Therapeutic Potential of Genistein: Preclinical Studies, Clinical Evidence, and Nanotechnology Application.

Genistein (4',5,7-trihydroxyisoflavone) is a natural plant-derived phytoestrogen that can be found, for example, in soybean seeds. Genistein is present mainly in the human diet and is a common precursor in the antimicrobial phytoalexins biosynthesis and phytoanticipins in vegetables. The interest in genistein has increased due to its pharmacological effects, including anti-cancer activity, neuroprotective effects, cardiovascular protection, anti-inflammatory effects, antioxidant activity, and prevention of obesity. The most challenging issue for improving genistein is its low oral bioavailability, which has led to many animal and human pharmacokinetic studies and numerous clinical trials. Several drug delivery systems have been developed to protect and stabilize genistein to overcome the challenge of low bioavailability. This work concerns a revision of the literature reporting nano and microformulations for genistein encapsulation, including lipid nanoparticles, liposomes, tocotrienol-rich nanoemulsions, polymeric nanoparticles, dextran complexes, chitosan complexes, and Fe3O4 nanoparticles with carboxymethylated chitosan. Regarding the enormous potential of genistein, several clinical trials and marketed formulations can be found in the market.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

The Influence of Circadian Rhythm on Cancer Cells Targeting and Transfection Efficiency of a Polycation-Drug/Gene Delivery Vector.

The conception of novel anticancer delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy. In this work, polyethylenimine (PEI) has been used to complex p53 encoded plasmid DNA (pDNA), and the anticancer drug methotrexate (MTX) has also been loaded into the vectors. To investigate the influence of circadian clock on drug/gene delivery efficiency, HeLa, C33A and fibroblast cells have been transfected with developed PEI/pDNA/MTX delivery vectors at six different time points. Phenomena as the cellular uptake/internalization, drug/gene delivery and p53 protein production have been evaluated. The cell-associated MTX fluorescence have been monitored, and p53 protein levels quantified. In HeLa and C33A cancer cells, significant levels of MTX were found for T8 and T12. For these time points, a high amount of p53 protein was quantified. Confocal microscopy images showed successful HeLa cell's uptake of PEI/pDNA/MTX particles, at T8. In comparison, poor levels of MTX and p53 protein were found in fibroblasts; nevertheless, results indicated rhythmicity. Data demonstrate the influence of circadian rhythm on both cancer-cells targeting ability and transfection performance of PEI/pDNA/MTX carriers and seemed to provide the optimum time for drug/gene delivery. This report adds a great contribution to the field of cancer chronobiology, highlighting the relationship between circadian rhythm and nanodelivery systems, and charting the path for further research on a, yet, poorly explored but promising topic.

Exploring the link between chronobiology and drug delivery: effects on cancer therapy.

Circadian clock is an impressive timing system responsible for the control of several metabolic, physiological and behavioural processes. Nowadays, the connection between the circadian clock and cancer occurrence and development is consensual. Therefore, the inclusion of circadian timing into cancer therapy may potentially offer a more effective and less toxic approach. This way, chronotherapy has been shown to improve cancer treatment efficacy. Despite this relevant finding, its clinical application is poorly exploited. The conception of novel anticancer drug delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy, instigating the incorporation of the circadian timing into clinical practice towards a more personalized drug delivery. This review focuses on the recent advances in the field of cancer chronobiology, on the link between cancer and the disruption of circadian rhythms and on the promising targeted drug nanodelivery approaches aiming the clinical application of cancer chronotherapy.

Kinetics of radium-223 and its effects on survival, proliferation and DNA damage in lymph-node and bone metastatic prostate cancer cell lines.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is associated with a very unfavorable prognosis. At this advanced stage of the disease, there are several therapeutic strategies approved in recent times, being one of them Radium-223 dichloride (Radium-223). However, its mechanisms of action and the process that conducts to cell death are not fully understood. Given this, our main goal is to characterize the radiobiological effects induced by Radium-223 and to evaluate its kinetics on metastatic Prostate Cancer (mPCa) cells. MATERIALS AND METHODS: In vitro studies were conducted using two mPCa cell lines, the LNCaP and PC3, the first being derived from lymph node metastasis and the second from bone metastasis. Kinetic studies were conducted to access the capacity of these cell lines to uptake, retain and internalize the Radium-223. For the assessment of radiobiological effects, cells were first exposed to different doses of Radium-223 and the clonogenic assay was done to evaluate cell survival and to determine lethal doses (LD50). Then, the effects were also evaluated in terms of proliferation, oxidative stress, morphological changes and cell damage. RESULTS: Radium-223 is uptaken by mPCa cells and reaches the nucleus, where it is retained over time. Irradiation decreases cell survival and proliferation, with LNCaP cells (LD50 = 1.73mGy) being more radiosensitive than PC3 cells (LD50 = 4.20mGy). Irradiated cells showed morphological changes usually associated with apoptosis and a dose-dependent increase in DNA damage. Moreover, activation of cell cycle checkpoints occurs through ATM/CHK2 pathway, which is involved in cell cycle arrest and cell death. CONCLUSIONS: The cytotoxic and anti-proliferative effects on both cell lines showed that Radium-223 can decrease the aggressiveness of tumor cells by decreasing the cell survival and proliferation and, also, by increasing the DNA damage. The similar results observed in both cell lines indicated that Radium-223 may have the potential to be used as a therapeutic option also for mCRPC patients with lymph node metastasis. The activation of DNA Damage Response pathways allows the possibility to understand the importance of these checkpoints as targets for new combined therapies.

