Dually Active Apigenin-Loaded Nanostructured Lipid Carriers for Cancer Treatment.

Purpose: Cancer is one of the major causes of death worldwide affecting more than 19 million people. Traditional cancer therapies have many adverse effects and often result in unsatisfactory outcomes. Natural flavones, such as apigenin (APG), have demonstrated excellent antitumoral properties. However, they have a low aqueous solubility. To overcome this drawback, APG can be encapsulated in nanostructured lipid carriers (NLC). Therefore, we developed dual NLC encapsulating APG (APG-NLC) with a lipid matrix containing rosehip oil, which is known for its anti-inflammatory and antioxidant properties. Methods: Optimisation, physicochemical characterisation, biopharmaceutical behaviour, and therapeutic efficacy of this novel nanostructured system were assessed. Results: APG-NLC were optimized obtaining an average particle size below 200 nm, a surface charge of -20 mV, and an encapsulation efficiency over 99%. The APG-NLC released APG in a sustained manner, and the results showed that the formulation was stable for more than 10 months. In vitro studies showed that APG-NLC possess significant antiangiogenic activity in ovo and selective antiproliferative activity in several cancer cell lines without exhibiting toxicity in healthy cells. Conclusion: APG-NLC containing rosehip oil were optimised. They exhibit suitable physicochemical parameters, storage stability for more than 10 months, and prolonged APG release. Moreover, APG-NLC were internalised inside tumour cells, showing the capacity to cause cytotoxicity in cancer cells without damaging healthy cells.

Studies on the Anticancer and Antioxidant Activities of Resveratrol and Long-Chain Fatty Acid Esters.

Resveratrol (RES) is gaining recognition as a natural bioactive compound. To expand the possible applications of RES with its enhanced bioactivity as well as to increase the health benefits of long-chain fatty acids, a lipophilization process of RES was performed using three fatty acids: palmitic acid (PA), oleic acid (OA), and conjugated linoleic acid (CLA). The obtained mono-, di-, and tri-esters of RES were evaluated for their anticancer and antioxidant properties against lung carcinoma (A549), colorectal adenocarcinoma (HT29), and pancreatic ductal adenocarcinoma (BxPC3) cell lines. Human fibroblast (BJ) cells were used as a control. Several parameters were investigated: cell viability and apoptosis, including the expression of major pro- and anti-apoptotic markers, as well as the expression of superoxide dismutase, a key enzyme of the body's antioxidant barrier. Three of the obtained esters: mono-RES-OA, mono-RES-CLA, and tri-RES-PA, which significantly reduced the tumor cell viability up to 23%, at concentrations 25, 10, 50 µg/mL, respectively, turned out to be particularly interesting. The above-mentioned resveratrol derivatives similarly increased the tumor cells' apoptosis by modifying their caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Moreover, among the mentioned esters, mono-RES-OA induced apoptosis of the analyzed cell lines most strongly, reducing the number of viable cells up to 48% for HT29 cells versus 36% for pure RES. Furthermore, the selected esters exhibited antioxidant properties towards the normal BJ cell line by regulating the expression of major pro-antioxidant genes (superoxide dismutases-SOD1 and SOD2) without the effect on their expression in the tumor, and therefore reducing the defense of cancer cells against increased oxidative stress induced by high ROS accumulation. The obtained results indicate that the esters of RES and long-chain fatty acids allow enhancement of their biological activity. The RES derivatives have the potential for being applied in cancer prevention and treatment, as well as for oxidative stress suppression.

Perspectives of the Application of Non-Steroidal Anti-Inflammatory Drugs in Cancer Therapy: Attempts to Overcome Their Unfavorable Side Effects.

