Elucidation of specific cytotoxic effects of dimethylacrylshikonin in melanoma cells

Despite much research in the last centuries, cancer is still the second most common cause of death in most developed countries and an increasing cause of death in developing countries. The incidence of malignant melanoma is rising faster than any other solid tumor type, and although it is responsible for only 4% of all skin cancers, it is responsible for 79% of all skin cancer deaths. Especially at an advanced stage, melanoma is still one of the most aggressive and incurable types of solid cancer. Therefore, improvements in the understanding of melanoma biology and the identification and development of novel therapeutic strategies are important research objectives. Today, about 79% of all approved anti-cancer drugs are natural products or derived from natural products. In a previous project funded by the Austrian Science Fund (FWF, P21114), constituents from the roots of Onosma paniculata Bur.&Franch. (Boraginaceae), especially dimethylacrylshikonin (DMAS), have emerged as extremely interesting candidates. We have shown that DMAS exhibited most promising effects in melanoma cell lines of different progression stage. There is evidence that DMAS affects several molecules involved in apoptosis, cell growth inhibition, proliferation, and survival which is of high interest for the elucidation of novel modes of action. From our and others results, we expect that DMAS has different molecular targets in melanoma cells, depending on mutational status and stage of progression. Therefore, we will use a panel of melanoma cell lines with these different characteristics and reveal differences in the cell death mode by using several functional assays covering apoptosis, necrosis, senescence and autophagy. In addition, four different cell lines will be chosen and subjected to microarray experiments to identify DMAS responsive pathways and genes. We expect to find classical pathways (e.g. activation of several caspases and the MEK-ERK pathway) to be altered upon DMAS treatment but also so far not identified DMAS responsive genes. Data obtained will be verified by RT-qRCR and investigated in all other cell lines as well and pathway analyses will be deepened using overexpression/silencing and/or ant-/agonists of key enzymes. From an already performed microarray experiment, we know that DMAS targets DHFR (dihydrofolate reductase), p62 (sequestosome 1), RNF181 (ring finger protein 181), PSMB2 (proteasome subunit, beta type, 2) and PSMC4 (proteasome 26s subunit, ATPase, 4) which have not been reported and closely studied yet. In the course of this project, these effects will be further investigated in more detail in parallel. Last but not least, we will modify the structure of DMAS to find even more active derivatives and investigate the effects of DMAS also in vivo. In summary, this study will reveal new insights into the modes of action of DMAS and derivatives in melanoma cell lines and contribute to the necessary search of novel targets in melanoma therapy.

Malignant melanoma (black skin cancer) is the deadliest type of skin cancer and is responsible for more than 75% of all skin cancer deaths. Worldwide, the number of new skin cancer cases is increasing rapidly; therefore, new therapy options are of great importance. There are currently only very limited therapy options, and, for some special forms of black skin cancer (NRAS mutation or metastatic malignant melanoma), there are hardly any effective drugs available. Therefore, the development of new drugs against this disease must has high priority. One way to discover new drugs is screening plant extracts for their cytotoxic effects. A broad screening of traditionally used Chinese medicinal plants was carried out beforehand and only the most effective substances were further investigated. In the current FWF project, we have very successfully tested the most potent substance, ß-ß- dimethylacrylshikonin (DMAS), against various melanoma cell lines. DMAS had been isolated from the roots of Onsoma paniculatum Bur. & Franch. (Boraginaceae) and was tested against different melanoma cell lines (reflecting the diversity of the melanoma), to detect the effects with different assays. The first step was the determination of IC50 values, which were always in a very low concentration range. DMAS induced apoptosis (controlled cell death) as well as necrosis (uncontrolled cell death). More detailed analyses were made using gene expression assays. Apoptosis was verified with different experiments, and one gene (NOXA) crystallized to play a central role in the effect of DMAS. Also, in vivo studies showed that DMAS has a very efficient effect on melanoma cells and that surrounding cells were hardly attacked. Many anti-cancer drugs originate from plants, but occur in nature only in small quantities. Therefore, it is important that the effective substance can be synthesized (to save natural sources), melanoma cells can be treated with low concentrations, controlled cell death (apoptosis) is induced with the substance and no side effects are induced in vivo. All these properties apply to DMAS, which, therefore, offers an innovative possibility for new therapeutic approaches for difficult to treat melanoma. In addition, in the course of this project, further 36 novel shikonin derivatives were synthesized and pharmacologically tested. In the course of these investigations, two additional very potent derivatives were found. Both also induced apoptosis, with IC50 values clearly below the IC50 values of DMAS. Finally, we could show that O. paniculata is often miss-identified. The reason is that the roots of related species look very similar and contain similar constituents. By using a chromatographic procedure (HPTLC), we were able to develop a method to distinguish the species and to identify a very useful marker substance.