Differential expression of immune-regulatory proteins C5AR1, CLEC4A and NLRP3 on peripheral blood mononuclear cells in early-stage non-small cell lung cancer patients.

Changes in gene expression profiling of peripheral blood mononuclear cells (PBMC) appear to represent the host's response to the cancer cells via paracrine signaling. We speculated that protein expression on circulating T-lymphocytes represent T-lymphocyte trafficking before infiltration into the tumor microenvironment. The possibility of using protein expression on circulating T-lymphocytes as a biomarker to discriminate early-stage non-small cell lung cancer (NSCLC) was explored. Four independent PBMC gene expression microarray datasets (GSE12771, GSE13255, GSE20189 and GSE3934) were analyzed. We selected C5AR1, CLEC4A and NLRP3 based on their significant protein expression in tumor-infiltrating lymphocytes, but not in normal lymphoid tissue. A validation study using automated flow cytometry was conducted in 141 study participants including 76 treatment-naive early-stage non-small cell lung cancer patients (NSCLC), 12 individuals with non-malignant pulmonary diseases, and 53 healthy individuals. Median ratios of C5AR1, CLEC4A and NLRP3 specific antibody staining to CD3 positive cells in early-stage NSCLC patients compared to healthy controls were 0.014 [0-0.37] vs. 0.01 [0-0.07, p = 0.13], 0.03 [0-0.87] vs. 0.02 [0-0.13, p = 0.10] and 0.19 [0-0.60] vs. 0.09 [0.02-0.31, p < 0.0001], respectively. Median fluorescence intensity (MFI) of CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ expression in early-stage NSCLC patients compared to healthy volunteers was 185 [64.2-4801] vs. 107.5 [27-229, p < 0.0001], 91.2 [42.4-2355] vs. 71.25 [46.2-103, p = 0.0005], and 1585 [478-5224] vs. 758.5 [318-1976, p < 0.0001], respectively. NLRP3:CD3 ratio, CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ MFI were significantly higher in early-stage NSCLC than healthy volunteers with an area under the ROC curve of 0.69-0.76. The CD3+NLRP3+ MFI provided the most distinguishable expression at 71.5% sensitivity and 70% specificity. Furthermore, CD3+NLRP3+ MFI potentially discriminated between early-stage NSCLC from malignant-mimic inflammation and infection pulmonary disease. Further validation in various pulmonary inflammatory disease might be warranted. Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes.

Regulation of periostin expression by Notch signaling in hepatocytes and liver cancer cell lines.

Notch signaling is involved in both differentiation of hepatocyte progenitors and hepatocellular carcinoma (HCC). The mechanism whereby Notch signaling regulates cellular transformation in hepatocytes is still controversial. This study investigated the impact of overexpressing truncated intracellular Notch1 (NICD1) on transcriptomic profiles of immortalized human hepatocytes. RNA sequencing and gene ontology enrichment analysis revealed that extracellular matrix organization and hyaluronan biosynthesis process gene sets are among those affected by Notch hyperactivation. The relationship between Notch signaling and periostin, an extracellular matrix protein highly expressed in HCC, were further studied. Modulating Notch signaling through NICD1 overexpression or treatment with a gamma secretase inhibitor resulted in increased or decreased periostin expression, respectively, in HCC and liver bile duct carcinoma cell lines. Based on The Cancer Genome Atlas database, mRNA levels of NOTCH1 and POSTN are positively correlated in tumor tissues but not in nontumor tissues. Two consensus RBPJ binding motifs were identified in the -3932/-3921 and + 2522/+2533 bp of POSTN regulatory regions, and NOTCH1 is associated with these binding sites in a liver bile duct carcinoma cell line. Taken together, these results indicate that Notch signaling directly regulates transcription of POSTN in hepatocytes and liver cancer cell lines and may be a candidate for drug targeting in liver cancer.

Facile and green synthesis of pullulan derivative-stabilized Au nanoparticles as drug carriers for enhancing anticancer activity.

