Anti-cancer potential of selenium-chitosan-polyethylene glycol-carvacrol nanocomposites in multiple myeloma U266 cells.

Multiple myeloma (MM) is an incurable cancer that is characterized by malignant plasma cell proliferation. Approximately 10% of all blood cancers are MM, and there is no standard curative therapy. In this work, we intended to synthesize, characterize, and assess the anticancer effects of selenium/chitosan/polyethylene glycol-carvacrol nanocomposites (SCP-Car-NCs) on MM U266 cells in vitro. Various characterization techniques were used to characterize the synthesized SCP-Car-NCs. Several in vitro free radical scavenging experiments were conducted to test the ability of synthesized SCP-Car-NCs to scavenge the different free radicals. The cytotoxicity of SCP-Car-NCs was assessed on Vero and U266 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. By using various fluorescence staining techniques, the amount of reactive oxygen species (ROS) generation, MMP, and apoptosis were measured. Using commercial test kits, the levels of oxidative stress and apoptotic biomarkers in control and treated U266 cells were assessed. The highest peak in the UV spectral analysis was found to be at 271 nm, demonstrating the development of SCP-Car-NCs. Fourier transform infrared analysis showed that the synthesized SCP-Car-NCs contained a variety of stretching and bonding. The X-ray diffraction study confirmed the crystallinity of SCP-Car-NCs. The dynamic light scattering analysis showed that the SCP-Car-NCs had an average size of 171 nm. The different free radicals, such as the 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, and peroxyl radicals, were significantly scavenged by the SCP-Car-NCs. According to the MTT assay results, the SCP-Car-NCs decreased the viability of U266 cells while having no impact on the proliferation of Vero cells. The SCP-Car-NCs significantly boosted ROS production, decreased the MMP level, and promoted apoptosis, as evidenced by the fluorescence staining experiments. In U266 cells treated with SCP-Car-NCs, the level of thiobarbituric acid reactive substances increased while superoxide dismutases and glutathione levels were reduced. In the SCP-Car-NCs treated U266 cells, it was found that the Bax, caspase-3, and -9 activities had increased while the Bcl-2 level had decreased. In conclusion, our findings show that SCP-Car-NCs treatment reduced the viability and increased apoptosis in the U266 cells, providing a new insight on SCP-Car-NCs' potential for usage in the future to treat MM.

Purification and characterization of a serine protease from organic dust and elucidation of its inductive effects on lung inflammatory mediators.

Organic dust inhalation is associated with the development of respiratory diseases. Serine protease activities in organic dusts were previously reported to contribute to the induction of lung inflammatory mediators however, the identities of the proteases and the mechanisms by which they induce inflammatory mediators are unknown. The goal of this study was to purify and characterize serine protease(s) from organic dust and elucidate mechanisms by which they induce lung inflammatory mediators. A serine protease was purified from poultry organic dust by benzamidine-agarose affinity chromatography. Mass spectrometry and amino-terminal sequence analysis identified the purified protease as chicken trypsin II-P29. Purified protease induced proinflammatory cytokine levels in Beas2B and NHBE epithelial and THP-1 macrophage cells. Treatment with the purified protease increased cellular and mitochondrial reactive oxygen species (ROS) generation. Induction of inflammatory mediators and ROS were suppressed by serine protease inhibitors and antioxidants. Purified protease activated protein kinase C (PKC), mitogen-activated protein kinase (MAPK)1/3 and MAPK14 signaling, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (Stat-3), and chemical inhibitors targeting these pathways suppressed induction of inflammatory mediators. Calcium mobilization studies showed that the purified protease activated protease-activated receptors (PAR) F2R, F2RL1, F2RL2, F2RL3, and F2R and F2RL1 knockdown suppressed the induction of inflammatory mediators. Intranasal instillation of purified protease increased lung chemokine (C-X-C motif) ligand (CXCL)1, interleukin (IL)-6, and tumor necrosis factor (TNF) levels in mice. Our studies have shown that chicken trypsin is a proinflammatory constituent of poultry organic dust, and induces lung inflammatory mediators via increased ROS and PAR activation in a cell signaling pathway involving PKC, MAPK1/3 and MAPK14, and NF-κB and Stat-3.NEW & NOTEWORTHY Inhalation of dust in industrial agricultural operations is linked to the development of lung diseases. Our studies have isolated for the first time a trypsin protease from poultry farm dust and have shown that it stimulates lung inflammation. The protease stimulates the production of oxidants and cell signaling pathways to increase inflammatory mediator production. Targeting trypsin protease in poultry farm environment may be a useful strategy to counter the harmful effects of dust.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