Non-steroidal anti-inflammatory drugs (NSAIDs) express anti-tumoral activity mainly by blocking cyclooxygenase-2 involved in the synthesis of prostaglandins. Therefore, in the last few decades, many have attempted to explore the possibilities of applying this group of drugs as effective agents for the inhibition of neoplastic processes. This review summarizes the evidence presented in the literature regarding the anti-tumoral actions of NSAIDs used as monotherapies as well as in combination with conventional chemotherapeutics and natural products. In several clinical trials, it was proven that combinations of NSAIDs and chemotherapeutic drugs (CTDs) were able to obtain suitable results. The combination with phospholipids may resolve the adverse effects of NSAIDs and deliver derivatives with increased antitumor activity, whereas hybrids with terpenoids exhibit superior activity against their parent drugs or physical mixtures. Therefore, the application of NSAIDs in cancer therapy seems to be still an open chapter and requires deep and careful evaluation. The literature's data indicate the possibilities of re-purposing anti-inflammatory drugs currently approved for cancer treatments.

Novel Strategies against Cancer: Dexibuprofen-Loaded Nanostructured Lipid Carriers.

The aim of this work was to design innovative nanostructured lipid carriers (NLCs) for the delivery of dexibuprofen (DXI) as an antiproliferative therapy against tumoral processes, and overcome its side effects. DXI-NLC samples were prepared with beeswax, Miglyol 812 and Tween 80 using high-pressure homogenization. A two-level factorial design 24 was applied to optimize the formulation, and physicochemical properties such as particle size, zeta potential, polydispersity index and entrapment efficiency were measured. Optimized parameters of DXI-NLCs exhibited a mean particle size of 152.3 nm, a polydispersity index below 0.2, and high DXI entrapment efficiency (higher than 99%). Moreover, DXI-NLCs provided a prolonged drug release, slower than the free DXI. DXI-NLCs were stable for 2 months and their morphology revealed that they possess a spherical shape. In vitro cytotoxicity and anticancer potential studies were performed towards prostate (PC-3) and breast (MDA-MB-468) cancer cell lines. The highest activity of DXI-NLCs was observed towards breast cancer cells, which were effectively inhibited at 3.4 µM. Therefore, DXI-NLCs constitute a promising antiproliferative therapy that has proven to be especially effective against breast cancer.

Elevating Phospholipids Production Yarrowia lipolytica from Crude Glycerol.

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.

Diclofenac Loaded Biodegradable Nanoparticles as Antitumoral and Antiangiogenic Therapy.

Cancer is identified as one of the main causes of death worldwide, and an effective treatment that can reduce/eliminate serious adverse effects is still an unmet medical need. Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has demonstrated promising antitumoral properties. However, the prolonged use of this NSAID poses several adverse effects. These can be overcome by the use of suitable delivery systems that are able to provide a controlled delivery of the payload. In this study, Diclofenac was incorporated into biodegradable polymeric nanoparticles based on PLGA and the formulation was optimized using a factorial design approach. A monodisperse nanoparticle population was obtained with a mean size of ca. 150 nm and negative surface charge. The release profile of diclofenac from the optimal formulation followed a prolonged release kinetics. Diclofenac nanoparticles demonstrated antitumoral and antiangiogenic properties without causing cytotoxicity to non-tumoral cells, and can be pointed out as a safe, promising and innovative nanoparticle-based formulation with potential antitumoral effects.

Evaluation of the Physico-Chemical Properties of Liposomes Assembled from Bioconjugates of Anisic Acid with Phosphatidylcholine.

The aim of this work was the evaluation of the physico-chemical properties of a new type of liposomes that are composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine. In particular, the impact of modified anisic acid phospholipids on the thermotropic parameters of liposomes was determined, which is crucial for using them as potential carriers of active substances in cancer therapies. Their properties were determined using three biophysical methods, namely differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Moreover, temperature studies of liposomes composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine provided information about the phase transition, fluidity regarding chain order, hydration and dynamics. The DSC results show that the main phase transition peak for conjugates of anisic acid with phosphatidylcholine molecules was broadened and shifted to a lower temperature in a concentration- and structure-dependent manner. The ATR-FTIR results and the results of measurements conducted using fluorescent probes located at different regions in the lipid bilayer are in line with DSC. The results show that the new bioconjugates with phosphatidylcholine have a significant impact on the physico-chemical properties of a membrane and cause a decrease in the temperature of the main phase transition. The consequence of this is greater fluidity of the lipid bilayer.

