The hedgehog signaling pathway plays an important role in vertebrate embryonic development, tissue homeostasis, and tumorigenesis. Constitutive activation of Hh signaling in various human tumors leads to GLI-mediated transcription and tumor progression. Based on the preliminary screening of a large library of known triterpenes that exhibited interesting Hh inhibitory activity, we designed and synthesized a new series of triterpenoid analogues containing aromatic heterocyclic substituents at position C-2 to enhance their interference with Hh signaling. In this study, we evaluated the effect of 15 synthesized triterpenoids on cell proliferation and Hh pathway activity in relevant cancer cell lines. Among these compounds, two derivatives, 11a and 11b, both featuring a furan ring at position C-2, demonstrated potent inhibitory effects on proliferation and induced cell death in nonsmall cell lung cancer (NSCLC) and prostate cancer cell lines exhibiting hyper-activated Hh signaling. Moreover, these compounds significantly reduced GLI-mediated transcription in cell-based reporter assays. Detailed immunoblot analyses revealed that compounds 11a and 11b decreased the expression of endogenous GLI1 protein and its target genes associated with tumor progression and proliferation, such as Cyclin D1, N-Myc, and Bcl-2, in A549 and DU-145 cancer cells. These findings suggest that the antiproliferative effects of 11a and 11b are mediated through inhibition of the Hh signaling pathway and are promising candidates for the development of new anticancer therapies targeting Hh-dependent tumors.
- Publikační typ
- časopisecké články MeSH
CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- CRISPR-Cas systémy genetika MeSH
- cystická fibróza * genetika metabolismus MeSH
- lidé MeSH
- protein CFTR * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Identifikace buněčných cílů aktivních látek má zásadní význam pro optimalizaci léčiv a minimalizaci jejich nežádoucích vedlejších účinků. Komplexní povaha biologických systémů ztěžuje tuto identifikaci, ale mikroskopické metody, zejména fenotypové testování, reprezentované metodou „Cell Painting“, představují cenný nástroj pro pochopení vlivu látek na úrovni buněk a organel. Tyto metody umožňují rychlé testování rozsáhlých knihoven látek a nabízejí unikátní pohled na mechanismus jejich účinku pozorováním chování buněk, pomocí hodnocení jejich morfologie, pohyblivosti, dělení a migrace. Mikroskopie živých buněk čelí výzvám, jako je fototoxicita, což vyžaduje pečlivý výběr fluorescenčních značek a optimalizaci podmínek. Mezi syntetickými fluorescenčními sondami pro mikroskopii živých buněk vynikají BODIPY barviva se svou syntetickou univerzálností a fotofyzikálními vlastnostmi, které zajišťují minimální poškození vzorku během biozobrazování.
Target identification of active substances is critical in optimizing drugs and minimizing side effects. The complex nature of biological systems presents challenges; to meet them, however, microscopic methods, particularly phenotypic screening, represented by "Cell Painting" method and fluorescent probes, can be used as valuable tools for understanding the impact of various substances at the cellular and organelle levels. These methods enable rapid testing of large libraries of compounds and offer unique insights into their mechanism of action by observing cell behavior, assessing cell morphology, motility, division, and migration. However, live cell microscopy faces challenges like phototoxicity, requiring a careful selection of fluorescent labels and optimized conditions. Among synthetic probes for live cell microscopy, BODIPY dyes stand out for their synthetic versatility and photophysical properties, providing minimal sample damage during bioimaging.
Photodynamic therapy (PDT) is a clinically-approved cancer treatment that is based on production of cytotoxic reactive oxygen species to induce cell death. However, its efficiency depends on distribution of photosensitizer (PS) and depth of light penetration through the tissues. Tendency of pathological cancer tissues to exhibit lower pH than healthy tissues inspired us to explore dual-targeted pH-activatable photosensitizers based on tunable near-infrared (NIR) boron-dipyrromethene (BODIPY) dyes. Our BODIPY PSs were designed to carry three main attributes: (i) biotin or cRGD peptide as an effective cancer cell targeting unit, (ii) amino moiety that is protonated in acidic (pH <6.5) conditions for pH-activation of the PS based on photoinduced electron transfer (PET) and (iii) hydrophilic groups enhancing the water solubility of very hydrophobic BODIPY dyes. Illumination of such compounds with suitable light (>640nm) allowed for high phototoxicity against HeLa (αvβ3 integrin and biotin receptor positive) and A549 (biotin receptor positive) cells compared to healthy MRC-5 (biotin negative) cells. Moreover, no dark toxicity was observed on selected cell lines (>10 μM) providing promising photosensitizers for tumour-targeted photodynamic therapy.