Bacterial extracellular vesicles isolated from organic dust induce neutrophilic inflammation in the lung.

Inhalation of organic dust is an occupational hazard leading to the development of respiratory symptoms and respiratory diseases. Bioaerosols from concentrated animal feeding operations are rich in bacteria and could carry bacterial extracellular vesicles (EVs) that could induce lung inflammation. It is not known if organic dust contains bacterial EVs and whether they modulate lung inflammation. Herein, we show that poultry organic dust contains bacterial EVs (dust EVs) that induce lung inflammation. Treatment of airway epithelial cells, THP-1-monocytes and -macrophages with dust EVs rapidly induced IL-8, IL-6, ICAM-1, proIL-1ß, and TNF-α levels. In airway epithelial cells, induction of inflammatory mediators was due to increased mRNA levels and NF-κB activation. Induction of inflammatory mediators by dust EVs was not inhibited by polymyxin B. Single and repeated treatments of mice with dust EVs increased lung KC, IL-6, and TNF-α levels without significantly altering IL-17A levels. Increases in cytokines were associated with enhanced neutrophil infiltration into the lung. Repeated treatments of mice with dust EVs increased lung mean linear intercept and increased collagen deposition around airways indicating lung remodeling. Peribronchial cell infiltrates and airway epithelial thickening were also observed in treated mice. Because bacterial EVs are nanometer-sized particles, they can reach and accumulate in the bronchiolar and alveolar regions causing lung injury leading to the development of respiratory diseases. Our studies have provided new evidence for the presence of bacterial EVs in organic dust and for their role as one of the causative agents of organic dust-induced lung inflammation and lung injury.

Organic dust induces inflammatory gene expression in lung epithelial cells via ROS-dependent STAT-3 activation.

Exposure to dust in agricultural and animal environments, known as organic dust, is associated with the development of respiratory symptoms and respiratory diseases. Inflammation is a key feature of lung pathologies associated with organic dust exposure, and exposure to organic dust induces the expression of several immune and inflammatory mediators. However, information on transcription factors and cellular and molecular mechanisms controlling the production of immune and inflammatory mediators induced by organic dust is limited. In this study, we have identified STAT-3 as an important transcription factor controlling the induction of expression of immune and inflammatory mediators by poultry dust extracts in airway epithelial cells and in mouse lungs and delineated the cellular pathway for STAT-3 activation. Poultry dust extract activated STAT-3 phosphorylation in Beas2B and normal human bronchial epithelial cells and in mouse lungs. Chemical inhibition and siRNA knockdown of STAT-3 suppressed induction of immune and inflammatory mediator expression. Antioxidants suppressed the increase of STAT-3 phosphorylation induced by poultry dust extract indicating that oxidative stress [elevated reactive oxygen species (ROS) levels] is important for the activation. Chemical inhibition and siRNA knockdown experiments demonstrated that STAT-3 activation is dependent on the activation of nonreceptor tyrosine-protein kinase 2 (TYK2) and epidermal growth factor receptor (EGFR) tyrosine kinases. Our studies show that poultry dust extract controls the induction of immune and inflammatory mediator expression via a cellular pathway involving oxidative stress-mediated STAT-3 activation by TYK2 and EGFR tyrosine kinases.

Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.