Treatment with onion bulb extract both prevents and reverses allergic inflammation in a murine model of asthma.

CONTEXT: Asthma presents a global health challenge. The main pharmacotherapy is synthetic chemicals and biological-based drugs that are costly, and have significant side effects. In contrast, use of natural products, such as onion (Allium cepa L., Amaryllidaceae) in the treatment of airway diseases has increased world-wide because of their perceived efficacy and little safety concerns. However, their pharmacological actions remain largely uncharacterized. OBJECTIVE: We investigated whether onion bulb extract (OBE) can (1) reverse established asthma phenotype (therapeutic treatment) and/or (2) prevent the development of the asthma phenotype, if given before the immunization process (preventative treatment). MATERIALS AND METHODS: Six groups of male Balb/c mice were established for the therapeutic (21 days) and five groups for the preventative (19 days) treatment protocols; including PBS and house dust mite (HDM)-challenged mice treated with vehicle or OBE (30, 60, and 100 mg/kg/i.p.). Airways inflammation was determined using cytology, histology, immunofluorescence, Western blot, and serum IgE. RESULTS: Therapeutic (60 mg/kg/i.p.) and preventative (100 mg/kg/i.p.) OBE treatment resulted in down-regulation of HDM-induced airway cellular influx, histopathological changes and the increase in expression of pro-inflammatory signaling pathway EGFR, ERK1/2, AKT, pro-inflammatory cytokines and serum IgE. DISCUSSION AND CONCLUSION: Our data show that OBE is an effective anti-inflammatory agent with both therapeutic and preventative anti-asthma effects. These findings imply that onion/OBE may be used as an adjunct therapeutic agent in established asthma and/or to prevent development of allergic asthma. However, further studies to identify the active constituents, and demonstrate proof-of-concept in humans are needed.

The effect of lactate dehydrogenase inhibitors on proliferation, motility and invasion of breast cancer cells in vitro highlights a new role for lactate.

Lactate dehydrogenase (LDH) is being increasingly recognized as a major factor in the progression of breast cancer. It was previously shown that short interfering RNA­mediated knockdown of either LDH­A or ­B isoform resulted in inhibition of cell motility due to reduced lactate levels in the extracellular environment. The aim of the present study was to determine the use of pharmacological LDH inhibitors to reduce aggressive behavior of breast cancer cells. The effect of LDH inhibitors was investigated in both estrogen receptor (ER)+ and ER­ breast cancer cell lines and in normal breast epithelial cells. Cell proliferation, motility and invasion were measured using MTT, wound healing and cultrex assays, respectively. Changes in several key mediators of mitogenic signaling important in breast cancer cells were determined using western blotting. Treatment with various inhibitors reported to block LDH activity resulted in significant reduction in extracellular lactate level, cell proliferation, motility and invasion. This was associated with changes in the levels of vimentin, E­cadherin, p38 MAPK, ERK1/2 and AKT. A couple of these inhibitors such as quercetin and lonidamine showed preferential inhibition of cancer cell proliferation compared with normal epithelial cell inhibition. These data extend initial findings, further underlining the importance of lactate as a major factor in breast cancer progression and indicate the practical use of various commercially available LDH inhibitors as promising therapeutic agents to oppose the processes leading to cancer progression.

Effect of minocycline, methyl prednisolone, or combination treatment on the colonic bacterial population in a state of colonic inflammation using the murine dextran sulfate sodium model.

