Effect of AM114 on apoptosis and proinflammatory cytokines in IL-1-beta treated human THP-1 derived macrophages
Authors: Selvarajan Chitra , Konda Mohan Vasanth , Ganesan Nalini , Kalaivani MK 
Published: May 2023

AbstractBackgroundChalcones and their derivatives are precursors of flavonoids and isoflavonoids abundantly present in edible plants. It displays a wide range of pharmacological activities, such as anticancer, antiinflammatory, antibacterial and antioxidant. AM-114 (3,5-Bis-[benzylidene-4-boronic acid]-1-methylpiperidin-4-one), a boronic-chalcone derivative exhibits potent anticancer activity through inhibition of the proteasome on human colon cancer cell line. However, the cytotoxic and anti-inflammatory effects of AM114 on THP-1 derived macrophages remain to be studied.Aim of the studyInflammation is a complex reaction managed by a variety of immune cells such as monocytes and macrophages. In response to inflammatory stimuli, the macrophages secrete increased proinflammatory cytokines, chemokines such as interleukin-6 (IL-6) and interleukin-8(IL-8). The proteasome complex is essential for several cellular processes including protein degradation, cellular differentiation and antigen presentation. In this study, human monocyte cell line THP-1 stimulated with interleukin-1-beta (IL-1-beta) was used as a model to investigate thein vitroeffects of AM114, a proteasome inhibitor (PI), on apoptosis and release of proinflammatory cytokines.Materials and methodsThe effect of AM114, with or without IL-1-beta stimulated THP-1derived macrophages, was assessed by comparing with aspirin. The cell viability was determined by MTT assay. The qualitative measurement of apoptosis was determined by acridine orange/ ethidium bromide (AO/EtBr), Hoechst 33342 staining and rhodamine 123 assays. The quantitative measurement of apoptosis was carried out to determine the caspase-3 activity by spectrofluorimetric method and DNA fragmentation by agarose gel electrophoresis. The release of proinflammatory cytokines and chemokines such as IL-6 and IL-8 was also measured by enzyme linked immunosorbent assay (ELISA) method. The ANOVA was used to compare the different groups.ResultsIn the present study the IC50concentration of AM114 and Aspirin on THP-1 cells were determined as 3.6 micromolar and 3900 micromolar respectively. The THP-1 derived macrophages treated with IL-1-beta showed insufficient apoptosis and increased release of proinflammatory cytokines. However the treatment of AM114, on stimulated THP-1 derived macrophages showed pronounced apoptotic features. The caspase-3 activity was increased 1.9 fold in AM114 with IL-1-beta treated THP-1 derived macrophages as compared to the unstimulated THP-1 derived macrophages pretreated with AM114. An increase of 3.2 fold in caspase-3 activity was observed in stimulated THP-1 derived macrophages pretreated with AM114 in comparison with stimulated THP-1 derived macrophages pretreated with aspirin. A significant increase of 5.0 and 3.0 fold DNA damage were observed in stimulated THP-1 derived macrophages treated with AM114 and aspirin as compared to unstimulated cells. Moreover AM114 treated cells showed significant decrease (p value less than 0.001) in the release of IL-6 and IL-8 on stimulated THP-1 derived macrophages.ConclusionThe results on the induction of apoptosis and suppression of proinflammatory cytokines suggest that AM114 is more effective than aspirin in stimulated THP-1 derived macrophages.