Page 21 - F. Cell biology

Indole-6-carboxaldehyde prevents oxidative stress-induced DNA damage and apoptosis in
C2C12 skeletal myoblasts by regulating the ROS-AMPK signaling pathway
2,3
1
Cheol Park , Yung Hyun Choi *
1 Division of Basic Sciences, College of Liberal Studies, Dong‐eui University, Busan 47340,
2 Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan 47227,
3 Anti-Aging Research Center, Dong-eui University, Busan 47340, Republic of Korea
BACKGROUND I6CA protected H 2 O 2 -induced mitochondrial
dysfunction in C2C12 cells
Myoblast damage due to oxidative stress is one of the main causes of skeletal muscle loss associated with the inhibition of
myopic differentiation and induction of muscle damage. Indole-6-carboxaldehyde (I6CA), a natural indole derivative
derived from the brown algae Sargassum thunbergii, is known to have several pharmacological activities. However, the
antioxidative effects of I6CA have not been identified. In this study, we investigated that the protective effect of I6CA and its
underlying mechanism in vitro using hydrogen peroxide (H 2 O 2 )-induced oxidative stress in a C2C12 mouse skeletal
myoblast. The findings revealed that pretreatment with I6CA protected H 2 O 2 -induced cytotoxicity and DNA damage by
blockage of ROS generation. Further studies have shown that I6CA suppressed C2C12 cells against H 2 O 2 -induced
apoptosis by preventing loss of mitochondrial membrane potential. I6CA attenuated H 2 O 2 -induced activation of AMPK and
ATP content. Additionally, the cytoprotective effects of I6CA against H 2 O 2 were eliminated by compound C, a specific
AMPK blocker. Collectively, the current results indicate that I6CA was able to protect C2C12 cells DNA damage and
apoptosis from oxidative stress by at least preserving mitochondrial homeostasis mediated through the ROS-AMPK
signaling pathway.
Keywords: Indole 6 carboxaldehyde; DNA damage; apoptosis; AMPK
AIM
Fig. 5. Inhibition of H 2 O 2 -induced mitochondrial dysfunction by I6CA in C2C12 cells.
The cells were treated with 400 μM I6CA or 10 mM NAC for 1 h and then exposed to 1
mM H 2 O 2 for 24 h. (A) The cells were collected and stained with JC-1. The JC-1
In this study, the antioxidant potential of I6CA against oxidative stress (H 2 O 2 )-induced cytotoxicity in fluorescence intensity was detected to evaluate the changes in the MMP using a flow
cytometer. (B) The percentage of cells with JC-1 monomers is indicated by bars, and
immortalized mouse myoblast C2C12 cells was evaluated. the data represent the mean ± SD of triplicate determinations (*** p< 0.001 compared
with the control group; ### p< 0.001 compared with the H 2 O 2 -treated group). (C) JC-1
fluorescence images of the cells treated with 1 mM H 2 O 2 in the presence or absence of
400 μM I6CA are shown. Red fluorescence indicates high membrane potential, and
RESULTS green fluorescence represents low membrane potential. Representative images were
captured using a fluorescence microscope (original magnification, ×400).
I6CA inhibited H 2 O 2 -induced cytotoxicity in I6CA attenuated H 2 O 2 -induced DNA damage in I6CA abolished the change in expression of
C2C12 cells C2C12 cells apoptosis regulatory factors caused by H 2 O 2
in C2C12 cells