Cold Atmospheric Plasma, a Novel Approach against Bladder Cancer, with Higher Sensitivity for the High-Grade Cell Line.

Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψm) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.

Synthesis of New Glycosylated Flavonoids with Inhibitory Activity on Cell Growth.

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell growth of these classes of compounds, the synthesis of four flavonoids (5, 6, 9, and 10) and one xanthone (7) containing one or more acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of 3,7-dihydroxyflavone (4) was performed using two synthetic methods: the Michael reaction and the Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds 5, 6, 7, 9, and 10 was investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-ß-glucopyranosyl) flavone (10) was the most potent compound in all tumor cell lines tested, with GI50 values < 8 µM and a notable degree of selectivity for cancer cells.

Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells.

Dendritic cells (DCs) are promising targets for drug delivery, as they can induce immunity or tolerance. The current study aims to examine the potential of using nanostructured lipid carriers (NLC) as delivery systems for human DC by evaluating nanoparticle internalization, cell labeling, and drug activity. NLC were formulated incorporating the fluorochrome fluorescein isothiocyanate (FITC-NLC) or the natural anti-inflammatory molecule resveratrol (rsv-NLC). Primary human DCs were differentiated from peripheral blood monocytes, and the innovative imaging flow cytometry technique was used to examine FITC-NLC internalization. The capacity of rsv-NLC to inhibit DC activation in response to proinflammatory cytokine tumor necrosis factor-α (TNF- α) was investigated by conventional flow cytometry. A combination of imaging and conventional flow cytometry was used to assess NLC cytotoxicity. The results obtained indicate that both NLC formulations were stable over time, with mean diameter <200 nm and highly negative zeta potential (about -30 mV). When DCs were placed in contact with NLC, imaging flow cytometry clearly showed that DCs efficiently internalized FITC-NLC, with nearly 100% of cells internalizing nanoparticles upon 1 hour of incubation. Both immature and mature DCs internalized NLC to high and comparable levels, and without cytotoxicity. Stimulating DC with TNF-α in the presence of rsv-NLC revealed that, using these nanoparticles, very small concentrations of rsv were sufficient to significantly decrease surface expression of activation marker CD83 (5 µM) and major histocompatibility complex-class II molecule human leukocyte antigen - antigen D related (10 µM), both upregulated in response to TNF-α stimulation. Rsv-NLC were compared with free rsv; at 5 µM, rsv-NLC were able to inhibit nuclear factor κ beta phosphorylation and significantly decrease the level of interleukin-12/23, both upregulated in response to TNF-α, while 10 µM free rsv were needed to promote a similar effect. Taken together, the results presented show that NLC are suitable carriers of fluorescent labels or bioactive molecules for human DCs, leading to inflammation modulation.

An extended dynamic model of Lactococcus lactis metabolism for mannitol and 2,3-butanediol production.

Biomedical research and biotechnological production are greatly benefiting from the results provided by the development of dynamic models of microbial metabolism. Although several kinetic models of Lactococcus lactis (a Lactic Acid Bacterium (LAB) commonly used in the dairy industry) have been developed so far, most of them are simplified and focus only on specific metabolic pathways. Therefore, the application of mathematical models in the design of an engineering strategy for the production of industrially important products by L. lactis has been very limited. In this work, we extend the existing kinetic model of L. lactis central metabolism to include industrially relevant production pathways such as mannitol and 2,3-butanediol. In this way, we expect to study the dynamics of metabolite production and make predictive simulations in L. lactis. We used a system of ordinary differential equations (ODEs) with approximate Michaelis-Menten-like kinetics for each reaction, where the parameters were estimated from multivariate time-series metabolite concentrations obtained by our team through in vivo Nuclear Magnetic Resonance (NMR). The results show that the model captures observed transient dynamics when validated under a wide range of experimental conditions. Furthermore, we analyzed the model using global perturbations, which corroborate experimental evidence about metabolic responses upon enzymatic changes. These include that mannitol production is very sensitive to lactate dehydrogenase (LDH) in the wild type (W.T.) strain, and to mannitol phosphoenolpyruvate: a phosphotransferase system (PTS(Mtl)) in a LDH mutant strain. LDH reduction has also a positive control on 2,3-butanediol levels. Furthermore, it was found that overproduction of mannitol-1-phosphate dehydrogenase (MPD) in a LDH/PTS(Mtl) deficient strain can increase the mannitol levels. The results show that this model has prediction capability over new experimental conditions and offers promising possibilities to elucidate the effect of alterations in the main metabolism of L. lactis, with application in strain optimization.