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Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors
Marina Stamenkovic1, Kristina Janjetovic2, Verica Paunovic1
1Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia.
Abstract:
We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling.
Insights
Lysosomal autophagy inhibitors like bafilomycin A1, chloroquine, and ammonium chloride induce cell death independently of autophagy. These agents trigger mitochondrial dysfunction and apoptosis through distinct molecular pathways involving lysosomal permeabilization and signaling.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Cancer Research
Background:
- Lysosomal autophagy inhibitors are investigated for their cytotoxic potential in cancer therapy.
- Understanding the precise molecular mechanisms of these inhibitors is crucial for optimizing their therapeutic application.
- Bafilomycin A1, chloroquine, and ammonium chloride are common agents used to inhibit lysosomal function.
Purpose of the Study:
- To comparatively analyze the molecular cytotoxic mechanisms of bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells.
- To elucidate the role of autophagy, lysosomal permeabilization, and specific signaling pathways in the observed cytotoxicity.
Main Methods:
- Comparative analysis of bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells.
- Assessment of oxidative stress, mitochondrial depolarization, caspase-dependent apoptosis, and lysosomal membrane permeabilization.
- Analysis of mRNA expression of apoptosis-related genes (Bcl-2 family, Pten, Puma, Bim, p53) and signaling pathway activation (mTOR, MAPK, ERK, JNK, AMPK).
Main Results:
- All agents induced oxidative stress, mitochondrial depolarization, and caspase-dependent apoptosis, independent of autophagy.
- Chloroquine, but not bafilomycin A1 or ammonium chloride, caused lysosomal membrane permeabilization.
- Distinct effects on Bcl-2 family mRNA expression and differential activation of MAPK/AMPK signaling pathways were observed among the inhibitors.
Conclusions:
- Lysosomal inhibitors induce autophagy-independent mitochondrial dysfunction and apoptosis.
- The agents exhibit distinct mechanisms regarding lysosomal permeabilization and modulation of Bcl-2 family proteins and signaling pathways.
- These mechanistic differences contribute to their varied cytotoxic effects and potential for synergistic or antagonistic interactions.
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