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Abstract and Figures

Opioids are widely administered to alleviate pain, including chronic pain in advanced cancer patients. Among opioids, morphine is one of the most clinically effective drugs for the palliative management of severe pain. In the last few decades, there has been a debate around the possible influence of opioids such as morphine on tumour growth and metastasis. Whilst several in vitro and in vivo studies suggest the possible modulatory effects of morphine on tumour cells, little is known about the impact of this analgesic drug on other mediators such as matrix metalloproteinases (MMPs) that play a key role in the control of cancer cell invasion and metastasis. MMP-9 has been considered as one of the principal mediators in regulation of not only the initial steps of cancer but during the invasion and spreading of cancer cells to distant organs. Herein, current studies regarding the direct and indirect effects of morphine on regulation of MMP-9 production are discussed. In addition, drawing from previous in vivo and in vitro studies on morphine action in regulating MMP-9 production, the potential roles of several underlying factors are summarised, including nuclear factor kappa-B and intracellular molecules such as nitric oxide.
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Opioids and matrix metalloproteinases: the influence of morphine
on MMP-9 production and cancer progression
Samira Khabbazi
&Mohammadhossein Hassanshahi
&Alireza Hassanshahi
&Yaser Peymanfar
&Yu-Wen Su
Cory J. Xian
Received: 7 November 2018 /Accepted: 9 January 2019 /Published online: 17 January 2019
#Springer-Verlag GmbH Germany, part of Springer Nature 2019
Opioids are widely administered to alleviate pain, including chronic pain in advanced cancer patients. Among opioids, morphine
is one of the most clinically effective drugs for the palliative management of severe pain. In the last few decades, there has been a
debate around the possible influence of opioids such as morphine on tumour growth and metastasis. Whilst several in vitro and
in vivo studies suggest the possible modulatory effects of morphine on tumour cells, little is known about the impact of this
analgesic drug on other mediators such as matrix metalloproteinases (MMPs) that play a key role in the control of cancer cell
invasion and metastasis. MMP-9 has been considered as one of the principal mediators in regulation of not only the initial steps of
cancer but during the invasion and spreading of cancer cells to distant organs. Herein, current studies regarding the direct and
indirect effects of morphine on regulation of MMP-9 production are discussed. In addition, drawing from previous in vivo and
in vitro studies on morphine action in regulating MMP-9 production, the potential roles of several underlying factors are
summarised, including nuclear factor kappa-B and intracellular molecules such as nitric oxide.
Keywords Opioids .Matrix metalloproteinases .MMP-9 .Morphine .NF-κB.Nitric oxide
AC Adenylyl cyclase
AP-1 Activator protein 1
BMM Bonemarrow-derivedmacrophages
CREB cAMP responsive element binding
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
cNOS Constitutive nitric oxide synthase
DRG Dorsal root ganglia
eNOS Endothelial nitric oxide synthase
ECM Extracellular matrix
GPCR G protein-coupled receptor
Gβγ Gbeta-gamma
iNOS Inducible nitric oxide synthase
IL-1βInterleukin 1 beta
IL-4 Interleukin 4
IL-6 Interleukin 6
IκB Nuclear factor-κB inhibitor
LPS Lipopolysaccharide
MMPs Matrix metalloproteinases
MMP-9 Matrix metalloproteinase 9
NF-κB Nuclear factor kappa B
NO Nitric oxide
NOS Nitric oxide synthase
nNOS Neuronal nitric oxide synthase
PKCδProtein kinase C-delta
PKA Protein kinase A
PLC Phospholipase C
PKC Protein kinase C
PI3K Phosphoinositide 3-kinase
PKB Protein kinase B
PEA3 Polyoma enhancer activator 3
Samira Khabbazi and Mohammadhossein Hassanshahi contributed
equally to this work.
*Cory J. Xian
School of Medical Science, The South Australian Health and
Medical Research Institute, The University of Adelaide,
Adelaide, SA 5001, Australia
School of Pharmacy and Medical Sciences, and University of South
Australia Cancer Research Institute, University of South Australia,
GPO Box 2471, Adelaide SA 5001, Australia
Department of Genetics, Faculty of Basic Sciences, Islamic Azad
University, Shahrekord, Iran
Naunyn-Schmiedeberg's Archives of Pharmacology (2019) 392:123133
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Thus, direct cytotoxic effects of local and systemic anesthetic agents such as lidocaine, ropivacaine, and propofol have been described suggesting anticancer effects (5,6). However, morphine reportedly activates matrix metalloproteinases that then would promote the dissemination of tumors (7). Moreover, perioperative immunomodulatory factors such as undernutrition, anemia, neoadjuvant chemotherapy, as well as the concomitant use of mechanistically distinct anesthetic agents during oncosurgery, render the translation of partially promising preclinical results into clinical practice difficult. ...
