Serum exosomal miR-16-5p functions as a tumor inhibitor and a new biomarker for PD-L1 inhibitor-dependent immunotherapy in lung adenocarcinoma by regulating PD-L1 expression.

OBJECTIVES: We aimed at investigating whether serum exosomal miR-16-5p could be utilized as an immunotherapy biomarker in lung adenocarcinoma (LUAD) patients administered by programmed cell death ligand-1 (PD-L1) inhibitors, and to evaluate its functions in LUAD progression. METHODS: Sixty LUAD sufferers and 20 healthy controls (HCs) were covered in this work. We applied both IHC and WB to examine PD-L1 level in clinical tissue samples and utilized WB to quantify PD-L1 expression in LUAD cells and LUAD xenograft tissues, respectively. Transmission electron microscopy (TEM), WB, and nanoparticle tracking analysis (NTA) were executed to confirm the exosomes isolated from serum specimens and cell culture media. To quantify of exosomal miR-16-5p level from serum and culture medium of cultured cell, qRT-PCR experiment was utilized. The connection between tissue PD-L1 level and serum exosomal miR-16-5p expression in PD-L1-positive sufferers administered by PD-L1 inhibitors was verified using Spearman correlation coefficient analysis. In addition, the overall survival (OS) and progression-free survival (PFS) rates among PD-L1 inhibitor managed sufferers were acquired through a follow-up visit. Finally, we used a group of assays, including 5-bromo-2'-dexoyuridine (BrdU) and colony formation test, wound healing experiment, flow cytometry, and nude mice xenograft experiment, to explore the functions of circulating exosomal miR-16-5p on LUAD cell proliferation, apoptosis, and migration, as well as tumor development, respectively. RESULTS: PD-L1 expression was positively related to T stage (tumor size stage), and PD-L1 inhibitor treatment reduced the PD-L1 expression and mitigated T stage in PD-L1-positive LUAD sufferers. For PD-L1-positive LUAD sufferers, elevated PD-L1 expression or reduced serum exosomal miR-16-5p level were linked to longer PFS and OS upon PD-L1 inhibitor treatment. The number of exosomes in patient's serum was more than that in the serum of healthy individuals, and PD-L1 inhibitor treatment decreased the number of serum-derived exosomes in PD-L1-positive LUAD sufferers. Exosome-derived miR-16-5p was downregulated in patient's serum and cell culture medium, and this was negatively linked to tumor stage and PD-L1 expression. Meanwhile, PD-L1 inhibitor treatment could increase the serum exosomal miR-16-5p expression, and the expression change of serum exosomal miR-16-5p was diametrically related to PD-L1 after the treatment. Moreover, the overexpression of PD-L1 accelerated tumor growth and decreased the exosomal miR-16-5p content in cell culture media, while exosomal miR-16-5p overexpression in cell culture media inhibited tumor development by decreasing the PD-L1 expression. Exosomal miR-16-5p overexpression in cell culture media also depressed LUAD cell proliferation and migration, and stimulated cell apoptosis, especially in the cells which cultured in the mediums with PD-L1 inhibitor in vitro. CONCLUSIONS: Serum exosomal miR-16-5p may be a latent tumor inhibitor and a new biomarker for PD-L1 inhibitor-dependent immunotherapy in LUAD by regulating the PD-L1 expression.

AQ-4, a deuterium-containing molecule, acts as a microtubule-targeting agent for cancer treatment.

The important physiological function of microtubules makes them an indispensable and clinically effective target of anti-tumor agents. Herein, we sought to design, synthesize, and evaluate a novel 4-anilinoquinazoline derivative and identify its anti-tumor activity in vitro and in vivo. The novel compound, N-(4-methoxyphenyl)-N-methyl-2-(methyl-d3)quinazolin-4-amine (AQ-4), was identified as a representative scaffold and potent microtubule-targeting agent. As a promising antimitotic agent, AQ-4 displayed remarkable anti-tumor activity with an average IC50 value of 19 nM across a panel of 14 human cancer cell lines. AQ-4 also exhibited nearly identical potent activities against drug-resistant cells, with no evidence of toxicity towards normal cells. A further target verification study revealed that AQ-4 targets the tubulin-microtubule system by significantly inhibiting tubulin polymerization and disrupting the intracellular microtubule spindle dynamics. According to the results of mechanism study, AQ-4 induced cell cycle arrest in the G2/M phase, promoting evident apoptosis and a collapses of mitochondrial membrane potential. The superior anti-tumor effect of AQ-4 in vivo suggests that it should be further investigated to validate its use for cancer therapy.

Silence of HDAC6 suppressed esophageal squamous cell carcinoma proliferation and migration by disrupting chaperone function of HSP90.

Esophageal carcinoma is aggressive in nature and its prognosis is largely dependent on the degree of invasion. Histone deacetylase 6 (HDAC6), as the most unique member of HDACs family, has the positive activity to promote initiation and progression of various cancers via targeting multiple non-histone proteins in cytoplasm. In this study, we found that HDAC6 was over-expressed in three esophageal cancer cell lines (KYSE140, KYSE170, KYSE180) when compared to non-carcinoma esophageal epithelial cell HEEC-1. Then two HDAC6 specific siRNAs and HDAC6 inhibitor tubastatin A greatly suppressed KYSE140 and KYSE180 cells proliferation and migration, and the inhibition of cell motility was accompanied by elevated acetylation of α-tubulin, a target of HDAC6. Consistently, the microtubulin skeleton was stabilized after HDAC6 knockdown or inhibition. In addition, acetylation status of HSP90, another HDAC6 target, was also increased towards HDAC6 knockdown or inhibition by co-immunoprecipitation assay. Besides, co-treatment of HSP90 inhibitor (PU-H71) and HDAC6 inhibitor (tubastatin A) induced a stronger cell migration inhibition compared to administration of either drug alone. Furthermore, cell proliferation of KYSE140 and KYSE180 were also compromised in response to combination of HDAC6 and HSP90 inhibitors. Additionally, co-administration of HSP90 inhibitor and HDAC6 inhibitor strongly inhibited tumor growth in vivo. Taken together, our results indicated that HDAC6 is a promising target by inhibiting HSP90 function in ESCC.

[Correlation of CD4+CD29+ regulatory T cells with recurrence and survival time in patients with non-small cell lung cancer].

OBJECTIVE: To investigate the correlation of CD4+CD29+ regulatory T cells (Treg) with tumor recurrence and survival time in patients with non-small cell lung cancer (NSCLC). METHODS: Fifty-nine patients with NSCLC treated with radical surgery were followed up for 5 years. Blood Treg cells were examined during the follow-up using flow cytometry (FCM). The sensitivity and specificity of Treg cells to predict recurrence of NSCLC were analyzed using receiver-operating characteristic (ROC) curve and compared with those of carcinoembryonic antigen (CEA) and cytokeratin21-1 (Cyfra21-1). The influences of gender, age, occupation and radiotherapy on survival time of the patients were analyzed with Kaplan-Meier method. RESULTS: Among the 59 patients, the shortest survival time was 23 months while the longest time was over 67 months. Nineteen patients had NSCLC recurrence, and 17 (28.81%) of them died of metastasis during the follow-up. The frequencies of blood Treg cells in patients who did not receive radiotherapy and in patients with tumor recurrence were significantly higher than those in patients receiving radiotherapy and in patients free of recurrence (P=0.000). ROC curves showed that the area under curve (AUC) lowered in the order of Treg cells, Cyfra21-1, CEA (P=0.002, 0.006 and 0.013, respectively) with 95% confidence interval (CI) of 0.649-0.981, 0.621-0.936 and 0.584-0.944, respectively. At the cut-off value of 7.53%, the sensitivity and specificity of Treg cells to predict NSCLC recurrence was 91.42% and 87.59%, respectively. The five-year survival rate of the 59 patients was 71.18% (42/59), and Kaplan-Meier analysis revealed a longer survival time in female patients (P=0.038), in patients below 50 years of age (P=0.013), in patients not engaging in mental work (P=0.029), and in patients receiving radiotherapy (P=0.003). CONCLUSION: Treg cells has a better efficiency than Cyfra21-1 and CEA to predict tumor recurrence in patients with NSCLC following radical surgery. The male gender, an age beyond 50 years, an occupation of mental work, and failure to receive radiotherapy are all risk factors for recurrence of NSCLC.

Lactoferrin protects against lipopolysaccharide-induced acute lung injury in mice.

Lactoferrin (LF) plays various anti-inflammatory roles in inflammation experimentally induced by lipopolysaccharides (LPS). But the protective effects of LF on LPS-induced acute lung injury (ALI) have not been elucidated. In this study, we aimed to study the effects of LF on ALI caused by LPS in mice. At 1h before or after LPS injection, an intraperitoneal injection of LF (5mg/body) was administered. Lung specimens and the bronchoalveolar lavage fluid (BALF) were isolated for histopathological examinations and biochemical analyses 12h after LPS exposure. We found that both prophylactic and therapeutic administration of LF significantly decreased the W/D ratio of the lung and protein concentration in the BALF. LF significantly reduced the pulmonary myeloperoxidase activity and the number of total cells in the BALF 12h after LPS challenge. LF treatment markedly attenuated lung edema, alveolar hemorrhage and inflammatory cells infiltration. Moreover, LF also decreased the production of TNF-α and increased interleukin-10 in the BALF. These results firstly indicate that LF may protect against LPS-induced ALI in mice.

[The protective effect of glutamine on endotoxemic intestinal injury and expression of heme oxygenase-1 in rats].

OBJECTIVE: To investigate the protective effect of glutamine (Glu) pretreatment on intestinal injury induced by endotoxin and expression of heme oxygenase-1 (HO-1) in rats. METHODS: Thirty-two male Sprague-Dawley (SD) rats were randomly divided into four groups (n=8 in each group): normal control group, model group, Glu group and Glu+zinc protoporphyrin (ZnPP) group. In model group, endotoxemia was produced by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). In Glu group, the rats received intragastrically 1 g/kg of Glu 12 hours before LPS intraperitoneal injection. In Glu+ZnPP group, the rats received 1 g/kg of Glu by gavage 12 hours before LPS intraperitoneal injection and ZnPP 10 mmol/kg intravenously via tail vein 1 hour before LPS injection. The distal ileum was harvested in full thickness 12 hours after LPS injection. The myeloperoxidase (MPO) activity, tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the intestine were determined, the pathologic changes were observed and expressed in Chiu grade. The expression of HO-1 was evaluated by immunohistochemistry method. RESULTS: Compared with normal control group, the Chiu grade, MPO activity, the content of TNF-α and IL-10 were significantly increased in model group [Chiu grade: 3.3±0.4 vs. 1.1±0.6, MPO activity (U/g): 0.40±0.08 vs. 0.26±0.07, TNF-α (ng/g): 25.2±6.9 vs. 6.5±2.8, IL-10 (ng/g): 27.6±10.2 vs. 5.7±2.9, all P<0.01], and the expression of HO-1 was decreased. Compared with model group, the Chiu grade, MPO activity, the content of TNF-α in Glu group were significantly decreased [Chiu grade: 1.6±0.5 vs. 3.3±0.4, MPO activity (U/g): 0.25±0.05 vs. 0.40±0.08, the content of TNF-α (ng/g): 13.4±3.2 vs. 25.2±6.9, all P<0.01], while the level of IL-10 (ng/g) elevated (47.3±5.5 vs. 27.6±10.2, P<0.01), and the expression of HO-1 was increased. There was no difference in above mentioned indexes between model group and Glu+ZnPP group. CONCLUSION: Glu pretreatment significantly ameliorates the expression of HO-1 of intestinal tissue induced by LPS in rats, and intestinal mucosa is protected with alleviation of inflammatory reaction in intestinal tract.

Effect of organic fractions on sorption properties of organic pollutants in sediments.

Humic substrates are a major fraction of sediment organic matters, and the sorption of hydrophobic organic chemicals by humic substrates influences their behavior and fate in sediment. In this paper, organic matters were divided into non-humic substrates and humic substrates. Non-humic substrates include acid leaching fraction, acid extracted fraction, and lipid; humic substrates were fractionated into Ca-binding-FA (fulvic acid), Ca-binding-HA (humic acid), oxide-FA, oxide-HA, and humin. To study the effect of organic fractions on sorption properties, sorption kinetic and equilibrium sorption experiments of phenanthrene and pentachlorophenol (PCP) in five sediments were carried out. The results showed that the contents of acid leaching fraction and oxide-binding-HA were the main fractions to affect the sorption rate constant, and for the sorption capacity of phenanthrene, humin was the major fraction, followed by oxide-binding-HA, oxide-binding-FA, and so on. While for PCP, the factors of influence on sorption capacity were mainly CEC, Ca-binding-FA, and Ca-binding-HA.

Estimation of apparent rate coefficients for phenanthrene and pentachlorophenol interacting with sediments.

To gain information on organic pollutants in water-sediment systems, a compartment model was applied to study the sorption course of phenanthrene and pentachlorophenol (PCP) in sediments. The model described the time-dependent interaction of phenanthrene and PCP with operationally defined reversible and irreversible (or slowly reversible) sediment fractions. The interactions between these fractions were described using first order differential equations. By fitting the models to the experimental data, apparent rate constants were obtained using numerical optimization software. The model optimizations showed that the amount of reversible phase increased rapidly in the first 10 d with the sorption time, then decreased after 10 d, while the amount of irreversible phase increased in the total sorption course. That suggested the mass transport between reversible phase and irreversible phase. The extraction efficiency with hot methanol ranged from 36% to 103% for phenanthrene and from 65% to 101% for PCP, with the trend of decreasing with sorption time.

Pollution characteristics of the recent sediments in the Hangzhou section of the Grand Canal, China.

Spatial distribution of heavy metals, arsenic and organic matter in recent sediments in the Hangzhou section of the Grand Canal and their relationships were analyzed. The results showed that the concentrations of heavy metals and organic matters varied widely along the canal, and the average geological accumulation factors decreased in the following orders: organic carbon(2.6), zinc(2.1), cadmium (2.0), copper(1.5), lead(1.1), nitrogen(0.9), mercury (0.8), phosphorus(0.4), arsenic(0.2) and chromium(0). Content of heavy metals and organic carbon in the top 10 cm layer were lower than that of lower layers, except for mercury and organic carbon in the S9 section. Contents of organic carbon in the top 50 cm layer of the mud sediments are significantly higher than those underneath. In the bottom mud layer, there is a concentration peak of the pollutants. In the mud sediments of the canal, cadmium mainly occurred in the Fe and Mn oxide fraction, copper in the organic fraction, lead in the Fe and Mn oxide fraction, and zinc in the carbonate and the Fe and Mn oxide fraction.

Irreversible sorption of pentachlorophenol to sediments: experimental observations.

The partitioning behavior of pentachlorophenol (PCP) in five sediments was studied using equilibrium sorption experiments and multiple cycles of sorption and desorption experiments. The results of the equilibrium sorption experiments showed that the isotherms of PCP on five sediments were linear and the partitioning coefficients (Kd) were proportional to the organic carbon content of the sediments. The average organic carbon content normalized partitioning coefficient (logK oc) of five sediments was 2.83 +/- 1.48. In multiple cycles of sorption and desorption experiments, the five sediments were found to exhibit statistically significant sorption-desorption hysteresis, and the hysteresis indices (HI) varied over a wide range (0.72 - 11.82). Correlations between the HI value and the percentage of lipid in the total organic matter in the sediment indicated that lipid was the main fraction to affect the hysteresis phenomenon, i.e., the higher the lipid percentage the greater the HI value. The hysteresis phenomenon was mostly caused by irreversible sorption of PCP on lipids, including entrapment by lipids, which induced the slow desorption rate from the sediment. Because of hysteresis in the sorption and desorption, the PCP ecological toxicity would be lower than expected.

Characterization of phenol biodegradation by Comamonas testosteroni ZD4-1 and Pseudomonas aeruginosa ZD4-3.

OBJECTIVE: To investigate the characteristic and biochemical mechanism about the phenol biodegradation by bacterial strains ZD 4-1 and ZD 4-3. METHODS: Bacterial strains ZD 4-1 and ZD 4-3 were isolated by using phenol as the sole source of carbon and energy, and identified by 16S rDNA sequence analysis. The concentrations of phenol and total organic carbon (TOC) were monitored to explore the degradation mechanism. The biodegradation intermediates were scanned at 375 nm by using a uv-vis spectrophotometer. The enzyme assays were performed to detect the activities of dioxygenases. RESULTS: Bacterial strains ZD 4-1 and ZD 4-3 were identified as Comamonas testosteroni and Pseudomonas aeruginosa by 16S rDNA sequence analysis, respectively. The growth of the two strains was observed on a variety of aromatic hydrocarbons. The strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and meta-pathways, respectively. In addition, the results of enzyme assays showed that the biodegradation efficiency of phenol by meta-pathways was higher than that by ortho-pathways. Finally, the results of induction experiment indicated that the catechol dioxygenases, both catechol 1,2-dioxygenase (C120) and catechol 2,3-dioxygenase (C230), were all inducible. CONCLUSION: The strains ZD 4-1 and ZD 4-3 metabolize phenol through ortho-pathways and meta-pathway, respectively. Furthermore, the biodegradation efficiency of phenol by meta-pathways is higher than that by ortho-pathways.

Phosphorus and copper leaching from dredged sediment applied on a sandy loam soil: column study.

In this paper, downward movement of phosphorus and copper as dredged sediment applied on sandy loam soil was studied by column leaching experiments. Three sediment application rate, (i.e., 1, 2 and 5-cm depth of sediments) were applied to the top of the soil columns. Two and a half months leaching experiments were conducted, which include a 15-day un-watered period. Concentrations of phosphorus and copper in the leachate and the vertical distribution of Olsen-P and diethylenetriaminepentaacetic acid (DTPA) extractable Cu in the soil columns were determined. The results showed that, un-watered period could increase the downward movements of phosphorus and copper. Sediment application significantly increased Olsen-P concentration in the top 15 cm of the soil columns, but has not significantly affected that in the deeper soil layer. The 1-cm depth sediment treatment did not increase the DTPA extractable Cu concentration in the whole soil column. The 5-cm depth sediment treatment, however, significant increased the DTPA extractable Cu in the deeper soil layers. This study suggested that the application of dredged sediment laden with P and Cu on sandy loam soil might cause the significant downward movement of phosphorus and copper.

Peroxynitrite is an important mediator in thermal injury-induced lung damage.

OBJECTIVE: Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the rat. INTERVENTIONS: In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS: The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS: Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.