The Anti-lung Cancer Effect And The Mechanism Of Novel Targeting Compound ZT-18 And Curzerene

Part IIn recent years, lung cancer is a high incidence and highly lethal tumor; most of the patients were in the middle stage or advanced in clinical diagnosis. However, there is no specific anti-cancer drug in advanced lung cancer; including tyrosine kinase inhibitor(TKI) drugs mostly appeared drug resistance and relapse. Therefore, the development of new targets anti-cancer drug is still taking a heavy burden and embarks on a long road. On the basis of the preliminary work, the energy metabolism of malignant tumor and amino acid transporter were proposed to explore and research on this paper around, we found that using the F18 labeled glutamate in the distribution of PET-CT showed aggregation effect, so this topic choice Glutamate as the target of the amino acid transporter to couple with the first substrate of tri-carboxylic acid cycle(TCA) Oxaloacetate, to design new compounds ZT-18.First chapter, the synthesis and characterization of ZT-18: taking H-Glu(Ot Bu)-Ot Bu?HCl and Et OOC-COCl as raw material, we used liquid phase synthesis method to synthesize the compound ZT-18, with the step of acylation first and then combined after. High Performance Liquid Chromatography, Mass Spectrometry, Hydrogen Spectrum and Carbon Spectrum technology were used to determinate the molecular weight and molecular structure, and the result is consistent with the target product. After separation and purification, the new solid compound ZT-18 with the purity of more than 96% was obtained.Second chapter, study on the stability of ZT-18: according to the stability Chinese pharmacopoeia standard of ZT-18 preliminary study. The main influencing factors test, accelerated test and long-term test of three aspects. The experimental results show that the ZT-18 is affected by temperature, humidity and light is large, easy to absorb moisture caking, but its content is relatively stable.Third chapter, pharmacodynamic study of ZT-18: the anti-tumor effect of ZT-18 in tumor bearing nude mice was studied. The results of MTT showed that ZT-18 did not show a significant inhibitory effect on lung cancer cells in vitro. ZT-18 in A549 lung cancer model in nude mice in vivo pharmacodynamics experiment showed a dose-dependent antitumor efficacy, indicating that ZT-18 may be as lung cancer target to compounds before drug forms in vivo play antitumor effect.Fourth chapter, study on the mechanism of anti-tumor action of ZT-18: the anti-tumor mechanism of ZT-18 in tumor bearing nude mice was studied. Blotting Western and PCR Real-time were used to verify the relationship between the expression of glutamate transporter GLAST in lung tissue and tumor tissue in nude mice bearing tumor. Real time PCR experiments showed that in A549 in nude mice bearing human tumor tissue, GLAST m RNA expression level and efficacy is inversely proportion relationship. At the same time, Blotting Western experiment also proved that GLAST in tumor bearing nude mice tumor tissue, the expression levels of protein is inversely related the anti-tumor effect.Part IIAs a commonly used traditional Chinese herbal medicine, Zedoary turmeric in the medical community appear with increasing frequency, reputation is also growing. The important role of zedoary turmeric on antitumor, antiviral, antibacterial and anti thrombosis etc have confirmed on clinical, it is a good prospects for the development of traditional Chinese medicine. Zedoary Volatile oil is an important component of turmeric rhizome, as antiviral, antitumor drug collection contains in 2010 "Chinese Pharmacopoeia". Zedoary oil preparation has been widely used in clinical treatment, its composition, pharmacological effects and correlation studies has also been hot research topic over the years. Curzerene is an important component of Zedoary Volatile Oil, with furandiene, anticancer principal component of zedoary turmeric oil, is a pair of heat sensitive materials. Now stage existing research about Curzerene is on the different extraction process, rarely involved in the pharmacology and pharmacodynamics of Curzerene, let alone its action mechanism. Therefore, this thesis intends to study Curzerene as the main research object, extracting from the Zedoary Volatile oil. We research the inhibition effect of Curzerene on lung adenocarcinoma cell line A549 and induction of apoptosis in vitro, and further research the relationship between the expression of GSTA1 and the anti-tumor mechanism.First chapter, the anti-tumor pharmacodynamics experiment of Curzerene in vitro: wu use the MTT method to study the effect of Curzerene induced apoptosis and proliferation of human lung adenocarcinoma cell line A549. Using Cisplatin as positive control drug, A549 cells were intervented in different concentrations of Curzerene at different time, showing different proliferation effect, with dose and time was positively related to dependency. 6.25, 12.5, 25, 50 and 100μmol/L concentration of Curzerene intervented after 48 h, A549 cell proliferation inhibition rate was 1.90%, 7.81%, 16.65%, 42.57%, 70.01% respectively. Effect of after 24 h, 48 h, 72 h respectively, the IC50 value is 236.60μM, 60.72μM, 57.17μM. Experimental study results show that the Curzerene on human lung adenocarcinoma cell line A549 has inhibitory effect on the proliferation, efficacy and dose and time were positive correlation.Second chapter, study on the mechanism of tumor cell apoptosis induced by Curzerene: the Hoechest33258 nuclear staining was used to observe the morphological changes of the A549 cells when the Curzerene intervented. Different concentrations of Curzerene intervented after 48 h, the blank control group of A549 cells showed blue fluorescence of morphologically normal cells. In contrast, the number of cells in high dose group is reduced greatly; the apoptotic cells increased and showed the landmark features, chromatin condensation, nuclear pyknosis, and apoptotic body. The total number of cells in each group was negatively correlated with the dose, while the ratio of apoptotic bodies was positively correlated. The experimental results show that Curzerene could induce A549 cells apoptosis.Third chapter, study on the mechanism of Curzerene induced apoptosis and cycle arrest of tumor cells: we use the flow cytometry to study the A549 cell apoptosis and the effect on cell cycle distribution of Curzerene. The experimental results show that the different concentrations of Curzerene could induce A549 cell apoptosis in different degree, the apoptosis rate and dose are positive correlation. And cell cycle experiments, Curzerene intervented after 48 h, the cell ratio of G2/M phase in the control group was 3.92%, with the increase of Curzerene dose, the ratio of G2/M phase cells become higher and higher, the highest dose was 11.78%, suggesting that the apoptosis of A549 induced by Curzerene could be cells arrest in G2/M phase.Fourth chapter, study on the correlation between the antitumor effects of Curzerene and the expression of GSTA1: Western Blot and Real-time PCR were used respectively to detect the expression of GSTA1 protein and m RNA, after the A549 cells were intervented by Curzerene 48 h, to study how Curzerene mediated A549 cell proliferation and apoptosis. The results showed that Curzerene down regulated the m RNA expression level of GSTA1 protein expression in A549 cells(P<0.05). The experimental results show that Curzerene could inhibit the expression of GSTA1 to promote cell apoptosis.In summary, the new small molecule targeting compound ZT-18 in this thesis, has a good anti-tumor effect in nude mice bearing lung cancer cell, has the prodrug characteristics, is a good development prospect of new compounds.Traditional Chinese medicine monomer Curzerene could inhibit proliferation, induce apoptosis and G2/M arrest on lung adenocarcinoma cell A549 in vitro, can also down regulated the expression of GSTA1 to promote cell apoptosis. Curzerene is expected to become a targeting traditional Chinese medicine for the treatment of lung cancer.