Marta Moskot

The Phytoestrogen Genistein Modulates Lysosomal Metabolism and Transcription Factor EB (TFEB) Activation

Background: Genistein is a potential drug for certain inherited lysosomal disorders. Results: Genistein influences molecular cross-talk in the cell responsible for lysosomal enhancement. Conclusion: Genistein potentiates lysosomal metabolism by activating transcription factor EB (TFEB). Significance: The explanation of genistein action offers more adequate therapeutic procedures for the treatment of some lysosomal storage diseases. Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

Biodegradation of nodularin and effects of the toxin on bacterial isolates from the Gulf of Gdańsk

Nodularin (NOD), a cyclic pentapeptide produced by the cyanobacterium Nodularia spumigena, is one of the most abundant natural metabolites occurring in the Baltic Sea. The present study investigated the role of this compound in the interactions between cyanobacteria and other bacteria. The toxin inhibited the growth of 15 out of 32 bacterial strains isolated from water and sediments of the Gulf of Gdańsk, southern Baltic Sea. Most of the bacteria sensitive to NOD belonged to the Proteobacteria phylum. Incubation of nodularin in the presence of the bacterial isolates did not reveal any NOD-degrading activity. However, natural microbial communities from sediment removed the toxin within 5-7 days. Analysis by liquid chromatography/hybrid quadrupole-time-of-flight mass spectrometry with turboion spray (QTOF-LC/MS/MS) revealed seven biodegradation products, including five novel ones. The results showed that not only freshwater microorganisms, but also those living in brackish waters, play an important role in cyanotoxin removal.

Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also in MPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease in MTOR transcript level was observed at these conditions.

Assessment of antibacterial effects of flavonoids by estimation of generation times in liquid bacterial cultures

Antibacterial activities of various flavonoids, a group of natural plant substances, have been reported previously, however, there are contradictory data, published by various authors, regarding sensitivity of particular bacterial species to these compounds. These problems arose apparently because of using different methods by various researchers. Here we tested sensitivity of several bacterial species (Gram-positive: Bacillus subtilis, Micrococcus luteus, Sarcina sp. and Staphylococcus aureus; and Gram-negative: Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens and Vibrio harveyi) to various flavonoids: genistein and daidzein (isoflavones), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol) by measurement of generation times of bacteria in liquid cultures. The presented results indicate that this simple method is adequate for unambiguous assessment of sensitivity of bacterial strains to flavonoids.

Metal and antibiotic resistance of bacteria isolated from the Baltic Sea.

The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzed and the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78%), while Alphaproteobacteria (8%), Actinobacteria (10%), and Bacteroidetes (4%) were less abundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the ability to produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relation between resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not to spectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as an aminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance to some antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycin and tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effects of NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrations of Mn2+ due to chemical interactions that potentially lead to precipitation.

Effects of flavonoids on expression of genes involved in cell cycle regulation and DNA replication in human fibroblasts

Flavonoids have been studied as potential agents in medicine for many years. Among them, genistein was found to be active in various biological systems, mainly in prevention of cancer. Our recent work supported the idea that genistein also impacts multiple cellular processes in healthy fibroblasts; however, its effects on cell cycle-related pathways remained to be elucidated. Thus, in this work, high throughput screening with microarrays coupled to real-time quantitative Reverse Transcription PCR analyses was employed to study the changes in expression of key genes associated with cell cycle regulation and/or DNA replication in response to genistein, kaempferol, daidzein, and mixtures of genistein and either kaempferol or daidzein. Among them, genistein was found as the most significantly modulating, in a time- and dose-dependent manner, compound of activity of studied genes, whose products are involved in different phases of the cell cycle and/or in regulatory processes important for DNA replication and cell growth. It considerably reduced the efficiency of expression of genes coding for MCM2-7 and MCM10 helicases, as well as some other proteins involved in the S phase control. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and GADD45A genes, as well as down-regulation of several mRNAs specific for this stage, demonstrated by transcriptomic assessments. We believe that studies described in this paper will be helpful in elucidating molecular mechanisms of action of genistein as modulator of cell cycle and inhibitor of DNA replication in humans.

Combined Therapies for Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) is a group consisting of over 50 disorders caused mostly by dysfunctions of lysosomal proteins and resultant accumulation of particular compounds inside cells and extracellular volumes in affected organisms. Genetic diseases are among the most difficult targets for medical treatment. Nevertheless, understanding of molecular bases of LSDs made it possible to develop novel procedures of treatment, employing molecular medicine. Although various therapeutic approaches have been proposed, and some of them were introduced into clinical practice, none of them was found to be effective in correcting all symptoms in treated patients. Central nervous system and skeleton appear to be the most difficult targets to be improved. Therefore, a proposal appeared that perhaps no single therapeutic procedure may be fully effective in treatment of LSD patients, and only combination of two or more approaches could be a successful therapy. In this review, we present and discuss current stage of various combination therapies for LSDs, based on already available published data.

Cell cycle is disturbed in mucopolysaccharidosis type II fibroblasts, and can be improved by genistein

Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by mutations resulting in deficiency of one of enzymes involved in degradation of glycosaminoglycans (GAGs). These compounds accumulate in cells causing their dysfunctions. Genistein is a molecule previously found to both modify GAG metabolism and modulate cell cycle. Therefore, we investigated whether the cell cycle is affected in MPS cells and if genistein can influence this process. Fibroblasts derived from patients suffering from MPS types I, II, IIIA and IIIB, as well as normal human fibroblasts (the HDFa cell line) were investigated. MTT assay was used for determination of cell proliferation, and the cell cycle was analyzed by using the MUSE® Cell Analyzer. While effects of genistein on cell proliferation were similar in both normal and MPS fibroblasts, fractions of cells in the G0/G1 phase were higher, and number of cells entering the S and G2/M phases was considerably lower in MPS II fibroblasts relative to control cells. Somewhat similar tendency, though significantly less pronounced, could be noted in MPS I, but only at longer times of incubation. However, this was not observed in MPS IIIA and MPS IIIB fibroblasts. Genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) was found to be able to partially correct the disturbances in the MPS II cell cycle, and to some extent in MPS I, at higher concentrations of this compound. The tendency to increase the fractions of cells entering the S and G2/M phases was also observed in MPS IIIA and IIIB fibroblasts treated with genistein. In conclusion, this is the first report indicating that the cell cycle can be impaired in MPS cells. The finding that genistein can improve the MPS II (and to some extent also MPS I) cell cycle provides an input to our knowledge on the molecular mechanisms of action of this compound.

Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways.

Activities of genes controlling sphingolipid metabolism in human fibroblasts treated with flavonoids

Natural flavonoids such as genistein, kaempferol and daidzein were previously found to be able to reduce efficiency of glycosaminoglycan synthesis in cells of patients suffering from mucopolysaccharidoses, inherited metabolic diseases with often brain disease symptoms. This feature was employed to test these compounds as potential drugs for treatment other neuronopathic lysosomal storage disorders, in which errors in sphingolipid metabolism occur. In this report, on the basis of DNA microarray analyses and quantitative real time PCR experiments, we present evidence that these compounds modify expression of genes coding for enzymes required for metabolism of sphingolipids in human dermal fibroblasts (HDFa). Expression of several genes involved in sphingolipid synthesis was impaired by tested flavonoids. Therefore, it is tempting to speculate that they may be considered as potential drugs in treatment of LSD, in which accumulation of sphingolipids, especially glycosphingolipids, occurs. Nevertheless, further studies on more advances models are required to test this hypothesis and to assess a therapeutic potential for flavonoids in this group of metabolic brain diseases.