Objectives: 

Some evidence suggest that perturbations in the control of free arachidonic acid (AA) levels within the cells may provide important cellular signals for the onset of apoptosis. Our previous results showed that melatonin (ML) inhibits AA release in transformed macrophages: RAW 264.7 cells. The objective of this study is to establish the effects of ML on the cell proliferation and on the cell apoptosis using RAW 264.7 activated by bacterial lipopolysaccharides (LPS).

Materials: 

Materials. Bromoenol lactone (BEL) from Cayman Chemical Co., ML, LPS, and all other reagents from Sigma. Cell culture. RAW 264.7(ATCC: TIB-71) cells were maintained in DMEM plus 10% FCS, 2mM Glu, 100 units/mL penicillin and 100 mg/mL streptomycin. The cells were incubated at 37 °C in atmosphere of 5 % CO2. Cellular proliferation. The cells, in 96-well plastic culture dishes, were treated with Cell Titer 96 from Promega, after 24 h of the appropriate stimulus addition. Absorbance at 490 nm was measured in a Versa Max Tunable dishes lector. Measurement of Apoptosis. Apoptosis was analyzed by labelling the cells with 4,6-diamine-2-fenilindol (DAPI) by the standard technique. Data Presentation. The data are presented as the Mean Value ±SD of at least 3 representative assays. T-test is applied.

Results: 

ML (1mM) inhibits cellular proliferation by 54 % of the control cells (P<0.001). Cellular proliferation LPS (200 ng/mL)-induced is twice the control value, and it is been reduced by ML to the control level (P<0.001). BEL 15 mM (an apoptosis-inductor in some cell lines), produces apoptosis in RAW 264.7 cells. ML produces similar increment of apoptosis as BEL does versus the control cell results.

Conclusions: 

ML significantly decreases cellular proliferation, and the cellular proliferation LPS-induced. ML induces apoptosis similar as BEL does on RAW 264.7 cells. These results suggest that ML can inhibit transformed cell proliferation by apoptosis and that the process may be linked to the ML inhibition of AA release.