Semaphorin 6 Family-An Important Yet Overlooked Group of Signaling Proteins Involved in Cancerogenesis.

According to recent evidence, some groups of semaphorins (SEMAs) have been associated with cancer progression. These proteins are able to modulate the cellular signaling of particular receptor tyrosine kinases (RTKs) via the stimulation of SEMA-specific coreceptors, namely plexins (plexin-A, -B, -C, -D) and neuropilins (Np1, Np2), which share common domains with RTKs, leading to the coactivation of the latter receptors. MET, ERBB2, VEGFR2, PFGFR, and EGFR, among others, represent acknowledged targets of semaphorins that are often associated with tumor progression or poor prognosis. In particular, higher expression of SEMA6 family proteins in cancer cells and stromal cells of the cancer niche is often associated with enhanced tumor angiogenesis, metastasis, and resistance to anticancer therapy. Notably, high SEMA6 expression in malignant tumor cells such as melanoma, pleural mesothelioma, gastric cancer, lung adenocarcinoma, and glioblastoma may serve as a prognostic biomarker of tumor progression. To date, very few studies have focused on the mechanisms of transmembrane SEMA6-driven tumor progression and its underlying interplay with RTKs within the tumor microenvironment. This review presents the growing evidence in the literature on the complex and shaping role of SEMA6 family proteins in cancer responsiveness to environmental stimuli.

Whole grain metabolite 3,5-dihydroxybenzoic acid is a beneficial nutritional molecule with the feature of a double-edged sword in human health: a critical review and dietary considerations.

Nonprocessed foodstuffs of plant origin, especially whole-grain cereals, are considered to be health-promoting components of the human diet. While most of their well-studied effects derive from their high fiber content and low glycemic index, the presence of underrated phenolic phytonutrients has recently been brought to the attention of nutritionists. In this review, we report and discuss findings on the sources and bioactivities of 3,5-dihydroxybenzoic acid (3,5-DHBA), which is both a direct dietary component (found, e.g., in apples) and, more importantly, a crucial metabolite of whole-grain cereal-derived alkylresorcinols (ARs). 3,5-DHBA is a recently described exogenous agonist of the HCAR1/GPR81 receptor. We concentrate on the HCAR1-mediated effects of 3,5-DHBA in the nervous system, on the maintenance of cell stemness, regulation of carcinogenesis, and response to anticancer therapy. Unexpectedly, malignant tumors take advantage of HCAR1 expression to sense 3,5-DHBA to support their growth. Thus, there is an urgent need to fully identify the role of whole-grain-derived 3,5-DHBA during anticancer therapy and its contribution in the regulation of vital organs of the body via its specific HCAR1 receptor. We discuss here in detail the possible consequences of the modulatory capabilities of 3,5-DHBA in physiological and pathological conditions in humans.

Lactate drives cellular DNA repair capacity: Role of lactate and related short-chain fatty acids in cervical cancer chemoresistance and viral infection.

The characteristic feature of a cancer microenvironment is the presence of a highly elevated concentration of L-lactate in the tumor niche. The lactate-rich environment is also maintained by commensal mucosal microbiota, which has immense potential for affecting cancer cells through its receptoric and epigenetic modes of action. Some of these lactate activities might be associated with the failure of anticancer therapy as a consequence of the drug resistance acquired by cancer cells. Upregulation of cellular DNA repair capacity and enhanced drug efflux are the most important cellular mechanisms that account for ineffective radiotherapy and drug-based therapies. Here, we present the recent scientific knowledge on the role of the HCA1 receptor for lactate and lactate intrinsic activity as an HDAC inhibitor in the development of an anticancer therapy-resistant tumor phenotype, with special focus on cervical cancer cells. In addition, a recent study highlighted the viable role of interactions between mammalian cells and microorganisms in the female reproductive tract and demonstrated an interesting mechanism regulating the efficacy of retroviral transduction through lactate-driven modulation of DNA-PKcs cellular localization. To date, very few studies have focused on the mechanisms of lactate-driven enhancement of DNA repair and upregulation of particular multidrug-resistance proteins in cancer cells with respect to their intracellular regulatory mechanisms triggered by lactate. This review presents the main achievements in the field of lactate impact on cell biology that may promote undesirable alterations in cancer physiology and mitigate retroviral infections.

Dual Targeting Ligands-Histamine H3 Receptor Ligands with Monoamine Oxidase B Inhibitory Activity-In Vitro and In Vivo Evaluation.

