Cepharanthine attenuates pulmonary fibrosis via modulating macrophage M2 polarization.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a group of chronic interstitial pulmonary diseases characterized by myofibroblast proliferation and extracellular matrix (ECM) deposition. However, current treatments are not satisfactory. Therefore, more effective therapies need to be explored. Cepharanthine (CEP) is a naturally occurring alkaloid that has recently been reported to have multiple pharmacological effects, particularly in chronic inflammation. METHODS: For in vivo experiments, first, a pulmonary fibrosis murine model was generated via tracheal injection of bleomycin (BLM). Second, the clinical manifestations and histopathological changes of the mice were used to verify that treatment with CEP might significantly reduce BLM-induced fibrosis. Furthermore, flow cytometric analysis was used to analyze the changes in the number of M2 macrophages in the lung tissues before and after treatment with CEP to explore the relationship between macrophage M2 polarization and pulmonary fibrosis. In vitro, we constructed two co-culture systems (THP-1 and MRC5 cells, RAW264.7 and NIH 3T3 cells), and measured the expression of fibrosis-related proteins to explore whether CEP could reduce pulmonary fibrosis by regulating macrophage M2 polarization and fibroblast activation. RESULTS: The results showed that the intranasal treatment of CEP significantly attenuated the symptoms of pulmonary fibrosis induced by BLM in a murine model. Our findings also indicated that CEP treatment markedly reduced the expression of fibrosis markers, including TGF-ß1, collagen I, fibronectin and α-SMA, in the mouse lung. Furthermore, in vitro studies demonstrated that CEP attenuated pulmonary fibrosis by inhibiting fibroblast activation through modulating macrophage M2 polarization and reducing TGF-ß1 expression. CONCLUSIONS: This study demonstrated the potential and efficacy of CEP in the treatment of pulmonary fibrosis. In particular, this study revealed a novel mechanism of CEP in inhibiting fibroblast activation by regulating macrophage M2 polarization and reducing the expression of fibrosis-associated factors. Our findings open a new direction for future research into the treatment of pulmonary fibrosis.

CircHN1 affects cell proliferation and migration in gastric cancer.

BACKGROUND: Increasing evidence indicates that circular RNAs (circRNAs) are dysregulated in human cancers. The biological roles of circRNAs in gastric cancer (GC) have not been well-characterized. METHODS: The GEO database was used to analyze circRNA expression profile in GC. The expression level of target circRNA in tumor tissues and adjacent non-tumor tissues was detected by reverse transcription-quantitative PCR. Gene transfection was used to manipulate the expression of circRNAs. The biological roles of circRNAs in cell proliferation, migration, and invasion were determined by cell counting, colony formation, transwell migration, Matrigel invasion, and mouse xenograft tumor assays. The interactions between circRNAs and miRNAs were verified by RNA immunoprecipitation and luciferase reporter assays. RESULTS: We found that circHN1 was upregulated in GC tissues and cell lines compared to adjacent non-tumor tissues and normal gastric epithelial cells. Additionally, circHN1 silencing significantly promoted GC cell growth, colony formation, migration, and invasion, whereas circHN1 overexpression had the opposite effects. CircHN1 overexpression also suppressed gastric cancer growth in the mouse xenograft tumor model. CircHN1 was mainly localized in the cytoplasm of GC cells and could bind to AGO2. MiR-1248 and miR-375 were predicted to interact with circHN1 by bioinformatic analyses. MiR-1248 and miR-375 overexpression inhibited the activity of the circHN1 luciferase reporter. CONCLUSION: CircHN1 is aberrantly expressed in GC and affects the proliferation and migration of gastric cancer cells by acting as miRNA sponge.

Tumor-Educated Neutrophils Activate Mesenchymal Stem Cells to Promote Gastric Cancer Growth and Metastasis.

In response to tumor signals, mesenchymal stem cells (MSCs) are recruited to tumor sites and activated to promote tumor progression. Emerging evidences suggest that in addition to tumor cells, non-tumor cells in tumor microenvironment could also interact with MSCs to regulate their phenotype and function. However, the mechanism for MSCs regulation in gastric cancer has not been fully understood. In this study, we reported that tumor-educated neutrophils (TENs) induced the transformation of MSCs into cancer-associated fibroblasts (CAFs) which in turn remarkably facilitated gastric cancer growth and metastasis. Mechanistic study showed that TENs exerted their effects by secreting inflammatory factors including IL-17, IL-23 and TNF-α, which triggered the activation of AKT and p38 pathways in MSCs. Pre-treatment with neutralizing antibodies to these inflammatory factors or pathway inhibitors reversed TENs-induced transformation of MSCs to CAFs. Taken together, these data suggest that TENs promote gastric cancer progression through the regulation of MSCs/CAFs transformation.

