Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.
- MeSH
- antigeny CD44 genetika metabolismus MeSH
- kolon metabolismus patologie MeSH
- kolorektální nádory diagnóza genetika metabolismus MeSH
- lidé MeSH
- nádorové biomarkery genetika metabolismus MeSH
- polypy tlustého střeva metabolismus patologie MeSH
- prognóza MeSH
- protein - isoformy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.
- MeSH
- antigeny CD44 analýza metabolismus MeSH
- antitumorózní látky aplikace a dávkování MeSH
- buněčné linie MeSH
- endocytóza MeSH
- fluorescenční barviva MeSH
- kyselina hyaluronová chemie metabolismus MeSH
- lidé MeSH
- lipidy chemická syntéza MeSH
- liposomy chemie terapeutické užití MeSH
- mikrofluidika MeSH
- nádory farmakoterapie MeSH
- systémy cílené aplikace léků metody MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".
- MeSH
- adheziva chemie MeSH
- aplikace bukální MeSH
- aplikace sublinguální MeSH
- léčivé přípravky aplikace a dávkování MeSH
- liposomy chemie MeSH
- lymfatické uzliny metabolismus MeSH
- myši MeSH
- nanočástice chemie MeSH
- nanovlákna chemie MeSH
- polyethylenglykoly chemie MeSH
- polyglactin 910 chemie MeSH
- prasata MeSH
- systémy cílené aplikace léků metody MeSH
- ústní sliznice metabolismus MeSH
- vakcinace metody MeSH
- vakcíny aplikace a dávkování MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100μM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.
- MeSH
- 2-aminopurin analogy a deriváty chemie farmakologie MeSH
- antivirové látky chemie farmakologie MeSH
- buněčné linie MeSH
- buňky MDCK MeSH
- Herpesviridae účinky léků MeSH
- organofosfonáty chemie farmakologie MeSH
- pseudovzteklina farmakoterapie MeSH
- psi MeSH
- replikace DNA účinky léků MeSH
- techniky in vitro MeSH
- transmisní elektronová mikroskopie MeSH
- Vero buňky MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pro-apoptotic analogues of vitamin E (VE) exert selective anti-cancer effect on various animal cancer models. Neither suitable formulation of α-tocopheryl succinate (α-TOS), representative semi-synthetic VE analogue ester, nor suitable formulations of the other VE analogues for clinical application have been reported yet. The major factor limiting the use of VE analogues is their low solubility in aqueous solvents. Due to the hydrophobic character of VE analogues, liposomes are predetermined as suitable delivery system. Liposomal formulation prevents undesirable side effects of the drug, enhances the drug biocompatibility, and improves the drug therapeutic index. Liposomal formulations of VE analogues especially of α-TOS and α-tocopheryl ether linked acetic acid (α-TEA) have been developed. The anti-cancer effect of these liposomal VE analogues has been successfully demonstrated in pre-clinical models in vivo. Present achievements in: (i) preparation of liposomal formulations of VE analogues, (ii) physico-chemical characterization of these developed systems and (iii) testing of their biological activity such as induction of apoptosis and evaluation of anti-cancer effect are discussed in this review.
- MeSH
- alfa-tokoferol aplikace a dávkování MeSH
- antitumorózní látky aplikace a dávkování chemie MeSH
- apoptóza účinky léků MeSH
- chemie farmaceutická MeSH
- lidé MeSH
- lipidy chemie MeSH
- liposomy MeSH
- nádory farmakoterapie patologie MeSH
- rozpustnost MeSH
- systémy cílené aplikace léků metody MeSH
- vitamin E aplikace a dávkování analogy a deriváty chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor microenvironment. CD44 contains two variable regions encoded by variable exons. Alternative splicing, which is often deregulated in cancer, can produce various isoforms of CD44 with properties that may have different tissue specific effects and therefore even diverse effects on cancer progression. This review summarizes and puts together all major regulators of alternative splicing of CD44 in cancer that have been documented so far and that have an experimentally proved effect on CD44 isoform switching. It is important to better understand the mechanisms of alternative splicing of CD44, where all the variability of CD44 originates, to be able to explain the isoform switching and occurrence of variant isoforms of CD44 (CD44v) in cancer.
- MeSH
- alternativní sestřih MeSH
- antigeny CD44 genetika metabolismus MeSH
- epitelo-mezenchymální tranzice MeSH
- lidé MeSH
- nádorové kmenové buňky metabolismus MeSH
- nádory metabolismus patologie MeSH
- protein - isoformy genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
α-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.
- MeSH
- ABC transportéry genetika fyziologie MeSH
- ABCA1 protein MeSH
- alfa-tokoferol terapeutické užití MeSH
- antitumorózní látky farmakologie MeSH
- chemorezistence * MeSH
- genový knockdown MeSH
- mitochondrie účinky léků MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory plic farmakoterapie MeSH
- nemalobuněčný karcinom plic farmakoterapie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.
- MeSH
- alfa-tokoferol farmakologie MeSH
- apoptóza fyziologie MeSH
- forkhead transkripční faktory genetika metabolismus MeSH
- genetická transkripce MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lymfom T-buněčný genetika metabolismus patologie terapie MeSH
- malá interferující RNA aplikace a dávkování genetika MeSH
- nádorové buněčné linie MeSH
- nádory plic genetika metabolismus patologie terapie MeSH
- nemalobuněčný karcinom plic genetika metabolismus patologie terapie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- promotorové oblasti (genetika) MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 biosyntéza genetika metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan alpha-tocopheryl succinate (alpha-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to alpha-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. alpha-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that alpha-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for alpha-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by alpha-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.
- MeSH
- alfa-tokoferol chemie toxicita MeSH
- antitumorózní látky chemie toxicita MeSH
- apoptóza MeSH
- Jurkat buňky MeSH
- lidé MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie účinky léků MeSH
- nádorový supresorový protein p53 genetika MeSH
- protein Bak chemie genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.
- MeSH
- alfa-tokoferol analogy a deriváty aplikace a dávkování farmakologie MeSH
- antitumorózní látky aplikace a dávkování farmakologie MeSH
- chemie farmaceutická MeSH
- lidé MeSH
- liposomy MeSH
- maleimidy aplikace a dávkování farmakologie MeSH
- melanom experimentální farmakoterapie patologie MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prsu farmakoterapie patologie MeSH
- polyethylenglykoly aplikace a dávkování farmakologie MeSH
- vitamin E analogy a deriváty aplikace a dávkování farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