- MeSH
- biotin * chemie MeSH
- buňky A549 MeSH
- cyklické peptidy chemie farmakologie MeSH
- fotochemoterapie * MeSH
- fotosenzibilizující látky * chemie farmakologie MeSH
- HeLa buňky MeSH
- infračervené záření MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- sloučeniny boru * chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Lck, a member of the Src kinase family, is a non-receptor tyrosine kinase involved in immune cell activation, antigen recognition, tumor growth, and cytotoxic response. The enzyme has usually been linked to T lymphocyte activation upon antigen recognition. Lck activation is central to CD4, CD8, and NK activation. However, recently, it has become clearer that activating the enzyme in CD8 cells can be independent of antigen presentation and enhance the cytotoxic response. The role of Lck in NK cytotoxic function has been controversial in a similar fashion as the role of the enzyme in CAR T cells. Inhibiting tyrosine kinases has been a highly successful approach to treating hematologic malignancies. The inhibitors may be useful in treating other tumor types, and they may be useful to prevent cell exhaustion. New, more selective inhibitors have been documented, and they have shown interesting activities not only in tumor growth but in the treatment of autoimmune diseases, asthma, and graft vs. host disease. Drug repurposing and bioinformatics can aid in solving several unsolved issues about the role of Lck in cancer. In summary, the role of Lck in immune response and tumor growth is not a simple event and requires more research.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Neurodegenerative disease (ND) incidence has recently increased due to improved life expectancy. Alzheimer's (AD) or Parkinson's disease (PD) are the most prevalent NDs. Both diseases are poly genetic, multifactorial and heterogenous. Preventive medicine, a healthy diet, exercise, and controlling comorbidities may delay the onset. After the diseases are diagnosed, therapy is needed to slow progression. Recent studies show that local, peripheral and age-related inflammation accelerates NDs' onset and progression. Patients with autoimmune disorders like inflammatory bowel disease (IBD) could be at higher risk of developing AD or PD. However, no increase in ND incidence has been reported if the patients are adequately diagnosed and treated. Autoantibodies against abnormal tau, β amyloid and α- synuclein have been encountered in AD and PD and may be protective. This discovery led to the proposal of immune-based therapies for AD and PD involving monoclonal antibodies, immunization/ vaccines, pro-inflammatory cytokine inhibition and anti-inflammatory cytokine addition. All the different approaches have been analysed here. Future perspectives on new therapeutic strategies for both disorders are concisely examined.
BACKGROUND: A population-based cervical cancer screening programme is implemented in the Czech Republic. However, participation is insufficient among women over 50 years. This study aimed to estimate the potential improvement in participation through directly mailed HPV self-sampling kits (HPVssk) compared with standard invitation letters in women aged 50-65 non-participating in screening. METHODS: The study recruited 1564 eligible women (no cervical cancer screening in the last 3 years or more, no previous treatment associated with cervical lesions or cervical cancer). Eight hundred women were mailed with an HPVssk (HPVssk group), and 764 women were sent a standard invitation letter (control group) inviting them to a routine screening (Pap test). The primary outcome was a comparison of the overall participation rate between study groups using a binominal regression model. RESULTS: The participation rate in the HPVssk group was 13.4% [95% confidence interval (CI) 11.2-15.9%; 7.4% of women returned the HPVssk and 6.0% attended gynaecological examination] and 5.0% (95% CI 3.6-6.8%) in the control group. Using the binominal regression model, the difference between the groups was estimated as 7.6% (95% CI 5.0-10.2%; P < 0.001). In the HPVssk group, 22% of women who returned HPVssk had a positive result and 70% of them underwent a follow-up examination. CONCLUSIONS: Compared with traditional invitation letters, the direct mailing of the HPVssk achieved a significantly higher participation rate, along with a notable HPV positivity rate among HPVssk responders. This approach offers a potentially viable method for engaging women who have not yet attended a cervical screening programme.
- MeSH
- časná detekce nádoru metody MeSH
- infekce papilomavirem * diagnóza MeSH
- lidé MeSH
- nádory děložního čípku * diagnóza prevence a kontrola MeSH
- plošný screening metody MeSH
- vaginální stěr MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Testing of pooled samples is an effective strategy for increasing testing capacity while saving resources and time. This study aimed to validate pooled testing and gather real-life data on its use for Covid-19 surveillance with a gargle lavage (GL) self-sampling strategy. METHODS: Two-stage pooled testing with pools of 6 and 12 samples was used for preventive testing of an asymptomatic population and Covid-19 surveillance in Czech schools. Both GL and nasopharyngeal swabs were used for sampling. RESULTS: In total, 61,111 samples were tested. The use of pooled testing for large-scale Covid-19 surveillance reduced consumable costs by almost 75% and increased testing capacity up to 3.8-fold compared to standard methods. RT-PCR experiments revealed a minimal loss of sensitivity (0-2.2%) when using pooled samples, enabling the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes with Ct values >35. The minor loss of sensitivity was counterbalanced by a significantly increased throughput and the ability to substantially increase testing frequencies. CONCLUSIONS: Pooled testing is considerably more cost-effective and less time-consuming than standard testing for large-scale Covid-19 surveillance even when the prevalence of SARS-CoV-2 is fluctuating. Gargle lavage self-sampling is a non-invasive technique suitable for sample collection without a healthcare worker's assistance.
- MeSH
- COVID-19 * diagnóza epidemiologie MeSH
- lidé MeSH
- nazofarynx * virologie MeSH
- odběr biologického vzorku * metody MeSH
- SARS-CoV-2 * genetika izolace a purifikace MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 průkazem nukleové kyseliny metody MeSH
- testování na COVID-19 metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