Conjugation with Phospholipids as a Modification Increasing Anticancer Activity of Phenolic Acids in Metastatic Melanoma-In Vitro and In Silico Studies.

Phenolic acids possess many beneficial biological activities, including antioxidant and anti-inflammatory properties. Unfortunately, their low bioavailability restricts their potential medical uses, as it limits the concentration of phenolic acids achievable in the organism. The conjugation with phospholipids constitutes one of the most effective strategies to enhance compounds bioavailability in biological systems. In the present study, the conjugates of anisic (ANISA) and veratric acid (VA) with phosphatidylcholine (PC) were investigated. Since both ANISA and VA are inhibitors of tyrosinase, a melanocyte enzyme, the expression of which increases during tumorigenesis, anticancer potential of the conjugates was tested in several metastatic melanoma cell lines. The conjugates proved to be antiproliferative, apoptosis-inducing and cell-cycle-affecting agents, selective for cancerous cells and not affecting normal fibroblasts. The conjugates substituted by ANISA and VA, respectively, at both the sn-1 and sn-2 positions of PC, appeared the most promising, since they were effective against the vast majority of metastatic melanoma cell lines. Additionally, the conjugation of phenolic acids to PC increased their antioxidant activity. Molecular modeling was employed for the first time to estimate the features of the investigated conjugates relevant to their anticancer properties and membrane permeation. Again, the conjugates substituted by phenolic acid at both the sn-1 and sn-2 positions of PC seemed to be presumably most bioavailable.

Biological Properties, Health Benefits and Enzymatic Modifications of Dietary Methoxylated Derivatives of Cinnamic Acid.

Methoxylated derivatives of cinnamic acid play an important role in the formation of the pro-health potential of food products. Numerous reports present them as molecules with strong antimicrobial, antidiabetic, anticancer as well as hepato-, cardio-, and neuroprotective activities. In the last three decades, many research groups have tried to extend the practical application of these molecules as therapeutic and antioxidant agents extensively studying the methods of their lipophilization as the solution of problems of their low oral bioavailability and rapid metabolism. This article summarizes the latest data of natural sources of occurrence, biological potential and bioavailability of methoxy derivatives of cinnamic acids. Metabolism and pharmacokinetics of this group of dietary compounds are also extensively discussed as well as reviewing the methods of their chemical and enzymatic lipophilization in the aspect of their use in food and pharmaceutical industries.

Recent Developments in Therapeutic and Nutraceutical Applications of p-Methoxycinnamic Acid from Plant Origin.

The p-methoxycinnamic acid (p-MCA) is one of the most studied phenylpropanoids with high importance not only in the wide spectrum of therapeutic activities but also its potential application for the food industry. This natural compound derived from plants exhibits a wide range of biologically useful properties; therefore, during the last two decades it has been extensively tested for therapeutic and nutraceutical applications. This article presents the natural sources of p-MCA, its metabolism, pharmacokinetic properties, and safety of its application. The possibilities of using this dietary bioactive compound as a nutraceutical agent that may be used as functional food ingredient playing a vital role in the prevention and treatment of many chronic diseases is also discussed. We present the antidiabetic, anticancer, antimicrobial, hepato-, and neuroprotective activities of p-MCA and methods of its lipophilization that have been developed so far to increase its industrial application and bioavailability in the biological systems.

Dexibuprofen Therapeutic Advances: Prodrugs and Nanotechnological Formulations.