The clinical symptoms of Parkinson's disease (PD) appear when dopamine (DA) concentrations in the striatum drops to around 20%. Simultaneous inhibitory effects on histamine H3 receptor (H3R) and MAO B can increase DA levels in the brain. A series of compounds was designed and tested in vitro for human H3R (hH3R) affinity and inhibitory activity to human MAO B (hMAO B). Results showed different activity of the compounds towards the two biological targets. Most compounds had poor affinity for hH3R (Ki > 500 nM), but very good inhibitory potency for hMAO B (IC50 < 50 nM). After further in vitro testing (modality of MAO B inhibition, permeability in PAMPA assay, cytotoxicity on human astrocyte cell lines), the most promising dual-acting ligand, 1-(3-(4-(tert-butyl)phenoxy)propyl)-2-methylpyrrolidine (13: hH3R: Ki = 25 nM; hMAO B IC50 = 4 nM) was selected for in vivo evaluation. Studies in rats of compound 13, in a dose of 3 mg/kg of body mass, confirmed its antagonistic effects for H3R (decline in food and a water consumption), decline in MAO B activity (>90%) in rat cerebral cortex (CTX), and an increase in DA content in CTX and striatum. Moreover, compound 13 caused a slight increase in noradrenaline, but a reduction in serotonin concentration in CTX. Thus, compound 13 is a promising dual-active ligand for the potential treatment of PD although further studies are needed to confirm this.

Higher Risk for Sensitization to Commonly Consumed Herbs among Adults and Youngsters Suffering from Birch, Mugwort or Grass Pollinosis.

Background: According to recent findings, mugwort and birch pollen-allergic patients represent a high-risk group for developing adverse allergic reactions to herbal spices due to cross-reacting allergens found in both pollen and raw herbs. Such associations are known as a pollen-plant food allergy syndrome. Objective: The aim of the study was to evaluate the extent of sensitization to commonly consumed herb species representing Lamiaceae, Apiaceae and Brassicaceae families in Polish patients with suspected birch, mugwort or grass pollen allergy. Materials and Methods: Data were obtained from 180 patients, adults and children with suspected allergy to aeroallergens. Skin prick tests (SPT) were performed with standard birch, mugwort, grass mixture or dust mite extracts. Prick by prick tests were performed with fresh extracts of popular herbs: basil, oregano, lemon balm, mint, salvia, rosemary, thyme, anise, caraway and mustard. Results: Twenty-nine percent of patients were characterized by concomitant positive skin prick reactions to both herbs and pollens extracts. The concomitant pollinosis significantly increased the risk of SPT reaction to all tested herbs in adults (odds ratio, OR = 2.15−7.35) and children (OR = 5.3−28). The extent of SPT responses to herbs from Lamiaceae + Apiaceae were strongly correlated with SPT responses to pollens in the pediatric group (r = 0.685/p < 0.001). Conclusion: The study demonstrates that youngsters suffering from pollinosis are at high risk of developing allergic reactions to herbs and highlights the importance of including native skin prick tests with herbs in the diagnostic work-up for suspected food allergy.

Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation.

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood-Brain Barrier Models.

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood-brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood-brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Polyphenol Extract from Evening Primrose (Oenothera paradoxa) Inhibits Invasion Properties of Human Malignant Pleural Mesothelioma Cells.

Extracts from the defatted evening primrose (Oenothera paradoxa Hudziok) seeds are the source of a range of stable polyphenolic compounds, including ellagic acid, gallic acid, and catechin. Our studies evaluate, for the first time, the influence of evening primrose isopropanol extract (EPE) on malignant pleural mesothelioma (MPM) cells. MPM is rarely diagnosed, its high aggressiveness and frequently noted chemoresistance limit its treatment schemes and it is characterized by low prognostic features. Here, we demonstrate that EPE inhibited MPM growth in a dose-dependent manner in cells with increased invasion properties. Moreover, EPE treatment resulted in cell cycle arrest in the G2/M phase and increased apoptosis in invasive MPM cell lines. Additionally, EPE strongly limited invasion and MMP-7 secretion in MPM cancer cells. Our original data provide evidence about the potential anti-invasive effects of EPE in MPM therapy treatment.

Impaired resolution of wheals in the skin prick test and low diamine oxidase blood level in allergic patients.

