Quaternary Ammonium Salts of Cationic Lipopeptides with Lysine Residues - Synthesis, Antimicrobial, Hemolytic and Cytotoxic Activities.

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.

Bradykinin and Neurotensin Analogues as Potential Compounds in Colon Cancer Therapy.

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential.

Triiodothyronine lowers the potential of colorectal cancer stem cells in vitro.

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, T3­treated 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 THRß1 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.

Comment on: Molecular Functions of Thyroid Hormone Signaling in Regulation of Cancer Progression and Anti-Apoptosis.

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Effects of 5-FU and anti-EGFR antibody in combination with ASA on the spherical culture system of HCT116 and HT29 colorectal cancer cell lines.

The aim of this study was to examine the effects of 5­fluorouracil (5­FU), anti­epidermal 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 cell­derived 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 re­create the colonospheres when sustained in serum­free medium. Flow cytometric analysis of the drug­treated HCT116 cell­derived spheres revealed changes in the fraction of cells expressing markers of cancer stem cells (CSCs), whereas the CSC phenotype of HT29 cell­derived colonospheres was affected to a lesser extent. All reagents enhanced the percentage of non­viable cells in the colonospheres despite the diminished fraction of active caspase­3­positive cells following treatment of the HT29 cell­derived spheres with anti­EGFR antibody. Increased autophagy, assessed by acridine orange staining, was noted following the incubation of the HT29­colonospheres with ASA and 5­FU in comparison to the control. Notably, the percentage of cyclooxygenase (COX)­2­positive cells was not affected by ASA, although its activity was markedly elevated in the colonospheres incubated with anti­EGFR 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 CSC­like cells. Thus, cancer cell­derived spheres may be used as a preferable model for in vitro anticancer drug testing.

Diversity of dendritic cells generated from umbilical cord or adult peripheral blood precursors.

Following the discovery of methods to generate large numbers of specific dendritic cells (DCs) ex vivo, the possibility of exploiting these cells in immunotherapeutic strategies will become a reality. It seems to be rationally to analyse the influence of the precursor source for further features and applications. For the needs of the given project DCs were derived from precursors derived from adult peripheral blood (APB) and umbilical cord blood (UCB). During some expansions of UCB CD34+ cells were separated giving non-adherent DCs (NA-DCs) or adherent DCs (A-DCs), whereas DCs derived from UCB precursors without separation gave rise to All-DCs. DC subpopulations were stimulated by lipopolysaccharides (LPS) or interferon-γ (IFN-γ), and afterwards the morphology, phenotype, and stimulatory properties were analysed. Our findings demonstrated that DCs generated from APB and UCB precursors were not equivalent and exhibited opposite features when expanded in comparable conditions. Additionally, all three subpopulations of UCB-derived DCs presented functional dissimilarities. Based on our results we concluded that the precursor source and the composition of media must be considered as crucial to the success of potential therapeutic application.

Cancer stem cells as targets for DC-based immunotherapy of colorectal cancer.

The therapy of colorectal cancer (CRC) patients is often unsuccessful because of the presence of cancer stem cells (CSCs) resistant to conventional approaches. Dendritic cells (DC)-based protocols are believed to effectively supplement CRC therapy. Our study was aimed to assess how the number and properties of CSCs isolated from tumor tissue of CRC patients will affect the biological characteristics of in vitro modified DCs. Similar procedures were conducted with the using of CRC HCT116 and HT29 cell lines. We found that the detailed configuration of CSC-like markers significantly influenced the maturation and activation of DCs after stimulation with cancer cells lysates or culture supernatants. This basic stimulatory effect was enhanced by LPS that is normally present in CRC CSCs niche. The increased number of CD29+ and CD44+ CSCs presented the opposite impact on treated DCs as showed by many significant correlations. The CD133+ CSCs seemed to impair the functions of DCs. The more CD133+ CSCs in tumor sample the lower number of activated DCs evidenced after stimulation. Moreover, our results showed superiority of the spherical culture model over the adherent one since spherical HCT116 and HT29 cells presented similar influence on DCs properties as CRC patients cancer cells. We concluded that the DCs features may depend directly on the properties of CSCs affected by progression status of tumor.

In vitro characterization of spheres derived from colorectal cancer cell lines.

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.

Therapeutic strategies against cancer stem cells in human colorectal cancer.

Colorectal cancer (CRC) is the third most frequent malignancy and represents the fourth most common cause of cancer-associated mortalities in the world. Despite many advances in the treatment of CRC, the 5-year survival rate of patients with CRC remains unsatisfactory due to tumor recurrence and metastases. Recently, cancer stem cells (CSCs), have been suggested to be responsible for the initiation and relapse of the disease, and have been identified in CRC. Due to their basic biological features, which include self-renewal and pluripotency, CSCs may be novel therapeutic targets for CRC and other cancer types. Conventional therapeutics only act on proliferating and mature cancer cells, while quiescent CSCs survive and often become resistant to chemotherapy. In this review, markers of CRC-CSCs are evaluated and the recently introduced experimental therapies that specifically target these cells by inducing CSC proliferation, differentiation and sensitization to apoptotic signals via molecules including Dickkopf-1, bone morphogenetic protein 4, Kindlin-1, tankyrases, and p21-activated kinase 1, are discussed. In addition, novel strategies aimed at inhibiting some crucial processes engaged in cancer progression regulated by the Wnt, transforming growth factor ß and Notch signaling pathways (pyrvinium pamoate, silibinin, PRI-724, P17, and P144 peptides) are also evaluated. Although the metabolic alterations in cancer were first described decades ago, it is only recently that the concept of targeting key regulatory molecules of cell metabolism, such as sirtuin 1 (miR-34a) and AMPK (metformin), has emerged. In conclusion, the discovery of CSCs has resulted in the definition of novel therapeutic targets and the development of novel experimental therapies for CRC. However, further investigations are required in order to apply these novel drugs in human CRC.

