NRT1.1B mediates rice plant growth and soil microbial diversity under different nitrogen conditions.

Interactions between microorganisms and plants can stimulate plant growth and promote nitrogen cycling. Nitrogen fertilizers are routinely used in agriculture to improve crop growth and yield; however, poor use efficiency impairs the optimal utilization of such fertilizers. Differences in the microbial diversity and plant growth of rice soil under different nitrogen application conditions and the expression of nitrogen-use efficiency-related genes have not been previously investigated. Therefore, this study investigates how nitrogen application and nitrogen-use efficiency-related gene NRT1.1B expression affect the soil microbial diversity and growth indices of two rice varieties, Huaidao 5 and Xinhuai 5. In total, 103,463 and 98,427 operational taxonomic units were detected in the soils of the Huaidao 5 and Xinhuai 5 rice varieties, respectively. The Shannon and Simpson indices initially increased and then decreased, whereas the Chao and abundance-based coverage estimator indices decreased after the application of nitrogen fertilizer. Nitrogen fertilization also reduced soil bacterial diversity and richness, as indicated by the reduced abundances of Azotobacter recorded in the soils of both rice varieties. Nitrogen application initially increased and then decreased the grain number per panicle, yield per plant, root, stem, and leaf nitrogen, total nitrogen content, glutamine synthetase, nitrate reductase, urease, and root activities of both varieties. Plant height showed positive linear trends in response to nitrogen application, whereas thousand-grain weights showed a negative trend. Our findings may be used to optimize nitrogen fertilizer use for rice cultivation and develop crop-variety-specific strategies for nitrogen fertilizer application.

Effect of Epichloë gansuensis Endophyte on Seed-Borne Microbes and Seed Metabolites in Achnatherum inebrians.

The seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.

Cuticular Wax Modification by Epichloë Endophyte in Achnatherum inebrians under Different Soil Moisture Availability.

The cuticular wax serves as the outermost hydrophobic barrier of plants against nonstomatal water loss and various environmental stresses. An objective of this study was to investigate the contribution of the mutualistic fungal endophyte Epichloë gansuensis to leaf cuticular wax of Achnatherum inebrians under different soil moisture availability. Through a pot experiment and gas chromatography-mass spectrometry (GC-MS) analysis, our results indicated that the hydrocarbons were the dominant components of leaf cuticular wax, and the proportion of alcohols, aldehydes, amines, and ethers varied with the presence or absence of E. gansuensis and different soil moisture availability. Amines and ethers are unique in endophyte-free (EF) A. inebrians plants and endophyte-infected (EI) A. inebrians plants, respectively. By transcriptome analysis, we found a total of 13 differentially expressed genes (DEGs) related to cuticular biosynthesis, including FabG, desB, SSI2, fadD, BiP, KCS, KAR, FAR, and ABCB1. A model is proposed which provides insights for understanding cuticular wax biosynthesis in the association of A. inebrians plants with E. gansuensis. These results may help guide the functional analyses of candidate genes important for improving the protective layer of cuticular wax of endophyte-symbiotic plants.

The effect of Epichloë endophyte on phyllosphere microbes and leaf metabolites in Achnatherum inebrians.

Upon exposure to the prevailing environment, leaves become increasingly colonized by fungi and bacteria located on the surface (epiphytic) or within (endophytic) the leaves. Many cool season grasses, including Achnatherum inebrians, host a seed-borne, intercellular, mutualistic Epichloë fungal endophyte, the growth of which is synchronized with the host grass. A study utilizing illumina sequencing was used to examine the epiphytic and endophytic microbial communities in Epichloë endophyte-infected and endophyte-free A. inebrians plants growing under hot dry field conditions. The presence of Epichloë endophyte increased the Shannon and decreased Simpson diversity of bacterial and fungal communities. Sphingomonas and Hymenobacter bacteria and Filobasidium and Mycosphaerella fungi were growing largely epiphytically, whereas Methylobacterium, Escherichia-Shigella, and the fungus Blumeria were mostly found within leaves with the location of colonization influenced by the Epichloë endophyte. In addition, leaf metabolites in Epichloë-infected and Epichloë-free leaves were examined using LC/MS. Epichloë was significantly correlated with 132 metabolites.

A phase 1b trial of the CXCR4 inhibitor mavorixafor and nivolumab in advanced renal cell carcinoma patients with no prior response to nivolumab monotherapy.