INTRODUCTION: Histamine is the major mediator of IgE- and non-IgE-mediated allergic reactions upon allergen or hapten contact. Reduced histamine degradation capacity was associated with atopic eczema as well as with non-immunological histamine intolerance. Higher blood serum histamine level concomitant with decreased intestinal diamine oxidase activity were observed in patients with food allergy. AIM: To evaluate the relationship between patients' blood diamine oxidase (DAO) activity/histamine status and their reactivity to time-resolved histamine skin prick test in respect to vulnerability to allergic diseases. MATERIAL AND METHODS: Fifty-three patients were examined with skin prick tests (SPT) and patch tests for suspected presence of either IgE- or non-IgE-mediated allergy. All individuals were skin prick tested with histamine and the resolution of the wheal was monitored for 50 min. Blood DAO activity and histamine concentration were measured with a radio-extraction radioimmunoassay. RESULTS: Time-resolved histamine skin prick testing revealed presence of wheals which were 35% larger in diameter in 47% of examined subjects at 20 min of the test. These patients exhibited significantly compromised time-course wheal resolution (wheal ≥ 3 mm at 50 min) compared to a group of patients with the normal-rate of wheal resolution (wheal = 0 mm at 50 min). Within a group of subjects exhibiting impaired wheal resolution, 61% of patients were diagnosed allergic compared to 50% in a group of patients with a normal rate of wheal resolution. Finally, allergic patients were characterized by a significantly lower DAO activity and higher histamine content compared to healthy subjects. CONCLUSIONS: The results of this study indicate that patients with IgE- or non-IgE-mediated allergy are likely to have low DAO blood activity and may concomitantly suffer from histamine intolerance. Furthermore, our results suggest that allergic patients are more likely to develop an excessive SPT reaction. Our results emphasize caution in interpretation of the SPT results in allergic patients with diagnosed histamine intolerance or histamine/DAO activity imbalance.

The Incidence of Delayed-Type Hypersensitivity Reactions to Apples Among Patients Allergic to Birch Pollen.

The major apple allergen Mal d 1 cross-reacts with the homologous birch pollen allergen Bet v 1 and causes immunoglobulin E (IgE)-mediated immediate-type allergic reactions. In some patients, delayed-type hypersensitivity to apples may develop within 72 hours without evidence of specific IgE or a positive skin prick test (SPT). The aim of the study was to evaluate the concomitance of delayed-type hypersensitivity reactions and immediate IgE-mediated reactions against high- and low-allergenic apple cultivars in patients with birch pollen allergy. Data were obtained from 45 adults with clinical symptoms of birch pollen allergy. Patients were exposed to apple pulp via atopy patch tests (APTs) and SPTs. Levels of IgE specific to Bet v 1 and Mal d 1 were measured with a radioallergosorbent test. Patients allergic to birch pollen showed the highest rate of positive SPT responses to Golden Delicious apples and the lowest rate to low-allergenic cultivar Grey French Reinette. Among these patients, 9% developed delayed hypersensitivity reactions to either Golden Delicious or Grey French Reinette apples; these reactions manifested clinically as erythema with papules (class ++). Fifty percent of APT-positive patients were concomitantly SPT-negative. Here, we show for the first time the clinical relevance of T cell-driven allergic reactions to apples. APTs may reveal type IV sensitization in patients who are negative for the corresponding type I sensitization tests. Thus, utilization of the APT procedure with fresh apple appears to be a valuable tool for the diagnosis of apple allergy and may improve the accuracy of food allergy diagnoses.

Tubulin beta 3 and 4 are involved in the generation of early fibrotic stages.

The endothelial-mesenchymal transition (EndMT) is a fundamental cellular mechanism that occurs under both physiological and pathological conditions and includes the fibrotic stages of numerous organs, namely, the skin, kidneys, heart, lungs and liver. Endothelial cells that undergo EndMT are one of the main source of (myo)fibroblasts in fibrotic tissues. A critical step in cellular transdifferentiation is morphological change, which is engineered by the reorganization of cytoskeletal elements such as microtubules. These dynamic structures consist of αß-tubulin heterodimers that are also involved in cellular movement and intracellular trafficking, processes modulated during EndMT. One fundamental mechanism that underlies microtubule stabilization is the regulation of the levels of α and ß-tubulin. However, little is known about the roles of specific tubulin isotypes in the development of EndMT-based diseases. This study provides the first evidence that the upregulation of TUBB3 and TUBB4 is coupled with increased cell migration in EndMT-induced HMEC-1 cells. Immunochemical analysis reveals that these tubulins are upregulated in the early stages of EndMT, and siRNA analysis indicates that they are engaged in the generation of mesenchymal behavior via the enhancement of cell migration. This modulation seems to be especially important in wound healing. Finally, cell surface analysis reveals that TUBB3 and TUBB4 are necessary for the transport and proper localization of N-cadherin within the plasma membrane. We believe that our results will be valuable for the development of effective new anti-fibrotic therapies.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Allergy to apple cultivars among patients with birch pollinosis and oral allergy syndrome.