S-(+) enantiomer of ibuprofen (IBU) dexibuprofen (DXI) is known to be more potent than its R-(-) form and exhibits many advantages over the racemic mixture of IBU such as lower toxicity, greater clinical efficacy, and lesser variability in therapeutic effects. Moreover, DXI potential has been recently advocated to reduce cancer development and prevent the development of neurodegenerative diseases in addition to its anti-inflammatory properties. During the last decade, many attempts have been made to design novel formulations of DXI aimed at increasing its therapeutic benefits and minimizing the adverse effects. Therefore, this article summarizes pharmacological information about DXI, its pharmacokinetics, safety, and therapeutic outcomes. Moreover, modified DXI drug delivery approaches are extensively discussed. Recent studies of DXI prodrugs and novel DXI nanoformulations are analyzed as well as reviewing their efficacy for ocular, skin, and oral applications.

Lipid Formulations and Bioconjugation Strategies for Indomethacin Therapeutic Advances.

Indomethacin (IND) is a drug which after successful clinical trials became available for general prescription in 1965 and from that time is one of the most widely used anti-inflammatory drug with the highest potencies in the in vitro and in vivo models. However, despite its high therapeutic efficacy in relieving the symptoms of certain arthritis and in treating gout or collagen diseases, administration of IND causes a number of adverse effects, such as gastrointestinal ulceration, frequent central nervous system disorders and renal toxicity. These obstacles significantly limit the practical applications of IND and make that 10-20% of patients discontinue its use. Therefore, during the last three decades many attempts have been made to design novel formulations of IND aimed to increase its therapeutic benefits minimizing its adverse effects. In this review we summarize pharmacological information about IND and analyze its new lipid formulations and lipid bioconjugates as well as discuss their efficacy and potential application.

Synthesis of novel phytol-derived γ-butyrolactones and evaluation of their biological activity.

The synthesis of phytol-derived γ-butyrolactones as well as their evaluation for deterrent activity towards peach-potato aphid Myzus persicae and antiproliferative activity against four selected cancer cell lines are reported. Products were obtained in good yields (19-96%) and their structures were fully characterized by spectroscopic data (NMR, HRMS). Four synthesized δ-halo-γ-lactones (4-7) are new and have not been previously described in the literature. In the choice test phytol (1) appeared deterrent to M. persicae, whereas modifications of its structure did not cause the avoidance of the treated leaves by the aphids. In contrast, aphids were attracted to the leaves treated with the new trans-δ-chloro-γ-lactone (6). Electrical Penetration Graph (EPG) technique applied to explore the aphid probing and feeding activity revealed that neither phytol nor lactone 6 affected aphid probing and the consumption of phloem sap, which means that both phytol and the lactone 6 might have acted as postingestive modifiers of aphid behavior. The results of in vitro antitumor assays showed that obtained phytol derivatives exhibit cytotoxic activity against studied cancer cell lines (leukemia, lung and colon carcinoma and its doxorubicin resistant subline). Halolactones 4-6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases.

Synthesis, Characterization, and In Vitro Cancer Cell Growth Inhibition Evaluation of Novel Phosphatidylcholines with Anisic and Veratric Acids.

Phenolic acids and its methoxy derivatives are known to induce caspase-mediated apoptosis activity and exhibit cytotoxic effect towards various cancer cell lines. However, their low stability and poor bioavailability in the human organism extensively restrict the utility of this group of compounds as anticancer and health-promoting agents. In this report, a series of eight novel phosphatidylcholines (3a-b, 5a-b, 7a-b, 8a-b) containing anisic or veratric acids (1a-b) at sn-1 and/or sn-2 positions were synthesized. The phenoylated phospholipids were obtained in good yields 28⁻66%. The structures of novel compounds were determined by their spectroscopic data. All synthesized compounds were evaluated for their antiproliferative activity towards six cancer cell lines and normal cell line Balb/3T3. Lipophilization of phenolcarboxylic acids significantly increased their anticancer properties. The asymmetrically substituted phenoylated phosphatidylcholines exhibited higher antiproliferative effect than free acids. Lysophosphatidylcholine (7b) effectively inhibited the proliferation of human leukaemia (MV4-11), breast (MCF-7), and colon (LoVo) cancer cell lines at concentrations of 9.5⁻20.7 µm and was from 19 to 38-fold more active than corresponding free veratric acid. The conjugation of anisic/veratric acids with the phosphatidylcholine have proved the anticancer potential of these phenolcarboxylic acids and showed that this type of lipophilization is an effective method for the production of active biomolecules.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

