Novel Apoptotic Protease Activated in Human Breast Cancer Cells After Poisoning Topoisomerase I

摘要

The goal of this grant was to clone the unknown protease activated by the active anti-breast cancer agent, beta-lapachone (beta-lap). The research team showed for the first time that beta-lap required NQ0l, a two-electron reductase elevated in many human breast cancers, for activation. The team then identified the unknown apoptotic protease activated in human breast cancer cells by beta-lap, defining biological endpoints specific for a non-caspase-mediated apoptotic event. The unknown protease: (a) is a non-caspase cysteine protease; (b) cleaves p53, lamin B, and PARP (atypically) in an NQ0l-dependent manner at a time co-incident with calpain activation (appearance of an 18 kDa active form and its movement into the nucleus by confocal microscopy); (c) is calcium- dependent (e.g., the proteolytic cleavage of PARP or p53 was blocked by co- administration of EUTA or EDTA), and the drug causes massive NQ0l-dependent calcium influx within 3 mins posttreatment with 5-8 micro beta-lap; and (d) inhibited by calpastatin, a specific endogenous inhibitor of calpain. We have, therefore, concluded that the unknown protease activated in NQ0l-expressing human breast cancer cells is calpain.