Protection against LPS-induced cartilage inflammation and degradation provided by a biological extract of Mentha spicata

<p>Abstract</p> <p>Background</p> <p>A variety of mint [<it>Mentha spicata</it>] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. RA has demonstrated significant anti-inflammatory activity <it>in vitro </it>and in small rodents; thus it was hypothesized that this plant would demonstrate significant anti-inflammatory activity <it>in vitro</it>. The objectives of this study were: a) to develop an <it>in vitro </it>extraction procedure which mimics digestion and hepatic metabolism, b) to compare anti-inflammatory properties of High-Rosmarinic-Acid <it>Mentha spicata </it>(HRAM) with wild-type control <it>M. spicata </it>(CM), and c) to quantify the relative contributions of RA and three of its hepatic metabolites [ferulic acid (FA), caffeic acid (CA), coumaric acid (CO)] to anti-inflammatory activity of HRAM.</p> <p>Methods</p> <p>HRAM and CM were incubated in simulated gastric and intestinal fluid, liver microsomes (from male rat) and NADPH. Concentrations of RA, CA, CO, and FA in simulated digest of HRAM (HRAM_sim) and CM (CM_sim) were determined (HPLC) and compared with concentrations in aqueous extracts of HRAM and CM. Cartilage explants (porcine) were cultured with LPS (0 or 3 μg/mL) and test article [HRAM_sim(0, 8, 40, 80, 240, or 400 μg/mL), or CM_sim(0, 1, 5 or 10 mg/mL), or RA (0.640 μg/mL), or CA (0.384 μg/mL), or CO (0.057 μg/mL) or FA (0.038 μg/mL)] for 96 h. Media samples were analyzed for prostaglandin E₂(PGE₂), interleukin 1β (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).</p> <p>Results</p> <p>RA concentration of HRAM_simand CM_simwas 49.3 and 0.4 μg/mL, respectively. CA, FA and CO were identified in HRAM_simbut not in aqueous extract of HRAM. HRAM_sim(≥ 8 μg/mL) inhibited LPS-induced PGE₂and NO; HRAM_sim(≥ 80 μg/mL) inhibited LPS-induced GAG release. RA inhibited LPS-induced GAG release. No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified.</p> <p>Conclusions</p> <p>Our biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAM_simis an effective inhibitor of LPS-induced inflammation in cartilage explants, and effects are primarily independent of RA. Further research is needed to identify bioactive phytochemical(s) in HRAM_sim.</p

Estrogen Receptor-Alpha 36 Mediates Mitogenic Antiestrogen Signaling in ER-Negative Breast Cancer Cells

It is prevailingly thought that the antiestrogens tamoxifen and ICI 182, 780 are competitive antagonists of the estrogen-binding site of the estrogen receptor-alpha (ER-α). However, a plethora of evidence demonstrated both antiestrogens exhibit agonist activities in different systems such as activation of the membrane-initiated signaling pathways. The mechanisms by which antiestrogens mediate estrogen-like activities have not been fully established. Previously, a variant of ER-α, EP–α36, has been cloned and showed to mediate membrane-initiated estrogen and antiestrogen signaling in cells only expressing ER-α36. Here, we investigated the molecular mechanisms underlying the antiestrogen signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of endogenous ER-α36. We found that the effects of both 4-hydoxytamoxifen (4-OHT) and ICI 182, 780 (ICI) exhibited a non-monotonic, or biphasic dose response curve; antiestrogens at low concentrations, elicited a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, antiestrogens inhibited cell growth. Antiestrogens at l nM induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at 5 µM induced Src-Y527 phosphorylation that inactivates Src. Antiestrogens at 1 nM also induced phosphorylation of the MAPK/ERK and activated the Cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at 5 µM. Knock-down of ER-α36 abrogated the biphasic antiestrogen signaling in these cells. Our results thus indicated that ER-α36 mediates biphasic antiestrogen signaling in the ER-negative breast cancer cells and Src functions as a switch of antiestrogen signaling dependent on concentrations of antiestrogens through the EGFR/STAT5 pathway

Tamoxifen stimulates arachidonic acid release from rat liver cells by an estrogen receptor-independent, non-genomic mechanism