BACKGROUND: Several reports demonstrated anti-inflammatory properties of minocycline in various inflammatory disorders including colitis. We have experimental evidence suggesting synergistic anti-inflammatory effect of minocycline with methyl prednisolone in reducing colitis severity in mice, but if this effect is in part related to modulating the composition of colonic microbiota is still unknown. METHODS: the effect of vehicle (V), minocycline (M), methyl prednisolone (MP), or combination (C) regimen on the composition of the microbiota of mice in a state of colon inflammation compared to untreated (UT) healthy mice was determined using 16s metagenomic sequencing, and the taxonomic and functional profiles were summarized. RESULTS: Overall, the bacterial flora from the phylum Firmicutes followed by Bacteroidota were found to be predominant in all the samples. However, the composition of Firmicutes was decreased relatively in all the treatment groups compared to UT group. A relatively higher percentage of Actinobacteriota was observed in the samples from the C group. At the genus level, Muribaculaceae, Bacteroides, Bifidobacterium, and Lactobacillus were found to be predominant in the samples treated with both drugs (C). Whereas "Lachnospiraceae NK4A136 group" and Helicobacter in the M group, and Helicobacter in the MP group were found to be predominant. But, in the UT group, Weissella and Staphylococcus were found to be predominant. Eubacterium siraeum group, Clostridia vadinBB60 group, Erysipelatoclostridium and Anaeroplasma genera were identified to have a significant (FDR p < 0.05) differential abundance in V compared to C and UT groups. While at the species level, the abundance of Helicobacter mastomyrinus, Massiliomicrobiota timonensis and uncultured Anaeroplasma were identified as significantly low in UT, C, and M compared to V group. Functional categories related to amino acid, carbohydrate, and energy metabolism, cell motility and cell cycle control were dominated overall across all the samples. Methane metabolism was identified as an enriched pathway. For the C group, "Colitis (decrease)" was among the significant (p = 1.81E-6) associations based on the host-intrinsic taxon set. CONCLUSION: Combination regimen of minocycline plus methyl prednisolone produces a synergistic anti-inflammatory effect which is part related to alternation in the colonic microbiota composition.

Minocycline synergizes with corticosteroids in reducing colitis severity in mice via the modulation of pro-inflammatory molecules.

Background: A few studies have highlighted the anti-inflammatory properties of minocycline in reducing colitis severity in mice, but its molecular mechanism is not fully understood. The aim of this study was to determine the anti-inflammatory properties of minocycline and the expression/activity profiles of molecules involved in pro-inflammatory signaling cascades, cytokines, and molecules involved in the apoptotic machinery. The synergistic effect between minocycline and corticosteroids was also evaluated. Methods: The effects of various treatment approaches were determined in mice using the dextran sulfate sodium (DSS) colitis model at gross and microscopic levels. The expression/activity profiles of various pro- or anti-inflammatory molecules were determined using Western blotting and polymerase chain reaction (PCR). Results: Minocycline treatment significantly reduced colitis severity using prophylactic and treatment approaches and produced a synergistic effect with budesonide and methylprednisolone in reducing the active state of colitis. This was mediated in part through reduced colonic expression/activity of pro-inflammatory molecules, cytokines, proteins involved in the apoptotic machinery, and increased expression of the anti-inflammatory cytokine IL-10. Conclusion: Minocycline synergizes with corticosteroids to reduce colitis severity, which could reduce their dose-dependent side effects and treatment cost. The reduction in colitis severity was achieved by modulating the expression/activity profiles of various pro- and anti-inflammatory signaling molecules, cytokines, and molecules involved in the apoptotic machinery.

α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling.

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.

Glucose deprivation reduces proliferation and motility, and enhances the anti-proliferative effects of paclitaxel and doxorubicin in breast cell lines in vitro.

