Screening Of Multiple-targeting Antitumor Active Natural Ingredients And Study On Antitumor Mechanism

This dissertation focused on the screening the multiple-targeting antitumor active natural components and studying the antitumor mechanisms. A multiple-targeting (EGFR、TNFR、Fas) lipid-raft chromatographic model was established to extract antitumor active components from Semen Descurainiae. Moreover, the mechanisms of proliferation inhibition, migration and pro-apoptosis effects of the antitumor active components on breast cancer in vitro were investigated. The in vivo acute toxicity of the antitumor active components was also analyzed. Furthermore, we shed light on the mechanism of in vivo inhibition of breast cancer cells caused by the antitumor active components in nude mouse. The whole study contains five parts as follows.Chapter1Review of the multiple-targeting lipid-raft chromatography and traditional Chinese medicines for antitumor therapyThis section reviewed the research progress about lipid-raft chromatography, including its current situation and construction principles and the antitumor mechanisms based on the traditional Chinese medicine science and the modern biology. We also elucidate the current research situation of Semen Descurainiae, from which the objective, the design of this dissertation as well as prospects were stemmed, providing a theoretic foundation for the development of this work.Chapter2Construction of the multiple-targeting lipid-raft chromatography and the screening of the antitumor active components from Semen DescurainiaeA novel multiple-targeting lipid-raft chromatographic model (rich in EGFR, TNFR and Fas) was established, thus providing a platform for multiple-targeting, fast, online screening the antitumor active components from the traditional Chinese medicine. Applying this chromatographic model to screen out active components from Semen Descurainiae, and MTT assay was used to assess the antitumor effects of water-saturated n-butanol extraction.The lipid-rafts rich in EGFR, TNFR and Fas were isolated from human mcf-7 cells, which were then anchored on the surface of silica beads to construct the multiple-targeting lipid-raft chromatographic model. The CdTe quantum dots (QDs) were prepared to tag EGFR, TNFR and Fas to visualize the fixed molecules on the silica surface. This chromatographic model was then employed to screen the antitumor components from different fractions of Semen Descurainiae, including the water extraction, ether extraction, ethyl acetate extraction, water-saturated n-butanol extraction and the raffinate. The antitumor active components were then applied to several different tumor cells at a series of concentrations (0.05ug/mL, O.1μg/mL,1μg/mL and5μg/mL), including MCF-7, HepG-2, SGC and SCLC, to evaluate their cell growth inhibition effects by MTT assay. In addition, its toxicity to rat fibroblasts was assessed by MTT method compared with5-Fu (1μg/mL,5μg/mL,10μg/mL,25μg/mL and50μg/mL). The results showed that the water-saturated n-butanol extraction of Semen Descurainiae was found to be retained on the MCF-7lipid-rafts chromatographic column during7.02-18.66min, which means that the fraction possessed anti-tumor activity. And results of MTT assay showed that the water-saturated n-butanol extraction of Semen Descurainiae had a better tumor inhibition effect on MCF-7(IC50:0.77ug/mL) than HepG2(IC50:1.55μg/mL), SGC(IC50:5.49μg/mL) and SCLC(IC50:6.09μg/mL), while displayed little cytotoxicity to rat fibroblasts.Chapter3Study of the inhibition mechanism in human MCF-7cellsA comprehensive study was carried out to investigate the in vitro antitumor mechanisms of the active components from Semen Descurainiae in terms of inhibiting proliferation, promoting apoptosis, controlling cells cycles, restraining migrations. Western blot and immunofluorescence were conducted to assess the inhibition effect on p-ERK, Bcl-2, Bax and PCNA proteins. Flow cytometry method, the DNA ladder tests as well as the transwell migration analysis were conducted to evaluate the apoptosis of breast cancer cell.The results of western blot demonstrated that p-ERK was significantly reduced by the active components (P<0.05) and the deeper inhibited with the higher concentration. It suggests that the active components inhibited the MCF-7cells through the MAPK signal pathway.Western blot was conducted to assess the inhibition effect on Bcl-2and Bax protein when MCF-7cells were treated with active components of Semen Descurainiae after24h. The