Fig. 6. Effects of I6CA on the expression of apoptosis regulators in H 2 O 2 -treated
C2C12 cells. The cells were treated with or without 400 μM I6CA for 1 h before
Fig. 3. Protection of H 2 O 2 -induced DNA damage by I6CA in C2C12 cells. The cells treatment with 1 mM H 2 O 2 for 24 h. (A) Cytochrome c levels were analyzed by Western
Fig. 1. Protective effect of I6CA on H 2 O 2 -induced cytotoxicity in C2C12 cells. The cells were treated with or without 400 μM I6CA for 1 h before treatment with 1 mM H 2 O 2 for blotting on mitochondrial and cytoplasmic fractions isolated from cells. Cytochrome
were treated with the various concentrations of I6CA for 24 h (A), or pretreated with or 24 h. (A) A comet assay was performed, and representative images were captured oxidase subunit VI (COX IV) and actin serve as protein loading controls for the
without the indicated concentrations of I6CA or 10 mM NAC for 1 h, and then cultured using a fluorescence microscope (original magnification, ×200). (B) The cell lysates mitochondria and cytosol, respectively. (B) Whole cell lysates were prepared, and Bax,
in the presence of 1 mM H 2 O 2 for 24 h (B and C). (A and B) The cell viability was were prepared, and p-γH2AX and γH2AX expression was identified by Western blot Bcl-2, caspase-9, caspase-3 and PARP expressions were identified by Western blot
determined by an MTT reduction assay. The results are expressed as the mean ± SD analysis. The equivalent loading of proteins in each well was confirmed by actin. (C) analysis. The equivalent loading of proteins in each well was confirmed by actin. (C
obtained from three independent experiments (* p < 0.05, and *** p < 0.001 compared The DNA samples of cells were subjected to assessment of the 8-OHdG levels. The and D) The activities of caspase-9 (C) and caspase-3 (D) in cell lysates were
with the control group; # p < 0.05 ### p < 0.01 and ### p < 0.001 compared with the measurements were made in triplicate, and the results are expressed as the mean ± measured using the respective substrate peptides. The measurements were made in
H 2 O 2 -treated group). (C) Representative images of the cells were captured by a phase- SD (*** p < 0.001 compared with the control group; ## p < 0.01 compared with the triplicate, and the results are expressed as the mean ± SD (*** p< 0.001 compared
contrast microscope (original magnification, 200×). H 2 O 2 -treated group). with the control group; ### p< 0.001 compared with the H 2 O 2 -treated group).
I6CA suppressed H 2 O 2 -induced ROS I6CA reduced H 2 O 2 -induced apoptosis in AMP-activated protein kinase (AMPK) was
generation in C2C12 cells C2C12 cells involved in the mitigation of H 2 O 2 -mediated
cytotoxicity by I6CA in C2C12 cells

Fig. 4. Inhibitory effect of I6CA on H 2 O 2 -induced apoptosis in C2C12 cells. The cells
were treated with or without 200 or 400 μM I6CA for 1 h before treatment with 1 mM
H 2 O 2 for 24 h. (A) The cells were stained with DAPI solution and stained nuclei were
observed using a fluorescence microscope (original magnification, ×200). Each image
Fig. 2. Attenuation of H 2 O 2 -induced ROS generation by I6CA in C2C12 cells. The cells is representative of at least three independent experiments. (B and C) The cells were Fig. 7. The relevance of the AMPK signaling system in the inhibition of H 2 O 2 -induced
were pretreated with 400 μM I6CA or 10 mM NAC for 1 h and then stimulated with or fixed and stained with annexin V-FITC and PI for flow cytometry analysis. (B) The cytotoxicity by I6CA in C2C12 cells. The cells were treated with 400 μM I6CA, 10 mM
without 1 mM H 2 O 2 for an additional 1 h. The medium was removed, and the cells were results show early apoptosis, defined as annexin V + and PI - cells (lower right quadrant), NAC or 5 μM compound C for 1 h, and then exposed to 1 mM H 2 O 2 for 24 h. (A) The
incubated with medium containing DCF-DA for 30 min. (A) ROS production was and late apoptosis, defined as annexin V + and PI + (upper right quadrant) cells, and cells were collected and the cellular ATP concentrations were measured using an ATP
measured using a flow cytometer, and representative profiles are shown. (B) The representative profiles are shown. (C) The percentages of apoptotic cells were determination kit. (B) Equal amounts of proteins were subjected to Western blot
measurements were made in triplicate, and the values are expressed as the mean ± determined by expressing the numbers of annexin V + cells as percentages of all the analysis of the listed proteins. Actin was used as an internal control. (C) The cell
SD (*** p< 0.001 compared with the control group; ### p< 0.001 compared with the present cells. The data represent the mean ± SD of three independent experiments viability was determined by MTT assay. (A and C) The results are expressed as the
H 2 O 2 -treated group). (C) DCF fluorescence images of cells cultured under the same (*** p < 0.001 compared with the control group; ### p < 0.001 compared with the H 2 O 2 - mean ± SD of three independent experiments (*** p < 0.001 compared with the control
conditions were captured by a phase-contrast microscope (original magnification, treated group). (D) DNA fragmentation was analyzed by extracting genomic DNA, group, ### p < 0.001 compared with the H 2 O 2 -treated group, &&&p < 0.001 compared
200×). Each image is representative of at least three independent experiments. electrophoresis in 1.5% agarose gel, and then visualizing by EtBr staining. with the I6CA and H 2 O 2 -treated group).
METHODS CONCLUSION REFERENCES
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Author email address process require further study. Nutr Food Res 53:1187-1204.
Cheol Park: parkch@deu.ac.kr, Yung Hyun Choi: choiyh@deu.ac.kr