... Until now, opioids have been the most commonly used analgesics for controlling acute pain. However, preclinical data indicate that opioids mediate pro-tumorigenic effects via the activation of matrix metalloproteinases and oncogenes like c-Myc as well as via an increase in DNA methylation (52)(53)(54). Of note, DNA methylation leads to the expression of the mu opioid receptor and predicts the response to endogenous endorphins and opioid analgesics (55). ...
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Defective silencing of tumor suppressor genes through epigenetic alterations contributes to oncogenesis by perturbing cell cycle regulation, DNA repair or cell death mechanisms. Reversal of such epigenetic changes including DNA hypermethylation provides a promising anticancer strategy. Until now, the nucleoside derivatives 5-azacytidine and decitabine are the sole DNA methyltransferase (DNMT) inhibitors approved by the FDA for the treatment of specific hematological cancers. Nevertheless, due to their nucleoside structure, these inhibitors directly incorporate into DNA, which leads to severe side effects and compromises genomic stability. Much emphasis has been placed on the development of less toxic epigenetic modifiers. Recently, several preclinical studies demonstrated the potent epigenetic effects of local anesthetics, which are routinely used during primary tumor resection to relief surgical pain. These non-nucleoside molecules inhibit DNMT activity, affect the expression of micro-RNAs and repress histone acetylation, thus exerting cytotoxic effects on malignant cells. The in-depth mechanistic comprehension of these epigenetic effects might promote the use of local anesthetics as anticancer drugs.
... [20][21][22] In this context, it would not be surprising that surgical interventions with their components of mental, nutritional, mechanic and inflammatory distress as well as the associated polypharmacy (including the administration of antibiotics, benzodiazepines, nonsteroidal anti-inflammatory drugs, general and local anesthetics, vasoactive amines, glucocorticoids…) would affect anticancer immunosurveillance, as suggested by statistically significant epidemiological associations. [23][24][25][26][27][28][29][30][31][32][33][34] Here, we investigated the possible impact of several local anesthetics that are currently used in the clinics on cancer immunosurveillance in preclinical models. For this, we determined the effects of six local anesthetics on immunogenic cancer cell stress pathways and determined their possible antineoplastic effects by injecting them into established tumors. ...
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Background: Retrospective clinical trials reported a reduced local relapse rate, as well as improved overall survival after injection of local anesthetics during cancer surgery. Here, we investigated the anticancer effects of six local anesthetics used in clinical practice. Results: In vitro, local anesthetics induced signs of cancer cell stress including inhibition of oxidative phosphorylation, and induction of autophagy as well as endoplasmic reticulum (ER) stress characterized by the splicing of X-box binding protein 1 (XBP1s) mRNA, cleavage of activating transcription factor 6 (ATF6), phosphorylation of eIF2α and subsequent upregulation of activating transcription factor 4 (ATF4). Both eIF2α phosphorylation and autophagy required the ER stress-relevant eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, best known as PERK). Local anesthetics also activated two hallmarks of immunogenic cell death, namely, the release of ATP and high-mobility group box 1 protein (HMGB1), yet failed to cause the translocation of calreticulin (CALR) from the ER to the plasma membrane. In vivo, locally injected anesthetics decreased tumor growth and improved survival in several models of tumors established in immunocompetent mice. Systemic immunotherapy with PD-1 blockade or intratumoral injection of recombinant CALR protein, increased the antitumor effects of local anesthetics. Local anesthetics failed to induce antitumor effects in immunodeficient mice or against cancers unable to activate ER stress or autophagy due to the knockout of EIF2AK3/PERK or ATG5, respectively. Uncoupling agents that inhibit oxidative phosphorylation and induce autophagy and ER stress mimicked the immune-dependent antitumor effects of local anesthetics. Conclusion: Altogether, these results indicate that local anesthetics induce a therapeutically relevant pattern of immunogenic stress responses in cancer cells.