BACKGROUND: Breast cancer chemotherapy with high dose alkylating agents is severely limited by their collateral toxicity to crucial normal tissues such as immune and gut cells. Taking advantage of the selective dependence of cancer cells on high glucose and combining glucose deprivation with these agents could produce therapeutic synergy. METHODS: In this study we examined the effect of glucose as well as its deprivation, and antagonism using the non-metabolized analogue 2-deoxy glucose, on the proliferation of several breast cancer cell lines MCF7, MDA-MB-231, YS1.2 and pII and one normal breast cell line, using the MTT assay. Motility was quantitatively assessed using the wound healing assay. Lactate, as the end product of anaerobic glucose metabolism, secreted into culture medium was measured by a biochemical assay. The effect of paclitaxel and doxorubicin on cell proliferation was tested in the absence and presence of low concentrations of glucose using MTT assay. RESULTS: In all cell lines, glucose supplementation enhanced while glucose deprivation reduced both their proliferation and motility. Lactate added to the medium could substitute for glucose. The inhibitory effects of paclitaxel and doxorubicin were significantly enhanced when glucose concentration was decreased in the culture medium, requiring 1000-fold lesser concentration to achieve a similar degree of inhibition to that seen in glucose-containing medium. CONCLUSION: Our data show that a synergy was obtained by combining paclitaxel and doxorubicin with glucose reduction to inhibit cancer cell growth, which in vivo, might be achieved by applying a carbohydrate-restricted diet during the limited phase of application of chemotherapy; this could permit a dose reduction of the cytotoxic agents, resulting in greater tolerance and lesser side effects.

MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines.

Background: MicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion. Methods: MicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR. Results: Each cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50-60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes. Conclusions: These data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.

Lactate Dehydrogenase A or B Knockdown Reduces Lactate Production and Inhibits Breast Cancer Cell Motility in vitro.

Background: Lactate dehydrogenase (LDH) plays an important role in cancer pathogenesis and enhanced expression/activity of this enzyme has been correlated with poor prognosis. In this study we determined the expression profile of LDH-A and B in normal as well as in endocrine-resistant and -responsive breast cancer cells and the effect of their knockdown on LDH activity, lactate production, proliferation and cell motility. Methods: Knockdown experiments were performed using siRNA and shRNA. The expression profile of LDH and signaling molecules was determined using PCR and western blotting. Intracellular LDH activity and extracellular lactate levels were measured by a biochemical assay. Cell motility was determined using wound healing, while proliferation was determined using MTT assay. Results: LDH-A was expressed in all of the tested cell lines, while LDH-B was specifically expressed only in normal and endocrine-resistant breast cancer cells. This was correlated with significantly enhanced LDH activity and lactate production in endocrine resistant breast cancer cells when compared to normal or endocrine responsive cancer cells. LDH-A or -B knockdown significantly reduced LDH activity and lactate production, which led to reduced cell motility. Exogenous lactate supplementation enhanced cell motility co-incident with enhanced phosphorylation of ERK1/2 and reduced E-cadherin expression. Also, LDH-A or -B knockdown reduced ERK 1/2 phosphorylation. Conclusion: Enhanced cell motility in endocrine resistant breast cancer cells is at least in part mediated by enhanced extracellular lactate levels, and LDH inhibition might be a promising therapeutic target to inhibit cancer cell motility.

The novel piperazino-enaminone JOAB-40 reduced colitis severity in mice via inhibition tumor necrosis factor-α and interleukin-1ß.

BRIEF INTRODUCTION: The synthetic compound enaminone E121 has an established role as a potent anti-tussive, bronchodilator and anti-inflammatory agent in asthma, cough, and colitis induced animal models. The addition of an N-alkylated piperazine motif to the terminal end of E121 lead to the generation of various analogues such as JOAB-40. JOAB-40 was shown to be more potent than the lead compound E121 in inhibiting the expression of the chemokine receptor CCR2, ERK1/2 phosphorylation, and the release of pro-inflammatory cytokines in vitro. MAIN OBJECTIVE OF THE STUDY: We hypothesize that JOAB-40 is more potent than the lead compound E121 in reducing colitis severity in mice in part through inhibiting the release of TNFα and IL-1ß. METHODS: Colitis was induced by dextran sulfate sodium (DSS) administration using prophylactic and treatment approaches. The severity of the inflammation was determined by the gross (macroscopic) and histological (microscopic) assessments. The levels of TNFα, IL-1ß, and IL-10 release in response to lipopolysaccharide (LPS) stimulation from the adherent murine macrophage cell line J774.2 in vitro, and the circulating levels of TNFα in vivo was measured by ELISA-based technique. SIGNIFICANT FINDINGS FROM THE STUDY: E121 administration (1-60 mg/kg) in mice with established colitis (treatment approach) did not reduce colitis severity. On the other hand, JOAB-40 administration significantly reduced colitis severity in mice when administered using two approaches; a) prophylactic (given along colitis induction), and b) treatment (given after colitis was established) with doses as low as 10 mg/kg. The degree of inhibition of TNFα and IL-1ß (but not IL-10) release from J774.2 cell line in response to LPS stimulation was more potent with JOAB-40 than E121. This was also observed in vivo in regards to the circulating levels of TNFα. RELEVANT CONTRIBUTION TO KNOWLEDGE: Our results indicate that JOAB-40 is more potent than E121 in reducing colitis severity in mice and may be a promising future therapeutic target for the management of inflammatory bowel disease (IBD).

