Water Extract of Samultang Reduces Apoptotic Cell Death by H2O2-Induced Oxidative Injury in SK-N-MC Cells

Gyoung Wan Lee; Min Sun Kim

doi:10.4196/kjpp.2009.13.3.139

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Lee and Kim: Water Extract of Samultang Reduces Apoptotic Cell Death by H2O2-Induced Oxidative Injury in SK-N-MC Cells

Original Article

Korean J Physiol Pharmacol 2009;13(3):139-145.

Published online: 17 June 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.3.139

Water Extract of Samultang Reduces Apoptotic Cell Death by H₂O₂-Induced Oxidative Injury in SK-N-MC Cells

Gyoung Wan Lee, Min Sun Kim

Department of Physiology, Wonkwang University School of Medicine, Iksan 570-749, Korea

Corresponding to: Min Sun Kim, Department of Physiology, Wonkwang University School of Medicine, 344-2, Shinyong-dong, Iksan 570-749, Korea. (Tel) 82-63-850-6779, (Fax) 82-63-852-6108, (E-mail) mskim@wku.ac.kr

Received 15 March 200916 June 2009 Accepted 19 June 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The purpose of this study was to evaluate the effects of the water extract of Samultang (SMT), a Chinese herb, on apoptotic cell death by H₂O₂-induced oxidative stress in SK-N-MC cells. A nuclear fragmentation was observed via fluorescence imaging 12 h after exposure to 30 μM H₂O₂ and DNA laddering was detected via agarose electrophoresis gel. In addition, increases in sub-G1 phase and cleavage of the PARP protein were observed. However, treatment with SMT for 2 h prior to H₂O₂ exposure significantly reduced apoptotic cell death induced by incubation with 30 μM H₂O₂ in SK-N-MC cells. Pre-incubation with water extract of SMT for 2 h prevented the H₂O₂-induced decrease in mitochondrial transmembrane potential. SMT also attenuated the increase in caspase-3 activity and the breakdown of PARP protein caused by H₂O₂-induced oxidative stress. These results suggest that the water extract of SMT provides inhibition of apoptotic cell death against oxidative injury in SK-N-MC cells.

Keywords: Samultang, Apoptosis, Oxidative injury, SK-N-MC

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Fig. 1.

Effects of H₂O₂ or SMT on cell viability in SK-N-MC cells. (A) Dose-response relationship of H₂O₂ on cell viability, (B) time-dependent relationship of H2O2 (30 μM) on cell viability, (C) dose-response relationship of SMT, (D) effect of SMT pretreatment on H₂O₂-induced cytotoxicity in SK-N-MC cells. The various concentrations of SMT were given 2 h before 30 μM H₂O₂ treatment. Cell viability was determined by MTT assay after 12 h H₂O₂ treatment. Values are mean±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05, ∗∗p<0.01).

Fig. 2.

Photographs of DAPI staining showing changes in DNA morphology of SK-N-MC cells. (A) Control cells not treated with H₂O₂, (B) cells treated with 30 μM H₂O₂ for 12 h, (C) cells treated with SMT (300 μg/ml) 2 h prior to 30 μM H₂O₂, (D) bar histogram showing percent of cells showing abnormal nuclear morphology counted in 3 randomly selected fields. A large amount of DNA was condensed or fragmented in cells treated only with H₂O₂ (arrows), but pretreatment with SMT significantly reduced DNA abnormalities. Values are mean±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05).

Fig. 3.

Flow cytometric analysis showing changes in cell cycle of SK-N-MC cells. (A) Control, (B) treatment with 30 μM H₂O₂, (C) SMT (300 μg/ml) treatment 2 h prior to exposure to H₂O₂ for 12 h, (D) bar histogram showing percent changes of sub-G1 phase in cell cycle. Values are mean± S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05).

Fig. 4.

Agarose gel electrophoresis of DNA extracted from SK-N-MC cells. Lane A, DNA marker (100 bp); Lane B, control; Lane C, treatment with 30 μM H2O2 for 12 h; Lane D, SMT (300 μg/ml) treatment 2 h prior to exposure to H₂O₂ for 12 h.

Fig. 5.

Flow cytometric analysis showing changes in the mitochondrial membrane potential of SK-N-MC cells. (A) Control, (B) treatment with 30 μM H₂O₂, (C) SMT (300 μg/ml) treatment 2 h prior to exposure to H₂O₂ for 12 h, (D) bar histogram showing the percent changes of relative fluorescence intensity (RFI) of mitochondrial membrane potential in control, H₂O₂-only treated, and SMT plus H₂O₂ treated SK-N-MC cells. Values are means±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05).

Fig. 6.

RT-PCR analysis of caspase-3 mRNA expression from the cDNA of SK-N-MC cells 3 h after H₂O₂ treatment. Control, cells not treated with H₂O₂; H₂O₂, cells treated with 30 μM H₂O₂ for 3 h; SMT-H₂O₂, cells treated with 300 μg/ml SMT 2 h prior to 30 μM H₂O₂. Values are means±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05).

Fig. 7.

Changes in caspase-3 enzyme activity from whole cell lysate of SK-N-MC cells. (A) Caspase-3 enzyme activity at 6 and 12 h after 30 μM H₂O₂ treatment, (B) effect of SMT (300 μg/ml) pretreatment on caspase-3 enzyme activity 12 h after H2O2 treatment. Values are means±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and H₂O₂ + SMT (∗p<0.05, ∗∗p<0.01).

Fig. 8.

Western blot analysis for expression of the PARP protein from whole cell lysates of SK-N-MC cells 12 h after H₂O₂ treatment. Control, cells not treated with H₂O₂; H₂O₂, cells treated with 30 μM H₂O₂ for 12 h; SMT-H₂O₂, cells treated with 300 μg/ml SMT 2 h prior to 30 μM H₂O₂. Values are means±S.D. of each triplet (n=3). ∗Denotes a significant difference between H₂O₂ and SMT-H₂O₂ (∗p<0.05, ∗∗p<0.01).

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