The Study Of Toxicology Activity And Its Mechanism By Quassinoid Compounds From Brucea Javanica

Brucea javanica?L.?Merr is a kind of evergreen shrubs,which belonging to the Simaroubaceae Family.In our country,it's mainly distributed in southern subtropical and tropical area,such as Yunnan,Guangxi and Hainan province.It has also been found in Indonesia,Malaysia,Myanmar,Philippines,Sri Lanka,Australia and other countries worldwide.Its mature fruits are known as"Yadanzi",also called yadanzi,laoyadan,kuzhenzi,kushenzi etc.In China,it has been used as a traditional chinese herbal medicine to the treatment of various ailments for many years.Until now,more than 150compounds have been isolated from the seeds and fruits of Brucea javanica.The major and predominant components among the compounds are quassinoids.Modern research has shown that quassinoid compounds possess multiple biological activities,such as anti-virus,anti-tumor,anti-parasite.It's pharmacological effects including anti-inflammatory,anti-ulcer,mainly used in treatment of diseases like leukemia,the malaria and dysentery.Brucea javanica oil emulsion has already been developed as commercially available drug for anti-tumor clinical applications for many years in China,and its clinical indications include primary tumors such as lung cancer,liver cancer and breast cancer.The present experiment investigated the effects of 7 purified quassinoid compounds extracted from the fruits of Brucea javanica on the proliferation of the human lung cancer cells,human gastric cancer cells,human endometrial cancer cells and human uterine cervical cancer cells proliferation for the first time.We screened out four compounds with obvious inhibitory activity and the mechanism of anti-proliferation of NCI-H292 cells was investigated in order to provide experimental basis and theoretical foundation for developing the high-efficiency and low-toxin drugs.Part one The inhibitory effects of 7 quassinoid compounds onproliferation of human tumor cellsObjective:To observe the inhibitory effects of 7 quassinoid compounds--?1?Bruceine B,?2?Bruceine A,?3?Bruceine C,?4?Bruceine D,?5?Dehydrobruceantinol,?6?Bruceaketolic acid,?7?Yadanzioside G on proliferation of human lung cancer cells,human gastric cancer cells,human endometrial cancer cells and human uterine cervical cancer cells;and to screen out the compounds with significantly inhibitory effects on proliferation of tumor cells,especially lung cancer cells.Methods:The proliferation of human lung cancer cells,human gastric cancer cells,human endometrial cancer cells and human uterine cervical cancer cells which treated with 7 quassinoid compounds were monitored by3-?4,5-dimethylthiazol-2-yl?-2,5-diphenyltetrazolium bromide?MTT?assay.Cell survival was calculated from the percentage of surviving cells.The concentration effect curve of experimental compounds on the tumor cells was used to calculate the half inhibitory concentration(IC₅₀)of medicine.Results:?1?100?mol/L 7 kinds of quassinoid compounds treated on the three kinds of human lung cancer cells?A549,NCI-H292 and HCC827?,compounds 1,2,3,4 showed very strong inhibition of proliferation activity to NCI-H292 cells,proliferation inhibition rates were 82.93%81.73%82.69%and 82.93%respectively;Yet A549 and HCC827 have not been inhibited above 50%;?2?100?mol/L 7 kinds of quassinoid compounds treated with human gastric cancer cells?AGS?and human uterine cervical cancer cells?HeLa?,compounds 1,2,3,4 showed very strong inhibition of proliferation activity,the proliferation inhibition rates were respectively 77.78%,79.24%,77.78%,79.24%and 76.09%,98.28%,98.52%,97.79%;?3?100?mol/L 7kinds of quassinoid compounds treated with three kinds of human endometrial cancer cells?HEC-1-A,HEC-1-B and RL95-2?,the inhibition rates were above 50%;?3?Compounds 5,6,7 showed no obvious inhibiting proliferation activity