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Abstract: Alzheimer's disease (AD) is the most common cause of dementia worldwide. Pathologi-cal deposits of neurotoxic proteins within the brain, such as amyloid-ß and hyperphosphorylated tau tangles, are the prominent features in AD. According to recent studies, the newly discovered brain lymphatic system was demonstrated to be crucial in the clearance of metabolic macromolecules from the brain. Meningeal lymphatic vessels located in the dura mater drain the fluid, macromole-cules, and immune cells from cerebrospinal fluid (CSF) and transport them, as lymph, to the deep cervical lymph nodes. The lymphatic system provides the perivascular exchange of CSF with inter-stitial fluid (ISF) and ensures the homeostasis of neuronal interstitial space. In this review, we aim to summarize recent findings on the role of the lymphatic system in AD pathophysiology and dis-cuss possible therapeutic perspectives, targeting the lymphatic clearance mechanisms within the brain.

Objective This study aimed to investigate the effect of low nanomolar concentrations of Aβ1–40 and Aβ25–35 on DNA double-strand breaks following NMDA activation of cells. Materials and methods After incubating the differentiated PC12 cells with Aβ25−35, Aβ1−40 or Aβ1−42 for 24 h, the culture was washed and stimulated for 15 min with NMDA. Then, tests were performed at four-time intervals from stimulation to assess the viability of the culture, the level of oxygen free radicals, and the γH2AX and pATM kinase. NMDAR1 expression was also evaluated by performing immunocytochemical staining. Results It was found that amyloid peptides in nanomolar concentrations reduce double-stranded DNA breaks after NMDA neuron activation. A slight antioxidant effect was also demonstrated when measured 120 min after NMDA cell activation. Conclusion The NMDA stimulation of PC12 cells led to a rapid increase in the number of double-stranded DNA breaks in the cells and is assumed to be the initial step in IEG activation and LTP induction. The effect of Aβ on the reduction of double-strand breaks after NMDA cell stimulation indicates that at concentrations similar to physiological amyloid peptides, it may reduce the mobilization of the neuronal response to stimuli, leading to inhibition of LTP induction and decreasing synaptic plasticity in the early stages of Alzheimer’s disease.

Metastatic colorectal cancer (CRC) remains a hard-to-cure neoplasm worldwide. Its curability declines with successive lines of treatment due to the development of various cancer resistance mechanisms and the presence of colorectal cancer stem cells (CSCs). Celastrol and resveratrol are very promising phytochemicals for colon cancer therapy, owing to their pleiotropic activity that enables them to interact with various biological targets. In the present study, the anticancer activities of both compounds were investigated in metastatic colon cancer cells (LoVo cells) and cancer stem-like cells (LoVo/DX). We showed that celastrol is a very potent anti-tumor compound against metastatic colon cancer, capable of attenuating CSC-like cells at the molecular and cellular levels. In contrast, resveratrol has a much greater effect on colon cancer cells that are expressing standard sensitivity to anticancer drugs, than on CSC-like cells. In addition, both polyphenols have different influences on the expression of

Background The development of a new type of wound dressing material that can support skin regeneration is an important challenge to improve treatment of chronic, non-healing wounds. Objectives The objective of this study was to compare the impact of flax fabrics from transgenic plants overexpressing phenolic acids and flavonoids (W92) and polyhydroxybutyrate (M48), as well as fabric from non-transgenic plant (Nike) on cultures of human skin cells. Material and methods Flax fabric pieces as well as water extracts from the fabrics were co-cultured with human skin cells: keratinocytes, fibroblasts, dermal microvascular endothelial cells, and with monocytoid cell line (THP1) for 48 h. Cell viability and proliferation were assessed with the sulforhodamine B colorimetric assay. Intracellular reactive oxygen species (ROS) was estimated with the 2'7 dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation method. Endothelial cell migration was measured with the scratch assay. The results were compared with the multi-criteria analysis (MCA) procedure. Results Tested flax fabrics released flavonoids and polyhydroxybutyrate to cell culture media, as it was determined by means of the high performance liquid chromatography (HPLC) method. Fabrics from transgenic plants W92 and M48 promoted proliferation of keratinocytes and fibroblasts. Water extracts from flax fabric diminished the proliferation of monocytoid cells, decreased oxidative burst in activated THP1 cells and accelerated the velocity of dermal microvascular cell migration. The MCA proved that the sum of beneficial effects estimated in human skin cell cultures was higher (by 47% and by 34% with W92 and M48, respectively) than that of non-transgenic flax fabric (Nike). Conclusions The W92 and M48 fabrics should be further studied as candidates for elaboration of new types of bandages, able to improve skin wound healing.