Onion Bulb Extract Downregulates EGFR/ERK1/2/AKT Signaling Pathway and Synergizes With Steroids to Inhibit Allergic Inflammation.

The treatment of allergic diseases, such as asthma, with both conventional and novel therapies presents a challenge both in terms of optimal effect and cost. On the other hand, traditional therapies utilizing natural products such as onion have been in use for centuries with demonstrated efficacy and safety but without much knowledge of their mechanims of action. In this study, we investigated if the anti-inflammatory effects of onion bulb extract (OBE) are mediated via the modulation of the EGFR/ERK1/2/AKT signaling pathway, and whether OBE can synergise with steroids to produce greater anti-inflammatory actions. Treatment with OBE inhibited the house dust mite (HDM)-induced increased phosphorylation of EGFR, ERK1/2 and AKT which resulted in the inhibition of HDM-induced increase in airway cellular influx, perivascular and peribronchial inflammation, goblet cell hyper/metaplasia, and also inhibited ex vivo eosinophil chemotaxis. Moreover, treatment with a combination of a low dose OBE and low dose dexamethasone resulted in a significant inhibition of the HDM-induced cellular influx, perivascular and peribronchial inflammation, goblet cell hyper/metaplasia, and increased the pERK1/2 levels, whereas neither treatment, when given alone, had any discernible effects. This study therefore shows that inhibition of the EGFR/ERK1/2/AKT-dependent signaling pathway is one of the key mechanisms by which OBE can mediate its anti-inflammatory effects in diseases such as asthma. Importantly, this study also demonstrates that combining OBE with steroids results in significantly enhanced anti-inflammatory effects. This action may have important potential implications for future asthma therapy.

Onion bulb extract can both reverse and prevent colitis in mice via inhibition of pro-inflammatory signaling molecules and neutrophil activity.

BACKGROUND: Onion is one of the most commonly used plants in the traditional medicine for the treatment of various diseases. We recently demonstrated the anti-inflammatory properties of onion bulb extract (OBE) in reducing colitis severity in mice when administered at the same time of colitis induction. However, whether onion can reverse established colitis or even prevent its development has not been investigated. HYPOTHESIS: To test 1. whether OBE can reduce colitis severity when given either before (preventative approach) or after (treatment approach) colitis induction and if so, 2. what are the mechanisms by which onion can achieve these effects. METHODS: Colitis was induced by dextran sulfate sodium (DSS) administration using treatment and preventative approaches. The severity of the inflammation was determined by the gross and histological assessments. The colonic level/activity of pro-inflammatory molecules and immune cell markers was assessed by immunofluorescence and western blotting analysis. In vitro neutrophil superoxide release and survival was assessed by chemilumenecense and Annexin-V/7AAD assays respectively. RESULTS: OBE treatment significantly reduced colitis severity in both approaches, the colonic expression/activity profile of pro-inflammatory molecules, inhibited WKYMVm-induced superoxide release, and increased spontaneous apoptosis of neutrophils in vitro. CONCLUSIONS: OBE can be used as an effective option in the prevention and/or the treatment of established colitis.

