Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos—e.g., proteins, RNAs, and lipids—that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system

Cell cycle analysis using time-lapse microscopy

Pośród wielu różnych technik mikroskopowych wykorzystywanych do badania procesów biologicznych, technika video-mikroskopii (ang. time-laps microscopy) daje unikalne możliwości prowadzenia obserwacji w czasie na poziomie pojedynczych komórek. Bez wątpienia, jednym z procesów, budzącym od lat szczególne zainteresowanie badaczy jest podział komórki. Prawidłowy rozdział materiału genetycznego, który ma miejsce w trakcie mitozy jest jednym z warunków zachowania stabilności genomu. Z drugiej strony, zaburzenia mitozy mogą prowadzić do aneulpoidii i w efekcie transformacji nowotworowej. Zmiany morfologiczne charakterystyczne dla komórki mitotycznej pozwalają na obserwowanie jej w trakcie podziału, a także na śledzenie losów komórek potomnych. Dodatkowo, wprowadzenie do komórek genów kodujących fluorescencyjnie wyznakowane białka, których ekspresja zmienia się w zależności od fazy cyklu, umożliwia stosunkowo precyzyjną obserwację komórek w poszczególnych fazach cyklu poprzedzających mitozę lub następujących po niej. Obserwacje żywych komórek i tworzenie filmów poklatkowych znajdują obecnie duże zastosowanie w badaniach prowadzonych na komórkach nowotworowych, mających na celu ocenę skuteczności działania istniejących chemioterapeutyków jak również poszukiwaniu nowych związków o takich właściwościach. Mogą stanowić również wskazówkę do tworzenia złożonych terapii wielolekowych, w trakcie których badane związki podane w określonej kolejności będą prowadziły do zatrzymania komórek w takiej fazie cyklu, w której są one bardziej wrażliwe na kolejny związek zastosowany w terapii. Możliwości wykorzystania techniki video-mikroskopii są bardzo duże i mogą mieć niezwykle istotny wpływ na rozwiązywanie różnorodnych problemów biologicznych.Among many different techniques used in microscopy in order to visualize biological processes, time-laps microscopy gives unique opportunity to observe living cells on single cell level in time. One of the important processes that gain special interest is mitosis. Equal and undisturbed distribution of genetic material into two daughter cell that take place during mitosis is prerequisite of genome stability. In contrary, mitosis disturbances give rise to chromosomal instability, aneuploidy and cancer. Morphological changes that characterize mitotic cells enables to monitor cell division as well as tracking the fate of daughter cells. Moreover, transfection of the cells with vectors coding fluorescent proteins which expression changes during cell cycle make possible to follow the cell cycle progression of individual cell. Time-lapse microscopy is used in cancer biology research in order to reveal the influence of chemotherapeutic drugs on the cell cycle of cancer cells. It is also used to find the new compounds or to set new chemotherapeutics drug regimes, that could lead to cell cycle alteration, lethal for the cancer cell. Time-laps microscopy is a very powerful technique that could help to resolve different biological issues

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. Methods: We treated human quiescent or proliferating CD8+ cells with 50 µM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Results: Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable

The Role of Curcumin in the Modulation of Ageing

It is believed that postponing ageing is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-ageing role of curcumin and discuss its senolytic potential

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers

Human dihydrofolate reductase and thymidylate synthase form a complex <i>in vitro</i> and co-localize in normal and cancer cells

<p>Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex <i>in vitro</i> and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation <i>in vivo</i>, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an <i>in silico</i> model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.</p