Lunasin, a bioavailable food peptide, exerts protective
effects against oxidative stress in human liver cells
S. Fernández-Tomé1, S. Ramos2, I. Cordero-Herrera2, I. Recio1, L. Goya2, B. Hernández-Ledesma1*
1 Instituto
de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Madrid, Spain. 2 Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN, CSIC), Madrid, Spain.
* b.hernandez@csic.es
BACKGROUND
METHODS
• Lunasin is a 43-amino acid peptide which chemopreventive properties
have been demonstrated both in vitro and in vivo studies1.
Effects on cell viability and biomarkers of redox status
Lunasin
Direct effects
• Bioavailability studies have shown that lunasin remains intact in a high
percentage during its passage through the gastrointestinal tract reaching
different target organs, such as liver, in an intact and active form2.
Analysis
t-BOOH
Chemical-induced
oxidative stress
• Reactive oxidative species (ROS) accumulate in cells playing a key role in
the induction and progression of several diseases related to the oxidative
damage to liver tissues3.
Lunasin
+
OBJECTIVES
t-BOOH
Chemo-protective
effects
HepG2
cells
To evaluate the potential chemo-protective effects of lunasin against
tert-butyl hydroperoxide (t-BOOH) induced oxidative stress in human liver
HepG2 cells.
Stability of lunasin
Lunasin:
incubation times (h)
Cell
supernatants
To investigate the stability of lunasin, and to identify lunasin derived
fragments released in HepG2 cultures.
RESULTS
Table 1. Direct effects of lunasin on non-stressed HepG2 cells. Cell viability was measured by the crystal
violet assay (n = 12). Fluorescence units correspond to intracellular ROS generation (n = 8). GSH
intracellular levels were calculated as nmoles of GSH per mg of protein (n = 6). All results are mean ± SD,
and are represented as percent of control non-stressed cells. *, significantly different from control cells.
Cell viability
(% control)
Intracellular ROS generation
(% control)
Intracellular GSH levels
(nmol/mg prot, % control)
Control
100.00 ± 8.41
100.00 ± 7.02
100.00 ± 7.85
0.5 μM lunasin
107.15 ± 2.73
76.17 ± 5.59 ***
118.83 ± 21.04
1 μM lunasin
111.37 ± 3.52
63.98 ± 5.61 ***
123.74 ± 10.04 *
5 μM lunasin
109.88 ± 3.68
63.61 ± 5.41 ***
122.13 ± 12.31 *
10 μM lunasin
107.26 ± 8.02
71.75 ± 10.82 ***
125.62 ± 6.18 *
A
***
50
150
###
###
###
###
100
50
0
0
0.5
1
5
t- BOOH
0.5
***
250
#
###
150
100
50
1
5
0.5
1
5
100
###
50
10
###
###
5.00E+07
###
***
4.00E+07
t- BOOH
0.5
E
***
300
250
200
#
##
##
150
1
5
10
3.00E+07
Lunasin (μM)
##
100
50
350
F
***
300
2.00E+07
250
200
###
150
1.00E+07
###
###
100
0.00E+00
###
50
0
0
C
t- BOOH
0.5
1
5
10
C
t- BOOH
0.5
Lunasin (μM)
Lunasin (μM)
Figure 2. Protective effects of lunasin on t-BOOH-stressed cells. (A) Cell viability
was measured by the crystal violet assay (n = 12). (B) Fluorescence units
correspond to intracellular ROS generation (n = 8). (C) GSH intracellular levels
were calculated as nmoles of GSH per mg of protein (n = 6). (D) GPx activity was
calculated as mUnits per mg of protein (n = 6). (E) CAT activity was calculated as
mUnits per mg of protein (n = 4). (F) Protein carbonyl content was calculated as
nmol per mg protein (n = 4). (G) Caspase-3 activity was calculated as Units per μg
of protein (n = 4). All results are mean ± SD, and are represented as percent of
control non-stressed cells (C). *, significantly different from control non-stressed
cells. # , significantly different from t-BOOH-stressed cells.
