Search

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

Conjugating an anticancer drug of high biological efficacy but large cytotoxicity with a “transporting” molecule of low toxicity constitutes a valuable approach to design safe drug delivery system. In the present study, doxorubicin (DOX) a drug of large cardiotoxicity was chemically conjugated to a C60-fullerene. The synthesized molecule, a fullerene-doxorubicin conjugate (Ful-DOX), was characterized using the 1H NMR and MALDI TOF mass spectrometry. The absorption and fluorescence spectra and dynamic light scattering of the conjugate were recorded in an aqueous solution, while the impact on viability of several cancer cell lines of the free DOX and the conjugate was compared using the SRB and WST-1 assays. A low antiproliferative activity of the conjugate as compared to the free DOX is a consequence of the presence of fullerene moiety in the former, which is also responsible for the conjugate aggregation in an aqueous solution. Unlike free DOX, these aggregates cannot pass through the nuclear membrane (as demonstrated by the confocal microscopy measurements), which makes them marginally cytotoxic.

Purpose Isothiocyanates (ITCs) are compounds derived from Brassica plants with documented anticancer activity. Molecular mechanisms of their selective activity against cancer cells are still underexplored. In this work, the impact of ITC on DNA replication and damage was compared between PC-3 prostate cancer cells and HDFa normal fibroblasts as well as PNT2 prostate epithelial cells. Methods Cells were treated with sulforaphane or phenethyl isothiocyanate. [3H]thymidine incorporation and the level of histone γH2A.X were estimated as indicators of DNA replication and double-strand breaks (DSB), respectively. Levels of HDAC3, CtIP, and p-RPA were investigated by immunoblotting. Comet assay was performed to visualize DNA damage. Results ITCs inhibited DNA replication in all tested cell lines, and this activity was independent of reactive oxygen species of mitochondrial origin. It was followed by DSB which were more pronounced in cancer than noncancerous cells. This difference was independent of HDAC activity which was decreased in both cell lines when treated with ITCs. On the other hand, it correlated with faster removal of DSB, and thus, transient activation of repair proteins in normal cells, while in PC-3 prostate cancer, cell DNA repair was significantly less effective. Conclusion DNA damage induced by ITCs is a consequence of the block in DNA replication which is observed in both, cancer and normal cells. Selective antiproliferative activity of ITCs towards cancer cells results from less efficient DNA repair in cancer cells relative to normal cells.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today&rsquo, s science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name &ldquo, Imunofan&rdquo, (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p &lt, 0.05) pro-proliferative activity (30&ndash, 40% and 20&ndash, 50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p &lt, 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p &lt, 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2–3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers—5-bromo-2′-deoxyuridine (BrdU)—on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ●OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions.

Background Isothiocyanates derived from the Brassicaceae plants possess chemopreventive and anticancer activities. One of them is sulforaphene (SF), which is abundant in Rhapanus sativus seeds. The underlying mechanism of its anticancer activity is still underexplored. Purpose SF properties make it an interesting candidate for cancer prevention and therapy. Thus, it is crucial to characterize the mechanism of its activity. Study design We investigated the mechanism of antiproliferative activity of SF in breast cancer cells differing in growth factor receptors status and lacking functional p53. Methods Viability of SKBR-3 and MDA-MB-231 breast cancer cells treated with SF was determined by SRB and clonogenic assays. Cell cycle, cell death and oxidative stress were analyzed by flow cytometry or microscopy. The levels of apoptosis and autophagy markers were assessed by immunoblotting. Results SF efficiently decreased the viability of breast cancer cells, while normal cells (MCF10A) were less sensitive to the analyzed isothiocyanate. SF induced G2/M cell cycle arrest, as well as disturbed cytoskeletal organization and reduced clonogenic potential of the cancer cells. SF induced apoptosis in a concentration-dependent manner which was associated with the oxidative stress, mitochondria dysfunction, increased Bax:Bcl2 ratio and ADRP levels. SF also potentiated autophagy which played a cytoprotective role. Conclusions SF exhibits cytotoxic activity against breast cancer cells even at relatively low concentrations (5–10 µM). This is associated with induction of the cell cycle arrest and apoptosis. SF might be considered as a potent anticancer agent.

Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC50 values after 48 h of treatment were ca. 3 μM for MCF-7 and PC-3 cells and 1 μM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells.

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

Antimicrobial peptides (AMPs) constitute a promising tool in the development of novel therapeutic agents useful in a wide range of bacterial and fungal infections. Among the modifications improving pharmacokinetic and pharmacodynamic characteristics of natural AMPs, an important role is played by lipidation. This study focuses on the newly designed and synthesized lipopeptides containing multiple Lys residues or their shorter homologues with palmitic acid (C16) attached to the side chain of a residue located in the center of the peptide sequence. The approach resulted in the development of lipopeptides representing a model of surfactants with two polar headgroups. The aim of this study is to explain how variations in the length of the peptide chain or the hydrocarbon side chain of an amino acid residue modified with C16, affect biological functions of lipopeptides, their self-assembling propensity, and their mode of action.