FasR and FasL in colorectal cancer.

Colorectal cancer (CRC) is one of the most common solid organ cancers prevalent worldwide causing, in spite of advancing therapeutic methodology, high rate of patient mortality, especially due to metastasis development. The cancer stem cell (CSC) theory of tumor growth indicates that CSCs within the tumor mass have great capacity to initiate and sustain tumor growth. Following the suggestion that Fas signaling can be engaged in apoptosis, tumor maintenance, senescence or DICE (death induced by CD95 or CD95L elimination), the attempts to broaden the knowledge concerning the relationships between CSCs features and FasR/FasL appeared to be necessary. The most important advantage of our study was the simultaneously analysis of CSCs from commonly used CRC lines (HCT116 and HT29) and tumor fragments collected from CRC patients. Moreover, the sphere-promoting expansion of CRC lines brought a specific three-dimensional specific environment for CSC exploration. We further investigated the function of Fas signaling in CRC lines depending on the culture mode as we incubated HCT116 and HT29 cells with anti-FasR agonistic antibodies. It appeared to act in a line-dependent and culture mode-dependent manner and influenced some particular features of CSCs such as spherogenicity, proliferation and phenotype. Additionally, the analysis of mRNA level showed that disease progression is associated with significantly increased expression of FasR and/or FasL. In conclusion, our observation seems to confirm that spherical model of cancer lines is more reliable for some sophisticated analysis because of their greater resemblance to the CSCs from human CRC samples in comparison to commonly used adherent cells, at least according to aspects of their biology analyzed in this study. That can be extended to the resemblance of in vitro sphere forming conditions to the in vivo environment. However, the greatest difference concerns the level of apoptosis, thus, this issue require further experiments.

The role of cancer stem cells in pathogenesis of colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer, accounting for about 10% of total adult malignancies worldwide. The majority of CRC cases are diagnosed at the late stage; thus the investigation of the pathogenesis of early-stage disease and its detection could prevent formation of metastasis, a leading cause of death. This review highlights the recent progress in the understanding of the development of cancer stem cells (CSCs) in the colon epithelium and mechanisms of their proliferation. Moreover, we describe the role of the CSCs in resistance to chemotherapy and formation of metastases. We present evidence for the importance of the interactions between CSCs and their environment in the propagation of the disease. It is hoped that further studies of colorectal cancer CSCs could be helpful in the early detection and improved therapy of this neoplasm.

Cytokine profiles during delivery affect cord blood hematopoietic stem and progenitors cells.

The study was aimed to determine the correlations between serum levels of cytokines (GM-CSF, IL-4, IL-10 and TNF) in maternal (MB) and cord blood (CB) and some features of cord blood hematopoietic stem and progenitor cells (CB HSPCs). Study material was MB and concomitant CB samples collected from 98 volunteers at the moment of delivery. The IL-4, IL-10, TNF and GM-CSF concentrations in serum and in supernatants from PMA-stimulated mononuclear cells isolated from both blood types were measured using BD Cytometric Bead Array Flex Set System. CB HSPCs (CD34(+)CD45(low)) proportion was also estimated by flow cytometry. The most relevant results concerned the tendency to down regulation of CB HSPCs number with an increase of IL-4, IL-10 and GM-CSF levels, only the TNF concentration seems to have no influence on HSPCs pole size. The strongest positive correlations were found between CD34(+)CD45(low) HSPCs number and IL-10 and GM-CSF in MB serum and GM-CSF and TNF from CB supernatants. The strongest negative correlations were found between CD34(+)CD45(low) HSPCs number and IL-4 and GM-CSF in CB serum and IL-10 in MB supernatants. Interestingly, we observed 'opposite correlation' between serum and supernatant from CB and MB. We concluded that elevated serum levels of IL-4, IL-10 and GM-CSF in CB are indicative of enhanced differentiation of HSPCs and characterize a normal perinatal development. Elevated levels of cytokines seem to stimulate differentiation of HSPCs what is advantageous for neonates during perinatal period.

Analogues of muramyl dipeptide (MDP) and tuftsin limit infection and inflammation in murine model of sepsis.

Pharmacological manipulation of the balance between pro- and anti-inflammatory mediators emerges as a key aspect of a successful treatment of sepsis. A murine model of septic shock was developed and chosen conjugates (1a, 1b, 8a, 8c) and analogs (T2) of muramyl dipeptide and tuftsin were tested in this model as prospective anti-bacterial drugs or adjuvants. The phagocytic activity of monocytes/macrophages was determined (flow cytometry, bacterial clearance from vital organs). To evaluate cytokines levels (TNFalpha, IFNgamma, IL6, IL10) we used real-time PCR. The most promising immunomodulatory properties were displayed by the analogue T2 and two conjugates: 8a, 8c.

Ex vivo expansion of CD4(+)CD25(+) T regulatory cells for immunosuppressive therapy.

Immunosuppressants are powerful drugs, capable of triggering severe adverse effects. Hence, there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models, they have been found to quench autoimmune diseases, maintain allogeneic transplants, and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery, not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles, and has opened the doors to the clinical application of Tregs. In the review, we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory, as well as some published reports, Treg adoptive therapy is a promising tool in immunosuppressive therapy, and should be considered for clinical trials.