Study On The Antitumor Activity And Main Chemical Compounds Of Total Alkaloids From Tripterygium Hypoglaucum (Levl.) Hutch

ObjectiveTripterygium hypoglaucum (levl.) Hutch (THH) is a perennial belonging to the genus Celastraceae. The root, root without bark, root bark, batch and stem are used in Chinese traditional medicine. The total alkaloids extracted from THH root without bark (THHta) showed significant antitumor activity in vitro by induction of cell cycle arrest and apoptosis. Further study on the antitumor activity in vivo and chemical compounds of THHta is necessary for the development of THHta and needs large amount of THHta. THHta were extracted from the root without bark of THH on pilot-scale (ZS-THHta). The in vitro and in vivo antitumor activity of ZS-THHta was investigated. Then the mechanism and main chemical compounds were investigated. Based on these work, we expected to provide scientific evidences for the development of THHta into a new antitumor traditional Chinese medicine.Methods(1) Pilot-scale extraction of THHta: Utilizing the same principle used for laboratory extraction of THHta, we cooperated with National Engineering Research Center of proprietary Chinese medicines (Benxi, Liaoning) to extract ZS-THHta.(2) Antitumor activity of ZS-THHta: The effect of ZS-THHta on the four sensitive tumor cell lines including HL60, MOLT-4, HCT116 and PC3 in vitro was examined by MTT assay. The soft agar colony forming assay and the models of nude mice bearing HCT116 xenografts were used to evaluate the anti-colon cancer activity of ZS-THHta.(3) The Ames assay was used to examine the mutagenic action of ZS-THHta.(4) The mechanisms involved antitumor activity of ZS-THHta: The effect of ZS-THHta on the cell cycle and apoptosis of HCT116 cells were investigated by the FCM assays. The expression of part of the cell cycle and apoptosis related proteins were determined by western blot and immunohistochemical staining.(5) Isolation and identification of the main chemical compounds of ZS-THHta: By means of chromatographic methods such as silica gel, TLC and HPLC, compounds were isolated from ZS-THHta. By physico-chemical data and spectral methods such as NMR and IR, compounds were identified.Results(1) The THHta extract yield from THH by pilot-scale extraction was 0.76‰.(2) ZS-THHta had remarkable dose-dependent and time-dependent inhibitory effect on HL60, MOLT-4, HCT116 and PC3 cell lines. The IC50 values of ZS-THHta for MOLT-4, HL60, HCT116 and PC3 cell lines were 1.62±0.12,9.01±1.10,19.11±6.34,50.91±11.41 ug/ml with 48h exposure to ZS-THHta. The IC50 values of ZS-THHta for HCT116 and PC3 cell lines were 3.59±0.86,21.62±1.88 ug/ml with 72h exposure to ZS-THHta. ZS-THHta could induce cell cycle arrest in G0/G1 phase and apoptosis in MOLT-4 cells.(3) The relative revertants of TA97,TA98,TA100 and TA102 strains induced by ZS-THHta all were no more than 2 times that of the control group with or without S9.(4) ZS-THHta could significantly inhibit the TPA induced transformation in JB CI41 cells (P < 0.01) in a dose-dependent manner. The inhibition ratios of 1.25, 2.5, 5.0 and 10μg/ml ZS-THHta were 54.4%,84.7%,97.8% and 99.8%.(5) ZS-THHta could significantly inhibit the anchorage independence growth of HCT116 cells (P < 0.01) in a dose-dependent manner. The inhibition ratios of 0.625, 1.25, 2.5, 5.0 and 10μg/ml ZS-THHta were 53.5%, 74.0%, 89.5%, 92.2% and 94.6% with the IC50 value of 0.44μg/ml.