Indigo naturalis and its component tryptanthrin exert anti-angiogenic effect by arresting cell cycle and inhibiting Akt and FAK signaling in human vascular endothelial cells.
BACKGROUND
Indigo naturalis has been used to treat inflammatory diseases and dermatosis, including psoriasis, since thousands of years in China. It has been proven effective in our previous clinical studies on treating psoriasis, but the active component and the mechanism of how indigo naturalis working still needs to be clarified. Since the dysregulated angiogenesis is known to play an important role in the pathogenesis of psoriasis, the anti-angiogenic effect of indigo naturalis and tryptanthrin, a pure component of indigo naturalis, was investigated.


METHODS
The in vivo angiogenesis was studied by chick chorioallantoic membrane assay. The in vitro studies were performed using human vascular endothelial cells. Cell viability was determined by MTT assay. Cell cycle distribution was revealed by flow cytometry. The cellular messenger (m)RNA or protein expression level was analyzed by real-time RT-PCR or Western blot, respectively. Transwell filter migration assay and matrix gel-induced tube formation method were applied to examine the angiogenic potential.


RESULTS
Indigo naturalis significantly inhibited the in vivo vascular endothelial growth factor (VEGF)-induced angiogenesis, as well as tryptanthrin. In vitro studies confirmed that indigo naturalis and tryptanthrin reduced the number of viable vascular endothelial cells. Tryptanthrin resulted in a cell cycle arrest and dose-dependently decreased the expressions of cyclin A, cyclin B, cyclin dependent kinase(CDK) 1 and 2, but not cyclin D and cyclin E, at both the mRNA and protein levels. The migration and tube formation of vascular endothelial cells were significantly inhibited by tryptanthrin in a dose-dependent manner. Result also showed that tryptanthrin could reduce the phosphorylated levels of both protein kinase B (PKB or Akt) and focal adhesion kinase (FAK).


CONCLUSIONS
All together, these results demonstrated the anti-angiogenic effect of tryptanthrin, the acting component of indigo naturalis and revealed the underlying mechanism by inhibiting the cell cycle progression, cell migration and tube formation, likely mediated through blocking the Akt and FAK pathways.
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC; Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan, ROC.
Hsin-Ning
HN
Chang
Department of Chinese Medicine, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Taiwan, ROC.
Sheng-Teng
ST
Huang
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan, ROC.
Yuan-Chieh
YC
Yeh
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC; Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Lin-Kou Medical Center, Tao-Yuan, Taiwan, ROC.
Hsin-Shih
HS
Wang
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Lin-Kou Medical Center, Tao-Yuan, Taiwan, ROC; Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, ROC.
Tzu-Hao
TH
Wang
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, ROC.
Yi-Hong
YH
Wu
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, ROC. Electronic address: jonghwei@mail.cgu.edu.tw.
Jong-Hwei S
JH
Pang
D027901:Acanthaceae / Q000737:chemistry*; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D006801:Humans; D051058:Oncogene Protein v-akt / Q000037:antagonists & inhibitors*; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D018797:Cell Cycle Proteins; D004365:Drugs, Chinese Herbal; D011799:Quinazolines; D042461:Vascular Endothelial Growth Factor A; C046243:tryptanthrine; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051058:Oncogene Protein v-akt; C530990:Qingdai compound
Akt; Angiogenesis; Cell cycle arrest; FAK; Psoriasis; Tryptanthrin