The aim of this study was to examine the effects of 5‑fluorouracil (5‑FU), anti‑epidermal growth factor receptor (EGFR) antibody and aspirin (ASA) on the characteristics of two CRC cell lines, HCT116 and HT29, maintained in a spherical culture system. We observed that the morphology of both the HCT116 and HT29 cell‑derived spheres was significantly impaired and the size of the colonospheres was markedly reduced following treatment with the aforementioned three drugs. In contrast to adherent cultures, the spherical cultures were more resistant to the tested drugs, as was reflected by their capacity to re‑create the colonospheres when sustained in serum‑free medium. Flow cytometric analysis of the drug‑treated HCT116 cell‑derived spheres revealed changes in the fraction of cells expressing markers of cancer stem cells (CSCs), whereas the CSC phenotype of HT29 cell‑derived colonospheres was affected to a lesser extent. All reagents enhanced the percentage of non‑viable cells in the colonospheres despite the diminished fraction of active caspase‑3‑positive cells following treatment of the HT29 cell‑derived spheres with anti‑EGFR antibody. Increased autophagy, assessed by acridine orange staining, was noted following the incubation of the HT29‑colonospheres with ASA and 5‑FU in comparison to the control. Notably, the percentage of cyclooxygenase (COX)‑2‑positive cells was not affected by ASA, although its activity was markedly elevated in the colonospheres incubated with anti‑EGFR antibody. On the whole, the findings of this study indicate that all the tested drugs were involved in different cellular processes, which suggests that they should be considered for the combined therapeutic treatment of CRC, particularly for targeting the population of CSC‑like cells. Thus, cancer cell‑derived spheres may be used as a preferable model for in vitro anticancer drug testing.
NK cells are a component of innate immunity which activity significantly correlates with health status. The aim of our study was to estimate a status of NK (natural killer) cells in the very old (mean age 92+/-2 ys) and old subjects (mean age 78+/-5 ys) as compared to a control group of young individuals (mean age 25+/-4 ys). NK cells were characterized by measurement of their cytotoxic activity, expression of intracellular interferon gamma, telomere length and telomerase activity in resting and activated cells. The results revealed that the oldest seniors did not differ from the other age groups in the number of NK cells and NK cytotoxic activity, however, they displayed the shortest telomeres and the lowest telomerase activity. Surprisingly, activated NK cells of the very old, similarly to the old subjects, were able to significantly increase intracellular level of IFNgamma. Moreover, activated with IL-2 NK cells of the old and oldest seniors showed increased telomerase activity. The results of our study suggest that the functional status of NK cells and their sensitivity to activation is well preserved until very advanced age and may contribute to longevity and successful ageing.
Novel Approaches in Cancer Study Different Faces of Fas Signaling in Cancer Cells Magdalena Szarynska* and Agata Olejniczak Kęder Department of Histology, Poland *Corresponding author:Magdalena Szaryńska, Debinki 1, 80-210, Gdansk, Poland Submission: September 10, 2019;Published: September 16, 2019 DOI: 10.31031/NACS.2019.03.000559 ISSN:2637-773XVolume3 Issue2
Spherical cultures (SCs) can be regarded in cancer research as a link between in vitro investigations on cancer lines and in vivo studies of tumor development. SCs are believed to mimic tumor architecture and to be enriched in cancer stem cell-like cells (CSC-like cells). In the present study we characterized colonospheres derived from colorectal cancer (CRC) cell lines, and we confirmed the ability of HCT116 and HT29 cell lines to form spheres within serum-free medium, however, the detailed analysis presented the major differences concerning their characteristics including morphology, phenotype, proliferative potential, distribution in the cell cycle phases and spherogenicity. Moreover, after we magnetically separated CD133+ and CD133- cells we could conduct the analogical analysis as we performed for the original cells. We observed that all cellular fractions unveiled sphere formation capacity, even when cultured in limited number of cells per well and only SCs originated from CD133+ fraction resembled morphologically the parental spheres. Both CD133+ and CD133- subsets derived from HCT116 line were more enriched in cells in G0/G1 phase of the cell cycle in comparison to their HT29 analogues. Additionally, proliferative potential also varied amongst all studied fractions. Surprisingly, 3-D invasion assay revealed that only HCT116-derived populations were able to migrate into extended regions of Matrigel Matrix confirming their higher aggressiveness. Our results provided comprehensive characterization of CRC cell lines culture in adherent and spherical forms and, what seems to be the most advantageous, the comparison of two distinct fractions after magnetic separation. As we found the specific features of cells presented line- and expansion mode-dependency, thus, such complete description might appear crucial before CRC lines would be involved into sophisticated assays, especially focused on potentially novel therapeutic agents targeting CSC-like cells.
The role of the innate immunity during human ageing is not well understood. The aim of the study was to estimate reactivity of NK (natural killer) cells in the very old (mean age 91 years) and old subjects (mean age 78 years) compared to young individuals (mean age 26 years) in respect to the indices of the oxidative stress (telomere length of NK cells, serum content of -H groups), serum total antioxidant status and serum concentrations of interleukin 6 and tumor necrosis factor-α (TNF-α). The activation state of NK cells, reflected by telomerase activity and intracellular interferon γ (IFNγ) content, was also measured. We found that length of telomeres in NK cells and serum concentration of -SH groups decreased both in the old and the oldest subjects as compared to young individuals. The oldest seniors, on the contrary to the old ones, revealed similar level of serum antioxidant status as the young subjects. The serum level of IL-6, not detectable in the young subjects, did not differ in the oldest and old seniors. TNF-α serum concentrations progressively increased with age. After stimulation, NK cells of both old groups showed higher intracellular levels of IFNγ than young subjects. IL-2-activated NK cells of the oldest seniors showed the highest increase of telomerase activity as compared to the other age groups. Serum level of IL-6 correlated positively with activation markers of NK cells. Moreover, in seniors but not in young subjects, the number of active, IFNγ-expressing NK cells, correlated positively with the serum content of the -SH groups. These findings indicate that sensitivity of NK cells to activation is maintained during ageing and this phenomenon may be related to the oxidative and inflammatory status of the elderly.