Lipase-Catalyzed Acidolysis of Egg-Yolk Phosphatidylcholine with Citronellic Acid. New Insight into Synthesis of Isoprenoid-Phospholipids.

The development of a biotechnological method for the production of new biologically active phosphatidylcholine containing monoterpene citronellic acid (CA) was the aim of this work. Incorporation of citronellic acid (CA) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed acidolysis process was studied. Isoprenoid acid CA was used as an acyl donor and five commercially available immobilized lipases were examined as biocatalysts. The effects of organic solvent, enzyme load, reaction time and molar ratio of substrates on the incorporation of citronellic acid (CA) into the phospholipids were evaluated. Modified phospholipid fraction enriched with CA in the sn-1 position (39% of incorporation) was obtained in high 33% yield using Novozym 435 as biocatalyst. In this study a biotechnological method for production of new phospholipid biopreparation enriched with citronellic acid, which can play an important role as a nutraceutical, was applied.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease.

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

Synthesis and biological evaluation of phosphatidylcholines with cinnamic and 3-methoxycinnamic acids with potent antiproliferative activity.

A series of eight novel phosphatidylcholines containing cinnamic or 3-methoxycinnamic acids (3a-b, 5a-b, 9a-b, 10a-b) at sn-1 and/or sn-2 positions were synthesized and tested for their antiproliferative activity in an in vitro model against representative six human cancer cell lines (MV4-11, A549, MCF-7, LoVo, LoVo/DX, HepG2) and a normal cell line BALB/3T3. The structures of the new compounds were confirmed by spectral analysis. Biological evaluation revealed that all the tested conjugates exhibited higher antitumor activity than the corresponding free aromatic acids. Compounds 3b and 9b turned out to be the most active, with IC50 values of 32.1 and 30.5 µM against the LoVo/DX and MV4-11 cell lines, respectively. Studies of the mechanism of the antitumor action were carried out for 1-palmitoyl-2-cinnamoyl-sn-glycero-3-phosphocholine (5a), and it was shown to be active toward almost all the tested types of cancer cells, showing that this compound could effectively arrest the cell cycle in G2/M and decrease the mitochondrial membrane potential of leukemia MV4-11 cells. The obtained results proved that the strategy of the incorporation of cinnamic and 3-methoxycinnamic acids into phospholipids could expand their potential application in industry, as well as could improve their antiproliferative activity and selectivity toward cancer cell lines.

Isoprenoid-phospholipid conjugates as potential therapeutic agents: Synthesis, characterization and antiproliferative studies.

The aim of this research was to extend application field of isoprenoid compounds by their introduction into phospholipid structure as the transport vehicle. The series of novel isoprenoid phospholipids were synthesized in high yields (24-97%), their structures were fully characterized and its anticancer activity was investigated in vitro towards several cell lines of different origin. Most of synthesized compounds showed a significantly higher antiproliferative effect on tested cell lines than free terpene acids. The most active phosphatidylcholine analogue, containing 2,3-dihydro-3-vinylfarnesoic acids instead of fatty acids in both sn-1 and sn-2 position, inhibits the proliferation of colon cancer cells at 13.6 µM.

Synthesis and Biological Evaluation of Novel Phosphatidylcholine Analogues Containing Monoterpene Acids as Potent Antiproliferative Agents.

The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and sn-2 positions is described. The structured phospholipids were obtained in high yields (59-87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-cancer cell line BALB/3T3 (normal mice fibroblasts). The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.