to 8 kinds of experimental tumor cells,the inhibition rates were below20%;?4?Through logarithm regression equation of proliferative rate of tumor cells on the concentration of compounds 1,2,3,4,we got the IC₅₀ values of the four compounds on the three human lung cancer cells.The IC₅₀ values of compounds 1,2,3,4 on NCI-H292 cells were 0.70 1.17,1.17 and 1.17?mol/L.Yet the IC₅₀ values were over 100?mol/L with the treatment of compounds 1,2,3,4 to A549 and HCC827 cells.Conclusions:Compounds 1,2,3 and 4 from Brucea javanica presented strong anticancer activity on NCI-H292,AGS and Hela cells.For C-20skeleton of quassinoids,the antitumor activity was perhaps associated with the carbonyl group in C-2,change the frame structure or increase the reactive group could significantly reduce its antitumor activity.The length of the side chain in the C-15 position had little effect on its cytotoxicity and anti-proliferation activity.Part two The mechanisms of inhibitory effects of quassinoidcompound Bruceine B on NCI-H292 cellsObjective:A large number of experiments in vivo and in vitro found that many ingredients in Chinese herbal medicine have obvious antitumor activity,and the effective components is accomplished by inducing tumor cell apoptosis.The first part of our study had confirmed that four quassinoid compounds inhibited the proliferation of human lung cancer cells NCI-H292cells significantly.In this part,we explored the antitumor mechanism of the quassinoid compound Bruceine B,and identified whether Bruceine B exert its antitumor activity of cell proliferation through apoptosis.Methods:?1?Flow cytometry was used to detect the apoptosis of NCI-H292 cells after the treatment of Bruceine B for 12 or 24 h at final concentration of 1 or 10?mol/L,which was determined by the method of Annexin?-FITC/PI stain.?2?In situ end labeling method TUNEL assay was used to examine the apoptosis of the cells after 12 h and 24 h in the NCI-H292cells,at final concentration of 10?mol/L with the treatment of Bruceine B.?3?Western blot was used to observe the changes of p53,Bax,Caspase-3,Caspase-8,Caspase-12 and GRP78.Results:?1?Effects of Bruceine B on apoptosis of NCI-H292 cells:After treated for 12 h with Bruceine B at final concentration of 1 or 10?mol/L,NCI-H292 cells'apoptosis rate respectively?16.30±0.29?%and?18.72±0.45?%,had significant difference compared with that?8.32±0.13?%of blank group,?P<0.05?;After treated for 24 h with Bruceine B at final concentration of 1 or 10?mol/L,NCI-H292 cells'apoptosis rate respectively?29.56±1.24?%and?32.47±1.87?%,had significant difference compared with that?9.01±0.33?%of blank group,?P<0.05?.?2?To further verify the occurrence of apoptosis with TUNEL assay:After treated for 12 h or 24 h with Bruceine B at concentration of 10?mol/L,the fluorescence microscopy positive cells number percentage of the total number of NCI-H292 cells were?14.89±1.27?%and?29.37±2.25?%,compared with control group?4.17±0.36?%,?P<0.05?.?3?The up-regulation effect of Bruceine B on p53,Bax,Caspase-3,Caspase-8,Caspase-12 and GRP78:After treated with Bruceine B at final concentration of 1 or 10?mol/L for 12 or 24 h,the expressions of p53,Bax,Caspase-3,Caspase-8,Caspase-12 and GRP78increase significantly compared with the control group?P<0.05?.Conclusions:The apoptosis of human lung cancer cells?NCI-H292cells?could be induced by quassinoid compound Bruceine B significantly;the expression of p53,Bax,Caspase-3 and Caspase-8 was increasely stimulated in NCI-H292 cells with Bruceine B,these results demonstrated that Bruceine B induced cell apoptosis through death receptor and mitochondria-dependent signaling pathways.Besides,in the early stage of low concentration,the effect of endoplasmic reticulum stress pathway GRP78 and Caspase-12 complex may also be one of the mechanisms for the apoptosis of NCI-H292 cells caused by Bruceine B.