"APVV-19-0094"
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Current antibiotics and chemotherapeutics are becoming ineffective because pathogenic bacteria and tumor cells have developed multiple drug resistance. Therefore, it is necessary to find new substances that can be used in treatment, either alone or as sensitizing molecules in combination with existing drugs. Peptaibols are bioactive, membrane-active peptides of non-ribosomal origin, mainly produced by filamentous fungi such as Trichoderma spp. This study focused on producing peptaibol-rich extracts from Trichoderma atroviride O1, cultivated on malt extract agar (MA) under circadian and constant darkness conditions for 13 days. Peptaibol production was detected by MALDI-TOF mass spectrometry after six days of cultivation. The extracts demonstrated antibacterial activity against Staphylococcus aureus strains, particularly the methicillin-resistant variant, but not against the Gram-negative Pseudomonas aeruginosa. Quorum sensing interference revealed that a peptaibol-rich extract suppressed Vibrio campbellii BAA-1119's AI-2 signaling system to a degree comparable with gentamycin. Beyond antibacterial properties, the extracts exhibited notable antiproliferative activity against human ovarian cancer cells and their adriamycin-resistant subline in both 2D and 3D models. Specifically, MA-derived extracts reduced ovarian cancer cell viability by 70% at 50 μg/mL, especially under light/dark regime of cultivation. Compared to previously published results for PDA-based extracts, MA cultivation shifted the biological effects of peptaibol-containing extracts toward anticancer potential. These findings support the idea that modifying fungal cultivation parameters, the bioactivity of secondary metabolite mixtures can be tailored for specific therapeutic applications.
- MeSH
- agar * chemie MeSH
- antibakteriální látky * farmakologie metabolismus MeSH
- Hypocreales MeSH
- kultivační média chemie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- nádorové buněčné linie MeSH
- peptaiboly * farmakologie metabolismus biosyntéza chemie MeSH
- proliferace buněk účinky léků MeSH
- protinádorové látky * farmakologie metabolismus MeSH
- Pseudomonas aeruginosa účinky léků MeSH
- Staphylococcus aureus účinky léků MeSH
- Trichoderma * metabolismus růst a vývoj chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The backbone of therapy for elderly patients with myelodysplastic syndromes and acute myeloid leukemia consists of hypomethylating agents 5-aza-2'-deoxycytidine (DAC) and 5-azacytidine (AZA). However, resistance frequently emerges during treatment. To investigate the mechanisms of resistance, we generated DAC-resistant variants of the acute myeloid leukemia cell lines, MOLM-13 and SKM-1, through their prolonged cultivation in increasing concentrations of DAC. The resistant cell variants, MOLM-13/DAC and SKM-1/DAC, exhibited cross-resistance to cytarabine and gemcitabine, but remained sensitive to AZA. Existing studies have suggested that the loss of deoxycytidine kinase (DCK) may play an important role in DAC resistance. DCK is critical for DAC activation, but the precise mechanisms of its downregulation remain incompletely understood. We identified a novel point mutation (A180P) in DCK, which results in acquired DAC resistance. Although the DCK mRNA was actively transcribed, the mutant protein was not detected in DAC-resistant cells. The transfection of HEK293 cells with the mutant DCK, combined with proteasomal inhibition, revealed rapid proteasomal degradation, establishing a mechanistic link between the A180P mutation and DCK loss, not previously described. This highlights the importance of also evaluating DCK at the protein and/or enzymatic activity levels in patients. The loss of functional DCK impairs the phosphorylation of deoxynucleosides, conferring resistance to DAC, gemcitabine, and cytarabine, but AZA, phosphorylated by uridine-cytidine kinase, remains effective and may represent a therapeutic alternative for patients with acquired DAC resistance.
- MeSH
- akutní myeloidní leukemie * genetika farmakoterapie MeSH
- azacytidin * farmakologie analogy a deriváty MeSH
- chemorezistence * genetika účinky léků MeSH
- cytarabin farmakologie MeSH
- decitabin * farmakologie MeSH
- deoxycytidin analogy a deriváty farmakologie MeSH
- deoxycytidinkinasa * genetika metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- protinádorové antimetabolity * farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The rising number of invasive fungal infections caused by drug-resistant Candida strains is one of the greatest challenges for the development of novel antifungal strategies. The scarcity of available antifungals has drawn attention to the potential of natural products as antifungals and in combinational therapies. One of these is catechins-polyphenolic compounds-flavanols, found in a variety of plants. In this work, we evaluated the changes in the susceptibility of Candida glabrata strain characterized at the laboratory level and clinical isolates using the combination of catechin and antifungal azoles. Catechin alone had no antifungal activity within the concentration range tested. Its use in combination with miconazole resulted in complete inhibition of growth in the sensitive C. glabrata isolate and a significant growth reduction in the azole resistant C. glabrata clinical isolate. Simultaneous use of catechin and miconazole leads to increased intracellular ROS generation. The enhanced susceptibility of C. glabrata clinical isolates to miconazole by catechin was accompanied with the intracellular accumulation of ROS and changes in the plasma membrane permeability, as measured using fluorescence anisotropy, affecting the function of plasma membrane proteins.
KlUpc2p, a transcription factor belonging to the fungal binuclear cluster family, is an important regulator of ergosterol biosynthesis and azole drug resistance in Kluyveromyces lactis. In this work, we show that the absence of KlUpc2p generates Rag- phenotype and modulates the K. lactis susceptibility to oxidants and calcofuor white. The KlUPC2 deletion leads to increased expression of KlMGA2 gene, encoding an important regulator of hypoxic and lipid biosynthetic genes in K. lactis and also KlHOG1 gene. The absence of KlUpc2p does not lead to statistically significant changes in glycerol, corroborating the expression of KlGPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase, that is similar in both the deletion mutant and the parental wild-type strain. Increased sensitivity of Klupc2 mutant cells to brefeldin A accompanied with significant increase in KlARF2 gene expression point to the involvement of KlUpc2p in intracellular signaling. Our observations highlight the connections between ergosterol and fatty acid metabolism to modulate membrane properties and point to the possible involvement of KlUpc2p in K. lactis oxidative stress response.