BACKGROUND: Oral allergy syndrome (OAS) is caused by cross-reacting allergens found in pollen, raw fruits, vegetables, and some tree nuts. The major apple allergen, Mal d 1, is a cause of food allergic reactions in birch pollen sensitized patients. OBJECTIVE: To explore the allergenicity of the most popular and commonly consumed apple cultivars in Poland in patients with birch pollen allergy with or without OAS. METHODS: Data were obtained from 46 adults with clinical symptoms of birch allergy and allergic rhinitis or rhinoconjunctivitis. Patients were divided into 2 groups according to the occurrence of OAS to apple. Skin prick tests (SPTs) were performed with pulp from the 11 most popular apple cultivars in Poland. Specific IgE (sIgE) to Bet v 1 was measured by radioallergosorbent test. RESULTS: Patients with OAS had more positive responses to apple SPT vs patients without OAS (odds ratios, 4.8-11.96). Patients with OAS had distinctive responses to apple cultivars. Szara Reneta and Cortland induced positive responses in 50% and 83% of patients, respectively. Patients with OAS vs patients without OAS who were allergic to apples vs nonallergic had 2-fold (P = .008) and 7-fold (P = .03) higher blood concentrations of sIgE Bet v 1, respectively. There were different profiles of correlations of sIgE Bet v 1 with wheal diameter for low and high allergenic cultivars in patients without and with OAS. CONCLUSION: We noted a substantial role for Bet v 1 sensitization in the allergic response based on evaluation of the allergenicity of 11 apple cultivars. The sIgE Bet v 1 and SPT results of patients with and without OAS allowed differentiation between low and high allergenic cultivars.

Lactate Stimulates IL-4 and IL-13 Production in Activated HuT-78 T Lymphocytes Through a Process That Involves Monocarboxylate Transporters and Protein Hyperacetylation.

Mucosal cells of the gastrointestinal and female reproductive tract are constantly exposed to l- and d-lactate of bacterial origin. These compounds not only protect the host from pathogen colonization but also modulate the activity of mucosal immune cells, thereby playing an important role in inflammatory host responses. In this study, we demonstrated that exposure of anti-CD3/CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin-activated HuT-78 T lymphocyte cells to 10-20 mM d-lactate significantly increased IL-4 and IL-13 production. Interestingly, the d-lactate isomer, exclusively produced locally by gut or cervicovaginal microbiota, was found to be more potent than the l-isomer. Interestingly, neither of the strong histone deacetylase inhibitors [structurally similar butyrate and suberoylanilide hydroxamic acid (SAHA)] was as effective in the stimulation of IL-13 production as d-lactate. Lactate transport through monocarboxylate transporters was required for lactate-enhanced IL-13 production in a manner that was not hydroxycarboxylic acid receptor 1-dependent. Furthermore, lactate treatment increased the acetylation of GATA-3, a critical regulator of Th1/Th2 differentiation and resulted in H3 and H4 histone hyperacetylation state, which is a characteristic feature of transcriptionally active chromatin. Both lactate isomers also enhanced IL4 and IL13 promoter-driven activity of reporter constructs in murine and human cells. Together, these findings demonstrate that a local millimolar concentration of l- or d-lactate may play an important role in the modulation of inflammation-mediated processes.

L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

BACKGROUND: The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells. METHODS: Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA. RESULTS: L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA). CONCLUSIONS: Our data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.

Interleukin-4 enhances PARP-dependent DNA repair activity in vitro.

Eukaryotic cells possess several DNA repair mechanisms, including homologous recombination and the non-homologous end-joining (NHEJ) system. There are two known NHEJ systems. The major mechanism depends on the catalytic unit of DNA-dependent protein kinase (DNA-PKcs) and DNA ligase IV, and an alternative mechanism (B-NHEJ) depends on poly(ADP-ribose) polymerase (PARP). These systems are upregulated by genotoxic agents. Interleukin 4 (IL-4) is an immunoregulatory cytokine that is secreted by immune cells upon contact with certain genotoxic compounds and is known to regulate several genes encoding components of DNA repair systems in human monocytes. We have investigated the possible effects of IL-4 on the DNA repair process within murine and human cells exposed to selected genotoxic compounds. In a series of experiments, including the comet assay, cell surface annexin V staining, analysis of histone H2AX phosphorylation, and a DNA end-joining assay, we observed that IL-4 decreased DNA damage in murine fibroblasts and human glioblastoma cells exposed to genotoxic agents and increased DNA ligation activity in the nuclei of these cells in a process that depended on PARP. These observations suggest that IL-4 is capable of upregulating the alternative NHEJ DNA repair mechanism in murine and human cells.

