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Potent anti-inflammatory activity of novel microtubule-modulating brominated noscapine analogs.

Zughaier S, Karna P, Stephens D, Aneja R - PLoS ONE (2010)

Bottom Line: Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties.Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability.This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America. szughai@emory.edu

ABSTRACT
Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-binding anti-inflammatory drug, noscapine and its semi-synthetic brominated analogs were examined for in vitro anti-inflammatory activity. Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability. Brominated noscapine analogs demonstrated anti-inflammatory properties in both TLR- and non-TLR induced in vitro innate immune pathway inflammation models, mimicking septic and sterile infection respectively. In addition, electron microscopy and immunoblotting data indicated that these analogs induced robust autophagy in human macrophages. This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.

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Related in: MedlinePlus

Dose-dependent anti-inflammatory effects of the brominated noscapine analogs.Nitric oxide release was measured in RAW 264.7 murine macrophages treated with 10, 25 and 50 µM of noscapine analogs for 4 hrs, then stimulated with the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4 and incubated overnight. Nitrite accumulation was detected by the Griess method. Nos: noscapine parent analog (10 µM Nos concentration, p value for LPS = 0.014, Pam3CSK4 = 0.0007; 25 µM Nos p value for LPS = 0.0001, Pam3CSK4 = 0.00001; 50 µM Nos p values<0.00001). Br-nos: brominated noscapine analog and TLR ligand (10 µM Br-nos p value for LPS = 0.001, Pam3CSK4 p<0.00001; 25 µM Br-nos p value for LPS = 0.0003, Pam3CSK4, p value<0.00001; 50 µM Br-nos p value<0.00001). Red-Br-nos: reduced brominated noscapine and TLR ligand (10, 25 and 50 µM Red-Br-nos p value<0.00001). Unstimulated cells were used as control and were treated with 25 and 50 µM of noscapine analogs. Error bars represent the SD from the mean of at least duplicate readings. These data is representative of 3 independent experiments. p values were calculated with reference to no drug values.
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pone-0009165-g004: Dose-dependent anti-inflammatory effects of the brominated noscapine analogs.Nitric oxide release was measured in RAW 264.7 murine macrophages treated with 10, 25 and 50 µM of noscapine analogs for 4 hrs, then stimulated with the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4 and incubated overnight. Nitrite accumulation was detected by the Griess method. Nos: noscapine parent analog (10 µM Nos concentration, p value for LPS = 0.014, Pam3CSK4 = 0.0007; 25 µM Nos p value for LPS = 0.0001, Pam3CSK4 = 0.00001; 50 µM Nos p values<0.00001). Br-nos: brominated noscapine analog and TLR ligand (10 µM Br-nos p value for LPS = 0.001, Pam3CSK4 p<0.00001; 25 µM Br-nos p value for LPS = 0.0003, Pam3CSK4, p value<0.00001; 50 µM Br-nos p value<0.00001). Red-Br-nos: reduced brominated noscapine and TLR ligand (10, 25 and 50 µM Red-Br-nos p value<0.00001). Unstimulated cells were used as control and were treated with 25 and 50 µM of noscapine analogs. Error bars represent the SD from the mean of at least duplicate readings. These data is representative of 3 independent experiments. p values were calculated with reference to no drug values.

Mentions: Noscapine and brominated noscapine analogs (Br-nos and Red-Br-nos) were first investigated in a TLR ligand induced septic inflammation model. Human (THP-1) and murine (RAW 264.7) macrophages were used that usually respond well to a diverse repertoire of pathogens and inflammatory mediators. Macrophages were treated with noscapine or brominated noscapine analogs at 10, 25 and 50 µM prior to stimulation or co-stimulation with highly purified TLR-ligands (meningococcal LPS, a TLR4-MD-2 ligand and the synthetic lipopetide Pam3CSK4, a TLR2 ligand). Pre-treatment of murine RAW 264.7 and human THP-1 macrophages with brominated noscapine analogs (50 µM) for 4 hr followed by a 12 hr stimulation with the TLR4-MD-2 ligand meningococcal LPS (doses 10-0.31 pM) resulted in a significant reduction in nitric oxide and TNFα release compared to cells that were treated with DMSO (untreated control) and stimulated with LPS or Pam3CSK4 (Figure 2). Similar reduction in nitric oxide and TNFα release was observed in time-course of drug pre-treatment i.e. 1, 2 and 12 hrs (data not shown). Brominated noscapine analogs displayed significantly higher anti-inflammatory activity compared to the parent drug noscapine. Similarly, co-treatment of human THP-1 and murine RAW 264.7 macrophages with brominated noscapine analogs and TLR-ligands LPS and Pam3CSK4 for 12 hr resulted in significant reduction of TNFα and nitric oxide release (Figure 3). However, no effects on cell viability as determined using trypan blue vital dye exclusion, microscopic morphology and LDH release were observed in these studies. THP-1 cells incubated for 48 hr with 50 µM of either DMSO, Nos, Br-nos or Rd-Br-nos released 5, 7, 8 and 10 IU/L of LDH, respectively. Noscapine or brominated noscapine analogs alone did not induce inflammatory responses or impair cellular viability in human or murine macrophages. The brominated noscapine analogs dampened TLR-mediated TNFα and nitric oxide (NO) release from human and murine macrophages in a dose-dependent manner (Figure 4). In summary, these data demonstrated significant innate immune anti-inflammatory activity of brominated noscapine analogs to TLR ligands compared to untreated controls or the parent drug noscapine.