Cancer stem cells (CSCs) play a key role in the development and progression of colorectal cancer (CRC), but the influence of triiodothyronine (T3) on the biological regulation of CSCs remains unclear. In the present study, it was reported that T3 exerts significant impact on CSCs of two CRC cell lines cultured in the form of colonospheres. It was observed that the incubation of colonospheres with T3 decreased the viability, proliferative and spherogenic potential of cancer cells (P<0.05). In addition, increased apoptotic rate of CRC cells treated with T3 was revealed. Furthermore, T3‑treated colonospheres were more likely to move into silenced pool in G0/G1 phase of the cell cycle. The smaller sizes of colonospheres observed after the treatment with T3 confirmed this conclusion. T3 could lower the proportion of primitive cells which supply the pool of proliferating cells within spheres. Thyroid receptors THRα1 and THRβ1 and two deiodinases (DIO2 and DIO3) were affected by T3 in manner depended on clinical stage of cancer and CRC cell line used for analysis. In summary, the present study uncovered a novel function of thyroid hormones signaling in the regulation of the CSCs of CRC, and these findings may be useful for developing novel therapies by targeting thyroid hormone functions in CRC cells.
Ultrashort cationic lipopeptides (USCLs) and quaternary ammonium salts constitute two groups of cationic surfactants with high antimicrobial activity. This study aimed to investigate the influence of quaternization of the amino group of the lysine side chain in USCLs on their antimicrobial, hemolytic and cytotoxic activities. To do this, two series of lipopeptides were synthesized, USLCs and their quaternized analogues containing trimethylated lysine residues - qUSCLs (quaternized ultrashort cationic lipopeptides). Quaternization was performed on a resin during a standard solid-phase peptide synthesis with CH3I as the methylating agent. According to our knowledge, this is the first study presenting on-resin peptide quaternization. The lipopeptides were tested for their antibacterial and antifungal activities against the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella aerogenes) bacteria and Candida glabrata yeast-like fungus. Most of the compounds proved to be active antimicrobial agents with enhanced activity against Gram-positive strains and fungi and a lower against Gram-negative species. In addition, the antimicrobial activity of lipopeptides was increasing with an increase in hydrophobicity but qUSCLs exhibited usually a poorer antimicrobial activity than their parent molecules. Furthermore, the toxicity against red blood cells and human keratinocytes was assessed. It's worth emphasizing that qUSCLs were less toxic than the parent molecules of comparative hydrophobicity. The results of the study proved that qUSCLs can offer a higher selectivity to pathogens over human cells than that of USCLs. Last but not least, quaternization of the peptides could increase their solubility and therefore their bioavailability and utility.
Mimo wielu lat poszukiwania skutecznych terapii, nowotwory stanowi± wci±? jedn±
z najczestszych przyczyn zgonow. Kluczem do rozwi±zania tego problemu wydaje sie
byae stworzenie protoko³ow leczenia zaprojektowanego dla ka?dego pacjenta indywidualnie,
lecz do tego potrzebna jest kompleksowa wiedza na temat genezy i progresji nowotworow.
Prze³omem dla postepu naszej wiedzy o patogenezie rozwoju nowotworow
by³o prawdopodobnie odkrycie nowotworowych komorek macierzystych (CSCs), ktore
sta³y sie podstaw± do sformu³owania nowych teorii opisuj±cych rozwoj chorob nowotworowych
bior±cych pod uwage z³o?ono¶ae ca³ej kancerogenezy oraz zro?nicowanie
stopnia wra?liwo¶ci komorek nowotworowych na ro?ne metody terapii. W czasie rozwoju
choroby nowotworowej prawdopodobnie dochodzi do wspo³wystepowania jednocze¶nie
zjawisk opisywanych przez dwie dominuj±ce teorie: w wyniku klonalnej ekspansji
dochodzi do selekcji CSCs, ktore nastepnie s± ¼rod³em ca³ej hierarchii heterogennych
komorek nowotworowych. Nowotworowe komorki macierzyste posiadaj± cechy typowe
dla prawid³owych komorek macierzystych. Prawdopodobnie powstaj± one z tkankowych
komorek macierzystych, jednak brakuje na to jednoznacznych dowodow. Analiza w³a¶ciwo¶ci
tych komorek pozwoli³a zrozumieae, dlaczego stosowane metody leczenia zawodzi³y
w tak wielu przypadkach, nie niszczy³y one bowiem tej populacji komorek. Odkrycie
CSCs pozwoli³o skoncentrowaae sie na poszukiwaniach nowych metod leczenia
skierowanych na pokonanie niewra?liwo¶ci CSCs na stosowane dot±d czynniki terapeutyczne.
Forum Medycyny Rodzinnej 2011, tom 5, nr 1, 47-56
W publikacji opisano role komorek dendrytycznych (DC) w regulowaniu ukladu immunologicznego. W ostatnich latach nastąpil przelom w postrzeganiu komorek dendrytycznych, ktore są jedną z kluczowych populacji komorek odpornościowych, nie tylko jako komorek prezentujących antygeny i indukujących uklad odpornościowy, lecz takze posiadających zdolnośc do wyciszania jego aktywności czy do aktywnego zabijania komorek nowotworowych.
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