(6) ZS-THHta could inhibit the HCT116 xenografts growth in nude mice. Compared with the control group, the tumor volumes were significantly inhibited from day 6 in the group treated with 200mg/kg ZS-THHta (P < 0.01) and from day 9 in the group treated with 100mg/kg ZS-THHta. The relative tumor proliferation rates (T/C, %) of 100mg/kg and 200mg/kg ZS-THHta were 18.5% and 11.9%. The tumor weights were1.82±0.49g (control), 0.72±0.27g (100mg/kg ZS-THHta) and 0.36±0.17g (200mg/kg ZS-THHta). Compared with the control group, the tumor weights of ZS-THHta treated groups were significantly less than that of the control group (P < 0.01). The inhibition rates of tumor weight were 60.4% (100mg/kg ZS-THHta) and 80.2% (200mg/kg ZS-THHta). (7) The body weights of tumor bearing nude mice were lost during the experiment. The vehicle treated mice lost 16.8% body weight, whereas ZS-THHta treated mice lost 13.9% (100mg/kg) and 24.2% (200mg/kg). The organ coefficiens of heart, liver, spleen and kidney had no difference between the treated groups and control group (P > 0.05). Histopathology observation of the liver, spleen and kidney of the groups showed no difference under light microscope.(8) ZS-THHta showed effects on the cell cycle and apoptosis of HCT116 cells. Treated with 0, 10, 20, 40μg/ml for 24h, the percentages of HCT116 cells in G0/G1 phase were 73.6%,81.4,85.6,83.0% respectively. THHta (10μg/ml) treated HCT116 cells could induce apoptosis in a time-dependent manner and the percentage of apoptotic cells was 20.4% after exposure to THHta for 36h.(9) ZS-THHta showed effects on the expression of cell cycle G1/S phase related proteins. HCT116 cells were treated with 0-10μg/ml ZS-THHta for 12h, 24h, 36h and 48h and then the proteins were extracted. It was showed ZS-THHta could down-regulate the expression of c-Myc from 12h to 48h in a dose- and time-dependent manner. Expsure to ZS-THHta (1.25-5.0μg/ml) for 36h up-regulated the expression of P21. Expsure to ZS-THHta (10μg/ml) for 36h and 48h down-regulated the expression of cyclin D1. Expsure to ZS-THHta for 36h and 48h down-regulated the expression of CDK4 and CDK6.(10) Exposure to ZS-THHta showed effects on the expression of apoptosis related proteins. The expression of cleaved forms of PARP, Caspase 3 and Caspase 8 were up-regulated in HCT116 cells exposured to ZS-THHta for 12h to 48h, especially exposure to ZS-THHta (10μg/ml) for 36h. Expsure to ZS-THHta (2.5-10μg/ml) for 36h and 48h down-regulated the expression of anti-apoptotic proteins Bcl 2, Bcl-xl and XIAP.(11) The immunohistochemical results showed ZS-THHta down-regulated the expression of PCNA and P53 and up-regulated the expression of P27 and P21 in HCT116 xenografts in vivo.(12) Ten compounds were isolated from THHta and nine of them were identified as canophyllal, oleanolic acid acetate, wilforlide B, wilforlide A, wilforine,wilfordine, wilforgine,β-sitosterol and daucosterol.ConclusionThe method of pilot-scale extraction of THHta was successful and the yield was higher than that of laboratory extraction. The antitumor activity of ZS-THHta in vitro was similar to that of laboratory extracted THHta. ZS-THHta had remarkable dose- and time-dependent inhibitory effect on HL60, MOLT-4, HCT116 and PC3 cell lines.ZS-THHta inhibited the growth of HCT116 xenograft in nude mice and exhibited significant anti-colon cancer activity. The mechanism involved the inhibitory effect of ZS-THHta on HCT116 cells may be associated with the induction of cell cycle arrest at G0/G1 phase and apoptosis. The cell cycle arrest induced by ZS-THHta in HCT116 cells may be mediated via reduction of c-Myc, CyclinD1, CDK4, CDK6 and accumulation of P21, at least partly. The induction of apoptosis by THHta in HCT116 cells related to reduction of anti-apoptotic protein Bcl 2, Bcl-xl and XIAP, activation of extrinsic and intrinsic pathways. Combined with the identified compounds and antitumor activity of THHta, further study should be focused on the development of a new antitumor traditional Chinese medicine in the second category.