Extracellular signal-regulated kinase, receptor interacting protein, and reactive oxygen species regulate shikonin-induced autophagy in human hepatocellular carcinoma
Authors: Yicheng Chen
Zhenxing Zhang
Wenhua Li
Ke Gong
Hong-Bing Shu
Journal: European Journal of Pharmacology (2014)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Autophagy; ERK; Hepatocellular carcinoma; RIP; Reactive oxygen species
Affiliations: College of Life Sciences, Wuhan University, Wuhan 430072, China., College of Life Sciences, Wuhan University, Wuhan 430072, China. Electronic address: .

Abstract:  Shikonin, a naphthoquinone derived from the Chinese medicinal plant Lithospermum erythrorhizon, shows potential to be a cancer chemotherapeutic agent. Our previous data demonstrate that high doses (about 6 μM) of shikonin induce apoptosis in human hepatocellular carcinoma (HCC) cells. Here, we discovered that a low dose of shikonin (2.5 μM) and a short treatment time (12h) induced autophagy, as evidenced by the upregulation of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, the formation of acidic autophagic vacuoles (AVOs), and the punctate fluorescence pattern of GFP-LC3 protein. Next, we investigated the mechanism and found reactive oxygen species accumulation after shikonin treatment. The reactive oxygen species scavengers NAC and Tiron completely blocked autophagy. We further found activation of ERK by generation of reactive oxygen species and inhibition of RIP pathway, which are at least partially connected to shikonin-induced autophagy. Moreover, experiments in vivo revealed similar results: shikonin caused the accumulation of reactive oxygen species and phospho-ERK and thus induced autophagy in a tumor xenograft model. These findings suggest that shikonin is an inducer of autophagy and may be a promising clinical antitumor drug. 

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PubPeer comments:
 In the last paragraph of the Discussion, there are some content and references that deviate from the subject of the article. ## 3. Discussion ... The pretreatment of various chemical, physical, physicochemical, and biological methods have been suggested to improve enzymatic hydrolysis; these techniques can be improved by the activities of extract [33,34,35]. In addition, the pretreatment and pyrolysis process require a high amount of external heat for (1) drying the washed biomass, (2) biomass torrefaction, and (3) biomass pyrolysis. Biomass drying and pyrolysis require a high amount of external heat for drying and torrefaction [36,37]. The plant also contains potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg), which will significantly affect the behaviors of extract activity. It is essential to remove the metals by adopting different methods [38]. References 33. Zhao, C.; Cao, Y.; Ma, Z.; Shao, Q. Optimization of liquid ammonia pretreatment conditions for maximizing sugar release from giant reed (Arundo donax L.). Biomass Bioenergy 2017, 98, 61u201369. 34. 34.Qiao, X.; Zhao, C.; Shao, Q.; Hassan, M. Structural Characterization of Corn Stover Lignin after Hydrogen Peroxide Presoaking Prior to Ammonia Fiber Expansion Pretreatment. Energy Fuels 2018, 32, 6022u20136030. 35. Chen, D.; Cen, K.; Cao, X.; Chen, F.; Zhang, J.; Zhou, J. Insight into a new phenolic-leaching pretreatment on bamboo pyrolysis: Release characteristics of pyrolytic volatiles, upgradation of three phase products, migration of elements, and energy yield. Renew. Sustain. Energy Rev. 2021, 136, 110444. 36. Chen, D.; Cen, K.; Cao, X.; Zhang, J.; Chen, F.; Zhou, J. Upgrading of bio-oil via solar pyrolysis of the biomass pretreated with aqueous phase bio-oil washing, solar drying, and solar torrefaction. Bioresour. Technol. 2020, 305, 123130. 37. Chen, D.; Chen, F.; Cen, K.; Cao, X.; Zhang, J.; Zhou, J. Upgrading rice husk via oxidative torrefaction: Characterization of solid, liquid, gaseous products and a comparison with non-oxidative torrefaction. Fuel 2020, 275, 117936. 38. Chen, D.; Cen, K.; Chen, F.; Ma, Z.; Zhou, J.; Li, M. Are the typical organic components in biomass pyrolyzed bio-oil available for leaching of alkali and alkaline earth metallic species (AAEMs) from biomass? Fuel 2020, 260, 116347. ,