Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress.
Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-induced ER stress, suggesting that protein synthesis is involved in ASK-induced ER stress. Moreover, we showed using salubrinal, an ER stress inhibitor that reactive oxygen species production, JNK activation, and Nur77 upregulation and its translocation to cytoplasm are necessary for ER-induced stress. Interestingly, we found that JNK inactivation suppresses ASK-induced ER stress, whereas Nur77 siRNA treatment does not, indicating that JNK is required for ASK-induced ER stress. Accordingly, we report that ASK induces ER stress, which is prerequisite for apoptosis of HBX-expressing hepatocellular carcinoma cells.
BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea.
Jeong
J
Moon
Department of Biological Sciences, Dong-A University, Busan 604-020, Republic of Korea.
Sang Seok
SS
Koh
BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea.
Waraporn
W
Malilas
BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea.
Il-Rae
IR
Cho
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand.
Chutima
C
Kaewpiboon
BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea.
Sirichat
S
Kaowinn
Hormone Research Center, School of Biological Science and Technology, Chonnam National University, 500-757, Republic of Korea.
Keesook
K
Lee
Department of Applied Nanoscience, Pusan National University, Busan 609-735, Republic of Korea.
Byung Hak
BH
Jhun
Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Republic of Korea.
Young Whan
YW
Choi
BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Republic of Korea. Electronic address: younghc@pusan.ac.kr.
Young-Hwa
YH
Chung
D000880:Anthraquinones / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D015854:Up-Regulation; D054334:Viral Regulatory and Accessory Proteins
D000880:Anthraquinones; D000970:Antineoplastic Agents; C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D034741:RNA, Small Interfering; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D048670:MAP Kinase Kinase 4; C073944:acetylshikonin
Acetylshikonin; ER stress; JNK; Nur77; Reactive oxygen species