádorů MeSH
- rodina transkripčních faktorů Snail genetika metabolismus MeSH
- stupeň nádoru MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.
- MeSH
- blastocysta cytologie metabolismus MeSH
- embryonální vývoj * MeSH
- inhibitor p27 cyklin-dependentní kinasy chemie metabolismus MeSH
- molekulová hmotnost MeSH
- NFKB inhibitor alfa chemie metabolismus MeSH
- proteiny asociované s kinázou S-fáze chemie metabolismus MeSH
- proteiny chemie metabolismus MeSH
- skot MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components--Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during preimplantation development. This paper reveals comprehensive expression profile, the basic but important knowledge necessary for further studying.
- MeSH
- blastocysta metabolismus ultrastruktura MeSH
- embryonální vývoj genetika MeSH
- F-box proteiny genetika metabolismus MeSH
- fertilizace in vitro MeSH
- genetická transkripce MeSH
- kulinové proteiny genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- oocyty cytologie růst a vývoj metabolismus MeSH
- proteinligasy komplexu SCF genetika metabolismus MeSH
- proteiny asociované s kinázou S-fáze genetika metabolismus MeSH
- signální transdukce MeSH
- skot MeSH
- spermie cytologie metabolismus MeSH
- substrátová specifita MeSH
- vývojová regulace genové exprese MeSH
- zinkové prsty genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma, body cavity-based lymphoma, and Castleman disease. vIRF-3 is a KSHV latent gene that is critical for proliferation of KSHV-positive lymphoid cells. Furthermore, vIRF-3 contributes to KSHV-associated pathogenesis by stimulating c-Myc transcription activity. Here we show that vIRF-3 can associate with Skp2, a key component of the SCF(skp2) ubiquitin ligase complex. Skp2 is a transcriptional co-factor for c-Myc that was shown to regulate the stability of c-Myc protein as well as c-Myc-dependent transcription. In this study, we show that vIRF-3 binds to the F-box of Skp2 and recruits it to c-Myc-regulated promoters to activate c-Myc-dependent transcription. Additionally, cells overexpressing vIRF-3 exhibit higher levels of c-Myc ubiquitylation, suggesting that ubiquitylation is necessary for c-Myc-mediated transcription. Moreover, vIRF-3 can stabilize the c-Myc protein by increasing its half-life. Collectively, these results indicate that vIRF-3 can effectively manipulate c-Myc stability and function and thus contribute to c-Myc-induced KSHV-associated lymphomagenesis.
- MeSH
- genetická transkripce genetika MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- hyperplazie velkých lymfatických uzlin genetika metabolismus virologie MeSH
- interferonové regulační faktory genetika metabolismus MeSH
- lidé MeSH
- lidský herpesvirus 8 genetika metabolismus MeSH
- lymfocyty metabolismus virologie MeSH
- proteiny asociované s kinázou S-fáze genetika metabolismus MeSH
- protoonkogenní proteiny c-myc genetika metabolismus MeSH
- stabilita proteinů MeSH
- ubikvitinace genetika MeSH
- vazba proteinů MeSH
- virové proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.
- MeSH
- androgenní receptory metabolismus MeSH
- antagonisté androgenů farmakologie MeSH
- beta-galaktosidasa metabolismus MeSH
- down regulace účinky léků MeSH
- fosfohydroláza PTEN metabolismus MeSH
- IGFBP-3 metabolismus MeSH
- kathepsin B metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prostaty genetika metabolismus patologie MeSH
- proteiny asociované s kinázou S-fáze genetika metabolismus MeSH
- průtoková cytometrie MeSH
- RNA interference MeSH
- signální transdukce účinky léků MeSH
- stárnutí buněk účinky léků MeSH
- vimentin metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR-dependent checkpoint response, followed by a later DDR-independent, but p27(Kip1)-dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA-PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two-step response was DDR-dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA-PKcs resulted in cell-cycle re-entry despite the increased levels of p27(Kip1) at all time points analyzed. We further investigated the regulation of p27(Kip1) protein levels in the particular setting. Our results showed that the protein status of p27(Kip1) is mainly determined by p38-MAPK, whereas the role of SKP2 is less significant in the doxoroubicin-treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy-induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.
- MeSH
- antitumorózní látky farmakologie MeSH
- ATM protein antagonisté a inhibitory MeSH
- buňky A549 MeSH
- chromony farmakologie MeSH
- doxorubicin farmakologie MeSH
- inhibitor p27 cyklin-dependentní kinasy fyziologie MeSH
- kofein farmakologie MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy p38 metabolismus MeSH
- morfoliny farmakologie MeSH
- poškození DNA MeSH
- proteinkinasa aktivovaná DNA antagonisté a inhibitory MeSH
- proteiny asociované s kinázou S-fáze metabolismus MeSH
- pyrony farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH