Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

BACKGROUND: Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/ß-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. METHODS: Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. RESULTS: DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. CONCLUSIONS: Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

BACKGROUND: Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. METHODS: In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. RESULTS: In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. CONCLUSIONS: Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

Recombinant Humanized IgG1 Antibody Promotes Reverse Cholesterol Transport through FcRn-ERK1/2-PPARα Pathway in Hepatocytes.

Hyperlipidemia-associated lipid disorders are considered the cause of atherosclerotic cardiovascular disease. Reverse cholesterol transport (RCT) is a mechanism by which excess peripheral cholesterol is transported to the liver and further converted into bile acid for excretion from the body in feces, which contributes to reducing hyperlipidemia as well as cardiovascular disease. We previously found that the recombinant humanized IgG1 antibody promotes macrophages to engulf lipids and increases cholesterol efflux to high-density lipoprotein (HDL) through ATP-binding cassette sub-family A1 (ABCA1), one of the key proteins related to RCT. In the present study, we explored other RCT related proteins expression on hepatocytes, including scavenger receptor class B type I (SR-BI), apolipoprotein A-I (ApoA-I), and apolipoprotein A-II (ApoA-II), and its modulation mechanism involved. We confirmed that the recombinant humanized IgG1 antibody selectively activated ERK1/2 to upregulate SR-BI, ApoA-I, and ApoA-II expression in mice liver and human hepatocellular carcinoma cell lines HepG2 cells. The rate-limiting enzymes of bile acid synthesis, including cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), exhibited a significant increase when treated with the recombinant humanized IgG1 antibody, as well as increased excretion of bile acids in feces. Besides, abolishment or mutation of peroxisome proliferator-activated receptor α (PPARα)/RXR binding site on SR-BI promoter eliminated SR-BI reporter gene luciferase activity even in the presence of the recombinant humanized IgG1 antibody. Knock down the neonatal Fc receptor (FcRn) on hepatocytes impaired the effect of recombinant humanized IgG1 antibody on activation of ERK1/2, as well as upregulation of SR-BI, ApoA-I, and ApoA-II expression. In conclusion, one of the mechanisms on the recombinant humanized IgG1 antibody attenuates hyperlipidemia in ApoE-/- mice model fed with high-fat-diet might be through reinforcement of liver RCT function in an FcRn-ERK1/2-PPARα dependent manner.

Recombinant Humanized IgG1 Antibody Protects against oxLDL-Induced Oxidative Stress and Apoptosis in Human Monocyte/Macrophage THP-1 Cells by Upregulation of MSRA via Sirt1-FOXO1 Axis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

Collagen VI antibody reduces atherosclerosis by activating monocyte/macrophage polarization in ApoE-/- mice.

Atherosclerosis (AS) has been regarded as an autoimmune disease. However, studies on immunotherapy against AS are limited. We previously found that IgG in AS patients serum binding to alpha 5 and 6 chain of collagen VI (COL6A5 or COL6A6) was significantly higher than that in healthy subjects, here we tried to identify whether they are AS-protective, and tried to develop human antibodies against them. ApoE-/- mice were immunized with COL6A5 or COL6A6 and COL6A6 was found a protective antigen against atherosclerosis. A phage display human single-chain antibody (scFv) library was constructed and COL6A6-specific scFv was obtained, and cloned into a modified pcDNA3 vector to express full-length human antibodies. ApoE-/- mice were fed a high-fat diet (HFD) for 20 weeks and administered three weekly injections of CVI monoclonal antibody (mAb) or isotype control antibody, CVI mAb was found to be able to reduce plaque area by 45 % via aorta oil red O staining. Flowcytometry method predicted that CVI mAb induced monocyte/macrophage polarization from M1 to M2. Furthermore, CVI mAb induced decreases of pro-inflammatory cytokines of MCP-1and IL-1ß, and increases of IL-4 and IL-10 levels in animal serum by using theLuminexassay. Overall, we found a novel atherosclero-related antigen - Collagen VI, and its protective fragment - Collagen VI alpha 6 chain (COL6A6) and proved that humanized antibody against COL6A6 therapy regresses atherosclerosis and induces monocyte/macrophage polarization from M1 to M2 in ApoE-/- mice animal model.

Methylene Blue versus Coil-Based Computed Tomography-Guided Localization of Lung Nodules.

BACKGROUND: Preoperative computed tomography (CT)-guided localization has been shown to significantly improve lung nodule video-assisted thoracoscopic surgery (VATS)-based wedge resection technical success rates. However, at present, there was insufficient research regarding the optimal approaches to localization of these nodules prior to resection. We aimed to compare the relative clinical efficacy of preoperative CT-guided methylene blue and coil-based lung nodule localization. METHODS: In total, 91 patients with lung nodules were subjected to either CT-guided methylene blue (n = 34) or coil (n = 57) localization and VATS resection from January 2014 to December 2018. We compared baseline data, localization-associated complication rates, as well as the technical success of localization and resection between these two groups of patients. RESULTS: In total, 42 lung nodules in 34 patients underwent methylene blue localization, with associated localization and wedge resection technical success rates of 97.6 and 97.6%, respectively. A total of 71 lung nodules in 57 patients underwent coil localization, with associated localization and wedge resection technical success rates of 94.4 and 97.2%, respectively. There were no significant differences in technical success rates of localization or wedge resection between these groups (p = 0.416 and 1.000, respectively). The coil group sustained a longer duration between localization and VATS relative to the methylene blue group (13.2 vs. 2.5 hours, p = 0.003). CONCLUSION: Both methylene blue and coil localization can be safely and effectively implemented for conducting the diagnostic wedge resection of lung nodules. The coil-based approach is compatible with a longer period of time between localization and VATS procedures.

In vivo Evaluation and Alzheimer's Disease Treatment Outcome of siRNA Loaded Dual Targeting Drug Delivery System.

BACKGROUND: To deliver drugs to treat Alzheimer's Disease (AD), nanoparticles should firstly penetrate through blood brain barrier, and then target neurons. METHODS: Recently, we developed an Apo A-I and NL4 dual modified nanoparticle (ANNP) to deliver beta-amyloid converting enzyme 1 (BACE1) siRNA. Although promising in vitro results were obtained, the in vivo performance was not clear. Therefore, in this study, we further evaluated the in vivo neuroprotective effect and toxicity of the ANNP/siRNA. The ANNP/siRNA was 80.6 nm with good stability when incubated with serum. In vivo, the treatment with ANNP/siRNA significantly improves the spatial learning and memory of APP/PS1 double transgenic mice, as determined by mean escape latency, times of crossing the platform area during the 60 s swimming and the percentage of the distance in the target quadrant. RESULTS AND CONCLUSION: After the treatment, BACE1 RNA level of ANNP/siRNA group was greatly reduced, which contributed a good AD treatment outcome. Finally, after repeated administration, the ANNP/siRNA did not lead to significant change as observed by HE staining of main organs, suggesting the good biocompatibility of ANNP/siRNA. These results demonstrated that the ANNP was a good candidate for AD targeting siRNA delivery.

The important role of apolipoprotein A-II in ezetimibe driven reduction of high cholesterol diet-induced atherosclerosis.

BACKGROUND AND AIMS: It has been well established that ezetimibe blocks cholesterol absorption to prevent the negative effects of a high-fat diet in atherosclerosis. However, the exact mechanism is unknown. Here we use a transgenic zebrafish, which expresses different fluorescent proteins on either endothelial cells or granulocytes and macrophages, to explore the specific mechanism of ezetimibe and its role in reducing atherosclerosis-related hypercholesteremia. METHODS: Zebrafish larvae were exposed to a control diet, high cholesterol diet (HCD) or a HCD with ezetimibe treatment. Both the control diet and high cholesterol diet were mixed with red or green fluorophore labeled cholesteryl ester to trace lipid distribution. Isobaric tags were used for relative and absolute quantification to examine protein expression profiles of zebrafish larvae in the different treatment groups. To knock down Apo A-II and investigate the role of Apo A-II in the anti-atherosclerotic function of ezetimibe, we used morpholinos to target zebrafish Apoa2 mRNA. To confirm ezetimibe regulatory role on Apo A-II expression, siRNA against HNF4, PPARα, and SREBP1 were transfected into HepG2 cells. RESULTS: The results show that ezetimibe increased the expression of Apo A-II but failed to reduce vascular lipid accumulation and macrophage recruitment induced by the HCD diet when Apo A-II was knocked down. Finally, we found that ezetimibe increased the expression of Apo A-II through HNF4 and PPARα transcriptional factors. CONCLUSIONS: Our data indicates that ezetimibe may not only prevents atherosclerosis by inhibiting cholesterol absorption in the intestine, but also by increasing the expression of Apo A-II in hepatocytes, thereby enhancing reverse cholesterol transport and removing excess cholesterol from the periphery.

Diagnosis and Treatment of 26 Cases of Abdominal Cocoon.

BACKGROUND AND AIMS: Abdominal cocoon (AC) is a rare abdominal disease with nonspecific clinical features, and it is difficult to be diagnosed before operation and hard to be treated in clinical practice. The aim of this study is to investigate the diagnosis and treatment of AC. METHODS: The clinical manifestations, findings during surgery, treatments, and follow-up results of 26 cases of AC were retrospectively studied from January 2001 to January 2015. RESULTS: All of 26 cases were diagnosed as AC definitely by laparotomy or laparoscopic surgery. Their clinical findings were various, with 7 intestines obstructed with bezoars and 4 intestines perforated by spiny material. Based on the existence of the second enterocoelia, all cases were categorized into 2 types: type I is absent of second enterocoelia (18 cases, 69.23%), while type II shows second enterocoelia (8 cases, 30.77%). Twenty cases (12 were type I and 8 were type II) underwent membrane excision and careful enterodialysis to release the small intestine entirely or partially, while the other 6 cases (all were type I) did not. In addition, all patients were treated with medical treatment and healthy diet and lifestyle. Finally, most of the patients recovered smoothly. CONCLUSIONS: AC can be categorized into two types; surgery is recommended for type II and part of type I with severe complications, but sometimes conservative therapy might be appropriate for type I. Laparoscopic surgery plays an important role in the diagnosis and treatment of AC. Furthermore, favorite health education, healthy diet and lifestyle are of significance in patients' recovery.

[The effects of NOX4 and TGF-ßinvolved in airway remodeling of Chronic Obstructive Pulmonary Disease].