H2S donor GYY4137 ameliorates paclitaxel-induced neuropathic pain in mice.

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect that largely affects the patient's quality of life and may limit the use of the drug as a chemotherapeutic agent for treating metastatic breast cancer and other solid tumors. Recently, a putative role for the gaseous mediator hydrogen sulfide (H2S) in nociception modulation has been suggested. The aim of the present study was to investigate the potential efficacy of the slow release H2S donor GYY4137 to alleviate and prevent PINP. Female BALB/c mice that were intraperitoneally (i.p.) injected with paclitaxel (2 mg/kg) for 5 consecutive days developed thermal hyperalgesia, cold and mechanical allodynia and had reduced of H2S, generation in the spinal cord and paw skin. Treatment of mice with established thermal hyperalgesia with GYY4137 or the analgesic positive control drug gabapentin produced antihyperalgesic activities. The antihyperalgesic activity of GYY4137 was antagonized by the ATP sensitive potassium channels (KATP channels) blocker glibenclamide. Co-treatment with GYY4137 and paclitaxel prevented the paclitaxel-induced decrease in H2S, generation as well as the paclitaxel-induced thermal hyperalgesia, cold allodynia and mechanical allodynia. GYY4137 enhanced paclitaxel's anti-proliferative effects against the breast cancer cell line MCF-7. The present results suggest that GYY4137 alleviates paclitaxel-induced thermal hyperalgesia, via KATP channels. GYY4137 prevents PINP possibly by blocking the paclitaxel-induced reduction in the generation of H2S, in the tissues, while enhancing the anti-cancer activity of paclitaxel, and therefore warrants further research as a candidate for prevention of PINP in clinical settings.

Aquaporin expression in breast cancer and their involvement in bleb formation, cell motility and invasion in endocrine resistant variant cells.

Estrogen receptor (ER)­silenced breast cancer cell lines exhibit endocrine resistance and morphological changes from an epithelial to a mesenchymal phenotype. These cells also display increased motility and invasive properties that are further accentuated by exposure to an alkaline pH, exhibiting dynamic plasma membrane blebbing and cytoplasmic streaming. These latter morphological changes are hypothesized to involve substantial water movement across the plasma membrane, contributing to bleb formation; this may involve aquaporin channel proteins (AQPs). AQP 1, 3, 4 and 5 expression/localization was examined via reverse transcription­quantitative PCR, western blotting and confocal microscopy in endocrine­sensitive (YS1.2) and ­resistant (pII and MDA­MB­231) breast cancer cells, as well as normal breast epithelial cells (MCF10A). The effects of osmotic changes on bleb formation were examined via live cell imaging. AQP3 protein expression was knocked down by small interfering RNA (siRNA) transfection, and the effect of its reduced expression on bleb formation, cell motility and invasion were determined via immunofluorescence, scratch and Cultrex assays, respectively. Expression of the four AQPs varied across the different cell lines, and exhibited nuclear, cytoplasmic and membranous localization. Osmotic changes affected the formation of blebs. In pII cells exposed to alkaline pH, AQP3 was observed to be redistributed from the nucleus into the newly formed blebs. siRNA­mediated knockdown of AQP3 in pII cells significantly reduced cellular blebbing induced by alkaline pH, as well as motility and invasion. These data suggested that AQP3, and potentially other aquaporins, may participate in the processes leading to blebbing of endocrine­resistant cells which is proposed to be a mechanism that drives tumor metastasis.

Hypoxic environment may enhance migration/penetration of endocrine resistant MCF7- derived breast cancer cells through monolayers of other non-invasive cancer cells in vitro.

