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

Background

Chalcones 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 study

Inflammation 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 methods

The 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.

Results

In 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.

Conclusion

The 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.