BACKGROUND: Tamoxifen is widely prescribed for the treatment of breast cancer. Its success has been attributed to the modulation of the estrogen receptor. I have previously proposed that the release of arachidonic acid from cells may also mediate cancer prevention. METHODS: Rat liver cells were radiolabelled with arachidonic acid. The release of [(3)H] arachidonic acid after various times of incubation of the cells with tamoxifen was measured. RESULTS: Tamoxifen, at micromolar concentrations, stimulates arachidonic acid release. The stimulation is rapid and is not affected by pre-incubation of the cells with actinomycin or the estrogen antagonist ICI-182,780. CONCLUSIONS: The stimulation of AA release by tamoxifen is not mediated by estrogen receptor occupancy and is non-genomic

Tamoxifen and the Rafoxifene analog LY117018: their effects on arachidonic acid release from cells in culture and on prostaglandin I(2 )production by rat liver cells

BACKGROUND: Tamoxifen is being used successfully to treat breast cancer. However, tamoxifen also increases the risk of developing endometrial cancer in postmenopausal women. Raloxifene also decreases breast cancer in women at high risk and may have a lower risk at developing cancer of the uterus. Tamoxifen has been shown to stimulate arachidonic acid release from rat liver cells. I have postulated that arachidonic acid release from cells may be associated with cancer chemoprevention. METHODS: Rat liver, rat glial, human colon carcinoma and human breast carcinoma cells were labelled with [(3)H] arachidonic acid. The release of the radiolabel from these cells during incubation with tamoxifen and the raloxifene analog LY117018 was measured. The prostaglandin I(2 )produced during incubation of the rat liver cells with μM concentrations of tamoxifen and the raloxifene analog was quantitatively estimated. RESULTS: Tamoxifen is about 5 times more effective than LY117018 at releasing arachidonic acid from all the cells tested. In rat liver cells only tamoxifen stimulates basal prostaglandin I(2 )production and that induced by lactacystin and 12-O-tetradecanoyl-phorbol-13-acetate. LY117018, however, blocks the tamoxifen stimulated prostaglandin production. The stimulated prostaglandin I(2 )production is rapid and not affected either by preincubation of the cells with actinomycin or by incubation with the estrogen antagonist ICI-182,780. CONCLUSIONS: Tamoxifen and the raloxifene analog, LY117018, may prevent estrogen-independent as well as estrogen-dependent breast cancer by stimulating phospholipase activity and initiating arachidonic acid release. The release of arachidonic acid and/or molecular reactions that accompany that release may initiate pathways that prevent tumor growth. Oxygenation of the intracellularly released arachidonic acid and its metabolic products may mediate some of the pharmacological actions of tamoxifen and raloxifene

p66 Shc and tyrosine-phosphorylated Shc in primary breast tumors identify patients likely to relapse despite tamoxifen therapy

INTRODUCTION: Shc adapter proteins are secondary messenger proteins involved in various cellular pathways, including those mediating receptor tyrosine kinase signaling and apoptosis in response to stress. We have previously reported that high levels of tyrosine-phosphorylated Shc (PY-Shc) and low levels of its inhibitory p66 Shc isoform are strongly prognostic for identifying both early node-negative and more advanced, node-positive, primary breast cancers with high risk for recurrence. Because aberrant activation of tyrosine kinases upstream of Shc signaling proteins has been implicated in resistance to tamoxifen – the most widely prescribed drug for treatment of estrogen receptor-positive breast cancer – we hypothesized that Shc isoforms may identify patients at increased risk of relapsing despite tamoxifen treatment. METHODS: Immunohistochemical analyses of PY-Shc and p66 Shc were performed on archival primary breast cancer tumors from a population-based cohort (60 patients, 9 relapses) and, for validation, an independent external cohort (31 patients, 13 relapses) in which all patients received tamoxifen as a sole systemic adjuvant prior to relapse. RESULTS: By univariate and multivariate analyses, the Shc proteins were very strong and independent predictors of treatment failure in both the population-based cohort (interquartile hazard ratio = 8.3, 95% confidence interval [CI] 1.8 to 38, P = 0.007) and the validating cohort (interquartile relative risk = 12.1, 95% CI 1.7 to 86, P = 0.013). CONCLUSION: These results suggest that the levels of PY-Shc and p66 Shc proteins in primary tumors identify patients at high risk for relapsing despite treatment with tamoxifen and therefore with further validation may be useful in guiding clinicians to select alternative adjuvant treatment strategies