... Matrix metalloproteinase 2 (MMP-2) and MMP-9 production can be time-and concentrationdependently inhibited by morphine in breast cancer cells [72]. MMPs participate in matrix degradation, embryogenesis, angiogenesis, wound healing, and tumor progression, promoting tissue remodeling [66,73,74]. Compared with the control group using normal saline (NS), mice injected intraperitoneally with morphine had reduced MMP-9 concentrations and inhibited TIMP-1 and TIMP-3/4, reducing the invasion and chemotactic potential of endothelial cells [75], which was consistent with the research of Afsharimani [76]. ...
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Purpose of Review Opioids are still the most effective and widely used treatments for acute and chronic pain in cancer patients. This review focuses on the impact of opioids and mu-opioid receptors (MOR) on tumor progression and providing new ideas for targeting the MOR in cancer treatment. Recent Findings Studies estimated that opioids facilitate tumor progression and are related to the worse prognosis in cancer patients. As the primary receptor of opioids, MOR is involved in the regulation of malignant transformation of tumors and participating in proliferation, invasion, metastasis, and angiogenesis. Summary MOR may be a new molecular marker of malignant tumors and thus become a new target for cancer therapy, which may be beneficial to the outcomes of cancer patients.
... Similarly, morphine precondition was found to prevent the deleterious effects of LPS on microglia cells (Gwak et al., 2010). Previous data could be explained by a mu Y. L. Chen et al., 2006;Eisenstein, 2019;Franchi et al., 2012;Khabbazi et al., 2019;P. Zhang et al., 2020). ...
Inflammatory pathway and disruption in glutamate homeostasis join at the level of the glia, resulting in various neurological disorders. In vitro studies have provided evidence that membrane proteins connexions (Cxs) are involved in glutamate release, meanwhile, excitatory amino‐acid transporters (EAATs) are crucial for glutamate reuptake (clearance). Moreover, pannexin‐1 (Panx‐1) activation is more detrimental to neurons. Their expression patterns during inflammation and the impacts of IκB kinase (IKK) inhibition, morphine, and galantamine on the inflammatory‐associated glutamate imbalance remain elusive. To investigate this, rats were injected with saline or lipopolysaccharide. Thereafter, vehicles, morphine, galantamine, and BAY‐117082 were administered in different groups of animals. Subsequently, electroencephalography, enzyme‐linked immunosorbent assay, western blot, and histopathological examinations were carried out and various indicators of inflammation and glutamate level were determined. Parallel analysis of Cxs, Panx‐1, and EAAts in the brain was performed. Our findings strengthen the concept that unregulated expressions of Cxs, Panx‐1, and EAATs contribute to glutamate accumulation and neuronal cell loss. Nuclear factor‐kB (NF‐κB) pathway can significantly contribute to glutamate homeostasis via modulating Cxs, Panx‐1, and EAATs expressions. BAY‐117082, via inhibition of IkK, promoted the anti‐inflammatory effects of morphine as well as galantamine. We concluded that NF‐κB is an important component of reshaping the expressions of Cxs, panx‐1, and EAATs and the development of glutamate‐induced neuronal degeneration.
... Therefore, these MMPs expression levels effectively reflect the aggressiveness of cancer cells and are related to poor prognosis in various cancers [25,26]. MMP9 is a critical member of MMP family which plays a key role in degrading basement membrane and has been proved to enhance tumor invasion and metastasis in many different types of cancer [27,28]. Some studies also reported that the invasive and metastatic abilities of breast cancer cells were reduced by inhibition of MMP9 [13,29]. ...
Natriuretic peptide receptor A (NPRA), one of the natriuretic peptide receptors, plays important roles in circulatory system. Recently some studies showed that NPRA was involved in tumorigenesis, however, its role in the development of breast cancer remains unclear. In this study, we observed that NPRA expression was upregulated in breast cancer tissues and NPRA high expression was associated with poor clinicopathological features. In addition, we found that patients with high NPRA expression had a worse 5-year survival and NPRA was an independent factor for predicting the prognosis of breast cancer patients. Knocking down NPRA expression reduced the proliferation, migration and invasion of breast cancer cells. Overexpressing NPRA was able to enhance the malignant behaviors of breast cancer cells. Furthermore, NPRA promoted the invasive phenotype through upregulating matrix metalloproteinase-9 (MMP9). Mechanistically, NPRA increased MMP9 expression through activating STAT3. We identified that NPRA might serve as a prognostic marker and p-STAT3 and MMP9 could be a potential target of NPRA in breast cancer patients.
