Effect of Lactacystin on the Sulindac-Induced Apoptosis Mechanisms in HT-29 Cells

Kim, Jung Min; Park, Ki Jae; Kim, Sung Heun; Choi, Hong Jo

Abstract

PURPOSE: One of possible mechanisms of the antineoplastic effect by nonsteroidal anti-inflammatory drugs (NSAIDs) is an induction of apoptosis. The NSAIDs-induced apoptosis appears to be caspase- and mitochondria-dependent. The ubiquitin-proteasome system, which is a fundamental non- lysosomal tool that cells use to process or degrade a variety of short-lived proteins, is known to be involved in apoptosis and to be located upstream of mitochondrial changes and caspase activation. The present study was conducted to explore the potential role of proteasome pathway in NSAIDs-induced apoptosis.
METHODS
We employed sulindac as a NSAID, and the lactacystin as a proteasome inhibitor to investigate the extent of the apoptosis in colon cancer cell line, HT-29 cells. The proteasome activity and the amount of apoptosis were quantified after cells were treated with 1 mM sulindac, 1micrometer lactacystin or both.
RESULTS
Sulindac treatment caused apoptosis of the HT-29 cells in a time-dependent manner with resultant changes in nuclear morphology. Western blots also showed caspase-3 activation and PARP cleavage after sulindac treatment. Not only single treatment with lactacystin decreased proteasome activity, but co-treatment with sulindac enhanced decrease in proteasome activity further (P<0.01). Treatment with lactacystin only did not induce apoptosis. However, lactacystin augmented the induction of sulindac-induced apoptosis (P<0.01). This synergistic effect was also proven by Western blot analyses, where co-treatment augmented the caspase-3 activation and PARP degradation.
CONCLUSIONS
The combination treatment of sulindac with a proteasome inhibitor lactacystin is suggested to be a very effective strategy for the induction of cancer cell apoptosis. Elucidation of the mechanism underlying the regression of colon cancers by combination of sulindac and lactacystin seems to be an immediate challenge in the near future.