Background The CXCR4 chemokine receptor promotes tumor survival through mechanisms that include suppressing antitumor immune responses. Mavorixafor (X4P-001) is an oral, selective, allosteric CXCR4 inhibitor that decreases the recruitment of immunosuppressive cells into the tumor microenvironment and increases activated cytotoxic Tcell infiltration. Methods Patients with metastatic clear cell renal cell carcinoma (ccRCC) unresponsive to nivolumab monotherapy received oral mavorixafor 400 mg daily plus 240 mg intravenous nivolumab every 2 weeks. Results Nine patients were enrolled, median age 65 years. At baseline 4 had progressive disease (PD) and 5 had stable disease (SD). One of 5 patients with SD at study entry on prior nivolumab monotherapy had a partial response (PR) on combination treatment; all 4 patients with PD at study entry had a best response of SD with the combination treatment (median duration: 6.7 months; range: 3.7-14.7). Four patients discontinued therapy due to treatment-related adverse events (AEs). Grade ≥ 3 drug-related AEs were elevated alanine and aspartate aminotransferase (2 patients each); and autoimmune hepatitis, chronic kidney disease, increased lipase, maculopapular rash, and mucosal inflammation (1 patient each). A robust increase in levels of chemokine (C-X-C motif) ligand 9 CXCL9 on mavorixafor appeared to correlate with clinical benefit. Conclusions The CXCR4 inhibition mediated by mavorixafor, in combination with PD-1 blockade to enhance antitumor immune responses in patients unresponsive to checkpoint inhibitor monotherapy, is worthy of further study. Mavorixafor and nivolumab combination therapy in patients with advanced ccRCC demonstrated potential antitumor activity and a manageable safety profile.Trial registration: ClinicalTrials.gov identifier: NCT02923531. Date of registration: October 04, 2016.

Effects of Epichloë gansuensis Endophyte on the Root and Rhizosphere Soil Bacteria of Achnatherum inebrians Under Different Moisture Conditions.

This study was conducted to explore effects of the systemic fungal endophyte Epichloë gansuensis on root and rhizosphere soil bacterial diversity of Achnatherum inebrians host plants growing under different moisture conditions. Soil properties of different treatments were compared using standard techniques. A total of 4371379 16S rRNA gene sequences were obtained and assigned to 5025 operational taxonomic units (OTUs). These OTUs in roots and rhizosphere soil were divided into 13 and 17 phyla, respectively, and the Actinobacteria and Proteobacteria were the most abundant phyla both in roots and rhizosphere soil. Shannon diversity and Chao1 richness index of bacteria in rhizosphere soil was significantly higher than in roots. E. gansuensis decreased the Shannon diversity of the root-associated bacterial community, and increased Shannon diversity and Chao1 richness index of the rhizosphere soil bacterial community of A. inebrians. Meanwhile, Chao1 richness of the rhizosphere soil bacterial community of A. inebrians significantly increased with the increase of the soil moisture level. Structural equation modeling also emphasized that E. gansuensis decreased the diversity of the root-associated bacterial community and increased the diversity of the rhizosphere soil bacterial community through decreasing soil available N. Additionally, soil moisture increased the diversity of the rhizosphere soil bacterial community through increased soil pH, C/N, and NN, and decreased soil AP. The E. gansuensis endophyte and soil moisture effects on root and rhizosphere soil bacterial diversity were likely to be from responses to modifications of the rhizosphere soil properties.

TRA-1-60-positive/CD45low cells found in the peripheral blood of prostate cancer patients with metastatic disease - A proof-of-concept study.

PURPOSE: Over 90% of all cancer related deaths are due to metastasis. However, current diagnostic tools can't reliably discriminate between invasive and localized cancers. PATIENTS AND METHODS: In this proof-of-concept study, we employed the embryonic stem cell marker TRA-1-60 (TRA+) to identify TRA + cells within the blood of prostate cancer patients and searched for TRA + cells in men with metastatic and localized cancers. We isolated whole peripheral blood mononuclear cells from 26 metastatic prostate cancer patients, from 13 patients with localized prostate cancer and from 17 healthy controls. Cells were stained for DAPI, CD45 and TRA + by immunofluorescence and imaged by epi-fluorescence microscopy. Imaged-based software was used both to identify TRA + cells, and to analyze CD45 levels in TRA+ and negative cells. RESULTS: We found high numbers of TRA + cells within the blood of metastatic cancer patients, whereas healthy individuals or men with localized prostate cancer showed none or very low numbers of TRA + cells. Further analysis of the CD45 levels of TRA + cells revealed a small population of TRA + cells with almost undetectable CD45 levels that were found frequently in metastatic prostate cancer patients. By excluding CD45 positive cells from the TRA + cell pool, we were able to refine the assay to be highly specific in identifying men with metastatic disease. In fact, the difference of CD45 levels between TRA+ and negative cells was a robust measure to distinguish between men with localized and metastatic prostate cancers in this small patient cohort. CONCLUSIONS: The data suggest that metastatic prostate cancer patient have significant numbers of TRA+/CD45low cells which might represent a potential tool for diagnostic assessment in the future.

