Shikonin Exerts Antitumor Activity Via Proteasome Inhibition In Vitro And In Vivo

BackgroundThe Ubiquitin-proteasome system (UPS) is the major intracellular proteidegradation system,which mediates degradation of various intracellular proteins antherefore is involved in regulating important life processes of the cell. Proteasome habecome an important target for many diseases,including cancer.So to find an effectivproteasome inhibitor is of great significance.China has rich resources of Chinese herbamedicine, so screening for natural proteasome inhibitors from the traditional Chinesmedicine has broad prospects. Our laboratory has established the proteasome inhibitoscreening platform, and we found that shikonin inhibited proteasome activity in vitro.ObjectivesTo further study the realationship between Shikonin's proteasome inhibition and itantitumor effect in vitro and in vivo.Methods and Results1.Shikonin inhibited proteasome function by using a stable transfectioGFPu-HEK293 cell lineCultured stable transfection GFPu-HEK293 cells were treated with different dose(0-8μmol/L) of shikonin for 8 hour,then the whole cell proteins were extracted fowestern blot assay and the results were analysed by using ECL-plus DeterminatioReagent and X-Kodak exposure imaging system. It was shown that the exogenous anendogenous proteasome substrates : Ub,GFP were all increased in concentration-dependent manner.2.Shikonin induced P388 cell death and proteasome inhibition P388 cells were treated with various concentrations of Shikonin (0-6μmol/L) for 6 hours respectively or treateded with the same dose of Shikonin (2μmol/L) for different time points (0-12h). Then the cell lysates were incubated with Chymotrypsin-like peptide substrates. It was found that shikonin can inhibit proteasome activity in P388 cells in a dose and time dependent manner. In the same condition ,the death rates were detected by flow cytometry (Annexin V-FITC staining). It was found that P388 cells could be induced to death by Shikonin also in a dose and time dependent manner. Additionally, western blot assay found that shikonin could induce ubiquitination protein aggregation and PARP cutting in a concentration and time dependent manner.Shikonin induced proteasome inhibition occurs prior to tumor cell death.3.Shikonin's effect on cumulative survival in KM mice bearing murine leukemiaMale KM mice were injected with murine leukemia P388 cells.After 24 hour,mice were randomly divided into 2 groups and treated with i.p.bolus injections of either drug vehicle (V) or 4 mg/kg/day shikonin (Shi 4) for 7 consecutive days.The mice were then kept for additional 60 days to determine the effect on mice survival of shikonin.It was found 4 mg/kg shikonin treatment significantly extended the survival period of KM mice bearing murine leukemia.Within 23 days,the entire control vehicle treated mice died.In a sharp contrast,the 9 of 10 mice treated with 4 mg/kg shikonin survived by the end of the experiment (60 days) only one mice died in 28 days.4. Shikonin suppressed solid tumor growth and induced proteasome inhibition in vivoMurine hepatoma H22 cells were injected s.c.in the left armpit of each Male KM mice.After 24 hour of injection,mice were randomly divided into 3 groups and treated with either vehicle or shikonin (4.0 or 8.0 mg/kg) for 7 consecutive days.Two days later, the mice were sacrificed, and the tumor tissues were weighed.The results showed that shikonin at 4mg/kg/day and 8mg/kg/day can suppresses tumor growth in vivo,inhibition rates were 42.4% and 55.9%.The proteasome inhibition by shikonin in H22 tumors was then examined by Western blotting analysis using whole tissue extracts and immunohistochemistry.Compared with vehicle control,shikonin treatment caused accumulation of ubiquitinated proteins,as well as IκB-α,Bax, p27 and Hsp70 proteins.In situ proteasome inhibition and increased p27 protein level in H22 tumors treated with shikonin was confirmed by tissue immunostaining.It was also found the existence of p85/PARP cleavage fragment and TUNEL positivity.Conclusions1.Shikonin could inhibit the proteasome activity and accumulate endogenous and exogenous proteasome subtrates.2.Shikonin's anti-tumor effect in vivo was associated with proteasome inhibition.