Potent anti-inflammatory activity of novel microtubule-modulating brominated noscapine analogs.

Zughaier S, Karna P, Stephens D, Aneja R - PLoS ONE (2010)

Dose-dependent anti-inflammatory effects of the brominated noscapine analogs.Nitric oxide release was measured in RAW 264.7 murine macrophages treated with 10, 25 and 50 µM of noscapine analogs for 4 hrs, then stimulated with the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4 and incubated overnight. Nitrite accumulation was detected by the Griess method. Nos: noscapine parent analog (10 µM Nos concentration, p value for LPS = 0.014, Pam3CSK4 = 0.0007; 25 µM Nos p value for LPS = 0.0001, Pam3CSK4 = 0.00001; 50 µM Nos p values<0.00001). Br-nos: brominated noscapine analog and TLR ligand (10 µM Br-nos p value for LPS = 0.001, Pam3CSK4 p<0.00001; 25 µM Br-nos p value for LPS = 0.0003, Pam3CSK4, p value<0.00001; 50 µM Br-nos p value<0.00001). Red-Br-nos: reduced brominated noscapine and TLR ligand (10, 25 and 50 µM Red-Br-nos p value<0.00001). Unstimulated cells were used as control and were treated with 25 and 50 µM of noscapine analogs. Error bars represent the SD from the mean of at least duplicate readings. These data is representative of 3 independent experiments. p values were calculated with reference to no drug values.
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Related In: Results  -  Collection

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pone-0009165-g004: Dose-dependent anti-inflammatory effects of the brominated noscapine analogs.Nitric oxide release was measured in RAW 264.7 murine macrophages treated with 10, 25 and 50 µM of noscapine analogs for 4 hrs, then stimulated with the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4 and incubated overnight. Nitrite accumulation was detected by the Griess method. Nos: noscapine parent analog (10 µM Nos concentration, p value for LPS = 0.014, Pam3CSK4 = 0.0007; 25 µM Nos p value for LPS = 0.0001, Pam3CSK4 = 0.00001; 50 µM Nos p values<0.00001). Br-nos: brominated noscapine analog and TLR ligand (10 µM Br-nos p value for LPS = 0.001, Pam3CSK4 p<0.00001; 25 µM Br-nos p value for LPS = 0.0003, Pam3CSK4, p value<0.00001; 50 µM Br-nos p value<0.00001). Red-Br-nos: reduced brominated noscapine and TLR ligand (10, 25 and 50 µM Red-Br-nos p value<0.00001). Unstimulated cells were used as control and were treated with 25 and 50 µM of noscapine analogs. Error bars represent the SD from the mean of at least duplicate readings. These data is representative of 3 independent experiments. p values were calculated with reference to no drug values.
Mentions: Noscapine and brominated noscapine analogs (Br-nos and Red-Br-nos) were first investigated in a TLR ligand induced septic inflammation model. Human (THP-1) and murine (RAW 264.7) macrophages were used that usually respond well to a diverse repertoire of pathogens and inflammatory mediators. Macrophages were treated with noscapine or brominated noscapine analogs at 10, 25 and 50 µM prior to stimulation or co-stimulation with highly purified TLR-ligands (meningococcal LPS, a TLR4-MD-2 ligand and the synthetic lipopetide Pam3CSK4, a TLR2 ligand). Pre-treatment of murine RAW 264.7 and human THP-1 macrophages with brominated noscapine analogs (50 µM) for 4 hr followed by a 12 hr stimulation with the TLR4-MD-2 ligand meningococcal LPS (doses 10-0.31 pM) resulted in a significant reduction in nitric oxide and TNFα release compared to cells that were treated with DMSO (untreated control) and stimulated with LPS or Pam3CSK4 (Figure 2). Similar reduction in nitric oxide and TNFα release was observed in time-course of drug pre-treatment i.e. 1, 2 and 12 hrs (data not shown). Brominated noscapine analogs displayed significantly higher anti-inflammatory activity compared to the parent drug noscapine. Similarly, co-treatment of human THP-1 and murine RAW 264.7 macrophages with brominated noscapine analogs and TLR-ligands LPS and Pam3CSK4 for 12 hr resulted in significant reduction of TNFα and nitric oxide release (Figure 3). However, no effects on cell viability as determined using trypan blue vital dye exclusion, microscopic morphology and LDH release were observed in these studies. THP-1 cells incubated for 48 hr with 50 µM of either DMSO, Nos, Br-nos or Rd-Br-nos released 5, 7, 8 and 10 IU/L of LDH, respectively. Noscapine or brominated noscapine analogs alone did not induce inflammatory responses or impair cellular viability in human or murine macrophages. The brominated noscapine analogs dampened TLR-mediated TNFα and nitric oxide (NO) release from human and murine macrophages in a dose-dependent manner (Figure 4). In summary, these data demonstrated significant innate immune anti-inflammatory activity of brominated noscapine analogs to TLR ligands compared to untreated controls or the parent drug noscapine.

Bottom Line: Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties.Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability.This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, United States of America. szughai@emory.edu

ABSTRACT
Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-binding anti-inflammatory drug, noscapine and its semi-synthetic brominated analogs were examined for in vitro anti-inflammatory activity. Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability. Brominated noscapine analogs demonstrated anti-inflammatory properties in both TLR- and non-TLR induced in vitro innate immune pathway inflammation models, mimicking septic and sterile infection respectively. In addition, electron microscopy and immunoblotting data indicated that these analogs induced robust autophagy in human macrophages. This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.

Show MeSH
Related in: MedlinePlus