Overexpression of the Pluripotent Stem Cell Marker Podocalyxin in Prostate Cancer.

BACKGROUND/AIM: Podocalyxin, a member of the CD34 family of cell surface sialomucins, is overexpressed in human embryonal carcinoma cell lines, as well as in several cancer types, and is associated with poor prognosis. Podocalyxin variants are associated with an increased risk and aggressiveness of prostate cancer. Herein podocalyxin protein expression in prostate cancer was characterized. MATERIALS AND METHODS: Expression of podocalyxin as well as of TRA-1-60 and TRA-1-81 antigens was assessed immunohistochemically in 84 radical prostatectomy specimens and in adjacent normal tissues. RESULTS: Podocalyxin expression and H-scores were considerably higher in prostate tumors compared to normal tissues. High TRA-1-60 and TRA-1-81 staining was detected, however, in a much smaller percentage of prostate tumors, while their expression and H-scores were low in normal tissues. Similar trends for all three proteins were observed in prostatic intraepithelial neoplasia. CONCLUSION: Overexpression of podocalyxin in prostate cancer renders the protein a putative immunohistochemical marker of prostate cancer that may contribute to stratification of patients for optimal treatment.

Increased numbers of CD23(+) CD21(hi) Bin-like B cells in human reactive and rheumatoid arthritis lymph nodes.

A unique population of CD23(+) CD21(high) B cells in inflamed nodes (Bin) has been shown to accumulate in lymph nodes (LNs) draining inflamed joints of TNF-transgenic (TNF-tg) mice. Bin cells contribute to arthritis flare in mice by distorting node architecture and hampering lymphatic flow, but their existence in human inflamed LNs has not yet been described. Here, we report the characterization of resident B-cell populations in fresh popliteal lymph nodes (PLNs) from patients with severe lower limb diseases (non-RA) and rheumatoid arthritis (RA) patients, and from banked, cryopreserved reactive and normal human LN single cell suspension samples. Bin-like B cells were shown to be significantly increased in reactive LNs, and strikingly elevated (>30% of total) in RA samples. Histopathology and immunofluorescence analyses were consistent with B follicular hyperplasia and histological alterations in RA vs. non-RA PLNs. This is the first description of Bin-like B cells in human inflamed LNs. Consistent with published mouse data, this population appears to be associated with inflammatory arthritis and distortion of LN architecture. Further analyses are necessary to assess the role of CD23(+) CD21(hi) Bin-like B cells in RA pathogenesis and arthritic flare.

Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice.

BACKGROUND: In this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis. METHODS: We used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice. RESULTS: LECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N(6)-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage. CONCLUSIONS: iNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.

The role of the lymphatic system in inflammatory-erosive arthritis.

Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it "expands" in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it "collapses" with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.

Power Doppler ultrasound phenotyping of expanding versus collapsed popliteal lymph nodes in murine inflammatory arthritis.

Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic "expanding" phase of the disease, and then suddenly "collapse" during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553 ± 0.007 vs. 0.008 ± 0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.

Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: characterization of Tnnt3(tm2a(KOMP)Wtsi) mice.

The troponin complex, which consists of three regulatory proteins (troponin C, troponin I, and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue-specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3(lacZ/+) mice are smaller than their WT littermates throughout development but do not display any gross phenotypes. Tnnt3(lacZ/lacZ) embryos are smaller than heterozygotes and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3(lacZ/lacZ) liver and kidney, which was not present in Tnnt3(lacZ/+) or WT, but no other gross tissue abnormalities. X-gal staining for Tnnt3 promoter-driven lacZ transgene expression revealed positive staining in skeletal muscle and diaphragm and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional-inducible gene deletion approach

Efficacy of B cell depletion therapy for murine joint arthritis flare is associated with increased lymphatic flow.

OBJECTIVE: B cell depletion therapy ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritis flare in tumor necrosis factor (TNF)-transgenic mice is associated with efferent lymph node (LN) "collapse," triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. The aim of this study was to examine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells. METHODS: We used contrast-enhanced magnetic resonance imaging, indocyanine green near-infrared imaging, and intravital immunofluorescence imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-transgenic mice. We conducted tests to determine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic draining and cell egress from arthritic joints. RESULTS: Unlike active lymphatics to normal and prearthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescence imaging demonstrated that CD11b+ monocyte/macrophages in lymphatic vessels afferent to expanding LNs travel at high velocity (mean±SD 186±37 µm/second), while these cells are stationary in lymphatic vessels afferent to collapsed popliteal LNs. B cell depletion therapy for arthritis flares in TNF-transgenic mice significantly decreased knee synovium volume (by 50% from the baseline level) and significantly increased lymphatic clearance compared with placebo (P<0.05). This increased lymphatic drainage restored macrophage egress from inflamed joints without recovery of the lymphatic pulse. CONCLUSION: These results support a novel mechanism in which B cell depletion therapy for joint arthritis flares lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.

