Preliminary Study On The Antitumor Activities Of The Natural Products D5, S3 And Their Derivatives

This thesis was based on studies conducted on the compound D5 using various microbiological techniques to do further research on the antitumor activity. The MTT method was applied to screen out the cell line which was the most sensitive to D5. The antitumor activities of the derivatives of D5 were also determined; the interaction between D5 and TR3, the possible target protein of D5, was tested by fluorescence quenching. The crystal TR3 was obtained after first optimizing the expression conditions of TR3 by orthogonal test. Moreover, the MTT method was used to screen out the most sensitive cell line to S3 and compare the activities of S3 and SA-1. FCS analysis was used to study the effects of S3 and SA-1 on the contents of ROS and the cell cycle distribution. Based on the above, the mechanism and acute toxicity of S3 were primarily studied.Several useful results were obtained from our study:1, The cytotoxicity of natural products D5 was primarily studied by the MTT method. Nine cell lines from human and mice were chosen to test the cytotoxicity of D5 in vitro. The results indicated that D5 had inhibition effects on special cell lines. The BGC-823 cell line, the IC50 of which was 3.550μg/mL, was the most sensitive among those cancer cell lines chosen, and D5 had lower toxicity on normal cell line, such as HL7702, Aml-12 and so on.2, Fusion protein GST-TR3 (LBD) was expressed in E.coli in order to study the interaction between D5 and TR3 (which maybe the target protein of D5), and fluorescence quenching was used to test this interaction. First, the concentration of IPTG (0.3mmol/L) and the density of the culture (OD₆₀₀=0.77) before induction were screened out as the most important factors in GST-TR3 expression by orthogonal test. The protein production reached 2.0mg/L in M9 medium after optimization (similar to LB medium). The result of fluorescence quenching, such as the constants of combination, the position of combination and so on suggested that D5 had obvious interaction with TR3 and provided scientific data for further study.3, The anti-tumor activities of the derivatives of D5 were tested also. The antitumor activities of 49 synthetic compounds, including the series of Zhn, Lix, Wj, Lwj and so on, were tested by MTT method with Zhn89 (D5) as control. Ten compounds were screened out for their good effects similar to those of D5. The antifungal activities of these 11 compounds (including D5) were tested against Saccharomyces cerevisiae and Candida albicans. The results indicated that D5, Zhn89, Zhn111, Zhn196a, Lwj126 and Wj94 has good inhibition effects on Saccharomyces cerevisiae with the MIC of about 100～140μM; only D5 had inhibition effect on Candida albicans with its MIC of 100μM.4, The cytotoxicity of natural products S3 was primarily studied for this thesis by MTT method. Ten cell lines from human and mice were chosen to test the cytotoxicity of S3 in vitro. The results indicated that S3 had inhibition effects on special cell lines. The Hela cell line with IC₅₀ of 1.275μg/mL, was the most sensitive of the cancer cell lines chosen. S3 also had toxicity to normal cell lines, such as HL7702, Aml-12 and so on.5, Furthermore, the mechanism of S3 on Hela cell line was thoroughly studied. It was discovered that S3 could induce Hela cell line apoptosis through morphological change, DAPI stain, FCS analysis, Western Blotting, and so on. Meanwhile, the results of acute toxicity test suggested that S3 belonged to the group of substance with low toxicity with its LD50 above 2000mg/kg in weight. S3 was worth developing.6, SA-1, the derivative of S3, had similar inhibitory effect on Hela cell line as S3 tested by MTT method. The ROS contents of S3 and SA-1 were measured by FCS, and the results indicated that SA-1 could obviously raise the ROS content but S3 could not. Meanwhile, SA-1 had no effect on cell cycle distribution because no apoptosis peak was captured by FCS analysis. Maybe the death pathway of Hela cell line was necrosis.The results of this thesis indicated that both D5 and S3 had good bioactivities, and studies of their derivatives suggested that structures of compounds played an important role in their bioactivities. Possibly, structural modification would be the new way develop the new drug with high efficiency and low side-effects.