In this work, we report for the first time AuNPs reduced/stabilized/capped with modified para-aminobenzoic acid-quat188-pullulan (PABA-QP) as excellent nanocarriers for delivery of doxorubicin to enhance the activity and safety of these systems. Spherical AuNPs@PABA-QP obtained by facile and green synthesis under optimum conditions were characterized by UV-VIS, TEM, EDS, SAED, XRD, ATR-FTIR and zeta-potential analyses and showed a narrow size distribution of 13.7 ±â€¯1.9 nm. DOX was successfully loaded onto AuNPs@PABA-QP via intermolecular interactions with high drug loading. DOX-AuNPs@PABA-QP (IC50 = 0.39µM) showed a 2.1-fold higher cytotoxicity against Chago cells than DOX alone (IC50 = 0.82µM), while exhibiting less cytotoxicity against normal cells (Wi-38). Moreover, DOX-AuNPs@PABA-QP also demonstrated high intracellular uptake by endocytosis, arrested in S and G2-M phases of the cell cycle (total S/G2-M increased to approximately 18.0%), induced excellent cytotoxicity, and increased the fraction of late-apoptotic cells (18.6%). Consequently, it is suggested that the novel combination of DOX-AuNPs@PABA-QP has the potential to be developed for human cancer treatment.

Notch signaling regulates the responses of lipopolysaccharide-stimulated macrophages in the presence of immune complexes.

Macrophages exhibit diverse effector phenotypes depending on the stimuli and their microenvironment. Classically activated macrophages are primed with interferon (IFN)γ and stimulated with pathogen-associated molecular patterns. They produce inflammatory mediators and inflammatory cytokines, such as IL-12. In the presence of immune complexes (ICs), activated macrophages have decreased IL-12 production and increased IL-10 production and presumably act as regulatory macrophages. Notch signaling has been shown to regulate the effector functions of classically activated macrophages. In this study, we investigated whether Notch signaling is active in lipopolysaccharide (LPS)-stimulated macrophages in the presence of ICs. LPS/IC stimulation increased the level of cleaved Notch1 in murine macrophages, while IC stimulation alone did not. Delta-like 4, but not Jagged1, was responsible for generating cleaved Notch1. The activation of Notch signaling by LPS/ICs depended upon NF-κB and MEK/Erk pathway activation. Macrophages with the targeted deletion of Rbpj, which encodes a DNA-binding protein central to canonical Notch signaling, produced significantly less IL-10 upon LPS/IC stimulation. A similar impact on IL-10 production was observed when Notch signaling was inhibited with a gamma-secretase inhibitor (GSI). Defects in NF-κB p50 nuclear localization were observed in GSI-treated macrophages and in Rbpj-/- macrophages, suggesting cross-regulation between the Notch and NF-κB pathways. Transcriptomic analysis revealed that Notch signaling regulates the transcription of genes involved in the cell cycle, macrophage activation, leukocyte migration and cytokine production in LPS/IC-stimulated macrophages. Taken together, these results suggest that the Notch signaling pathway plays an important role in regulating the functions of macrophages activated by LPS and ICs.

Hepatitis B Virus HBx Activates Notch Signaling via Delta-Like 4/Notch1 in Hepatocellular Carcinoma.

Hepatitis virus B (HBV) infection is one of the major causes of hepatocellular carcinomas (HCC). HBx protein encoded in HBV genome is one of the key viral factors leading to malignant transformation of infected cells. HBx functions by interfering with cellular functions, causing aberration in cellular behaviour and transformation. Notch signalling is a well-conserved pathway involved in cellular differentiation, cell survival and cell death operating in various types of cells. Aberration in the Notch signalling pathways is linked to various tumors, including HCC. The role of HBx on the Notch signalling in HCC, however, is still controversial. In this study, we reported that HBV genome-containing HCC cell line HepG2 (HepG2.2.15) expressed higher Notch1 and Delta-like 4 (Dll4), compared to the control HepG2 without HBV genome. This upregulation coincided with increased appearance of the cleavage of Notch1, indicating constitutively activated Notch signalling. Silencing of HBx specifically reduced the level of Dll4 and cleaved Notch1. The increase in Dll4 level was confirmed in clinical specimens of HCC lesion, in comparison with non-tumor lesions. Using specific signalling pathway inhibitors, we found that MEK1/2, PI3K/AKT and NF-κB pathways are critical for HBx-mediated Dll4 upregulation. Silencing of HBx clearly decreased the level of phosphorylation of Akt and Erk1/2. Upon silencing of Dll4 in HepG2.2.15, decreased cleaved Notch1, increased apoptosis and cell cycle arrest were observed, suggesting a critical role of HBx-Dll4-Notch1 axis in regulating cell survival in HCC. Furthermore, clonogenic assay confirmed the important role of Dll4 in regulating cell survival of HBV-genome containing HCC cell line. Taken together, we reported a link between HBx and the Notch signalling in HCC that affects cell survival of HCC, which can be a potential target for therapy.