Structure Modification Of Ganoderic Acids And Study On Anti-tumor Mechanism Of Novel Ganoderic Acid Derivatives

Ganoderma lucidum is a well-known traditional Chinese medicinal fungus and has been used to prevent and treat various diseases for thousands of years in China. Recent pharmacological researches have revealed that triterpenoids including ganoderic acids (GAs) possess interesting anti-tumor activities. A two-stage culture process of G. lucidum developed in our lab has shown great potential for efficient production of GAs, which provides a materials base for their pharmacological study. So far, six GAs, namely, GA-T,-Me,-Mk,-Mf,-S and7-O-ethyl GA-O, have been purified from the G. lucidum mycelia in our lab. Our previous studies demonstrated that GA-T and GA-Me exhibited inhibitory effects against various tumor cell lines, and induced apoptosis and inhibited metastasis in mice. However, the anti-tumor function of the other four GAs remains unclear and thus needs to be revealed. In another aspect, chemical modification of natural product is a useful approach to develop more powerful molecules for cancer treatment. Nevertheless, no information has bee reported on the modification of GAs for obtaining better anticancer agents.In this dissertation, by employing MTT assay, we discovered triterpenoid-enrich fraction displayed cytotoxicity while non-triterpenoid fraction did not. This result confirmed that similar to the fruiting body of G. lucidum, triterpenoids are the major anti-tumor constituents in its fermentation mycelia. Subsequently, six GAs (GA-T,-Me,-Mk,-Mf,-S and7-O-ethyl GA-O) were tested in vitro for their cytotoxic effects on human tumor cell lines (SW1990, HeLa,95D and HO-8910) and all of them showed significant cytotoxicity against the tested tumor cells. Among the six GAs, GA-T had the most cytotoxicity to tumor cells and its cytotoxic effect was dependent on the dosage. Moreover, its low cytotoxic effect on normal skin fibroblast cell line HF suggested that it had a well selectivity between normal and cancer cells. In addition, the apoptotic function of a pair of isomers, GA-Mf and GA-S were also investigated and compared. GA-Mf had more potent in inducing HeLa cells apoptosis than that of GA-S. What is more, GA-Mf caused cell cycle arrest in the G1phase, while GA-S caused cell cycle arrest in the S phase. The difference of this pair of GAs in anticancer effect was possibly ascribed to the location change of substituents and the α/β configuration of C-3position. GAs contain various substituents such as hydroxyl, carboxyl and acetyl groups. These substituents not only endow the versatile bioactivities of this class of compounds, but provide excellent sites for chemical modification. Currently, there are no reports on the chemical structure and activity relationship (SAR) of GAs. Using GA-T as template, a series its derivatives were semi-synthesized by esterification, amidation and deacetylation. The structures of derivatives including four novel compounds, namely, TLTO-Ee, TLTO-Pe, TLTO-A and GA-T1, were elucidated by NMR and MS.By using cervical cancer cell line HeLa as a model, comparative study on the anti-tumor activity and mechanism of GA-T and its carboxyl-terminal derivatives (TLTO-A, TLTO-Me, TLTO-Ee and TLTO-Pe) were carried out. The cytotoxic effect of the five compounds on HeLa cells was found to rank in the order of TLTO-A> GA-T-TLTO-Me-TLTO-Ee≈TLTO-Pe. However, all the tested compounds arrested the HeLa cells at the G1phase, which indicated that the differentia of substituent group at the C-26position do not change the ability of GA to arrest the cell cycle. Furthermore, they decreased the mitochondrial membrane potential and increased the activities of pro-apoptotic factors caspase-3and caspase-9. Accordingly, the apoptosis induction was presumed to occur through the mitochondrial pathway. TLTO-A showed the most potent effect on pro-apoptosis among the tested compounds. These results suggested that the carboxyl group of GA-T is not the main active group and is suitable for its further structural modification.This dissertation also explored the contribution of acetylation of GAs to their anti-tumor activity. Four structurally related GAs were used in this study, including GA-T, GA-Mk, GA-T1and GA-T2with3,2,2and1acetyl groups present in their mother core, respectively. The results showed that their cytotoxicity against HeLa cells was:GA-T> GA-Mk GA-T1> GA-T2. The mechanism of their inducing apoptosis of HeLa cells was subsequently investigated. GAs suppressed the antioxidant defense system by decreasing glutathione (GSH) level and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX). Meanwhile, they increased the intracellular and mitochondrial reactive oxygen species (ROS) level, resulting in activation of the pathway of mitochondria-mediated apoptosis. Additionally, the acetyl groups of GAs may contribute to their cytotoxicity and their potency for ROS-mediated apoptosis. On the other hand, significant decrease of ROS level and caspase-3activity was observed by addition of exogenous antioxidants such as N-acetylcysteine (NAC), catalase (CAT) and diphenyleneiodonium chloride (DPI). For the first time, these results revealed that ROS may play a key role in the inducing apoptosis of HeLa cells by GAs.Taken together, this dissertation investigated the chemical modification of GA-T and studied the anti-tumor activity and mechanism of its novel derivatives. These results may provide valuable information on SAR of GAs on anti-tumor effect and the chemotherapy agents’ development.