Résumé La chirurgie est le traitement le plus fréquent pour les tumeurs solides. Présentant des spécificités et des techniques propres à chaque organe, elle est le plus souvent associée à des thérapies conventionnelles ou ciblées aux effets indésirables conséquents. Les patients qui en bénéficient présentent eux-aussi des caractéristiques singulières, telles que des facteurs de risque et des addictions à maîtriser, le besoin d’une préhabilitation, un accès aux voies aériennes complexe ou encore une douleur difficile à contrôler. Bien que nécessaire à la rémission ou à la reconstruction de séquelles esthétiques, la chirurgie est paradoxalement immunodépressive. L’inflammation, la douleur et le stress glucocorticoïde peropératoire affectent la réponse immune antitumorale et la manipulation directe de la tumeur favorise la dissémination des cellules cancéreuses. Il semble nécessaire de favoriser une prise en charge périopératoire qui pourrait renforcer les effecteurs du système immunitaire et diminuer les cellules tumorales résiduelles responsables des récidives métastatiques (immunonutrition, chirurgie mini-invasive, contrôle de la douleur…). La publication de travaux précliniques démontrant les mécanismes par lesquels les agents d’anesthésie pourraient avoir une activité immunomodulatrice laisse suggérer que le choix des produits d’anesthésie pourrait jouer un rôle sur le pronostic oncologique. Connaître les caractéristiques des différents traitements médicaux et chirurgicaux anti-cancéreux, maîtriser les facteurs périopératoires immunosuppresseurs et privilégier les produits d’anesthésie anti-tumoraux sont des pratiques encouragées afin d’optimiser la rémission et éviter les rechutes. Le but de cette mise au point est de donner un aperçu de ces particularités qui pourraient utilement faire l’objet d’un enseignement spécifique en onco-anesthésie inexistant à ce jour.
Opioids have been used for medicinal purposes as an analgesic and recreational purposes as a euphorigenic throughout human history. Cancer patients are often treated with different doses of opioids concurrently with anti-cancer drugs for pain relief without exhibiting excessive adverse effects. The intersection of the biology of pain, opioid therapy, and disease progression represents the crux of the matters and is of potentially great importance in cancer care. For more than 20 years, multiple investigations have focused on the stimulatory effects of opioids on cancer cell growth, while in-depth studies on the inhibitory effects on cancer cell growth development have usually been neglected. This paper reviews the evidence regarding opioid therapies and their anti-cancer effects on various malignancies. Likewise, we have a glimpse into the molecular mechanisms necessary for pinpointing their positive or negative impacts on malignancies to raise awareness and stimulate more excellent dialogue regarding their carcinogenic/anticarcinogenic roles.
Angiogenesis plays a vital role in tumor progression and metastasis. To better understand the role of anesthesia in tumor biology, we previously reported that bupivacaine displayed the inhibitory effects in endothelial cells. In this work, we demonstrated that fentanyl, an opioid medication commonly used in cancer patients, stimulated tumor angiogenesis. We found that fentanyl at nanomolar concentrations significantly stimulated capillary network formation of human lung tumor-associated endothelial cell (HLT-EC) in a similar manner as vascular endothelial growth factor (VEGF), and furthermore that the stimulatory effect of fentanyl was mainly involved in early stage of HLT-EC vascular structure assembly. Particularly, fentanyl significantly increased HLT-EC growth and migration. Fentanyl also protected HLT-EC from apoptosis induced by growth factor withdrawal. In contrast, the same concentrations of fentanyl did not affect human lung cancer cell growth and survival. Fentanyl stimulated migration of some but not all tested human lung cancer cells. Mechanism analysis suggested that fentanyl activates multiple pro-angiogenic signaling pathways, including VEGFR2/FAK/PI3K/Akt and small GTPases. Our work systematically demonstrates that fentanyl stimulates tumor angiogenesis via activating multiple pro-angiogenic signaling pathways. Our findings highlight the potential adverse effect of fentanyl in cancer patients.
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Opinion statement: So far, opioids have been successfully used to reduce cancer pain in patients in order to improve their quality of life. However, the use of opioids leads to numerous side effects such as constipation, drowsiness, nausea, itching, increased sweating and hormonal changes. In this review, we described the action of opioids in several molecular pathways significant for maintenance of the intestinal homeostasis including the impact on the intestinal epithelium integrity, changes in microbiome composition, modulation of the immune system or induction of apoptosis and inhibition of angiogenesis. We summed up the role of individual opioids in the processes involved in the growth and development of cancer and elucidated if targeting opioid receptors may constitute novel therapeutic option in colon cancer.