Recent findings have improved our understanding of the multifactorial nature of AD. While in early asymptomatic stages of AD, increased amyloid-β synthesis and tau hyperphosphorylation play a key role, while in the latter stages of the disease, numerous dysfunctions of homeostatic mechanisms in neurons, glial cells, and cerebrovascular endothelium determine the rate of progression of clinical symptoms. : The main driving forces of advanced neurodegeneration include increased inflammatory reactions in neurons and glial cells, oxidative stress, deficiencies in neurotrophic growth and regenerative capacity of neurons, brain insulin resistance with disturbed metabolism in neurons, or reduction of the activity of the Wnt-β catenin pathway, which should integrate the homeostatic mechanisms of brain tissue. In order to more effectively inhibit the progress of neurodegeneration, combination therapies consisting of drugs that rectify several above-mentioned dysfunctions should be used. It should be noted that many widely-used drugs from various pharmacological groups, "in addition" to the main therapeutic indications, have a beneficial effect on neurodegeneration and may be introduced into clinical practice in combination therapy of AD. : There is hope that complex treatment will effectively inhibit the progression of AD and turn it into a slowly progressing chronic disease. Moreover, as the mechanisms of bidirectional communication between the brain and microbiota are better understood, it is expected that these pathways will be harnessed to provide novel methods to enhance health and treat AD.

In developing and developed countries, an increasing elderly population is observed. This affects the growing percentage of people struggling with neurodegenerative diseases, including Alzheimer’s disease. Nevertheless, the pathomechanism of this disease is still unknown. This contributes to problems with early diagnosis of the disease as well as with treatment. One of the most popular hypotheses of Alzheimer’s disease is related to the pathological deposition of amyloid-β (Aβ) in the brain of ill people. In this paper, we discuss issues related to Aβ and its relationship in the development of Alzheimer’s disease. The structure of Aβ and its interaction with the cell membrane are discussed. Not only do the extracellular plaques affect nerve cells, but other forms of this peptide as well.

Transport of proteins, transcription factors, and other signaling molecules between the nucleus and cytoplasm is necessary for signal transduction. The study of these transport phenomena is particularly challenging in neurons because of their highly polarized structure. The bidirectional exchange of molecular cargoes across the nuclear envelope (NE) occurs through nuclear pore complexes (NPCs), which are aqueous channels embedded in the nuclear envelope. The NE and NPCs regulate nuclear transport but are also emerging as relevant regulators of chromatin organization and gene expression. The alterations in nuclear transport are regularly identified in affected neurons associated with human neurodegenerative diseases. This review presents insights into the roles played by nuclear transport defects in neurodegenerative disease, focusing primarily on NE proteins and NPCs. The subcellular mislocalization of proteins might be a very desirable means of therapeutic intervention in neurodegenerative disorders.

Background. Despite the wide range of medical dressings available commercially, there is still a search for better biomaterials for use in the treatment of especially difficult-to-heal wounds. For several years, attention has been paid to the use of substances, compounds, and even whole plants in medicine. Flax is a plant that has been used as a dressing for thousands of years. Therefore, we decided to test flax fibres that had previously been genetically modified as a potential wound dressing. Materials and Methods. In this study, two modified flax fibres and their combinations were tested on cell lines (mice fibroblast, normal human dermal fibroblast, normal human epidermal keratinocytes, human dermal microvascular endothelial cell, epidermal carcinoma cancer cells, monocyte cells). In the tests, fibres of the traditional flax (Nike) were used as a control. Several experiments were performed to assess cell proliferation and viability, the number of apoptotic cells, the cell cycle, genotoxicity, the level of free oxygen radicals, and determination of the number of cells after 48 hours of incubation of cell cultures with the tested flax fibres. Results. The obtained results confirm the positive influence of flax on the used cell lines. Both traditional fibres (Nike) and genetically modified fibres increased the proliferation of fibroblast cells and keratinocytes, reduced the level of free oxygen radicals, and influenced the repair of DNA damage. At the same time, the tested flax fibres did not have a proproliferative effect on the neoplastic cell line. Interestingly, genetic modifications had a stronger impact on the proliferative activity of fibroblasts, keratinocytes, and microvascular endothelium compared to the traditional flax fibre used. Conclusions. In this study, the positive properties of the tested flax fibres on cell lines were proved. In the next stage, it is worth carrying out in vivo tests of tested genetically modified flax fibres.

