Study On The Antitumor Activity Of ITCs And The Apoptosis Mechanism Of A549 Cells Inducing By MTBITC

The morbidity and mortality of lung cancer show an upward trend in recent years. Epidemiological studies reveal that high dietary intake of cruciferous vegetables containing isothiocyanates (ITCs) can lower the risk from cancers. A lot of literatures reported the composition and content of ITCs in cruciferous vegetables, but few involved the preparative separation of ITCs, which are the material foundation of bioactive studies. Anticancer activity of more than 20 ITCs was studied, but structure-activity relationship of ITCS has not been clarified. Also the studied of mechanism of ITCs-induced apoptosis and the signal transduction have not been reported. The present dissertation focuses on the isolation and identification of five ITCs from four cruciferous vegetables, the effects of ITCs on human cancer cells, the structure-activity relationship of ITCs, and the effects of MTBITC (4-(methylthio)-3-butenyl isothiocyanate) on the signal transduction pathways in A549 cells apoptosis. The main results are listed as follows:1. The hydrolyzation of glucosinolate to obtain ITCs from dried radish seeds was optimized as following:temperature,25℃; time,8 hours; quantity of activator,0.06 mg/g and pH,3.71. The yield rate of MTBITC under the above hydrolyzation condition was 3.53 mg/g.2. The acetone extracts of hydrolysates of four cruciferous vegetables seeds were extracted and separated by chromatographic methods on silica gel, Sephadex LH-20 and Pre-HPLC to yield 5 compounds:MTBITC (Ⅰ), Iberverin (Ⅱ), Sulforaphane (Ⅲ), Iberin (Ⅳ) and 3-BITC(Ⅴ).3. The inhibition activity of five ITCs agaist human cancer cells A549, LAC, HepG2 and HELA were tested. The results demonstrated that MTBITC, Sulforaphane and Iberin could significantly inhibit the growth of the four human cancer cell lines in a dose-manner, the cell growth inhibition rates of MTBITC, Sulforaphane and Iberin were higher than Iberverin and 3-BITC, and the inhibitory activity of MTBITC to A549 was the strongest.4. The relationship between the electronic structure and anticancer activity was analyzed with the parameters of the geometry, Mulliken populations and the atomic frontier electron densities which were optimized by DMol3 module. The results showed that carbon atom in the group of N=C=S of the ITCs is the main electrophonic reaction site, and sulfonyl or sulfur in the side chain was the secondary site. The analysis also clued that the anticancer activities of ITCs was relative to the negative charge populations, delocalization of frontier molecular orbital and the energy gaps of HOMO-LUMO.5. Effects of MTBITC on A549 cells were investigated by MTT method. The results showed that MTBITC suppressed the growth of A549 cells in-vitro with both dose-and time-dependant manners, and the inhibition of MTBITC on A549 cell proliferation belong to the apoptosis induction, not necrosis. Conventional morphological signs of apoptosis could be found when the cells were treated with MTBITC, through the methods including Giemsa stain, Hoechst 33258 fluorescence stain and transmission electron microscope. MTBITC treatment significantly increased the apoptotic sub-Gl fraction in dose-dependent manner by PI staining. The result of flow cytometry (FCM) exhibited induced apoptosis was blocked at phases of S and G2/M, and MTBITC-induced cell apoptosis is mainly at the earlier stage of apoptosis. On the basis of Molecular docking, we found that MTBITC covaiently interacted with the Cys347 residue and formed hydrogen bonds with Lys352 in the lipophilic pockets, which disrupted the tubulin polymerization and induced the cancer cells apoptosis.6. The mechanism of apoptosis induced by MTBITC was investigated by testing the change of mitochondrial membrane potential (△(?)m), reactive oxygen species (ROS) and intracellular calcium with FCM, the expression of mRNA of apoptosis-related gene by RT-PCR, and the activities of Caspase-3,-8,-9 by caspase colorimetric assay. The results indicated that MTBITC treatment decreased mitochondrial membrane potential by down-regulating the rate of Bcl-2/Bax and Bcl-xL/Bax, increased ROS and intracellular calcium and activated Caspase-9. Therefore, mitochondrial pathway and Bcl-2 gene family could involve in the mechanism of the A549 cells apoptosis induced by MTBITC. Further investigation indicated that the mRNA expression of Fas, FasL and FADD were up-regulated in a time-dependent manner, Caspase-3 and-8 were activated. These results demonstrated that MTBITC induced apoptosis of A549 cells via Fas signal transduction pathway.All the above results indicated the apoptosis of A549 cells was induced by MTBITC by changing the valencebonds of tubulin to block cells entering S and G2/M phases via Fas signal transduction pathway and mitochondria pathway. And the mechanism of A549 cells apoptosis induced by MTBITC should be relative to the calcium-dependent ROS pathway. Those results will provide scientific information for developing healthcare products using cruciferous vegetables or developing medicines for cancer treatment.