CXCL5 promotes gastric cancer metastasis by inducing epithelial-mesenchymal transition and activating neutrophils.

Deregulated expression of chemokines in tumor microenvironment contributes to tumor metastasis by targeting distinct cells. Epithelial-derived neutrophil-activating peptide-78 (ENA78/CXCL5) is upregulated in many cancers and involved in tumor progression. The role and underlying mechanism of CXCL5 in gastric cancer (GC) metastasis remain unclear. In this study, we reported that the expression of CXCL5 was elevated in tumor tissues and positively associated with lymphatic metastasis and tumor differentiation. Stimulation by recombinant human CXCL5 (rhCXCL5) induced epithelial-mesenchymal transition (EMT) in GC cells through the activation of ERK pathway, which enhanced their migration and invasion abilities. The culture supernatant from tumor tissues also enhanced the migration and invasion abilities of GC cells, however, this effect was reversed by pre-treatment with CXCL5 neutralizing antibody. Further studies showed that rhCXCL5 could induce the expression of IL-6 and IL-23 in neutrophils through the activation of ERK and p38 signaling pathways, which in turn facilitated GC cell migration and invasion. The culture supernatant from tumor tissues showed similar effects on neutrophils in a CXCL5-dependent manner. Blockade of IL-6 and IL-23 with neutralizing antibodies reversed the induction of EMT and the increased migration and invasion abilities in GC cells by CXCL5-activated neutrophils. Moreover, CXCL5 activated neutrophils could promote gastric cancer metastasis in vivo. Taken together, our results indicate that CXCL5 acts on gastric cancer cells to induce EMT and mediates pro-tumor activation of neutrophils, which synergistically promotes the metastatic ability of GC cells.

Extracellular Vesicles From Gastric Cancer Cells Induce PD-L1 Expression on Neutrophils to Suppress T-Cell Immunity.

Neutrophils are prominent components of solid tumors and exhibit distinct phenotypes in different tumor milieu. We have previously shown that tumor extracellular vesicles (EVs) could induce pro-tumor activation of neutrophils; however, the role of tumor EV-elicited neutrophils in tumor immunity remains unclear. Herein, we reported that gastric cancer cell-derived EVs (GC-EVs) induced the expression of programmed death-ligand 1 (PD-L1) on neutrophils. GC-EVs transported high-mobility group box-1 (HMGB1) to activate signal transducer and activator of transcription 3 (STAT3) and upregulate PD-L1 gene expression in neutrophils. Blocking STAT3 pathway and silencing HMGB1 reversed GC-EV-induced PD-L1 expression on neutrophils. GC-EV-elicited neutrophils suppressed T cell proliferation, activation, and function in vitro, which could be antagonized by a specific PD-L1 antibody. Furthermore, GC tissue-derived EVs also showed similar effects. Taken together, our results indicate that EVs from the GC microenvironment induce PD-L1 expression on neutrophils to suppress T-cell immunity, which provides a new insight into the pro-tumor roles of neutrophils in GC and sheds light on the multifaceted roles of EVs in orchestrating an immunosuppressive microenvironment.

Circular RNA CCDC66 promotes gastric cancer progression by regulating c-Myc and TGF-ß signaling pathways.

