Molecular and Cellular Biochemistry
in 100 μL of binding bufer, and stained with 5 μL of FITC
Annexin V labeling reagent and 5 μL of PI for 15 min in
dark. Finally, the samples were assessed with a FACSCanto
fow cytometer using Cell Quest software. In our study,
apoptotic cells included cells with early (Annexin V+, PI−)
and late (Annexin V+, PI+) apoptosis, while viable cells
were negative for both Annexin V and PI.
Statistical analysis
All results are presented as mean SEM from at least three
independent experiments. Statistical evaluation was per-
formed with one-way ANOVA and Dunnett’s post hoc test
using GraphPad Prism Software Version 4.03 (San Diego,
CA, USA). Diferences were considered to be signifcant for
p values≤0.05.
Western blot analysis of p53, Bax, and Bcl‑xl protein
levels
Results
The immunoblot assay was used to determine the level of
p53, Bax, and Bcl-xl proteins. Briefy, after 24 h culture with
various concentrations of methoxy stilbenes or RSV, cell
lysates were obtained by extracting HL-60 and THP-1 cells
with RIPA bufer. The concentration of total proteins was
determined using the Bradford protein assay (Bio-Rad Labo-
ratories, Hercules, CA, USA). Fifty micrograms of total pro-
tein from each concentration was resolved by electrophoresis
using 12% or 10% SDS-PAGE, followed by electrotransfer
onto a polyvinyl difuoride transfer membrane (Immobilon-
P, Millipore). After blocking with 10% skimmed milk, the
proteins were probed with anti-human p53, Bax, and Bcl-
xl antibodies. The β-actin protein was used as an internal
control. As the secondary antibodies in the staining reac-
tion, alkaline phosphatase-labeled anti-goat IgG, anti-mouse
IgG, or anti-rabbit IgG was used. Bands were visualized
with the Bio-Rad AP Conjugate Substrate Kit NBT/BCIP.
The amount of the immunoreactive product in each lane was
determined using the Quantity One software (Bio-Rad Labo-
ratories). Values were calculated as relative absorbance units
(RQ) per mg protein.
Efect of RSV and methoxy stilbenes on cell viability
The efect of the tested methoxy stilbenes and RSV on the
viability of HL-60 and THP-1 cells was assessed using the
MTT assay. Table 1 presents the IC50 values obtained after
icant diferences were noted in the efect of these compounds
on the viability of HL-60 and THP-1 cells. The IC50 values
in HL-60 cells were approximately two times lower than
those obtained in THP-1 cells. Moreover, the cytotoxicity of
RSV in comparison with its methoxy analogs in HL-60 cells,
particularly after 48 h treatment, did not difer signifcantly.
In contrast, THP-1 cells were more susceptible to the cyto-
toxic efect of RSV analogs than to the parent compound.
The highest cytotoxicity was shown by 3,4,4ʹ-tri-MS fol-
lowed by 3,4,2ʹ,4ʹ-tetra-MS.
The efect of RSV and methoxy stilbenes on cell
cycle distribution
To assess the effect of RSV and methoxy stilbenes on
cell cycle distribution, the HL-60 and THP-1 cells were
treated with these compounds for 24 h, and the cell cycle
Table 1 Efect of resveratrol and its derivatives on human HL-60 and THP-1 cell viability (IC50; μM)
Tested compounds
IC50 (μM)
HL-60
24 h
THP-1
24 h
48 h
48 h
3,5,4′-trihydroxy-trans-stilbene (Resveratrol)
3,4,4′-trimethoxy-trans-stilbene (3,4,4′-tri-MS)
3,4,2′-trimethoxy-trans-stilbene(3,4,2′-tri-MS)
3,4,2′,4′-tetramethoxy-trans-stilbene (3,4,2′,4′-tetra-MS)
3,4,2′,6′-tetramethoxy-trans-stilbene (3,4,2′,6′-tetra-MS)
3,4,2′,4′,6′-pentamethoxy-trans-stilbene (3,4,2′4′,6′-penta-MS)
59.98 1.54
51.36 2.09*
58.27 1.47
55.35 2.23
61.32 3.80
56.85 1.17
46.84 1.92
41.32 1.70
46.34 1.89
43.01 2.68
40.00 3.04
44.52 2.40
130.05 1.37#
97.25 3.11*,#
121.41 2.08#
117.35 3.68*,#
127.60 3.24#
118.52 3.05#
79.02 1.63##
62.81 1.83*,##
75.20 2.64##
70.48 2.45##
77.26 3.17##
77.71 1.91##
The cells were treated for 24 and 48 h with diferent concentrations of methoxy stilbenes or resveratrol and IC50 values were obtained by plot-
ting log (% inhibition/100−% inhibition) vs log (concentration), where % inhibition=(100−viability) based on means SEM from three inde-
pendent experiments
#
*p<0.05 compared to resveratrol treated cells; p<0.05 compared to HL-60 cells treated for 24 h; ##p<0.05 compared to HL-60 cells treated
for 48 h
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