Cytotoxicity mechanism of α-MMC in normal liver cells through LRP1 mediated endocytosis and JNK activation.
Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein isolated from Momordica charantia, is a potential drug candidate with strong anti-tumor activity. However, α-MMC has a severe hepatotoxicity when applied in vivo, which may greatly hinders its use in clinic in the future. The biological mechanism of hepatotoxicity induced by α-MMC is largely unknown, especially the mechanism by which α-MMC enters the hepatocytes. In this study, we investigated α-MMC-induced cytotoxicity in normal liver L02 cell line as well as the mechanism underlying it. As expected, α-MMC is more toxic in L02 cells than in various normal cells from other organs. The cytotoxic effect of α-MMC on L02 cells is found to be mediated through cell apoptosis as detected by flow cytometry and fluorescence microscopy. Importantly, α-MMC was shown to bind to a specific receptor on cell membrane, as the density of the cell membrane receptor is closely related to both the amount of α-MMC endocytosed and the cytotoxicity in different cell lines. By using LRP1 competitive inhibitor α2-M or siRNA targeting LRP1, we further identified that LRP1 protein served as the membrane receptor for α-MMC. Both α2-M and siRNA targeting LRP1 can significantly inhibit α-MMC's endocytosis as well as its cytotoxicity in L02 cells. In addition, it was found that α-MMC can activate the JNK signalling pathways via LRP1 in L02 cells. As JNK activation often leads to cell apoptosis, the activation of JNK may play an important role in α-MMC-induced cytotoxicity. To our knowledge, this is the first report showing that LRP1 mediates the cytotoxicity of α-MMC through (1) endocytosis and induced apoptosis and (2) the activation of the JNK pathway. Our findings shed light on the fundamental mechanism of hepatotoxicity of α-MMC and offer reference to understand its mechanism of lymphocytotoxicity and neurotoxicity.
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China.
Ling
L
Wang
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China. Electronic address: shenfubing@163.com.
Fubing
F
Shen
Isotope Lab, College of Preclinical and Forensic Medicine, Sichuan University, Chengdu, PR China.
Min
M
Zhang
Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Qianchuan
Q
He
Department of Cardiology (II), Xiangyang Central Hospital, Hubei Province, Xiangyang 441021, PR China.
Hui
H
Zhao
Department of Public Health, Chengdu Medical College, Chengdu 610500, PR China.
Xiaoping
X
Yu
Center of Science and Research, Chengdu Medical College, Chengdu 610500, PR China.
Shuxia
S
Yang
Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Yang
Y
Liu
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China.
Nianhua
N
Deng
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China.
Juecun
J
Zheng
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China.
Lixia
L
Zhu
School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, PR China.
Xiaolan
X
Liu
D000020:Abortifacient Agents, Nonsteroidal / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology*; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000008:administration & dosage; D054788:Ribosome Inactivating Proteins / Q000633:toxicity*
D000020:Abortifacient Agents, Nonsteroidal; C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D034741:RNA, Small Interfering; C039582:MMC protein, Momordica charantia; D054788:Ribosome Inactivating Proteins
Apoptosis; Cytotoxicity; LRP1 receptor; Normal embryonic liver cell line L02; α-MMC