The concept of developing target-specific drugs for treatment of cancer has not been as successful as initially envisioned. The success rate of oncology drugs from first-in-man to registration during 1991�C2000 was only around 5% for 10 major high content screening pharma companies. A major causes of attrition in the clinic is lack of drug efficacy. This realization has lead to a renewed interest in the use of bioassays for drug development in the field of oncology. One attractive screening endpoint is apoptosis since this form of cell death is induced by many clinically used anticancer agents. Natural products have been used as source of novel therapeutics for many years. Natural products have been selected during evolution to interact with biological targets and their high degree of chemical diversity make them attractive as lead structures for discovery of new drugs. A number of plant-derived anticancer drugs have received FDA approval for marketing: taxol, vinblastine, vincristine, topotecan, irinotecan, etoposide and teniposide. Antibiotics from Streptomyces species, including 
bleomycins, dactinomycin, mitomycin, and the anthracyclines daunomycin and doxorubicin are important anticancer agents. More recently developed anticancer agents such as the Hsp90 inhibitor geldanamycin was also isolated from Streptomyces. Marine organisms have also been used as source for the search of anticancer agents. Interesting compounds, including bryostatin, Afatinib ecteinascidin and dolastatin, have been identified. Although being the source of lead compounds for the majority of anticancer drugs approved by the Food and Drug Administration, natural products have largely been excluded from modern screening programs. We here used a high-throughput method for apoptosis detection to screen a library of natural compounds using a human colon carcinoma cell line as screening target. One of the most interesting hits in this screen was thaspine, an alkaloid from the cortex of the South American tree Croton lechleri. We show that thaspine is a topoisomerase inhibitor which is active on cells overexpressing drug efflux transporters. Thaspine has previously been described to have anti-tumor activity in the mouse S180 sarcoma model. To examine whether in vivo anti-tumor activity is associated with induction of apoptosis, SCID mice carrying HCT116 xenografts were treated with thaspine and tumor sections were stained with an antibody to active caspase-3. Positivity was observed in tumor tissue at 48 hours after treatment with 10 mg/kg thaspine. We also utilized caspase-cleaved CK18 as a plasma biomarker for tumor apoptosis. When applied to human xenografts transplanted to mice, this method allows determination of tumor apoptosis independently of host toxicity. Most forms of cancer therapeutics induce the mitochondrial pathway of apoptosis. This pathway is associated with opening of the mitochondrial permeability transition pore. We examined whether thaspine induced a decrease in HCT116 mitochondrial membrane potential using the fluorescent probe tetramethyl-rhodamine ethyl ester. Mitochondria in thaspine-treated cells underwent a shift to lower DyM values. A hallmark of the mitochondrial apoptosis pathway is release of cytochrome c from mitochondria to the cytosol. Thaspine was found to induce a decrease in the levels of mitochondrial cytochrome c and an increase of the levels in the cytosol. The Bcl-2 family proteins Bak and Bax are key regulators of the mitochondrial apoptosis pathway. During apoptosis, the conformation of these proteins is altered. Experiments using conformation-specific antibodies showed that thaspine induce conformational activation of both Bak and Bax.