Huntington’s disease (HD) is a monogenic disorder, caused by mutations in the HTT gene which result in expansion of CAG triplets. The product of the mutated gene is misfolded huntingtin protein that forms aggregates leading to impairment of neuronal function, neurodegeneration, motor abnormalities and cognitive deficits. No effective cure is currently available for HD. Here we studied effects of genistein (trihydroxyisoflavone) on a HD cellular model consisting of HEK-293 cells transfected with a plasmid bearing mutated HTT gene. Both level of mutated huntingtin and number of aggregates were significantly decreased in genistein-treated HD cell model. This led to increased viability of the cells. Autophagy was up-regulated while inhibition of lysosomal functions by chloroquine impaired the genistein-mediated degradation of the mutated huntingtin aggregates. Hence, we conclude that through stimulating autophagy, genistein removes the major pathogenic factor of HD. Prolonged induction of autophagy was suspected previously to be risky for patients due to putative adverse effects; however, genistein has been demonstrated recently to be safe and suitable for long-term therapies even at doses as high as 150 mg/kg/day. Therefore, results presented in this report provide a basis for the use of genistein in further studies on development of the potential treatment of HD.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM&mdash, a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH&mdash, the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

It is well established that mTORC1 suppresses autophagy by phosphorylation and inactivation of proteins involved in autophagosome formation. However, the role of its substrate, p70S6 kinase1 (S6K1), in autophagy is quite controversial. In some models S6K1 activity correlates with autophagy suppression, however, some other studies show that S6K1 promotes rather than inhibits this process. Here, we investigated the role of S6K1 in prostate cancer cells (PC-3) and non-cancerous, mouse embryonic fibroblasts (MEF), either treated with autophagy inducer sulforaphane, an isothiocyanate derived from cruciferous plants, or deprived of serum. Our results indicate that constitutively active S6K1 decreases the level of LC3 processing and foci formation by autophagosomal vacuoles in cells treated with sulforaphane. On the other hand, presence of S6K1 is necessary for autophagosome maturation under conditions of autophagy induced by either sulforaphane or serum deprivation. Diminished level of S6K1 or lack of S6 kinases results in both, accumulation of autophagosomes and drop in the autophagolysosome number, and thus disturbs autophagy flux under stress conditions. Moreover, lack of S6 kinases reduces cell survival under stress conditions.

Accumulation of mutated huntingtin aggregates in cells is the main cause of Huntington’s disease. Autophagy allows for removing protein aggregates and prevents their accumulation in normal cells. In our study we determined whether sulforaphane (SFN), a natural agent present in cruciferous plants and known inducer of autophagy in cancer cells, is able to modulate the amount of mutated huntingtin in normal human fibroblasts expressing the exogenous mutated protein. We observed reduction in the exogenous mutant huntingtin level as well as a decrease in the amount of its aggregates in normal human fibroblasts treated with sulforaphane. Simultaneously, we observed autophagy induction. Activation of autophagy by SFN in normal human fibroblasts only marginally affected their viability and was accompanied by an inhibition of a major negative regulator of autophagy, mTORC1. Autophagy induction and mTORC1 inhibition was preceded by activation of AMPK kinase, a known inhibitor of mTORC1 and thus autophagy activator. The autophagy induction by SFN coincided with a block in protein synthesis which might be, together with the induction of autophagy, the molecular mechanism leading to reduction of the mutant huntingtin amount in cells by sulforaphane. Summarising, these results indicate that sulforaphane might potentially be used in therapy of diseases caused by aggregation of mutated proteins, including Huntington’s disease.

Background Expansions of CAG codons in the first exon of the HTT gene, encoding huntingtin, result in production of the protein that forms aggregates in cells, being the cause of cellular dysfunctions. Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)−4H-1-benzopyran-4-one) is a naturally occurring isoflavone that has a number of biological activities. Recent studies performed by our group (Moskot et al . 2014, J. Biol. Chem. 289:17054–69; Moskot et al . 2015, Sci. Rep. 5:9378) indicated that genistein stimulates biogenesis of lysosomes and production of lysosomal hydrolases. Aims The aim of this work was to determine if genistein can cause effective degradation of huntingtin’s aggregates, and if yes, what is the molecular mechanism of this phenomenon. Methods Human embryonic kidney (HEK) cells were transfected with a plasmid expressing the first exon of huntingtin which codes for 74 glutamine repeats (pEGFP-Q74). The analyses were performed by using fluorescence microscopy, Western-blotting, and cell survival assays. Results We found that the presence of genistein in culture medium resulted in a significant decrease in the number of mutant huntingtin aggregates, as well as in the reduction of their volume. Biochemical analyses indicated that the amount of mutant huntingtin decreased considerably in genistein-treated cells relative to controls. Under these conditions, survival of such cells increased by a few times. Genistein was able to stimulate the process of autophagy, as demonstrated by an increased level of the LC3-II protein in cells in the presence of this isoflavone in the culture medium. Inhibition of lysosomal functions resulted in inability of genistein to stimulate huntingtin degradation. Conclusions Genistein stimulates the autophagy process which leads to degradation of mutant huntingtin. Therefore, one might suggest that genistein could be considered as a novel compound inhibiting the Huntington’s disease.