Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells

You Cheng Hseu, Elong Lin, Jing Yi Chen, Yi Ru Liua, Chun Yin Huang, Fung Jou Lu, Jiunn Wang Liao, Ssu Ching Chen, Hsin Ling Yang

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 μg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (ΔΨm), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.

Original languageEnglish
Pages (from-to)243-258
Number of pages16
JournalEnvironmental Toxicology
Volume24
Issue number3
DOIs
StatePublished - Jun 2009

Keywords

  • Apoptosis
  • Cell cycle arrest
  • Humic acid
  • Smooth muscle cells

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