Potential mechanisms for apoptosis induced by both the NSAIDs and thioredoxin, including inhibition of the transcription factor NF-κB

Abstract

This work describes an investigation into potential mechanisms by which both the non-steroidal anti-inflammatory drugs (NSAIDs) and the redox protein thioredoxin (Trx) may modulate sensitivity to apoptosis in colon carcinoma via the transcription factor NF-κB. NSAIDs are chemopreventive against colon cancer. NSAIDs appear to inhibit tumor growth by a mechanism distinct from cyclooxygenase inhibition. All NSAIDs tested induced apoptosis in HT-29 colon cells at concentrations near their respective anti-proliferative IC₅₀ concentrations. In a search for potential apoptotic mechanisms of the NSAIDs, indomethacin was found to decrease intracellular concentrations of diacylglycerol (DAG) in both FBS and TGFα-stimulated cells. This decrease did not derive from inhibition of either phospholipase D or phosphatidylinositol-specific phospholipase C. Thus, the specific mechanism by which indomethacin decreases DAG levels remains undefined. Signaling via the DAG/protein kinase C (PKC) pathway regulates both growth and apoptosis. DAG stimulates PKC, which in turn, indirectly stimulates the transcription factor NF-κB. Activation of NF-κB protects cells from apoptosis. Ibuprofen was observed to inhibit NF-κB activity only at high concentrations, while aspirin and indomethacin did not inhibit NF-κB. Human Trx redox-regulates transcription factors that affect cell growth, including NF-κB. At low concentrations of FBS, stable overexpression of wildtype Trx (wTrx) in HT-29 cells resulted in faster anchorage-independent growth compared to control, while the growth of clones overexpressing a redox inactive mutant Trx (mTrx) was inhibited. Stable overexpression of wTrx increases the sensitivity of HT-29 cells to both constitutive and etoposide-induced apoptosis. Overexpression of wTrx also inhibits constitutive NF-κB activity, while mTrx overexpression stimulates NF-κB. However, the Trx-mediated increase in apoptotic sensitivity is not linked to Trx-mediated inhibition of constitutive NF-κB activity. During cell differentiation, Trx exclusion from the nucleus is associated with both cell differentiation and increased sensitivity to apoptosis. However, Trx is localized to both the cytoplasm and nucleus many primary tumors, and is associated with both increased growth rate and resistance to apoptosis. In HT-29 colon cells, Trx is localized exclusively to the cytoplasm. This may alter the ability of Trx to redox-regulate certain transcription factors, thereby increasing the apoptotic sensitivity of cells.