Cancer incidence is increasing and novel anticancer drugs with new mechanisms of action are essential for future chemotherapeutic treatment. Natural products have historically played an important role in the development of anti-cancer drugs and have potential to do so also in the future. In this thesis two classes of natural products are identified as possible drug lead candidates, and the mechanisms of their action are elucidated. Initially, in a screening of a compound library for cytotoxic effects in colon cancer cells, natural products with potent activity were identified. Based on their potency, and on previously reported activities in cancer cells, two main groups of compounds, cardiac glycosides (CGs) and gambogic acid (GA) analogues, were selected for further in-depth studies. The concentration-dependent cytotoxicity was confirmed in cell lines of different origin. Cardiac glycosides were mainly evaluated for their activity in colon cancer cells and in leukemic cells, whereas the GA analogues were studied using a resistance-based panel of ten human cancer cell lines. Using activity profiles and the ChemGPS-NP model, the compounds were compared, structurally and mechanistically, to standard chemotherapeutic drugs. The results from these analyses suggested that the CGs and the GA analogues act by mechanisms different from those of antimetabolites, alkylating agents, topoisomerase I and II inhibitors, or tubulin-active agents. By analysis of drug-induced gene expression, one GA analogue, dihydro GA, was identified as a possible inhibitor of the ubiquitin-proteasome system (UPS), and the CGs showed similarities to protein synthesis inhibitors. Starting from these hypotheses, we further investigated the mechanisms of actions on a molecular level. The results showed that GA and dihydro GA act as inhibitors of the 20S proteasome chymotrypsin activity, leading to accumulation of ubiquitinated proteins. The CGs were confirmed to inhibit protein synthesis in colon cancer cell lines. However, interestingly, in leukemia cell lines, it seemed that the CGs act through a different, yet unexplored, mechanism of action. The leukemic cells (pre-B and T-ALL) were particularly susceptible to the cytotoxic effects of CGs, including at concentrations that may be achievable in the clinic.

<p>The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. </p><p>The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. </p><p>The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species.</p><p>In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.</p>

The development of resistance by tumour cells to chemotherapeutic agents is a major problem in cancer treatments. One way to counter this is to find compounds with cytotoxic mechanisms other than those of drugs in clinical use today. The biological and chemical diversity encountered in Nature provide opportunities to discover completely new chemical classes of compounds. Some of these may represent previously unknown anticancer agents, and in some cases, novel, potentially relevant cytotoxic mechanisms. The selection of plants for the cytotoxic investigation in this project was designed to cover large parts of the angiosperm system, providing a broad representation of species. Extracts of the plants were subjected to a polypeptide fractionation protocol, followed by bioassay-guided isolation, yielding series of fractions with increasing purity and cytotoxicity. The cytotoxicity assay included tumour cells from patients and a cell-line panel including ten different cell lines representing several types of resistant and non-resistant tumours. This screening strategy allowed fractions and compounds acting with novel mechanisms to be detected at an early stage. The compounds isolated represent substantial chemical diversity and originate from diverse parts of the phylogenetic spectrum examined. They include the highly potent cytotoxic alkaloid, thiobinupharidine, the structure of which was determined by NMR techniques. Furthermore, two types of compound were shown to have previously unreported cytoxic activity: cyclotides (small macrocyclic polypeptides, in this case from violets) and polypeptides, possibly of thionine type, of loranthaceaeous mistletoes (collected in Panama). The well known cardiac glycosides from the foxglove, Digitalis, were identified as being responsible for the anti-tumour activity of this species. In conclusion, the results obtained in this project show that selection based on phylogenetic information, together with a robust and reliable method to detect cytotoxicity, can be a useful approach for exploring the plant kingdom for cytotoxic substances.

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