Taxol induces caspase-10-dependent apoptosis.

Taxol (paclitaxel) is known to inhibit cell growth and trigger significant apoptosis in various cancer cells. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. In this study we investigated death receptors, FAS-associated death domain protein (FADD), the activation of caspases-10 and -8 as well as the downstream caspases, and reactive oxygen species (ROS) in taxol-induced apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line. Pretreating the cells with neutralizing antibodies to Fas, tumor necrosis factor (TNF)-alpha receptor 1, or TNF-related apoptosis-inducing ligand receptors (DR4 and DR5) did not affect taxol-induced apoptosis, but transfection of the cells with a dominant negative FADD plasmid resulted in inhibition of taxol-induced apoptosis, revealing that taxol induces apoptosis independently of these death receptors but dependently on FADD. Furthermore, the drug induced activation of caspases-10, -8, -6, and -3, cleaved Bcl-2, Bid, poly(ADP-ribose) polymerase, and lamin B, and down-regulated cellular levels of FLICE-like inhibitory protein (FLIP) and X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, despite the release of cytochrome c from the mitochondria in taxol-treated cells, caspase-9 was not activated. Inhibitors of caspases-8, -6, or -3 partially inhibited taxol-induced apoptosis, whereas the caspase-10 inhibitor totally abrogated this process. Taxol-induced apoptosis was also associated with decreased mitochondrial membrane potential (Deltapsim) and a significant increase in ROS generation. However, increased ROS production was not directly involved in taxol-triggered apoptosis. Therefore, these results demonstrate for the first time that taxol induces FADD-dependent apoptosis primarily through activation of caspase-10 but independently of death receptors.

A P-glycoprotein- and MRP1-independent doxorubicin-resistant variant of the MCF-7 breast cancer cell line with defects in caspase-6, -7, -8, -9 and -10 activation pathways.

BACKGROUND: Several mechanisms are known to cause resistance to chemotherapy in cancer cells, but the mechanisms of drug resistance due to a lack of apoptosis are not well elucidated. MATERIALS AND METHODS: To understand the mechanisms of resistance to apoptosis induced by doxorubicin (DOX), we developed a DOX-resistant variant of MCF-7 referred to as MCF-7/Adr-20, measured growth inhibition by methylene blue cell survival assay, quantitated apoptosis by annexin V binding assay and detected activation of caspases-6, -7, -8, -9 and -10 in these cells. RESULTS: The resistant cells expressed 20-fold resistance to apoptosis induced by DOX compared to MCF-7 cells. MCF-7/Adr-20 cells did not express MDR1 mRNA or its product P-glycoprotein and they did not overexpress MRP-1. Treating MCF-7 cells with 0.01, 0.1 and 1 microM DOX for 72 h induced 8, 14 and 28% apoptosis, respectively. However, only 1 microM DOX was able to trigger about 8% apoptosis in MCF- 7/Adr-20 cells. Moreover, apoptosis triggered by 0.01 and 0.1 microM DOX in MCF-7 cells was mainly caspase-dependent, but at 1 microM about 70% of apoptosis was caspase-dependent. Western blot analysis revealed that caspase-7 was activated at 0.1 and 1 microM DOX treatment and caspases-6, -8, -9 and 10 were only activated at 1 microM DOX treatment in MCF-7 cells, but none of the caspases checked were activated in MCF-7/Adr-20 cells. Moreover, DOX at 0.01 and 0.1 microM induced p53 and p21(WAF-1/CIP-1) to the same extent in both MCF-7 and MCF-7/Adr-20 cells. Therefore, while DOX triggers growth arrest and induces p53 and p21(WAF-1/CIP-1) in these cells, defects in activation of the initiator and executioner caspases play a major role in resistance to apoptosis triggered by DOX.