Tamoxifen enhances the cytotoxic effects of nelfinavir in breast cancer cells

Introduction: The HIV protease inhibitor nelfinavir is currently under investigation as a new anti-cancer drug. Several studies have shown that nelfinavir induces cell cycle arrest, endoplasmic reticulum stress, autophagy, and apoptosis in cancer cells. In the present article, the effect of nelfinavir on human breast cancer cells is examined and potential combination treatments are investigated. Methods: The effects of nelfinavir and tamoxifen on the human breast cancer cell lines MCF7, T47 D, MDA-MB-453, and MDA-MB-435 were tested by analysing their influence on cell viability (via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay), apoptosis (annexin binding, poly(ADP-ribose) polymerase cleavage), autophagy (autophagy marker light chain 3B expression), endoplasmic reticulum stress (binding protein and activating transcription factor 3 expression), and the occurrence of oxidative stress (intracellular glutathione level). Results: Nelfinavir induced apoptosis in all four breast cancer cell lines tested, although the extent of autophagy and endoplasmic reticulum stress varied among the cell lines. The concentration of nelfinavir needed for an efficient induction of apoptosis in breast cancer cells could be reduced from 15 mu g/ml to 6 mu g/ml when combined with tamoxifen. At a concentration of 6 mu g/ml, tamoxifen substantially enhanced the endoplasmic reticulum stress reaction in those cell lines that responded to nelfinavir with binding protein (BiP) upregulation (MCF7, T47D), and enhanced autophagy in cell lines that responded to nelfinavir treatment with autophagy marker light chain 3B upregulation (MDA-MB-453). Although tamoxifen has been described to be able to induce oxidative stress at concentrations similar to those applied in this study (6 mu g/ml), we observed that nelfinavir but not tamoxifen reduced the intracellular glutathione level of breast cancer cells within hours of application by up to 32%, suggesting the induction of oxidative stress was an early event and an additional cause of the apoptosis induced by nelfinavir. Conclusions: The results demonstrate that nelfinavir may be an effective drug against breast cancer and could be combined with tamoxifen to enhance its efficacy against breast cancer cells. Moreover, the cytotoxic effect of a tamoxifen and nelfinavir combination was independent of the oestrogen receptor status of the analysed breast cancer cells, suggesting a potential benefit of a combination of these two drugs even in patients with no hormone-responsive tumours. We therefore recommend that clinical studies on nelfinavir with breast cancer patients should include this drug combination to analyse the therapeutic efficacy as well as the safety and tolerability of this potential treatment option

Tamoxifen mechanically deactivates hepatic stellate cells via the G protein-coupled estrogen receptor

Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.Peer reviewe

CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways

BACKGROUND: CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. METHODS: The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. RESULTS: Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. CONCLUSION: These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively

B cell and/or autoantibody deficiency do not prevent neuropsychiatric disease in murine systemic lupus erythematosus

Background: Neuropsychiatric lupus (NPSLE) can be one of the earliest clinical manifestations in human lupus. However, its mechanisms are not fully understood. In lupus, a compromised blood-brain barrier may allow for the passage of circulating autoantibodies into the brain, where they can induce neuropsychiatric abnormalities including depression-like behavior and cognitive abnormalities. The purpose of this study was to determine the role of B cells and/or autoantibodies in the pathogenesis of murine NPSLE. Methods: We evaluated neuropsychiatric manifestations, brain pathology, and cytokine expression in constitutively (JhD/MRL/lpr) and conditionally (hCD20-DTA/MRL/lpr, inducible by tamoxifen) B cell-depleted mice as compared to MRL/lpr lupus mice. Results: We found that autoantibody levels were negligible (JhD/MRL/lpr) or significantly reduced (hCD20-DTA/MRL/lpr) in the serum and cerebrospinal fluid, respectively. Nevertheless, both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice showed profound depression-like behavior, which was no different from MRL/lpr mice. Cognitive deficits were also observed in both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice, similar to those exhibited by MRL/lpr mice. Furthermore, although some differences were dependent on the timing of depletion, central features of NPSLE in the MRL/lpr strain including increased blood-brain barrier permeability, brain cell apoptosis, and upregulated cytokine expression persisted in B cell-deficient and B cell-depleted mice. Conclusions: Our study surprisingly found that B cells and/or autoantibodies are not required for key features of neuropsychiatric disease in murine NPSLE