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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
Published on: July 6, 2014
A SILAC-based proteomics elicits the molecular interactome of alisertib (MLN8237) in human erythroleukemia K562 cells
Li-Ping Shu1, Zhi-Wei Zhou2, Dan Zi1
1Guizhou Provincial Key Laboratory for Regenerative Medicine, Tissue Engineering and Stem Cell Research Center, Laboratory Animal Center, Department of Immunology, Guiyang Medical UniversityGuiyang, Guizhou 550004, People's Republic of China; Department of Pharmaceutical Sciences, College of Pharmacy, University of South FloridaTampa, FL 33612, USA.
Abstract:
Alisertib (MLN8237, ALS), an Aurora kinase A (AURKA) inhibitor, exerts potent anti-tumor effects in the treatment of solid tumor and hematologic malignancies in preclinical and clinical studies. However, the fully spectrum of molecular targets of ALS and its anticancer effect in the treatment of chronic myeloid leukemia (CML) are not clear. This study aimed to examine the proteomic responses to ALS treatment and unveil the molecular interactome and possible mechanisms for its anticancer effect in K562 cells using stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data identified that ALS treatment modulated the expression of 1541 protein molecules (570 up; 971 down). The pathway analysis showed that 299 signaling pathways and 459 cellular functional proteins directly responded to ALS treatment in K562 cells. These targeted molecules and signaling pathways were mainly involved in cell growth and proliferation, cell metabolism, and cell survival and death. Subsequently, the effects of ALS on cell cycle distribution, apoptosis, and autophagy were verified. The flow cytometric analysis showed that ALS significantly induced G2/M phase arrest and the Western blotting assays showed that ALS induced apoptosis via mitochondria-dependent pathway and promoted autophagy with the involvement of PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways in K562 cells. Collectively, this study provides a clue to quantitatively evaluate the proteomic responses to ALS and assists in globally identifying the potential molecular targets and elucidating the underlying mechanisms of ALS for CML treatment, which may help develop new efficacious and safe therapies for CML treatment.
Insights
Alisertib (ALS) effectively targets cancer cells by modulating protein expression, inducing cell cycle arrest, apoptosis, and autophagy. This study reveals ALS mechanisms for treating chronic myeloid leukemia (CML).
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Alisertib (MLN8237, ALS) is an Aurora kinase A (AURKA) inhibitor with demonstrated anti-tumor activity.
- The precise molecular targets and anticancer mechanisms of ALS in chronic myeloid leukemia (CML) remain incompletely understood.
Purpose of the Study:
- To investigate the proteomic responses to ALS treatment in K562 cells.
- To identify molecular interactomes and elucidate the anticancer mechanisms of ALS in CML.
Main Methods:
- Utilized stable-isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis.
- Performed pathway and functional enrichment analysis on differentially expressed proteins.
- Verified effects on cell cycle, apoptosis, and autophagy using flow cytometry and Western blotting.
Main Results:
- ALS treatment modulated 1541 proteins (570 upregulated, 971 downregulated).
- Affected proteins were involved in cell growth, metabolism, survival, and death pathways.
- ALS induced G2/M phase arrest, mitochondria-dependent apoptosis, and autophagy via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling.
Conclusions:
- This study provides a quantitative proteomic evaluation of ALS effects in K562 cells.
- Identified potential molecular targets and elucidated mechanisms underlying ALS's anticancer activity in CML.
- Findings may contribute to developing novel and safer CML therapies.