Regulation of human osteoclast development by dendritic cell-specific transmembrane protein (DC-STAMP).

Osteoclasts (OC) are bone-resorbing, multinucleated cells that are generated via fusion of OC precursors (OCP). The frequency of OCP is elevated in patients with erosive inflammatory arthritis and metabolic bone diseases. Although many cytokines and cell surface receptors are known to participate in osteoclastogenesis, the molecular mechanisms underlying the regulation of this cellular transformation are poorly understood. Herein, we focused our studies on the dendritic cell-specific transmembrane protein (DC-STAMP), a seven-pass transmembrane receptor-like protein known to be essential for cell-to-cell fusion during osteoclastogenesis. We identified an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of DC-STAMP, and developed an anti-DC-STAMP monoclonal antibody 1A2 that detected DC-STAMP expression on human tumor giant cells, blocked OC formation in vitro, and distinguished four patterns of human PBMC with a positive correlation to OC potential. In freshly isolated monocytes, DC-STAMP(high) cells produced a higher number of OC in culture than DC-STAMP(low) cells and the surface expression of DC-STAMP gradually declined during osteoclastogenesis. Importantly, we showed that DC-STAMP is phosphorylated on its tyrosine residues and physically interacts with SHP-1 and CD16, an SH2-domain-containing tyrosine phosphatase and an ITAM-associated protein, respectively. Taken together, these data show that DC-STAMP is a potential OCP biomarker in inflammatory arthritis. Moreover, in addition to its effect on cell fusion, DC-STAMP dynamically regulates cell signaling during osteoclastogenesis.

LPK-26, a novel kappa-opioid receptor agonist with potent antinociceptive effects and low dependence potential.

Analgesics such as morphine cause many side effects including addiction, but kappa-opioid receptor agonist can produce antinociception without morphine-like side effects. With the aim of developing new and potent analgesics with lower abuse potential, we studied the antinociceptive and physical dependent properties of a derivate of ICI-199441, an analogue of (-)U50,488H, named (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (LPK-26). LPK-26 showed a high affinity to kappa-opioid receptor with the Ki value of 0.64 nM and the low affinities to micro-opioid receptor and delta-opioid receptor with the Ki values of 1170 nM and >10,000 nM, respectively. It stimulated [(35)S]GTPgammaS binding to G-proteins with an EC50 value of 0.0094 nM. In vivo, LPK-26 was more potent than (-)U50,488H and morphine in analgesia, with the ED50 values of 0.049 mg/kg and 0.0084 mg/kg in hot plat and acetic acid writhing tests, respectively. Moreover, LPK-26 failed to induce physical dependence, but it could suppress naloxone-precipitated jumping in mice when given simultaneously with morphine. Taken together, our results show that LPK-26 is a novel selective kappa-opioid receptor agonist with highly potent antinociception effects and low physical dependence potential. It may be valuable for the development of analgesic and drug that can be used to reduce morphine-induced physical dependence.

Morphine inhibits doxorubicin-induced reactive oxygen species generation and nuclear factor kappaB transcriptional activation in neuroblastoma SH-SY5Y cells.

Morphine is recommended as a first-line opioid analgesic in the pain management of cancer patients. Accumulating evidence shows that morphine has anti-apoptotic activity, but its impact on the therapeutic applications of antineoplastic drugs is not well known. The present study was undertaken to test the hypothesis that morphine might antagonize the pro-apoptotic activity of DOX (doxorubicin), a commonly used antitumour drug for the treatment of neuroblastoma, in cultured SH-SY5Y cells. In the present study we demonstrated that morphine suppressed DOX-induced inhibition of cell proliferation and programmed cell death in a concentration-dependent, and naloxone as well as pertussis toxin-irreversible, manner. Further studies showed that morphine inhibited ROS (reactive oxygen species) generation, and prevented DOX-mediated caspase-3 activation, cytochrome c release and changes of Bax and Bcl-2 protein expression. The antioxidant NAC (N-acetylcysteine) also showed the same effects as morphine on DOX-induced ROS generation, caspase-3 activation and cytochrome c release and changes in Bax (Bcl-2-associated X protein) and Bcl-2 protein expression. Additionally, morphine was found to suppress DOX-induced NF-kappaB (nuclear factor kappaB) transcriptional activation via a reduction of IkappaBalpha (inhibitor of nuclear factor kappaB) degradation. These present findings support the hypothesis that morphine can inhibit DOX-induced neuroblastoma cell apoptosis by the inhibition of ROS generation and mitochondrial cytochrome c release, as well as by blockade of NF-kappaB transcriptional activation, and suggests that morphine might have an impact on the antitumour efficiency of DOX.