1
5
10
2
Lunasin (μM)
250
(U/μg prot, % control)
t- BOOH
6.00E+07
C
0
C
C
10
Caspase-3 activity
0
(mU/mg prot, % control)
350
200
7.00E+07
Lunasin (μM)
CAT
(mU/mg prot, % control)
GPx
Lunasin (μM)
D
150
Peak Area
(AU)
0
C
10
(nmol/mg prot, % control)
t- BOOH
Protein carbonyl content
C
300
(nmol/mg prot, % control)
100
***
GSH
###
###
B
200
(% control)
###
###
ROS generation
(% control)
Viable cells
250
A
D
Figure 1. Morphological analysis of HepG2 cells. Representative images of (A) non-stressed cells pre-incubated
with medium for 20h, (B) t-BOOH-induced (400 μM, 3h) cells pre-incubated with medium for 20h, and t-BOOHinduced (400 μM, 3h) cells pre-incubated for 20h with (C) 0.5 μM lunasin, and (D) 5 μM lunasin.
Treatment of non-stressed cells with lunasin (0.5-10μM) for 20h did not damage cell
integrity, decreased intracellular ROS generation, and enhanced cytosolic levels of GSH.
150
C
B
6
12
20
Incubation time (h)
G
***
200
Lunasin
150
###
0
0.5
1
F3, f(29-43)
F4, f(26-43)
F5, f(25-43)
###
50
t- BOOH
F2, f(30-43)
###
100
C
F1, f(32-43)
5
10
Lunasin (μM)
Pre-treatment of t-BOOH-induced cells with lunasin (0.5-10μM) for 20h restored cell viability and GSH levels,
quenched intracellular ROS and carbonyl groups generation, regulated antioxidant enzymes activities, and
prevented the apoptotic effects induced by disruption of the redox steady-state.
Figure 3. Stability of peptide lunasin in medium added to HepG2 cells and identification
of lunasin derived fragments. Relative amount (expressed as peak area) was calculated
from extracted ion chromatogram of the molecular ion of lunasin m/z 1257.5 (charge +4),
f(32-43) (F1) m/z 1324.5 (charge +1), f(30-43) (F2) m/z 1565.5 (charge +1), f(29-43) (F3)
m/z 1693.8 (charge +1), f(26-43) (F4) m/z 1034.1 (charge +2), and f(25-43) (F5) m/z 1102.7
(charge +2) in medium incubated with 10 μM lunasin and collected after 0, 2, 6, 12, and
20 h-incubation.
Content of lunasin in the medium of HepG2 cells notably decreased with the
incubation time, while five lunasin derived fragments were generated.
CONCLUSIONS
 Lunasin prevented the increased ROS generation and the depletion of GSH, modulated the GPx and CAT activities, and evoked a decline in
carbonyl groups and a recovery from cell death by apoptosis.
 Five major lunasin-derived fragments released by cell metabolism have been identified as part of cellular response to lunasin treatment.
 Lunasin and its derived-fragments, at physiological concentrations, might confer a significant chemoprotection against oxidative stressassociated liver disorders.
REFERENCES
(1) Hernández-Ledesma, B., Hsieh, C.-C., & de Lumen, B. O. (2013) Chemopreventive properties of peptide lunasin: a review. Protein Peptide Lett. 20, 424-432.
(2) Hsieh, C.-C., Hernández-Ledesma, B., Jeong, H.J., Park, J.H, & de Lumen, B.O. (2010) Complementary roles in cancer prevention: protease inhibitor makes the
cancer peptide lunasin bioavailable. PLoS ONE 5, e8890.
(3) Vitaglione, P., Morisco, F., Caporaso, N., & Fogliano, V. (2004) Dietary antioxidant compounds and liver health. Crit. Rev. Food Sci. Nutr. 44, 575-86.
ACKNOWLEDGEMENTS
This work was supported by projects AGL2010-17579, AGL2011-24643, CSD2007-00063 from Programa Consolider-Ingenio from the Spanish Ministry
of Education and Science (CICYT), FP7-SME-2012-315349 (FOFIND), and FEDER-INNTERCONECTA-GALICIA (ENVELLEFUN). The authors are participants
in the FA1005 COST Action INFOGEST on food digestion. I. C. -H. and S. F. -T. acknowledge Ministry of Economy and Competitiveness (MINECO) for
their FPI fellowships, and B. H. -L. acknowledges MINECO for her “Ramon y Cajal” contract.