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Lymphatic metastasis is facilitated by lymphangiogenic growth factor VEGFC that is secreted by some primary tumors. We previously identified tumor necrosis factor superfamily 15 (TNFSF15), a blood vascular endothelium‐derived cytokine, in lymphatic endothelial cells, as a key molecular modulator during lymphangiogenesis. However, the effect of TNFSF15 on tumor lymphatic metastasis and the underlying molecular mechanisms remain unclear. We report here that TNFSF15, which is known to inhibit primary tumor growth via suppressing angiogenesis, can promote lymphatic metastasis through facilitating lymphangiogenesis in tumor. Mice bearing tumors induced by A549 cells stably overexpressing TNFSF15 exhibited a significant increase in densities of lymphatic vessels and a marked enhancement of A549 tumor cells in newly formed lymphatic vessels in the primary tumors as well as in lymph nodes. Treatment of A549 cells with TNFSF15 results in up‐regulation of VEGFC expression, which can be inhibited by siRNA gene silencing of death domain‐containing receptor‐3 (DR3), a cell surface receptor for TNFSF15. Additionally, TNFSF15/DR3 signaling pathways in A549 cells include activation of NF‐κB during tumor lymphangiogenesis. Our data indicate that TNFSF15, a cytokine mainly produced by blood endothelial cells, facilitates tumor lymphangiogenesis by up‐regulating VEGFC expression in A549 cells, contributing to lymphatic metastasis in tumor‐bearing mice. This finding also suggests that TNFSF15 may be of potential as an indicator for prognosis evaluation. This article is protected by copyright. All rights reserved.
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Opioids modulate the tumor microenvironment with potential functional consequences for tumor growth and metastasis. We evaluated the effects of morphine administration on the circulating proteolytic profile of tumor-free mice. Serum from morphine-treated (1 or 10 mg/kg, i.p. every 12 h) or saline-treated mice was collected at different time points and tested ex vivo in endothelial, lymphatic endothelial, and breast cancer cell migration assays. Serum from mice that were treated with 10 mg/kg morphine for 3 d displayed reduced chemotactic potential for endothelial and breast cancer cells, and elicited reduced cancer cell invasion through reconstituted basement membrane compared with serum from saline controls. This was associated with decreased circulating matrix metalloproteinase 9 (MMP-9) and increased circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3/4 as assessed by zymography and reverse zymography. By using quantitative RT-PCR, we confirmed morphine-induced alterations in MMP-9 and TIMP expression and identified organs, including the liver and spleen, in which these changes originated. Pharmacologic inhibition of MMP-9 abrogated the difference in chemotactic attraction between serum from saline-treated and morphine-treated mice, which indicated that reduced proteolytic ability mediated the decreased migration toward serum from morphine-treated mice. This novel mechanism may enable morphine administration to promote an environment that is less conducive to tumor growth, invasion, and metastasis.-Xie, N., Khabbazi, S., Nassar, Z. D., Gregory, K., Vithanage, T., Anand-Apte, B., Cabot, P. J., Sturgess, D., Shaw, P. N., Parat, M.-O. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.
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Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages.
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The feasibility and clinical implication of drug monitoring of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) need further investigation. This study aimed to determine what predicts serum concentrations of morphine in cancer patients receiving continuously intravenous morphine, the relationships between serum concentration of morphine/its metabolites and urinary concentrations, and the relation between morphine concentrations and with clinical outcomes. We collected serum and urine samples from 24 patients with advanced cancer undergoing continuously intravenous morphine therapy. Serum samples were obtained at day one. Spot urine samples were collected once daily on three consecutive days. Pain and adverse drug events were assessed using the Korean version of MD Anderson Symptom Inventory. A total of 96 samples (72 urine and 24 serum samples) were collected. Median dose of morphine was 82.0 mg/24 h. In a multivariate analysis, total daily morphine dose was the most significant predictors of both serum and urine concentration of morphine. Morphine, M6G, and M3G in serum and urine were statistical significantly correlated (correlation coefficient = 0.81, 0.44, 0.56; p values < 0.01, 0.03, 0.01, respectively). Spot urine concentrations of morphine and its metabolites were highly correlated to those of serum. Total dose of daily morphine was related to both serum and urine concentration of morphine and its metabolites.
