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Polypeptide antibiotic actinomycin D induces Mcl-1 uncanonical downregulation in lung cancer cell apoptosis
Chia-Ling Chen1, Po-Chun Tseng2, Yen-Po Chao3
1School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 110, Taiwan.
Aims:
Actinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation.
Main Methods:
Human adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles.
Key Findings:
We found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis.
Significance:
All of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.
Insights
Actinomycin D induces cancer cell death by downregulating Mcl-1 expression through a proteasome-dependent pathway. This study reveals a novel mechanism for Actinomycin D
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Death Pathways
Background:
- Actinomycin D (Act D) is a clinical antibiotic used to inhibit tumor growth by interfering with RNA elongation.
- Mcl-1 is an anti-apoptotic protein implicated in cancer cell survival.
- The molecular mechanisms underlying Act D's cytotoxic effects, particularly its impact on Mcl-1, require further elucidation.
Purpose of the Study:
- To investigate the effects of Actinomycin D on Mcl-1 expression in human adenocarcinoma cells.
- To elucidate the molecular mechanisms responsible for Act D-induced Mcl-1 downregulation and subsequent apoptosis.
Main Methods:
- Human adenocarcinoma A549 cells were treated with Actinomycin D.
- Apoptosis was assessed using propidium iodide staining and flow cytometry.
- Mcl-1 expression, mitochondrial potential, and caspase activation were analyzed by Western blotting and other assays.
- The role of proteasomal degradation was investigated using the proteasome inhibitor MG132.
Main Results:
- Actinomycin D induced significant cell growth inhibition and apoptosis in A549 cells.
- Treatment led to DNA damage, p53-independent cell death, decreased Mcl-1 expression, and activation of the caspase-9/caspase-3 pathway.
- Proteasomal degradation mediated Act D-induced Mcl-1 downregulation, as evidenced by MG132 reversal.
- Cullin-Ring E3 ligase partially contributed to Mcl-1 downregulation.
Conclusions:
- Actinomycin D exhibits pro-apoptotic effects by promoting an uncanonical downregulation of Mcl-1.
- The proteasome pathway plays a crucial role in mediating Act D-induced Mcl-1 reduction.
- These findings highlight a novel mechanism for Actinomycin D's anti-cancer activity.
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