results showed that the active component of Semen Descurainiae inhibit Bcl-2protein and increase Bax protein with concentration dependence, which means that the promoting apoptosis of the active components was under controlling the Bcl-2family. The results of FITC and PI double parameter points illustrated that the early apoptosis of MCF-7cells caused by active components of Semen Descurainiae had dose-time dependency. DNA electrophoresis was employed to examine the DNA fragmentation in MCF-7cells. As a result, MCF-7showed significant DNA fragmentation and subsequent apoptosis when treated with the active components of Semen Descurainiae and5-Fu for72h, indicating the apoptosis of MCF-7.Flow cytometry analysis was applied to study the effect of active component on the cell line cycle. The resulted revealed that the control group had a higher percentage of cells in S phase, suggesting that the more DNA was synthesized, the more tumor cells were proliferated. The proportion of MCF-7cells in G0/G1would increase obviously and significantly decreased in S phase under the high concentration, also it showed a dose-dependent manner. They all showed that the active component blocked the MCF-7cells in the cell line cycle of G0/G1, which caused the cells of G1phase were arrested. Western blot and immunofluorescence were employed to detect effect of active components of Semen Descurainiae on PCNA protein expression. The low concentration showed no inhibitions at any time, while the concentrations of medium and the high made a significant inhibition of the expression of the PCNA, which indicates the active component interfered with the synthesis of DNA during the S phase.The transwell migration analysis was conducted to observe the effects generated by the active components of Semen Descurainiae and5-Fu on breast cancer cells. The assay revealed that the MCF-7had a significantly reduced cell migration (P<0.05) when treated with active components of Semen Descurainiae and5-Fu, respectively, and both with a dose-dependent manner. Chapter4Study on the oral acute toxicity of the active components from Semen DescurainiaeThe oral and intraperitoneal injection acute toxicity of the active components of Semen Descurainiae was evaluated by acute toxic class method improved Karber method. The LD50of oral-taking and intraperitoneal injection was calculated, which provided a basis of dose in the inhibition of tumor xenograft studies. Finally, the acute toxicity was evaluated by the organ index of the active component from Semen Descurainiae by oral-taking and intraperitoneal injection.The findings resulted from the acute toxic class method showed that the LD50of intragastric administration of the active components of Semen Descurainiae was4000-5000mg/kg. Active components of Semen Descurainiae is low toxic substances by oral-taking.The results of improved Karber method demonstrated that the LD50of intraperitoneal injection of the active components of Semen Descurainiae was505.36mg/kg, with the95%average confident limit of LD50ranging from590.32mg/kg to432.61mg/k. No acute toxicity when the dose of intraperitoneal injection was250mg/kg.Chapter5Study on the growth inhibitions of MCF-7in nude mouseThe nude mouse with human MCF-7cells was constructed, and the inhibition effect by oral-taking and intraperitoneal injection was evaluated in vivo. Moreover, western blot was applied to test the expression of Bcl-2and Bax proteins to evaluate the antitumor mechanism preliminarily.MCF-7cells were inoculated subcutaneously in the right flanks of nude mice. After the tumor formation, the active components of Semen Descurainiae were administrated by oral-taking (various doses100mg/kg,200mg/kg,400mg/kg) and intraperitoneal injection (various doses25mg/kg,50mg/kg,100mg/kg). As a control,5-Fu was delivered by intraperitoneal injection (20mg/kg). Four weeks later, the tumors were stripped, measured and weighed. Western blot was then employed to determine the expression level of Bcl-2and Bax. The active components of Semen Descurainiae delivered by both intraperitoneal injection and oral administration showed obvious inhibition on tumor volume and weight (P<0.05), similar to the tumor inhibition effect in the5-Fu group (P<0.05). Both the active components of Semen Descurainiae and5-Fu inhibited down-regulated expression of the Bcl-2, up-regulated expression of Bax (P<0.05). The intraperitoneal injection groups of active components of Semen Descurainiae inhibited Bcl-2with a dose-dependent manner.