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Endogenously released Smac is insufficient to mediate cell death of human lung carcinoma in response to etoposide
Babett Bartling1, Rolf Lewensohn, Boris Zhivotovsky
1Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Abstract:
Cytotoxic agents eliminate tumor cells via different mechanisms including apoptosis, although this process is not equally efficient in all kinds of cancer cells. Thus, small cell lung carcinomas (SCLCs) are more sensitive than non-small cell lung carcinomas (NSCLCs) to therapy-induced killing. During apoptosis, several apoptogenic proteins release from the mitochondria. Among these proteins is Smac/DIABLO. Overexpression of Smac effectively potentiates apoptosis by neutralizing the caspase-inhibitory function of the inhibitors of apoptosis proteins (IAPs). However, the physiological relevance of endogenously released Smac in the promotion of malignant cell death is still unclear. Analysis of a panel of human lung cancer cell lines revealed that there is no altered Smac expression in NSCLC and SCLC that might initially impair the drug-induced cell death. Upon engagement of the mitochondrial pathway of apoptosis, etoposide provoked cytosolic accumulation of Smac along with cytochrome c and loss of the mitochondrial membrane potential. Most of these events as well as nuclear apoptotic changes required caspase activation in SCLC, but not in NSCLC. Unexpectedly, pan-caspase inhibition had no effect on Smac release. Co-treatment of SCLC with the IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis in a concentration-dependent manner, whereas Smac downregulation by small interfering RNA (siRNA) did not influence caspase-3/-7 activities, nuclear morphological changes, DNA fragmentation, and plasma membrane integrity. Release of cytochrome c and mitochondrial protease Omi/HtrA2 is still detectable at these conditions. These data suggest that Smac deficiency may be compensated for by action of redundant determinants to kill cancer cells. Thus, translocation of endogenous Smac into cytosol does not play a critical role in cell death of human lung carcinoma after etoposide treatment.
Insights
Smac/DIABLO protein release is not critical for etoposide-induced apoptosis in lung cancer cells. Even when Smac/DIABLO is downregulated, cancer cells still undergo apoptosis, suggesting redundant cell death pathways in non-small cell lung carcinomas.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Apoptosis, or programmed cell death, is a key mechanism for eliminating tumor cells, but its efficiency varies across cancer types.
- Small cell lung carcinomas (SCLCs) are generally more sensitive to apoptosis-inducing therapies than non-small cell lung carcinomas (NSCLCs).
- Smac/DIABLO is an apoptosis-promoting protein released from mitochondria during cell death, which counteracts inhibitors of apoptosis proteins (IAPs).
Purpose of the Study:
- To investigate the physiological role of endogenously released Smac/DIABLO in promoting malignant cell death in human lung cancer.
- To determine if Smac/DIABLO deficiency contributes to the differential sensitivity of SCLC and NSCLC to apoptosis-inducing drugs.
Main Methods:
- Analysis of Smac/DIABLO expression in human lung cancer cell lines (SCLC and NSCLC).
- Treatment of cell lines with etoposide to induce apoptosis and monitoring of Smac/DIABLO and cytochrome c release, mitochondrial membrane potential, and caspase activation.
- Manipulation of Smac/DIABLO levels using small interfering RNA (siRNA) and assessment of apoptosis markers.
- Inhibition of caspases and use of Smac-N7 peptide to evaluate Smac/DIABLO function.
Main Results:
- No significant alteration in Smac/DIABLO expression was found in NSCLC or SCLC cell lines.
- Etoposide treatment induced Smac/DIABLO cytosolic accumulation and mitochondrial membrane potential loss in both SCLC and NSCLC.
- While caspase activation was required for etoposide-induced apoptosis in SCLC, it was not essential in NSCLC.
- Pan-caspase inhibition did not affect Smac/DIABLO release.
- Smac/DIABLO downregulation via siRNA did not inhibit etoposide-induced apoptosis, suggesting compensation by other cell death pathways.
Conclusions:
- The translocation of endogenous Smac/DIABLO into the cytosol is not a critical determinant of etoposide-induced cell death in human lung carcinoma.
- Lung cancer cells possess redundant mechanisms for apoptosis that can compensate for Smac/DIABLO deficiency.
- Differential sensitivity of SCLC and NSCLC to apoptosis may involve factors beyond Smac/DIABLO levels or function.
