Natural Product Anticancer Drug Discovery and Mechanistic Studies on Hapolosin and Silvestrol

Cancer is one of the major health concerns for people and will be for the foreseeable future. Chemotherapy, as well as surgery and radiation, is an important clinical method to successfully treat cancer. Within the chemotherapeutic agents, natural products and chemicals derived from or related to natural products constitute an important part of the oncologist’s armamentarium. The studies in this thesis focus on screening natural products for chemotherapeutic candidates and clarifying their mechanisms. This thesis contains three parts. The first part describes screening natural products for activity against human cancer cells. The second part is focused on elucidating the mechanism of action of hapalosin and hapalosin B, chemicals capable of reversing multidrug resistance. The third section of the thesis contains a study on the mechanism of anticancer activity of silvestrol, a promising chemotherapeutic candidate. The collected natural materials, from tropical plants, aquatic cyanobacteria, or filamentous fungi, were tested in human cancer cells to screen for inhibitors of cancer cell growth and proteasome activity. Screening began with evaluation of crude extracts. Active extracts were separated into several fractions the fractions were tested again. The fractionation and bioassay procedure were repeated until active pure compounds were obtained. This process is referred to as bioassay guided fractionation. Hapalosin B and silvestrol were the two most interesting compounds that were evaluated during this project. Hapalosin B, as well as hapalosin, was isolated from species of the cyanobacterium Nostoc, using a bioactivity guided strategy. Its analogue, hapalosin, can reverse multidrug resistance in cancer cells overexpressing P-gp protein, a plasma membrane transporter that pumps a variety of substances, including anti-cancer drugs, out of the cell. Experiments presented here extend these findings by demonstrating that hapalosin B has similar activity as hapalosin: the hapalosins can reverse multidrug resistance in cells that overexpress P-gp. Furthermore, the hapalosins were found to inhibit P-gp activity in a non-competitive manner and not to affect the expression of P-gp protein production. Silvestrol was isolated from the fruits and twigs of Aglaia silvestris (M. Roemer) Merrill and has shown potent (low nM) anticancer activity. It has been reported that it interferes with the assembly of the eIF4F complex, which inhibits initiation of protein synthesis. Studies were conducted to further these findings. Apoptosis is induced by silvestrol through inhibition of the PI3K/AKT pathway; which further suppresses protein synthesis by inhibiting the activity of p70S6K. Silvestrol also induces autophagy and its upstream genes expression.