Novel anticancer compounds from Chinese plant extracts

Cancer is the cause of 12% of the world`s mortality and the second-leading cause of death in Austria and Germany. Natural products are an important source for new anti-tumour compounds. In a pilot project the applicant and the project partners Joanneum Research Graz, the Institute of Toxicology, University of Mainz, and the Kunming Institute of Botany (Chinese Academy of Sciences) identified out of 256 tested plant extracts 23 extracts with inhibitory activity against the human leukaemia cell line CCRF-CEM. Of those, 8 extracts from 5 plants were selected which appeared most promising because of extraordinary bioactivity (more than 90 % inhibition of cell growth) and lacking literature data related to active principles. These active extracts have been fractionated by chromatographic methods and tested again. So, 42 highly active fractions have been identified. The specific hypothesis is that the project team wants to isolate compounds from extracts which have already shown strong activity in cytotoxicity assays. This is the approach which led also to the isolation of paclitaxel or camptothecin and is called "activity guided isolation". It means that each active extract will be separated into fractions, which then will be tested again in the cytotoxicity assay. The tests necessary for activity guided isolation (CCRF-CEM leukaemia cells, MDA-MB-231 breast cancer cells, HCT116 colon cancer cells, and U251 CNS cancer cells) have been established at the Institute of Pharmaceutical Sciences, Dept. Pharmacognosy in Graz. MCF-7, MX-1, and MDA-MB-231 breast cancer cells, SK-OV-3 ovarian cancer cells, AN2CA endometrium carcinoma cells, HeLa cervix carcinoma cells, A549, LX1, and LXF289 lung cancer cells, PC-3, DU-145, and DAN-G prostate cancer cells, DLD1, HCT 116, and CX1 colon cancer cells, U87-MG, U937, and U251 CNS cancer cells, MeWo, MEL 154, and SK-MEL28 melanoma cells, and ACHN, MRI-H-121, and 769 renal cell carcinoma cells are available and will be tested at the German Cancer Research Centre. Active fractions will be separated again into further fractions, which again will be tested, and so on, until we end up with pure compounds. The active pure compounds will then be studied mechanistically in the different further assays. Regularly the approach of "activity guided isolation" leads to the active constituents and so there is no need for alternative strategies. However, in fact it could happen that during isolation activity gets lost. This usually can mean that the active compounds are unstable. This can be detected by a thorough analysis of the extract before and after fractionation which will directly hint to the missing and therefore potentially active compound(s). If such instability of constituents would occur, more soft isolation techniques (e.g. solvent partition chromatography or Sephadex chromatography) would have to be applied. A second explanation of loss of activity could be that synergistic effects of different constituents are responsible for activity. This can be tested by combination of inactive fractions which resulted from an active extract. If synergistic activity is detected it would be an even more interesting aspect from a mechanistic point of view, which then could be studied. Active fractions which shall be investigated will be obtained from the following plants: Hydnocarpus anthelmintica, (Flacourtiaceae, extract HA- E1), Eleutherococcus senticosus (Araliaceae, extract ES-E1), Lonicera japonica extract (Caprifoliaceae, extract LJ- E1), Caesalpinia sappan (Caesalpiniacea, extracts CS-E1 and CS-E2), Confidential plant (Euphorbiaceae, extracts BJ-E1, BJ-E2 and BJC-E3). Active compounds will be further investigated in a subsequent project in growth inhibition assays using cell lines from solid tumours for validation (MCF-7, SK-OV-3, A549, PC-3, DLD1, U87, MeWo). Cell lines most sensitive to test substances in vitro will be grown as xenograft tumours on nude mice to prove the activity in vivo. In addition, the toxicity of the test substances to normal tissues will be investigated. Test substances which are active in vitro and in vivo will be investigated for their molecular modes of action in low and high density microarrays with cancer related genes.

Estimated 700 to 800 plants are used in traditional Chinese medicine (TCM) to treat cancer or related syndromes. Several well established tumour therapeutics such as paclitaxel and camptothecin have already been derived from plants or have a direct relationship to TCM. In preceding investigations, a database including over 560 species used to treat cancer or ulcers has been created by our group. In this project, seven of these plants, which have been selected based on an initial screening, were investigated towards their anti-cancer activities. These plants were Bischofia javanica Blume, Caesalpinia sappan L., Eleutherococcus senticosus (Rupr. & Maxim.) Harms., Hydnocarpus anthelminthica Pierre ex Laness., Onosma paniculata Burr. & Franch., Periploca sepium Bunge, and Saussurea lappa Clarke. The goal was the isolation and identification of the compounds responsible for cytotoxic effects. Of each plant, extracts of different polarities were tested whether they inhibited the growth of several cancer cell lines in vitro. The active extracts were then subjected to an activity-guided fractionation. By means of several chromatographic techniques, the extracts were fractionated, and obtained active fractions were further analyzed and processed. Obtained pure compounds were identified using several spectroscopic techniques such as NMR, MS and CD. The most promising candidate identified was Onosma paniculata, a plant whose roots are traditionally used to treat especially cancer, cuts, dermatitis, and burns. Five active shikonin derivatives could be isolated and identified. The main and most active compound, dimethylacrylshikonin, was further investigated towards its mode of action within cancer, especially melanoma cell lines. Experiments conducted so far indicate, that it leads to programmed cell death and targets proteins involved in cell death, survival and drug resistance. This compound and its mode of action will be the object of a follow-up project. Active compounds were also isolated from Caesalpinia sappen (brazilin, sappanchalcone and sappanol), Periploca sepium (periplocin, glucosyl divostroside, periplocymarin, periplogenin, periplocoside M and others), and Saussurea lappa (costunolide and dehydrocostus lactone).