Studies On The Active Constituents Of Actinidia Chinensis And The Synergistic Effects Of Dehydrobruceine B Combined With Cisplatin On Proliferation Of Lung Cancer Cells

Actinidia argute is the dry root of Actinidia chinensis Planch.It is bitter taste and cold-natured,with the functions of heat and dampness,drainage and detoxification,scattered stasis.Clinically used mainly for hepatitis,damp heat jaundice,dysentery,rheumatoid arthritis,gastrointestinal cancer.In this research,we studied the chemical constituents of the root of Actinidia chinensis,and evaluated the anti-tumor activity of the obtained triterpenoids.By using silica gel chromatography,C18 reverse phase column chromatography,Sephadex LH-20 gel column chromatography and HPLC,18 compounds were isolated from the ethyl acetate fraction of the ethanol extract of Actinidia chinensis,the structures are identified as:ursolic acid(1),2a,3a,24-trihydroxyolean-12-en-28-oic acid(2),corosolic acid(3),cleomiscosin A(4),isolariciresinal(5)7,8-trans-3,5'-dimethoxy-4',7-epoxy-8,3'-neolignane-5,9,9'-triol(6),7R,8R-threo-4,7,9,9'-tetrahy?droxy-3,3'-dimethoxy-8-O-4'-neolignan(7),7S',8S,-threo-4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan(8),quercetin(9),3.5,7.3',5'-pentahydroxyflavan(10),fraxetin(11),scopoletin(12),/?-sitosterol(13),daucosterol(14),vanillic acid(15),methyl caffeate(16),2,6-dihydroxy-1,4-benzenedicarboxylic acid dimethyl ester(17),2,3-dihydroxypropyl stearate(18).Compounds 4,5,6,7,8,10,11,12,16,17,18 are first reported from this plant.Triterpenoids are the main anti-cancer ingredients of Actinidia chinensis.The cytotoxic activities of triterpenoids 1,2 and 3 were screened by using MTT assay,the results showed that both compounds 2 and 3 exhibited proliferation inhibition on lung cancer NCI-H292 cells with IC50 values of 29.28 ?M and 12.20 ?M respectively.Meanwhile,compound 3 had a moderate inhibitory effect on proliferation of human lung cancer A549 cells with IC50 value of 33.83 ?M.Compound 2(TLG-2),which could be obtained from the root of Actinidia chinensis a lot,exihibited moderate cytotoxicity on lung cancer cell line NCI-H292 and no cytotoxicity on human umbilical vein cell line EA.hy926.We choose compound 2 for further evaluation.MTT assay and real-time cell growth assay(RTCA)showed that TLG-2 could inhibit the proliferation of lung cancer NCI-H292 cells in a dose-and time-dependent manner;PI single staining results showed that TLG-2 could not cause cell cycle changes in NCI-H292 cells;DAPI staining and Annexin V/PI double staining showed that TLG-2 could induce apoptosis of lung cancer NCI-H292 cells.Further researches found that TLG-2 could induce intracellular ROS accumulation and mitochondrial membrane potential(MMP)decrease.Western blotting analysis showed that TLG-2 could increase the protein expression of Bax,decrease the protein levels of Bcl-2 and Bcl-xL,induce the release of cytochrome c,and further activate caspase cascade,resulting in apoptosis.The results of immunoblotting and immunofluorescence showed that TLG-2 could induce the translocation of AIF,the breakage of DNA,and eventually causing apoptosis.In conclusion,TLG-2 can trigger apoptosis of NCI-H292 cells through mitochondrial-dependent pathway.Dehydrobruceine B(DHB)is a quassinoid purified from the seed of Bruceca javanica.Previous study has showed that DHB induced apoptosis of two kinds of human lung cancer cell lines A549 and NCI-H292.The aim of this study was to investigate the effect of DHB on the apoptosis of A549 cells induced by cisplatin(CDDP)at the subtoxic concentration.The results of RTCA showed that DHB could enhance CDDP-induced cell proliferation inhibition;DAPI staining and Annexin V/PI double staining showed that DHB could promote CDDP-induced apoptosis;Combination of DHB and CDDP could induce the decrease of MMP,the release of cytochrome c and AIF;Further studies showed that the combination treatment could induce the changes of apoptosis-related proteins,such as the increase of Bax,the decrease of Bcl-2 and Bcl-xL,the increase of cleaved caspase-9,cleaved caspase-3,and cleaved PARP.These results suggest that DHB can enhance CDDP-induced apoptosis through mitochondrial-mediated apoptotic pathway.High expression of Nrf2 in tumor cells can lead to tumor cell resistance to chemotherapeutic agents.We detected that DHB could be able to reduce the expression of Nrf2 in A549 cells in a time-and dose-dependent manner,it also decreased the expression of Nrf2 downstream proteins in a dose-dependent manner.The results of real time RT-PCR showed that DHB could inhibit the expression of Nrf2 and its downstream genes dose-dependently,which may contribute to the increase of intracellular ROS level,consequently,induced ROS-mediated mitochondria apoptosis.These results suggest that DHB can be used in combination with CDDP to the treatment of lung cancer.