Identification of acetylshikonin as the novel CYP2J2 inhibitor with anti-cancer activity in HepG2 cells.
BACKGROUND
Acetylshikonin is one of the biologically active compounds derived from the root of Lithospermum erythrorhizon, a medicinal plant with anti-cancer and anti-inflammation activity. Although there have been a few previous reports demonstrating that acetylshikonin exerts anti-cancer activity in vitro and in vivo, it is still not clear what is the exact molecular target protein of acetylshikonin in cancer cells.


OBJECTIVE
The purpose of this study is to evaluate the inhibitory effect of acetylshikonin against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines.


METHODS
The inhibitory effect of acetylshikonin on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its cytotoxicity against human hepatoma HepG2 cells was also evaluated.


METHODS
Astemizole, a representative CYP2J2 probe substrate, was incubated in HLMs in the presence or absence of acetylshikonin. After incubation, the samples were analyzed by liquid chromatography and triple quadrupole mass spectrometry. The anti-cancer activity of acetylshikonin was evaluated on human hepatocellular carcinoma HepG2 cells. WST-1, cell counting, and colony formation assays were further adopted for the estimation of the growth rate of HepG2 cells treated with acetylshikonin.


RESULTS
Acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation activity (Ki= 2.1µM) in a noncompetitive manner. The noncompetitive inhibitory effect of acetylshikonin on CYP2J2 enzyme was also demonstrated using this 3D structure, which showed different binding location of astemizole and acetylshikonin in CYP2J2 model. It showed cytotoxic effects against human hepatoma HepG2 cells (IC50= 2μM). In addition, acetylshikonin treatment inhibited growth of human hepatocellular carcinoma HepG2 cells leading to apoptosis accompanied with p53, bax, and caspase3 activation as well as bcl2 down-regulation.


CONCLUSIONS
Taken together, our present study elucidates acetylshikonin displays the inhibitory effects against CYP2J2 in HLMs and anti-cancer activity in human hepatocellular carcinoma HepG2 cells.
Department of Bio and Chemical Engineering, Hongik University, Sejong 30016, Republic of Korea.
See-Hyoung
SH
Park
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
Nguyen Minh
NM
Phuc
Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Jongsung
J
Lee
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
Zhexue
Z
Wu
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
Jieun
J
Kim
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea; New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea.
Hyunkyoung
H
Kim
New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea.
Nam Doo
ND
Kim
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea.
Taeho
T
Lee
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea. Electronic address: kssong@knu.ac.kr.
Kyung-Sik
KS
Song
BK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea. Electronic address: dstlkh@knu.ac.kr.
Kwang-Hyeon
KH
Liu
D000880:Anthraquinones / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D056945:Hep G2 Cells / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy*; D008862:Microsomes, Liver / Q000187:drug effects*; D008517:Phytotherapy; D010936:Plant Extracts / Q000627:therapeutic use
D000880:Anthraquinones; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D010936:Plant Extracts; D003577:Cytochrome P-450 Enzyme System
Acetylshikonin; Anti-cancer; CYP2J2; Inhibition