Screening On 171 Natural Natural Products For Proteasomes Inhibitive Activity

OBJECTIVE:Based on that the ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth.To establish a primary screening model for the proteasomes inhibitors in vitro from 171 natural products,and to evaluate effects on tumor growth and cause cells to undergo apoptosis of those inhibitors in vitro and in vivo.METHOD:SMMC-7721 cells were cultured,and total protein of SMMC -7721 cells were extracted and added to the fluorescence substrate,and the rate of proteasome activity was determined.Proteasome activity is related with the fluorescence value produced by proteolysis of the specific substrate by the proteasomes.Active products after primary screening were chosen to further examine the antitumor activity in vitro.Modified MTT was adopted for Cell Cytotoxicity Assays to evaluate the suppressing effect on proliferation of the cell line of human liver tumor at 24h,48h and 72h,the IC50 were calculated.The natural products with high cytotoxicity were selected for further studies:The effects on cell cycle progression were analyzed by Flow Cytometry(FCM).At the same time,Western Blot technique was used for the further evaluation.The anti-tumor effects of these natural products on SMMC-7721 cell bearing in nude mice were also evaluated.RESULT:The results of primary screening of 171 natural products showed that GYH013,KZ4,KZ7,SOC18,L1 and L17 inhibited the proteasomal chymotrypsin-like activity.The IC50 values were 4.36ug/ml,1.88ug/ml, 1.31ug/ml,4.11ug/ml,11.36ug/ml and 2.18ug/ml respectively.The test proteasome inhibitors were exposed to SMMC-7721 cell line,the growth and viability of the treated cells was compared with that of control cells by means of MTT The results showed that the IC50 of compound KZ4,KZ7, L1 and L17 were＜10ug/ml at all testing time(24,48,72h).The results also showed that the linear correlation(r＞0.80)(P＜0.01)could be found between the inhibition ratio and the change of dose of these natural products at all testing time.and not between the inhibition ratio and the change of time(P＞0.05)at 0.1～100ug/ml.Moreover,exposure of such cells to the 4 test proteasome inhibitors caused them to be blocked in the G2/M phase of the cell cycle and subsequently undergo apoptosis,and ahead of G0/G1 phases appears the apoptotic peak of hypodiploid.Western Blot method proved the 4 test natural products has inhibiting action on proteasome.Treatment of the 4 test proteasome inhibitors to mice bearing the SMMC-7721 tumor by i.p,a significant decrease in tumor burden was observed in vivo.With KZ7 2.5mg/kg/d and 0.5mg/kg/d,resulted in T/C (%):6.09%and 9.35%respectively.CONCLUSION:The method of fluorescence can be used for screening proteasome inhibitors from natural products in batches.GYH013,KZ4,KZ7, SOC18,L1 and L17 could inhibited the proteasomal chymotrypsin-like activity.KZ4,KZ7,L1,L17 showed concentration dependant inhibiting activity at the tumor cell line in vitro.Compound KZ4,KZ7,L1 and L17 reduced tumor growth in murine tumor models and arrested the cell cycle into G2/M phases,which may be one of the mechanisms of antitumor action.