Celastrol (tripterine) a pentacyclic triterpenoid extracted from the roots of Tripterygium wilfordii Hook f., exhibits potent antioxidant and anti-inflammatory activity and also exerts important anti-cancer effects, as induction of apoptosis and lowering the level of drug resistance of several cancers. Increased level of cellular resistance to cytostatic drugs is typical for colorectal cancers, and largely determines the failure of chemotherapy for this tumor. The purpose of our research was to evaluate the chemopreventive effect of celastrol on cultures of colon cancer cells resistant to doxorubicin (LOVO/DX). With the use of flow cytometry we have shown that celastrol reduces the cell size of the SP (side population; subpopulation of cancer cells enriched with cancer stem cells), increases frequency of apoptosis and binds to Pgp protein in cell membranes inhibiting its transport function. The inhibition of the Pgp transport function has been shown to increase the accumulation of rhodamine-123 and standard cytostatic- doxorubicin in LOVO/DX cells. Our results prove that celastrol exhibits significant chemopreventive and chemosensitizing activities on drug resistant colon cancer cells. Celastrol appears to be a good candidate for adjuvant medicine that can improve the effectiveness of standard cytostatic therapy in humans.

BackgroundThe p53 protein is a transcription factor for many genes, including genes involved in inhibiting cell proliferation and inducing apoptosis in genotoxically damaged and tumor-transformed cells. In more than 55% of cases of human cancers, loss of the essential function of p53 protein is found. In numerous reports, it has been shown that small molecules (chemical compounds) can restore the suppressor function of the mutant p53 protein in tumor cells. The aim of this study was to evaluate the potential anticancer activity of three newly synthesized olivacine derivatives.MethodsThe study was performed using two cell lines—CCRF/CEM (containing the mutant p53 protein) and A549 (containing a non-mutant, wild-type p53 protein). The cells were incubated with olivacine derivatives for 18 h and then assays were carried out: measurement of the amount of p53 and p21 proteins, detection of apoptosis, cell cycle analysis, and rhodamine 123 accumulation assay (evaluation of P-glycoprotein inhibition). Multiple-criteria decision analysis was used to compare the anticancer activity of the tested compounds.ResultsEach tested compound caused the reconstitution of suppressor activity of the p53 protein in cells with the mutant protein. In addition, one of the compounds showed significant antitumor activity in both wild-type and mutant cells. For all compounds, a stronger effect on the level of the p53 protein was observed than for the reference compound—ellipticine.ConclusionsThe observed effects of the tested new olivacine derivatives (pyridocarbazoles) suggest that they are good candidates for new anticancer drugs.

Neuroinflammation is considered to be one of the potential causes for the development of neurodegenerative diseases, including Alzheimer&rsquo, s disease. In this study, we evaluated the effect of four newly synthesized pyrrolo[3,4-d]pyridazinone derivatives on the neuron-like PC12 cells under simulated inflammation conditions by preincubation with lipopolysaccharide (LPS). Our novel derivatives are selective cyclooxygenase-2 (COX-2) inhibitors and have similar effects to nonsteroidal anti-inflammatory drugs (NSAIDs). We assessed viability (LDH assay), metabolic activity (MTT assay), DNA damage (number of double-strand breaks measured by fast halo assay), and the neuronal features of cells (average neurite length and neurite outgrowth measured spectrofluorimetrically). DCF-DA and Griess assays were also performed, which allowed determining the impact of the tested compounds on the level of oxygen free radicals and nitrites. LPS administration significantly negatively affected the results in all tests performed, and treatment with the tested derivatives in most cases significantly reduced this negative impact. Multiple-criteria decision analysis indicated that overall, the best results were observed for compounds 2a and 2b at a concentration of 10 &micro, M. The new derivatives showed intense activity against free oxygen radicals and nitrites. Reduced reactive oxygen species level also correlated with a decrease in the number of DNA damage. The compounds improved neuronal features, such as neurite length and outgrowth, and they also increased cell viability and mitochondrial activity. Our results suggest that derivatives 2a and 2b may also act additionally on mechanisms other than 3a and 3b.

Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

Wstep. Ceramid (cer) jest zazwyczaj produkowany na drodze hydrolizy sfingomieliny przez sfingomielinazy (SMase). Zbadano, ze aktywnośc chemoprewencyjna — propoptotyczna i chemouwrazliwiająca — piperazynowej pochodnej fenotiazyny (Pht), flufenazyny (FPh) i jej nowo syntezowanych analogow — związkow 1b i 3f — zalezy od stymulacji lizosomalnej, kwaśnej sfingomielinazy (aSMase), niezaleznej od Zn2+, jednego z enzymow uczestniczących w powsta­waniu endogennego cer. Innym waznym szlakiem tworzenia cer w komorce jest synteza de novo z udzialem syntetazy ceramidowej (CerS). Celem pracy byla ocena aktywności chemoprewencyjnej FPh i jej analogow: związkow 1b i 3f po nieodwracalnym zablokowaniu CerS. Material i metody. Aktywnośc chemoprewencyjną badanych związkow (10 μM, 2 godz.) oceniano w hodowlach limfocytow ludzkich uszkodzonych genotoksycznie przez inkubacje z benzo[ a ]pirenem (+B[ a ]P; 7,5 μM, 48 godz.) i po preinkubacji z selektywnym inhibitorem CerS — fumonizyną B1 (+FB1; 20 μM, 1,5 godz.). W ocenie efektow badanych związkow zastosowano metody: mikroskopii fluorescencyjnej, spektrofotometrii i spektrofluorymetrii. Istotnośc statystyczną uzyskanych rezultatow sprawdzono za pomocą testu t -Studenta. Wyniki. W hodowlach limfocytow w obecności inhibitora CerS (+B[ a ]P; +FB1) inkubacja hodowli z analogami 1b i 3f prowadzila do istotnie nizszego (p < 0,05) odsetka komorek w apoptozie i akumulacji rodaminy 123 (Rod-123) w po­rownaniu z hodowlą bez inhibitora CerS (+B[ a ]P; -FB1). W przypadku macierzystego związku FPh wplyw na hodowle limfocytow nie zalezal od obecności inhibitora CerS. Wnioski. Efekty proapoptotyczny i chemouwrazliwiający analogow 1b i 3f są uwarunkowane aktywacją CerS . Nato­miast dla macierzystej FPh wykazano, ze jej aktywnośc chemoprewencyjna nie zalezy od stymulacji szlaku syntezy de novo cer z udzialem CerS .

Polyunsaturated fatty acids, sterols, and hydrophilic phenolic compounds are components of flax oil that act as antioxidants. We investigated the impact of flax oil from transgenic flax in the form of emulsions on stressed Chinese hamster pulmonary fibroblasts. We found that the emulsions protect V79 cells against the H2O2and the effect is dose dependent. They reduced the level of intracellular reactive oxygen species and protected genomic DNA against damage. The rate of cell proliferation increased upon treatment with the emulsions at a low concentration, while at a high concentration it decreased significantly, accompanied by increased frequency of apoptotic cell death. Expression analysis of selected genes revealed the upregulatory impact of the emulsions on the histones, acetylases, and deacetylases. Expression of apoptotic, proinflammatory, and anti-inflammatory genes was also altered. It is thus suggested that flax oil emulsions might be useful as a basis for biomedical products that actively protect cells against inflammation and degeneration. The beneficial effect on fibroblast resistance to oxidative damage was superior in the emulsion made of oil from transgenic plants which was correlated with the quantity of antioxidants and squalene. The emulsions from transgenic flax are promising candidates for skin protection against oxidative damage.

Aim of the study: To evaluate the inhi -bitory effect of 17 new analogues of FPhon the Pgp transport function, by esti-mation of the rhodamine 123 (Rod-123)accumulation inside cultured lympho-cytes.Material and methods: Lymphocyte werecultured in the presence of a lectin (PHA;2%, v/v), incubated with benzo[ α]pyrene(B[α]P; 7.5 μM, 48 h) to induce genotox-ic damage and to increase Pgp expressionin the cells. Lymphocytes cultured with-out the tested compounds were consid-ered as controls. Results: It was established that 10 ana-logues of FPh, among 17 tested, signifi-cantly increased Rod-123 accumulation in lymphocytes at the concentration of10 μM. As compared to the control cul-tures the Pgp transport function was the most strongly inhibited by 1a, 1b, 1d,3f, 3h and 3i analogues (approximatelyby 25%).Conclusions: FPh analogues 1a, 1b, 1d, 3f,3h and 3i should be further studied aspromising candidates for adjuvant can-cer chemotherapeutics.Key words: fluphenazine analogues,MDR, P-glycoprotein, MDR modulators.