: Tumor progression is a complex, multistage process by which a normal cell undergoes genetic changes that result in phenotypic alterations and the acquisition of the ability to spread and colonize distant sites in the body. Although many factors regulate malignant tumor growth and spread, interactions between a tumor and its surrounding microenvironment result in the production of important protein products that are crucial to each step of tumor progression. The matrix metalloproteinases (MMPs) are a family of degradative enzymes with clear links to malignancy. These enzymes are associated with tumor cell invasion of the basement membrane and stroma, blood vessel penetration, and metastasis. They have more recently been implicated in primary and metastatic tumor growth and angiogenesis, and they may even have a role in tumor promotion. This review outlines our current understanding of the MMP family, including the association of particular MMPs with malignant phenotypes and the role of MMPs in sp...
Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-kappa B) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-kappa B activation and consequent iNOS gene expression, we studied the effects of NO donors [(+/-)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1 beta-induced NF-kappa B activation and I kappa B-alpha degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1 beta-induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappa B site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-beta-induced NF-kappa B activation and its nuclear translocation, as demonstrated with immunocytochemical study. These effects were independent of guanylate cyclase activation; an inhibitor of soluble guanyiate cyclase (1H-oxadiazolo-1,2,4-[4,3-alpha]quinoxaline-1-one) had no effect on NOR3-induced inhibition of NF-kappa B activation or iNOS mRNA expression by IL-1 beta, and a cGMP derivative (8-bromo-cGMP) failed to mimic the effects of NO donors. Western blot analysis using anti-I kappa B-alpha and anti-phospho-I kappa B-alpha antibodies revealed that IL-1 beta induced a transient degradation of I kappa B-alpha preceded by a rapid appearance of phosphorylated I kappa B-alpha, both of which were completely blocked by NOR3, A proteasome inhibitor (MG115) blocked IL-1 beta-induced transient degradation of I kappa B-alpha and stabilized the appearance of phosphorylated I kappa B-alpha stimulated by IL-1 beta. NOR3 inhibited the appearance of IL-1 beta-induced phosphorylated I kappa B-alpha even in the presence of MG115. Our results indicate that an inhibitory action by NO on cytokine-induced NF-kappa B activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of I kappa B-alpha via the cGMP-independent pathway in rat VSMCs.
AIM: To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca²⁺ entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts. METHODS: NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca²⁺ entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca²⁺]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca²⁺ entry in adhesion was determined using matrigel-coated culture plates. RESULTS: 8-bromo-cGMP inhibited the thapsigargin-induced Ca²⁺ entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1μmol/L). A similar effect on the Ca²⁺ entry was observed in 3T3 cells in response to a NO donor, (±)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca²⁺ entry was also observed, indicating NO/cGMP-regulated Ca²⁺ entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca²⁺ concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates. CONCLUSION: NO/cGMP sensitive store-operated Ca²⁺ entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion. Keywords: cGMP, Nitric oxide, Protein kinase G, Store-operated Ca²⁺ entry, Matrix metalloproteinase, Fibroblast Citation: Huang Y, Lu MQ, Li H, Xu C, Yi SH, Chen GH. Occurrence of cGMP/nitric oxide-sensitive store-operated calcium entry in fibroblasts and its effect on matrix metalloproteinase secretion. World J Gastroenterol 2006; 12(34): 5483-5489
The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases, The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor alpha (TNF alpha)-inducible NO synthesis, because by affecting NF-kappa B activation it inhibits inducible Ca2+-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNF alpha-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF alpha induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNF alpha-inducible nitrite release, as determined by Griess reaction, Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF alpha-elicited NF-kappa B activation, This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-kappa B availability.
To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-kappa B, we stimulated human vascular endothelial cells with tumor necrosis factor-alpha in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-kappa B activation by tumor necrosis factor-alpha. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-kappa B, suggesting tonic inhibition of NF-kappa B under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-kappa B inhibitor, I kappa B alpha, by preventing its degradation from NF-kappa B. NO also increased the mRNA expression of I kappa B alpha, but not NF-kappa B subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the I kappa B alpha promoter suggested transcriptional induction of I kappa B alpha by NO. me propose that the induction and stabilization of I kappa B alpha by NO are important mechanisms by which NO inhibits NF-kappa B and attenuate atherogenesis.