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NSAID-induced apoptosis in Rous sarcoma virus-transformed chicken embryo fibroblasts is dependent on v-src and c-myc
X Lu1, D W Fairbairn, W S Bradshaw
1Department of Zoology, Brigham Young University, Provo, UT 84602, USA.
Abstract:
Mounting epidemiological and experimental evidence implicates non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.
Insights
Non-steroidal anti-inflammatory drugs (NSAIDs) induce cancer cell death (apoptosis) through a src/myc pathway. Bcl-2 expression inhibits this NSAID-driven apoptosis, suggesting a novel therapeutic target for cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Non-steroidal anti-inflammatory drugs (NSAIDs) show anti-tumorigenic properties.
- NSAID treatment of src-transformed fibroblasts induces apoptosis, a key mechanism in cancer cell death.
- The proto-oncogene pp60v-src antagonizes apoptosis in many cell systems.
Purpose of the Study:
- To quantitate apoptosis induced by NSAIDs using the comet assay.
- To investigate the role of cyclooxygenase (COX) enzymes and apoptosis-associated genes in NSAID-induced cell death.
- To elucidate the specific molecular pathways, including src, myc, and bcl-2, involved in NSAID-mediated apoptosis.
Main Methods:
- Utilized the comet assay to detect and quantitate DNA cleavage, indicating apoptosis.
- Analyzed cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) mRNA levels during cell death.
- Examined the expression of apoptosis-associated genes: c-rel, p53, bcl-2, and c-myc.
- Investigated the effect of bcl-2 overexpression and antisense c-myc oligonucleotides on NSAID-induced apoptosis.
Main Results:
- Diclofenac treatment of pp60v-src-activated fibroblasts induced 100% lethality, significantly enhancing apoptosis.
- Cyclooxygenase-2 (COX-2) mRNA increased uniformly with all tested apoptotic NSAIDs, while COX-1 remained unchanged.
- Overexpression of bcl-2 inhibited diclofenac-induced apoptosis by 90%, and antisense c-myc reduced cell death by 50% while decreasing drug-induced myc expression.
- NSAID treatment following pp60v-src activation persistently induced myc protein and mRNA by over 20-fold.
Conclusions:
- NSAID-induced apoptosis in src-transformed fibroblasts is mediated via a src/myc-dependent pathway.
- Bcl-2 expression negatively regulates NSAID-induced apoptosis, highlighting its potential as a therapeutic target.
- These findings provide a deeper understanding of the molecular mechanisms underlying NSAID anti-tumorigenic effects.