Transfection with 9 different siRNA pools showed that Bid and Bik siRNA significantly reduced thaspin-induced cytokeratin 18 caspase-cleavage 
in HCT116 cells, suggesting that these proteins are regulators of apoptosis elicited by this compound. Topoisomerase inhibition was tested using in vitro enzyme assays. The results showed that thaspine inhibits both topoisomerase I and II activity at the apoptotic concentration. Furthermore, thaspine was found to have a reduced cytotoxic effect on the viability on CEM/VM-1, a cell line selected for resistance to the topoisomerase II inhibitor teniposide compared to the parental cell line CCRF-CEM. CEM/ VM-1 harbors a mutated topoisomerase II gene which mediates a specific resistance to topoisomerase II inhibitors, but not general multidrug resistance. CCRF-CEM are not resistant to camptothecin. The resistance to thaspine was not as pronounced as seen for etoposide, known to be a non-intercalating topoisomerase II inhibitor, but well in line with the intercalating topoisomerase inhibitors doxorubicin and mitoxantrone. These data further suggest that thaspine is a topoisomerase inhibitor. Thaspine induced an accumulation of HCT116 cells in the S and G2/M phases of the cell cycle. For comparison, the topoisomerase II inhibitor etoposide induced G2/M accumulation, whereas camptothecin induced some S-phase arrest. We here screened a collection of natural products for their capacity to induce apoptosis of colon carcinoma cells. Natural products are known to have a high chemical diversity, a necessity for drug discovery in the oncology field. This approach lead to the identification of 20 agents that induced strong increases in the levels of caspase-cleaved cytokeratin 18 in colon carcinoma cells. Several of these compounds are well known to have anti-tumor activity. Of the remaining compounds we noted thaspine, an alkaloid present in the cortex of the South American tree Croton lechleri. Thaspine is of interest since Croton lechleri is used in traditional medicine. A red latex, Dragon��s blood, is extracted from the tree cortex and used by tribes of the Amazonian basin for several purposes, including wound healing, as an anti-inflammatory agent, and to treat cancer. Thaspine was previously reported to be cytotoxic, anti-angiogenic, and to have antitumor activity. Consistent with these previous reports, we found that thaspine treatment induced caspase activation in tumor tissue and release of human caspase-cleaved CK18 from tumor cells into the blood of SCID mice. Our connectivity map analysis showed that thaspine induced a similar gene expression pattern as the topoisomerase inhibitors ellipticine and camptothecin. Direct measurements of enzyme activity confirmed that both topoisomerase I and II were inhibited by relevant concentrations of thaspine. Furthermore, CEM/VM-1 cells, which express a mutated form of topoisomerase II resistant to inhibitors of this enzyme, showed increased resistance to thaspine. Topoisomerases are enzymes which have important roles in DNA metabolism by CT99021 252917-06-9 adjusting the number of supercoils in the DNA molecule – a key requirement for Dabrafenib transcription and replication. Topoisomerase I is capable of introducing single strand breaks in DNA, while topoisomerase II can break both strands. A variety of clinically used anticancer drugs inhibit the action of topoisomerase I or topoisomerase II. The topoisomerase I inhibitors topotecan and irinotecan are among the most effective drugs used to treat colorectal, small cell lung and ovarian cancer.