Phenothiazines belong to the oldest, synthetic antipsychotic drugs, which do not have their precursor in the world of natural compounds. Apart from their fundamental neuroleptic action connected with the dopaminergic receptors blockade, phenothiazine derivatives also exert diverse biological activities, which account for their cancer chemopreventive-effect, as: calmodulin- and protein kinase C inhibitory-actions, anti-proliferative effect, inhibition of P-glycoprotein transport function and reversion of multidrug resistance. According to literature data on relations between chemical structure of phenothiazines and their biological effects, the main directions for further chemical modifications have been established. They are provided and discussed in this review paper.

Purpose. To determine, in vitro, the susceptibility to apoptosis of lymphocytes from patients with peripheral arterial disease (PAD) in the presence of a low culture medium serum concentration, and to evaluate the correlation of the degree of apoptosis and the serum lipid levels. Methods. Lymphocytes were isolated from the venous blood of PAD patients with lower limb ischemia secondary to obliterative atherosclerosis of Fountain stage IIb. None of the patients had received hypo-lipemic therapy. The lymphocytes were incubated for 48 hr in media containing reduced concentrations of fetal calf serum. The study group consisted of 10 patients (7 men and 3 women), with a mean age of 67.0 ± 4.0 yr. The control group consisted of ten healthy volunteers, of the same mean age and sex proportion as the study group. Results. The percentage of non-apoptotic lymphocytes was lower (by 17%) and the percentage of late apoptotic lymphocytes was higher (by 33%) in the PAD patients than in the healthy donors when comparing the slopes of regression lines describing the relation between frequency of apoptotic lymphocytes in culture media containing reduced concentration of fetal calf serum The percentage of late apoptotic lymphocytes was correlated with the levels of total cholesterol (rs=0.93; P < 0.01) and LDL cholesterol (rs=0.80; P < 0.01) , and negatively correlated with the level of triglycerides (rs=-0.71; P < 0.05). Conclusion. The results of this study of lymphocyte apoptosis are important in understanding of the disease pathogenesis and should be taken into account in elaboration of treatment strategies.

Todralazine–HCl (TDR), analytical grade, was kindly supplied by Polfa cleus assay and the sister chromatid exchange test were (Pabianice, Poland). Benzo[a]pyrene (B[a]P), cytochalasin B, mitomycin C, used to demonstrate the antimutagenicity of todralazine. 5-bromo-2-deoxyuridine, phorbol 12-myristate 13-acetate (PMA) and Todralazine lowered the level of free radicals generated by dimethylsulfoxide (DMSO) were purchased from Sigma (St Louis, MO). human granulocytes in vitro in the presence of benzo[a] The blood cell separation solutions (Histopaque-1077 and Histopaquepyrene and also in the presence of the granulocyte activator 1119), as well as components of the cell culture medium [RPMI 1640, fetal calf serum (FCS) and L-glutamine] were also obtained from Sigma. and tumor promoter phorbol myristate acetate. These Phytohemagglutinin (PHA-M) was obtained from Gibco (Gaithersburg, MD). results, together with our previous data obtained in the The stains acridine orange, ethidium bromide, azur II, eosin B, trypan blue, standard bacterial Ames test, strongly suggest that todrala- Giemsa solution and nitroblue tetrazolium (NBT) were purchased from Sigma. zine is a good antimutagen in vitro and deserves further The other reagents used for buffers and culture medium preparation were from POCH (Gliwice, Poland). research on its inhibitory action on mutagenesis and carcinogenesis. Blood cell separation Heparinized blood was obtained by venipuncture from four male volunteers aged 40–50 who were heavy smokers (30 cigarettes/day). The blood donors were picked from a group of hypertonic patients, periodically examined at

The membrane interactions can play a vital role in chemosensitizing activity of fluphenazine dihydrochloride, an inhibitor of P-glycoprotein transport function. In in vitro testings of the influence of fluphenazine dihydrochloride on model dipalmitoylphosphatidylcholine membranes and on human lymphocyte cultures, genotoxically damaged by benzo[a]pyrene, differential scanning calorimetry and rhodamine 123 retention test were applied. The results of both testings shown that the membrane interactions of fluphenazine dihydrochloride in model membrane structure and the chemosensitizing effect of this compound in examined human lymphocyte cultures, depend on its concentration in a sample.