Hemin-induced reactive oxygen species triggers autophagy-dependent macrophage differentiation and pro-inflammatory responses in THP-1 cells.

The toxic effect of oxidized-heme, also known as hemin, is implicated in developing adverse clinical outcome in various hematolytic diseases. To simulate and reconstruct the molecular events associated with hemin exposure on circulating monocytes, we employed a THP-1 cell line based in vitro model. Flow cytometry and Western blot analyses were subsequently applied. Hemin-treated THP-1 produced ROS in a dose-dependent manner which resulted in 10-30 % of cell death primarily through apoptosis. Surviving cells induced autophagy which too was ROS-dependent, as revealed by application of N-acetyl-L-cysteine. Hemin-mediated autophagy promoted differentiation of CD14+ THP-1 cells into CD11b+ macrophages. Application of 3-methyladenine, reinforced that differentiation of THP-1 was an autophagy-dependent process. It was revealed that despite a higher polarization towards M2-macrophage, synthesis of pro-inflammatory cytokines namely TNF-α, IL-1A, IL-2, IL-8 and IL-17A predominated. IL-6, a pleiotropic cytokine, was also elevated. It may thus be surmised that hemin-induced pro-inflammatory response in THP-1 is downstream to ROS-dependent autophagy and monocyte differentiation. This finding is translationally meaningful as hemin is already approved by FDA for amelioration of acute porphyria and is actively considered as a therapeutic agent for other diseases. This study underscores the need of further research untangling the reciprocal regulation of inflammatory signaling and autophagy under oxidative stress.

Reciprocal interplay between asporin and decorin: Implications in gastric cancer prognosis.

Effective patient prognosis necessitates identification of novel tumor promoting drivers of gastric cancer (GC) which contribute to worsened conditions by analysing TCGA-gastric adenocarcinoma dataset. Small leucine-rich proteoglycans, asporin (ASPN) and decorin (DCN), play overlapping roles in development and diseases; however, the mechanisms underlying their interplay remain elusive. Here, we investigated the complex interplay of asporin, decorin and their interaction with TGFß in GC tumor and corresponding normal tissues. The mRNA levels, protein expressions and cellular localizations of ASPN and DCN were analyzed using real-time PCR, western blot and immunohistochemistry, respectively. The protein-protein interaction was predicted by in-silico interaction analysis and validated by co-immunoprecipitation assay. The correlations between ASPN and EMT proteins, VEGF and collagen were achieved using western blot analysis. A significant increase in expression of ASPN in tumor tissue vs. normal tissue was observed in both TCGA and our patient cohort. DCN, an effective inhibitor of the TGFß pathway, was negatively correlated with stages of GC. Co-immunoprecipitation demonstrated that DCN binds with TGFß, in normal gastric epithelium, whereas in GC, ASPN preferentially binds TGFß. Possible activation of the canonical TGFß pathway by phosphorylation of SMAD2 in tumor tissues suggests its role as an intracellular tumor promoter. Furthermore, tissues expressing ASPN showed unregulated EMT signalling. Our study uncovers ASPN as a GC-promoting gene and DCN as tumor suppressor, suggesting that ASPN can act as a prognostic marker in GC. For the first time, we describe the physical interaction of TGFß with ASPN in GC and DCN with TGFß in GC and normal gastric epithelium respectively. This study suggests that prevention of ASPN-TGFß interaction or overexpression of DCN could serve as promising therapeutic strategies for GC patients.