The response of cancer cells to hypoxic conditions found within the interior of a tumor mass is mediated through the hypoxia inducible factor (HIF) cascade and is thought to promote metastasis. However, given their distant proximity from blood vessels as compared to normoxic cells at the vascularised tumor periphery, it is uncertain if these cells can migrate through the tumor mass to gain access. Hypoxia was simulated by exposure to cobalt chloride or deferoxamine in normal (MCF10A) and cancerous [estrogen receptor (ER)-ve (pII), and ER +ve (YS1.2/ EII)] cells. In this report, HIF1α expression and localization was measured using western blotting, ELISA, and immunofluorescence, cell proliferation by MTT assay, motility and invasion by wound healing, live cell imaging, matrigel and co-culture in chambered slides. We found that the expression and nuclear translocation of HIF1α was significantly elevated by hypoxia, which inhibited cell proliferation, but significantly increased motility of pII cells and their penetration into and through a dense layer of adjacent EII cells, as well as their selective emergence out of a co-culture. These data suggest that endocrine resistant pII cancer cells, having undergone epithelial to mesenchymal transition are able to penetrate through other cell layers, with possible enhancement in response to hypoxia.

Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma.

The angiotensin-(1-7) [Ang-(1-7)]/MAS1 receptor signaling axis is a key endogenous anti-inflammatory signaling pathway. However, the mechanisms by which its mediates the anti-inflammatory effects are not completely understood. Using an allergic murine model of asthma, we investigated whether Ang-1(1-7)/MAS1 receptor axis a): inhibits allergic inflammation via modulation of Src-dependent transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling effectors such as ERK1/2, and b): directly inhibits neutrophil and/or eosinophil chemotaxis ex vivo. Ovalbumin (OVA)-induced allergic inflammation resulted in increased phosphorylation of Src kinase, EGFR, and ERK1/2. In addition, OVA challenge increased airway cellular influx, perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyperresponsiveness (AHR). Treatment with Ang-(1-7) inhibited phosphorylation of Src kinase, EGFR, ERK1/2, the cellular and histopathological changes and AHR. Ang-(1-7) treatment also inhibited neutrophil and eosinophil chemotaxis ex vivo. These changes were reversed following pre-treatment with A779. These data show that the anti-inflammatory actions of Ang-(1-7)/ MAS1 receptor axis are mediated, at least in part, via inhibition of Src-dependent transactivation of EGFR and downstream signaling molecules such as ERK1/2. This study therefore shows that inhibition of the Src/EGRF/ERK1/2 dependent signaling pathway is one of the mechanisms by which the Ang-(1-7)/ MAS1 receptor axis mediates it anti-inflammatory effects in diseases such as asthma.

Onion bulb extract reduces colitis severity in mice via modulation of colonic inflammatory pathways and the apoptotic machinery.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of nutraceutical-based products has increased in recent years due to their demonstrated efficacy and their good safety profile. Onion is one of the most commonly used plants in the traditional medicine for the management of various conditions including inflammatory and gastrointestinal diseases. However, little is known regarding the molecular mechanism of the anti-inflammatory effects of onion particularly in inflammatory bowel disease (IBD). AIM OF THE STUDY: To test the anti-inflammatory effects of onion bulb extract (OBE) in an IBD mouse model and the molecular mechanisms responsible for these effects such as modulation of the expression and/or the activity profile of various pro-inflammatory molecules. MATERIALS AND METHODS: Colitis was induced in mice by dextran sulfate sodium (DSS) daily administration for 5 days. Animals were sacrificed, colons were removed and the severity of the inflammation was determined by the gross and histological assessments. The colonic level/activity of various cytokines and chemokines were measured using proteome profiling-based assay, western blotting, and immunofluorescence techniques. RESULTS: DSS-induced colitis was significantly reduced by the daily OBE treatment and 5-aminosalicylic acid (5-ASA, positive control), particularly at 100-200 mg/kg doses, at both the gross and histological levels. OBE was also shown to reduce colonic expression and activity of several pro-inflammatory molecules and signaling pathways, such as mitogen activated protein kinase family, mammalian target of rapamycin, cyclooxygenase-2, and tissue inhibitors of metalloproteinases. In addition, OBE reduced the expression of interferon-γ, various C-C and C-X-C chemokines, and molecules involved in the apoptotic machinery such as cytochrome c, caspase-3 and -8, B-cell lymphoma-extra-large and -2. CONCLUSIONS: OBE showed anti-inflammatory actions in IBD mouse model, which is attributed, in part, to the modulation of the expression and the activity of important pro-inflammatory molecules and signaling pathways involved in the inflammatory response. These data suggest that OBE may be a promising lead in the therapeutic management of IBD.