Orexins protect neuronal cell cultures against hypoxic stress: an involvement of Akt signaling.

Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

Effect of an angiotensin II type 1 receptor blocker on caveolin-1 expression in prostate cancer cells.

INTRODUCTION: Caveolin-1, the major structural protein of caveolae, interacts directly with the AT1 receptor. The biological functions of caveolin-1 in cancer are compound, multifaceted, and depend on cell type, tumour grade and cancer stage. The AT1-R-caveolin complex in caveolae may coordinate angiotensin II (Ang II) induced signalling. The aim of this study was to determine the effect of the angiotensin II receptor type 1 blocker candesartan on caveolin expression in human metastatic prostate adenocarcinoma cells PC-3. MATERIAL AND METHODS: WST-1 and BrdU assays were used as indicators of cell viability and proliferation after angiotensin II and/or candesartan stimulation. Real-time RT-PCR and western blot were used to study the effect of Ang II and/or candesartan on the expression of Cav-1 and AT1-R in PC-3 cells. RESULTS: We found that the expression of caveolin-1 mRNA in the PC-3 cells treated with CV was significantly decreased in comparison with the control (2.9 ±0.17, 4.7 ±0.6, p < 0.05), whereas a higher caveolin-1 mRNA expression was observed in those after Ang II treatment (6.0 ±0.43, 4.7 ±0.6, p < 0.05). Protein analysis indicate that the expression of caveolin-1 protein in the PC-3 cells treated with candesartan was significantly decreased when compared with the control (0.69 ±0.05, 1.6 ±0.12, p < 0.05), whereas higher caveolin-1 protein expression was observed after Ang II treatment (2.5 ±0.20, 1.6 ±0.12, p < 0.05). CONCLUSIONS: These results provide new information on the action of candesartan and may improve the knowledge about AT1 receptor inhibitors, which can be potentially useful in prostate cancer therapy.

Procyanidins from Japanese quince (Chaenomeles japonica) fruit induce apoptosis in human colon cancer Caco-2 cells in a degree of polymerization-dependent manner.

Plant proanthocyanidins, including procyanidins, display various biological activities. Here we report an inhibition of human colon cancer Caco-2 cell growth by the extract from Japanese quince fruit and the procyanidin-rich fractions of the extract. We observed that the amount of apoptotic Caco-2 cells increased by 52.1% vs. control after 72-h incubation with 50 µg extract/mL, as assessed by flow cytometry and image cytometry. Under the same experimental conditions the corresponding values for human colon cancer HT-29 cells and for rat normal intestinal IEC-6 cells were 5.0% and 8.1%, respectively. The extract fractions enriched with higher oligomers exhibited the highest proapoptotic activity. In conclusion, the Japanese quince procyanidins exhibited proapoptotic activity in Caco-2 cells within a submilimolar concentration range.

Polyphenols from evening primrose ( Oenothera paradoxa ) defatted seeds induce apoptosis in human colon cancer Caco-2 cells.

Polyphenols extracted from evening primrose seeds (industrial waste product) were studied as apoptosis inducers in human colorectal adenocarcinoma Caco-2 and HT-29 cell lines and in rat normal intestinal IEC-6 cells. The extract dose-dependently inhibited the growth of Caco-2, HT-29, and IEC-6 cells. However, nuclear DNA fragmentation characteristic of apoptosis was observed only in Caco-2. After 72 h of incubation with the extract at 150 µM gallic acid equivalents (44.1 µg extract/mL), Caco-2 cell numbers decreased to 19% of control and 48.8% of the cells were identified by flow cytometry as apoptotic. Under the same conditions only 8% of HT-29 cells and 12.6% of IEC-6 cells exhibited hypodiploid DNA content. The effects of the extract and its fractions on phosphatidylserine exposure and cell membrane integrity were assessed by high content screening image cytometry. The fractions strongly and dose-dependently reduced Caco-2 cell numbers, whereas HT-29 and IEC-6 cells were affected to lesser extents.