The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.

Understanding the Role of Self-Assembly and Interaction with Biological Membranes of Short Cationic Lipopeptides in the Effective Design of New Antibiotics.

This study investigates short cationic antimicrobial lipopeptides composed of 2-4 amino acid residues and C12-C18 fatty acids attached to the N-terminal part of the peptides. The findings were discussed in the context of the relationship among biological activity, self-assembly, stability, and membrane interactions. All the lipopeptides showed the ability to self-assemble in PBS solution. In most cases, the critical aggregation concentration (CAC) much surpassed the minimal inhibitory concentration (MIC) values, suggesting that monomers are the main active form of lipopeptides. The introduction of ß-alanine into the peptide sequence resulted in a compound with a high propensity to fibrillate, which increased the peptide stability and activity against S. epidermidis and C. albicans and reduced the cytotoxicity against human keratinocytes. The results of our study indicated that the target of action of lipopeptides is the bacterial membrane. Interestingly, the type of peptide counterion may affect the degree of penetration of the lipid bilayer. In addition, the binding of the lipopeptide to the membrane of Gram-negative bacteria may lead to the release of calcium ions necessary for stabilization of the lipopolysaccharide layer.

Influence of Hypoxia on Radiosensitization of Cancer Cells by 5-Bromo-2'-deoxyuridine.

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.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

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.

S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.

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.

Double-Headed Cationic Lipopeptides: An Emerging Class of Antimicrobials.

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.

Imunofan-RDKVYR Peptide-Stimulates Skin Cell Proliferation and Promotes Tissue Repair.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today'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 "Imunofan" (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 < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 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 < 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).

Mechanism of selective anticancer activity of isothiocyanates relies on differences in DNA damage repair between cancer and healthy cells.

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.

Dihydroxy-Substituted Coumarins as Fluorescent Probes for Nanomolar-Level Detection of the 4-Amino-TEMPO Spin Label.

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-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-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).

Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells.

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.

Correction of Huntington's Disease Phenotype by Genistein-Induced Autophagy in the Cellular Model.

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.

Sulforaphene, an isothiocyanate present in radish plants, inhibits proliferation of human breast cancer cells.

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 (5-10µM). This is associated with induction of the cell cycle arrest and apoptosis. SF might be considered as a potent anticancer agent.

S6K1 controls autophagosome maturation in autophagy induced by sulforaphane or serum deprivation.

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.