COL­3 enhances the anti­proliferative and pro­apoptotic effects of paclitaxel in breast cancer cells.

Paclitaxel, a chemotherapeutic agent used in the treatment of breast cancer and other solid tumor types, including ovarian and lung, causes a dose­dependent neuropathic pain, which limits its use. Chemically modified tetracycline­3 (COL­3) has anticancer properties and was previously reported to inhibit neuroinflammation and protect against paclitaxel­induced neuropathic pain (PINP) in mice models. However, it is not known whether it affects the anticancer activities of paclitaxel. Thus, the aim of the present study was to evaluate the effect of COL­3 on the anticancer activity of paclitaxel on the breast cancer cell lines MCF­7 (estrogen receptor­positive), pII [estrogen receptor­negative (ER­ve)] and MDA­MB­231 (ER­ve). Cell proliferation, apoptosis and cell cycle stage were determined using an MTT assay, Annexin V/7­aminoactinomycin D and flow cytometry. The expression of various signaling molecules was determined with ELISA­based proteome profiling and western blotting. Additionally, the degree of cell invasion was determined with a Matrigel assay and caspase­3 activity was determined with a colorimetric assay. Treatment with paclitaxel or COL­3 alone inhibited cell proliferation in a concentration­dependent manner in all cell lines. The anti­proliferative effects of paclitaxel and COL­3 in combination varied from synergism against MDA­MB­231 and pII cells to notably additive and slight antagonism against MCF­7 cells. In the highly proliferative and invasive pII cells, the observed synergistic anti­proliferative effect was partially through the induction of apoptosis via modulation of caspase­3 levels and activity, and P70S6K phosphorylation, but not cell cycle arrest. COL­3 inhibited the invasion of pII cells in a concentration­dependent manner partially through inhibiting total matrix metalloproteinase activity. The combination regimen significantly inhibited the expression of two proteases, ADAM metallopeptidase with thrombospondin type 1 motif 1 and proteinase 3. In conclusion, the combination of paclitaxel and COL­3 indicated additive to synergistic anti­proliferative effects on breast cancer cells mediated partially via the induction of apoptosis. The combination regimen could further inhibit invasion and metastasis. Thus, COL­3 could be a beneficial adjunct to a paclitaxel­based anticancer regimen to improve therapeutic outcome and reduce the adverse effects of paclitaxel, primarily PINP.

ß-Caryophyllene, a CB2-Receptor-Selective Phytocannabinoid, Suppresses Mechanical Allodynia in a Mouse Model of Antiretroviral-Induced Neuropathic Pain.

