Curcumin-induced apoptosis in 3T3L1 cells
723
kinase (AMPK)) in adipocytes and cancer cells (19) and cause apoptosis in several
cell lines (20, 21). The safety of curcumin was proved previously (17, 25- 27)
The current study was performed to investigate the underlying molecular
mechanism of curcumin inhibition of 3T3-L1 adipocytes. The study will aid further
molecular signals involved in 3T3L1 adipocytes apoptosis and supports the
potential use of Curcumin therapy in obesity resistance.
2. MATERIALS AND METHODS
2.1. Cell culturing and treatment.
The 3T3-L1 cells were obtained from American Type Culture Collection
(ATCC-CL-173) and maintained at 37°C in a 5% CO2-moistened environment.
3T3-L1 cells were cultured in Dulbecco’s minimum essential medium (DMEM) at
a density of 5 × 105 and supplemented with L- glutamine, 10% heat-inactivated
fetal bovine serum (FBS), and penicillin-streptomycin (Gibco, Invitrogen
Corporation, Carlsbad, CA, USA). After cells reached 70% confluence, Then cells
were collected and sub cultured using a standard trypsin /EDTA method (28 - 30 ).
Different concentrations of curcumin were dissolved in DMSO for cells treatment
as well as with 40 µM for different periods (h).
2.2. Cell viability assay.
MTT ( 3-(4, 5- dimethylthiazol-2-yl)-2, 5- diphenyl tetrasodium bromide)
reagent was used for detecting the cell death induce by curcmin treatment
according to the previously reported method (31). Cells were treated with
different doses of curcumin for 24 h, followed by the addition of MTT reagents and
finally DMSO . After discarding unbound stain, the crystalised formazan was
dissolved and spectrophotometrically measured at 570 nm. Growth inhibition %
was presented, with control growth as 100%, viability% was calculated
(sample/control × 100) (32). Cell culturing was repeated to evaluate the dose-
dependent effect of curcumin using concentrations of (5, 10, 20,
40, 60, and 80 μM) for 24 h. Ultimately, 40 μM curcumin was used to evaluate the
time-dependent effect of treatment.
2.3. Flow cytometric analysis of cell cycle status.
After treatment with (40 µM) curcumin for different time periods (4, 8 ,12,
and 24 h), cell cycle progress was examined by fluorescence-activated cell sorting
(FACS). In brief, cells were collected by centrifugation. For fixation cells were
vortexed slowly with 70% ethanol and kept at 4°C overnight, stained with
propidium iodide (PI) reagents, the stained DNA content was then measured using
a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as previously
described(29).