OBJECTIVE: To investigate the role of nicotinamide adenine dinucleotide phosphate 4 (NADPH4,NOX4) and transforming growth factor-beta (TGF-ß) involve in pathogenesis of airway remodeling in chronic obstructive pulmonary disease (COPD). METHODS: Lung tissues from 36 COPD patients and 19 patients with normal lung function were enrolled in this study. The volume of airway smooth muscle (ASM)mass was evaluated. The expressions of NOX4, collagen Ⅳ (Col Ⅳ) and TGF-ß were tested by a semi-quantitative morphological and/or immunohistochemistry staining method and Western blot, and their correlations with pulmonary functions were analyzed. RESULTS: ①Index of the percentage of the thickness of ASM/external diameter of small airway (WT%) and the percentage of the area of ASM/transverse area of small airway (WA%) were significantly higher in the COPD group than those in controls(P<0.05).②In COPD patients,epithelial cells metaplasia were found and α-SMA and Col Ⅳwere expressed in a part of epithelial cells. The expressions of α-small muscle actin (α-SMA) and Col Ⅰ were increased in COPD patients in comparison with the patients without obstructive airway disorders(P<0.05).③The expression of NOX4 in ASM and epithelial cells of COPD patients was significantly higher in comparison with the patients without COPD. The expression of NOX4 in ASM of small airway were statistically different among different COPD grade (P<0.05). Correlation analysis demonstrated that the level of NOX4 protein in ASM of small airway was inversely associated with pulmonary functions. ④The expression of TGF-ß in COPD was significantly higher than that in patients without COPD. ⑤ Correlation analysis demonstrated that the level of NOX4 protein in ASM of small airway, WT% and WA% were inversely associated with pulmonary functions. CONCLUSIONS: ①The airway remodeling of COPD is characterized by increasing hyperplasia of small airway smooth muscle.②Remodeling of airway smooth muscle associats with severity of airflow limitation in COPD patients. ③The expressions of NOX4, TGF-ß and α-SMA in COPD epithelial cells and small airway smooth muscle cells are significantly enhanced. The expressions of NOX4, α-SMA and TGF-ß are positively correlated with the severity of chronic obstructive pulmonary air flow, suggesting that TGF-ß and NOX4 signaling may be involved in the development of chronic obstructive pulmonary disease airway remodeling.

oxLDL antibody inhibits MCP-1 release in monocytes/macrophages by regulating Ca2+ /K+ channel flow.

oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP-1 release in monocytes/macrophages through their TLR-4 (Toll-like receptor 4) and ERK MAPK pathway and is calcium/potassium channel-dependent. Using blocking antibodies against CD36, TLR-4, SR-AI and LOX-1, only TLR-4 antibody was found to have an inhibitory effect and ERK MAPK-specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL-induced MCP-1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage-dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP-regulated potassium channel (K+ATP ) inhibitor, inhibit oxLDL-induced MCP-1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel - inward rectifier channel (Kir ) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL-induced Ca2+ /Kir channel alterations. The interfering of oxLDL-induced MCP-1 release by its monoclonal antibody is through its FcγRIIB (CD32). Using blocking antibodies against FcγRI (CD64), FcγRIIB (CD32) and FcγRIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL-induced MCP-1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL-induced ERK MAPK activation and ensuing Ca2+ /Kir channel alterations, and MCP-1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL-induced ERK MAPK pathway and consequent MCP-1 release.

A Dual Targeting Drug Delivery System for Penetrating Blood-Brain Barrier and Selectively Delivering siRNA to Neurons for Alzheimer's Disease Treatment.

BACKGROUND: Alzheimer's disease (AD) is one of most serious threats to human beings, however, the treatment is hindered by blood-brain barrier and poor intra-brain cell selectivity. METHODS: In this study, we developed a novel dual targeting drug delivery system by modification of NL4 peptide and apolipoprotein A-I (ApoA-I) onto dendrimer particles that may efficiently deliver siRNA into neuron cells to down-regulate BACE1 and inhibit Aß formation. The constructed ANNP/ siRNA was approximately 79.26 nm with a spherical structure and a zeta potential of 3.53 mV. At N/P ratio of 10, the siRNA could be completely packaged into particles to avoid degradation by RNAase. RESULTS: In vitro, the modification with ApoA-I considerably increased bEnd.3 cell uptake and NL-4 considerably increased PC12 cell uptake. As a result, ANNP/siRNA showed higher uptake in both the cells. In addition, ANNP/siRNA could efficiently penetrate through bEnd.3 monolayers, which was 2.4-fold higher than unmodified complexes. In PC12 cells, the ANNP/siRNA could escape from endosomes and transport into cytoplasm after 8 h incubation, resulting in 87.5% BACE1 gene knockdown capacity, which was better than PEI. Additionally, the particles showed low cytotoxicity to both bEnd.3 and PC12 cells. CONCLUSION: In conclusion, this study preliminarily demonstrated that ApoA-I and NL4 dual modified dendrimer nanoparticles were efficient carriers for siRNA delivery to AD bearing brain.