Neuropathic pain associated with nucleoside reverse transcriptase inhibitors (NRTIs), therapeutic agents for human immunodeficiency virus (HIV), responds poorly to available drugs. Smoked cannabis was reported to relieve HIV-associated neuropathic pain in clinical trials. Some constituents of cannabis (Cannabis sativa) activate cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors. However, activation of the CB1 receptor is associated with side effects such as psychosis and physical dependence. Therefore, we investigated the effect of ß-caryophyllene (BCP), a CB2-selective phytocannabinoid, in a model of NRTI-induced neuropathic pain. Female BALB/c mice treated with 2'-3'-dideoxycytidine (ddC, zalcitabine), a NRTI, for 5 days developed mechanical allodynia, which was prevented by cotreatment with BCP, minocycline or pentoxifylline. A CB2 receptor antagonist (AM 630), but not a CB1 receptor antagonist (AM 251), antagonized BCP attenuation of established ddC-induced mechanical allodynia. ß-Caryophyllene prevented the ddC-induced increase in cytokine (interleukin 1 beta, tumor necrosis factor alpha and interferon gamma) transcripts in the paw skin and brain, as well as the phosphorylation level of Erk1/2 in the brain. In conclusion, BCP prevents NRTI-induced mechanical allodynia, possibly via reducing the inflammatory response, and attenuates mechanical allodynia through CB2 receptor activation. Therefore, BCP could be useful for prevention and treatment of antiretroviral-induced neuropathic pain.

Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells.

BACKGROUND: The Na+/K+-ATPase (NKP) is an important ion transporter also involved in signal transduction. Its expression profile is altered in various tumours including that of the breast. We studied the effect of inhibiting NKP activity in non-tumorigenic breast cell line and in estrogen receptor positive and negative breast cancer cells. METHODS: Expression and localization of NKP and downstream signaling molecules were determined by RT-PCR, western blotting and immunofluorescence. Cell proliferation, apoptosis and cell cycle stage were determined using MTT, annexin V and flow cytometry. Cell motility and invasion were determined using wound healing and matrigel assays. Total matrix metalloproteinase (MMP) was determined by a fluorescence-based assay. RESULTS: NKP was mainly localized on the cell membrane. Its baseline expression and activity were enhanced in breast cancer compared to the non-tumorigenic breast cell line. Ouabain and 3,4,5,6-tetrahydroxyxanthone (TTX) treatment significantly inhibited NKP activity, which significantly reduced cell proliferation, motility, invasion and pH-induced membrane blebbing. EGF stimulation induced internalization of NKP from the cell membrane to the cytoplasm. Ouabain inhibited EGF-induced phosphorylation of Rac/cdc42, profillin, ERK1/2 and P70S6K. CONCLUSIONS: The NKP may offer a novel therapeutic target in breast cancer patients who have developed metastasis, aiming to improve therapeutic outcomes and enhance survival rate.

Src-dependent EGFR transactivation regulates lung inflammation via downstream signaling involving ERK1/2, PI3Kδ/Akt and NFκB induction in a murine asthma model.

The molecular mechanisms underlying asthma pathogenesis are poorly characterized. In this study, we investigated (1) whether Src mediates epidermal growth factor receptor (EGFR) transactivation; (2) if ERK1/2, PI3Kδ/Akt and NF-κB are signaling effectors downstream of Src/EGFR activation; and (3) if upstream inhibition of Src/EGFR is more effective in downregulating the allergic inflammation than selective inhibition of downstream signaling pathways. Allergic inflammation resulted in increased phosphorylation of EGFR, Akt, ERK1/2 and IκB in the lung tissues from ovalbumin (OVA)-challenged BALB/c mice. Treatment with inhibitors of Src (SU6656) or EGFR (AG1478) reduced EGFR phosphorylation and downstream signaling which resulted in the inhibition of the OVA-induced inflammatory cell influx in bronchoalveolar lavage fluid (BALF), perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyper-responsiveness. Treatment with pathway-selective inhibitors for ERK1/2 (PD89059) and PI3Kδ/Akt (IC-87114) respectively, or an inhibitor of NF-κB (BAY11-7085) also reduced the OVA-induced asthmatic phenotype but to a lesser extent compared to Src/EGFR inhibition. Thus, Src via EGFR transactivation and subsequent downstream activation of multiple pathways regulates the allergic airway inflammatory response. Furthermore, a broader upstream inhibition of Src/EGFR offers an attractive therapeutic alternative in the treatment of asthma relative to selectively targeting the individual downstream signaling effectors.