Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells.
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 microg/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 (Delta Psi m), 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.
Hseu
You-Cheng
YC
Department of Cosmeceutics, China Medical University, Taichung, Taiwan.
Lin
Elong
E
Chen
Jing-Yi
JY
Liua
Yi-Ru
YR
Huang
Chun-Yin
CY
Lu
Fung-Jou
FJ
Liao
Jiunn-Wang
JW
Chen
Ssu-Ching
SC
Yang
Hsin-Ling
HL
D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D024161:Collagen Type XI / Q000378:metabolism; D019938:Cyclin D1 / Q000187:drug effects / Q000235:genetics; D019927:Cyclin E / Q000187:drug effects / Q000235:genetics; D051357:Cyclin-Dependent Kinase 2 / Q000187:drug effects / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4 / Q000187:drug effects / Q000235:genetics; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D016193:G1 Phase / Q000187:drug effects*; D006812:Humic Substances / Q000633:toxicity*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D016160:Retinoblastoma Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C499319:COL11A2 protein, human; D024161:Collagen Type XI; D019927:Cyclin E; D006812:Humic Substances; D019253:Proto-Oncogene Proteins c-bcl-2; D016160:Retinoblastoma Protein; D051028:bcl-2-Associated X Protein; D019938:Cyclin D1; D045304:Cytochromes c; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D020169:Caspases