Background: CircRNAs play important roles in cancer development and progression and have the potential to serve as cancer biomarkers. The aim of this study was to investigate the role of circular RNA CCDC66 (circCCDC66) in gastric cancer and to reveal the underlying mechanisms. Methods: The expression of circCCDC66 in GC tissues and cell lines was examined by qRT-PCR. The correlation between circCCDC66 expression level and clinicopathological characteristics was analyzed. The biological roles of circCCDC66 in GC cell apoptosis, proliferation, migration and invasion were determined by flow cytometry, cell counting, cell colony formation, wound healing, transwell migration and matrigel invasion assays. The role of circCCDC66 in GC growth was further confirmed by mouse xenograft tumor model. Western blot and qRT-PCR were used to explore the effects of circCCDC66 on epithelial-mesenchymal transition (EMT)-related gene and protein expression. Results: CircCCDC66 expression was elevated in both GC tissues and cell lines compared to adjacent normal tissues and normal gastric epithelial cell line. The upregulation of circCCDC66 in GC tissues was related to tumor stage and lymphatic metastasis. CircCCDC66 knockdown significantly inhibited GC cell proliferation, migration and invasion and induced cell apoptosis in GC cells. On the contrary, circCCDC66 overexpression had the opposite effects. In addition, circCCDC66 knockdown suppressed the tumorigenesis of GC cells in nude mice. Furthermore, circCCDC66 knockdown inhibited the activation of c-Myc and TGF-ß signaling pathways and reversed EMT in GC cells. c-Myc and TGF-ß interference blocked circCCDC66-mediated promotion of gastric cancer cell proliferation, migration and invasion. Conclusion: CircCCDC66 promotes GC growth and metastasis by activating c-Myc and TGF-ß signaling pathways, suggesting that it may serve as a potential biomarker for GC.

miR­498 inhibits the growth and metastasis of liver cancer by targeting ZEB2.

MicroRNAs (miRNAs) play critical roles in the growth, metastasis and therapeutic resistance of liver cancer. Accumulating evidence suggests that miR­498 is aberrantly expressed in several human malignancies. However, the role and underlying mechanism of miR­498 in liver cancer remain unclear. In the present study, we investigated the potential roles and clinical value of miR­498 in liver cancer. We found that the miR­498 expression level was significantly lower in liver cancer patient tissues than that in healthy control tissues. The expression of miR­498 was also decreased in liver cancer cell lines compared to that noted in a normal human normal liver cell line. miR­498 overexpression markedly inhibited liver cancer cell proliferation, migration and invasion. miR­498 overexpression induced cell cycle arrest and apoptosis while it suppressed epithelial­mesenchymal transition (EMT) in liver cancer cells. Bioinformatic analysis and luciferase reporter assay further identified zinc finger E­box binding homeobox 2 (ZEB2) as a novel target of miR­498. Furthermore, ZEB2 knockdown recapitulated the inhibitory effects of miR­498 overexpression in liver cancer cells. ZEB2 overexpression rescued the inhibition of liver cancer cell proliferation, migration, and invasion by miR­498, indicating that ZEB2 acts as a downstream effector of miR­498 in liver cancer cells. Thus, we demonstrated that miR­498 suppresses the growth and metastasis of liver cancer cells, partly at least, by directly targeting ZEB2, suggesting that miR­498 may serve as a potential biomarker for the diagnosis and therapy of liver cancer.

LINC00978 promotes the progression of hepatocellular carcinoma by regulating EZH2-mediated silencing of p21 and E-cadherin expression.

Long non-coding RNAs (lncRNAs) have been suggested as important regulators of cancer development and progression in hepatocellular carcinoma (HCC). Nevertheless, the clinical value and biological roles of LINC00978 in HCC remain unclear. In this study, we detected the expression of LINC00978 in tumor tissues and serum of HCC patients, examined the roles of LINC00978 in HCC progression and elucidated the underlying molecular mechanisms. We found that LINC00978 expression was upregulated in tumor tissues and serum of HCC patients. Higher serum levels of LINC00978 could distinguish HCC patients from hepatitis and liver cirrhosis patients and healthy controls. LINC00978 knockdown inhibited HCC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. Overexpression of LINC00978 led to the opposite effects. LINC00978 knockdown also inhibited HCC growth and metastasis in mouse tumor models. Mechanistically, LINC00978 bound to EZH2 and mediated its accumulation at the promoter region of p21 and E-cadherin genes, leading to the trimethylation of H27K3 and the inhibition of p21 and E-cadherin expression. Moreover, the simultaneous depletion of p21 and E-cadherin expression reversed the inhibitory effects of LINC00978 knockdown on HCC cell proliferation, migration, and invasion. Taken together, these findings suggest that LINC00978 promotes HCC progression by inhibiting p21 and E-cadherin expression via EZH2-mediated epigenetic silencing. LINC00978 may represent a novel biomarker for HCC diagnosis, prognosis, and therapy.