Korean J Gynecol Oncol.  2008 Mar;19(1):48-56. 10.3802/kjgo.2008.19.1.48.

Induction of apoptosis by Hibiscus protocatechuic acid in human uterine leiomyoma cells

• Shon WK
• Cho CH
• Ramachandran S
• Song DK
• Shin SJ
• Kwon SH
• Cha SD

• KMID: 1979501
• DOI: http://doi.org/10.3802/kjgo.2008.19.1.48

Abstract

OBJECTIVE
Hibiscus protocatechuic acid (PCA) is a food-derived polyphenol antioxidants used as a food additive and a traditional herbal medicine. In this study, PCA was to determine its effect on cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells.
METHODS
The effect of PCA on cell proliferation and cell cycle progression was examined in the primary cultured human uterine leiomyoma cells. MTT reduction assay was carried out to determine the viability of uterine leiomyoma cells. Cell cycle analysis for Hibiscus protocatechuic acid treated leiomyoma cells was done by FACS analysis. DNA fragmentation assay was performed to determine fragmentation rate by PCA in leiomyoma cells. Western blot analysis was done using anti pRB, anti-p21(cip1/waf1), anti-p53, anti-p27(kip1), anti-cyclinE, anti CDK2 antibodies to detect the presence and expression of these proteins in PCA treated myoma cells.
RESULTS
PCA induced growth inhibition in a dose dependent manner, treatment with 5 mmol/L PCA blocked 80% cell growth. FACS results showed that there was increased the percentage of cells in sub G1. DNA fragmentation assay by ELISA was done to find the rate of apoptosis. Apoptosis took place but in a dose dependent manner. From Western blot analysis it revealed PCA induced the expression of p21(cip1/waf1) and p27(kip1) increasingly and was not mediated by p53. Caspase-7 pathway was activated and dephosphorylation of pRB took place.
CONCLUSION
In Conclusions, PCA, a polyphenol antioxidant, inhibited cell proliferation and induced cell cycle arrest at sub G1 phase by enhancing the production of p21cip1/waf1 and p27kip1. These results indicate that PCA will be a promising agent for use in chemopreventive or therapeutics against human uterine leiomyoma.

Keyword

Hibiscus protocatechuic acid; Uterus; Leiomyoma; Apoptosis

MeSH Terms

Antibodies
Antioxidants
Apoptosis
Blotting, Western
Caspase 7
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
DNA Fragmentation
Enzyme-Linked Immunosorbent Assay
Food Additives
G1 Phase
Herbal Medicine
Hibiscus
Humans
Hydroxybenzoates
Hypogonadism
Leiomyoma
Mitochondrial Diseases
Myoma
Ophthalmoplegia
Passive Cutaneous Anaphylaxis
Proteins
Uterus
Antibodies
Antioxidants
Caspase 7
Food Additives
Hydroxybenzoates
Hypogonadism
Mitochondrial Diseases
Ophthalmoplegia
Proteins

Figure

• 

  Fig. 1. Antiproliferative effect of PCA on human uterine leiomyoma cells. Growth inhibition in uterine leiomyoma cells treated with an indicated dose for indicated hs. Cell viability was measured using Cell Titer Cell Proliferation Assay and expressed as % cell survival of control cells. Data represent the mean±SE of three independent experiments (Key: *p<0.05).

• 

  Fig. 2. Growth inhibition in uterine leiomyoma cells treated with 2 mmol/L for indicated hs. Cell viability was measured using Cell Titer cell Proliferation Assay and expressed as % of control culture conditions (Key: *p<0.05).

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  Fig. 3. Effect of PCA treatment on the cell cycle profile. After treatment with an indicated dose of PCA for 0 or 24 hours, uterine leiomyoma cells were collected, fixed, stained with PI and analyzed by flow cytometry. The values represent the number of cells in a phase of the cell cycle as a percentage of total cells.

• 

  Fig. 4. DNA fragmentation assay of PCA on cell growth of human uterine leiomyoma cells. Cells were seeded at a density of 5,000 cells/well in 96-well culture plate. Cells were then treated with a indicated concentrations of PCA for 24 hrs. Apoptosis was estimated by an ELISA method. *p<0.05.

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  Fig. 5. Effect of the PCA treatment on the cell cycle related gene expression. Tubulin Beta was shown as an internal control.

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  Fig. 6. Effect of PCA on caspase activation, PARP, pRB, and Bcl-2 expression. Tubulin Beta was shown as an internal control.

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