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

• Lee GW
• Kim MS

• KMID: 2071663
• DOI: http://doi.org/10.4196/kjpp.2009.13.3.139

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 micrometer 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 micrometer 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.

Keyword

Samultang; Apoptosis; Oxidative injury; SK-N-MC

MeSH Terms

Apoptosis
Asian Continental Ancestry Group
Caspase 3
Cell Death
DNA
Drugs, Chinese Herbal
Electrophoresis
Humans
Membrane Potentials
Optical Imaging
Oxidative Stress
Sepharose
Water
Caspase 3
DNA
Drugs, Chinese Herbal
Sepharose
Water

Figure

• 

  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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