Am J Transl Res 2015;7(11):2442-2461 www.ajtr.org /ISSN:1943-8141/AJTR0016309
Original Article A SILAC-based proteomics elicits the molecular interactome of alisertib (MLN8237) in human erythroleukemia K562 cells Li-Ping Shu1,2, Zhi-Wei Zhou2, Dan Zi1,2, Zhi-Xu He1, Shu-Feng Zhou2 Guizhou Provincial Key Laboratory for Regenerative Medicine, Tissue Engineering and Stem Cell Research Center, Laboratory Animal Center, Department of Immunology, Guiyang Medical University, Guiyang, Guizhou 550004, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33612, USA 1
Received September 16, 2015; Accepted November 4, 2015; Epub November 15, 2015; Published November 30, 2015 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. Keywords: Alisertib, human erythroleukemia cells, cell cycle, apoptosis, autophagy, SILAC
Introduction Myeloproliferative neoplasms are a group of clonal hematopoietic malignancies that include chronic myeloid leukemia (CML), polycythemia vera, essential thrombocythemia, and primary myelofibrosis, with a characteristics of excessive proliferation of myeloid/erythroid lineage cells [1, 2]. CML accounts for 10-15% among those neoplasms [3, 4]. Almost all CML patients have a chromosomal abnormality known as the Philadelphia chromosome producing an abnormal protein called BCR-ABL that signals the bone marrow to keep generating abnormal white blood cells [3, 4]. Currently, CML therapies include surgery, chemotherapy, radiother-
apy, immunotherapy, and target therapy [1]. Imatibnib, a tyrosine kinase inhibitor (TKI), is the first targeted drug approved by FDA in 2001 and has become the “Gold standard” treatment for CML, due to its activity to specifically inhibit BCR-ABL protein. Other targeted therapeutics also included dasatinib, nilotinib, bosutinib, and ponatinib. Although there is great advances been made in the treatment of CML, many patients still develops resistance to TKI (e.g. imatinib) treatment mainly due to the mutations in ABL kinase. The drug resistance substantially compromises the clinical therapeutic outcome in CML treatment. Therefore, it is imperative to develop more efficacious and safe drug for the treatment of CML.
Proteomic responses to alisertib in K562 cells Aurora kinase A (AURKA), a member of a family of serine-threonine kinases, regulates mitosis [5]. The role of AURKA in the pathogenesis of cancer has been attracted increasing attention and AURKA has been proposed to be a therapeutic target in cancer treatment [6, 7]. Currently, the AURKA inhibitor alisertib (MLN8237, ALS, Figure 1A) is being tested in various Phase I and Phase II clinical trials for advanced solid tumors and hematologic malignancies [8-13]. ALS selectively inhibits AURKA and has been shown in preclinical studies to induce cell cycle arrest, polyploidy, and mitotic catastrophe in various types of tumour cells and induce tumour regression [14-16]. Notably, it has been reported that aberrant activity and expression of AURKA has been implicated in the pathogenesis of leukemia and that AURKA may function as a target for leukemia targeted therapy [1720]. In particular, it has been shown that ALS was active in resistant CML and significantly increased the efficacy of nilotinib [21]. However, the molecular interactome of ALS in CML treatment has not been investigated yet.
FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN). Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad Inc. (Hercules, CA). The proteomic quantitation kit, ionic detergent compatibility reagent (IDCR), Pierce BCA protein assay kit, and Western blotting substrate were obtained from Thermo Scientific Inc. (Hudson, NH). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and the other primary antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA).
Due to the lack of comprehensive and global understanding on the proteomic responses to ALS in the treatment of CML, it is challengable to evaluate the anticancer effect of ALS and to explore the underlying mechanism for its cancer cell killing effect. It therefore needs a practical approach to unveil the full spectrum of molecular targets of ALS in CML treatment. Stable-isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach to uncover the global proteomic responses to drug treatment and other interventions [22-24]. Particularly, it can be used to systemically and quantitatively evaluate and explore the target network of drugs, assess drug toxicity, and identify new biomarkers for the diagnosis and treatment of important diseases, including cancer [23-25]. In this regard, we evaluated the proteomic responses and validated the molecular targets of ALS in K562 cells using a combination of proteomic and functional approaches, with a focus on the effect of ALS on cell cycle progression, apoptosis, and autophagy.
For proteomic analysis, K562 cells were cultured in DMEM/F12 for SILAC with (heavy) or without (light) stable isotope labeled amino acids (13C6 L-lysine and 13C6 15N4 L-arginine) and 10% dialyzed FBS. Cells were cultured in SILAC medium for six cell doubling times to achieve a high level (>98%) of labeled amino acid incorporation. Then, the cells were grown in “light” media were treated with 0.05% DMSO for 24 h to function as the negative control; cells grown in “heavy” media were treated with predetermined ALS for 24 h. All the experiments were performed three times independently.
Materials and methods Chemicals and reagents ALS and all cell culture required materials were purchased from Sigma-Aldrich (St. Louis, MO). 2443
Cell line and cell culture The human erythroleukemia cell line K562 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/F12 medium supplemented with 10% heat-inactivated FBS. The cells were maintained at 37°C in a 5% CO2/95% air humidified incubator. ALS was dissolved in DMSO and the final concentration of DMSO was at 0.05% (v/v).
Proteomic response to ALS treatment analyzed by SILAC-based approach Digestion and desalting SILAC protein samples: Prior to the quantitative proteomic analysis, the protein samples were subject to digestion and desalting which were performed using SILACbased approach as previously described [2426]. The desalted samples were concentrated and resuspended in 0.1% formic acid prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS and statistical analysis: The concentrated samples (5 μL) were subject to the hybrid linear ion trap-Orbitrap (LTQ Orbitrap XL, Thermo Scientific Inc., Hudson, NH) as previAm J Transl Res 2015;7(11):2442-2461
Proteomic responses to alisertib in K562 cells
Figure 1. Chemical structure of ALS and the cytotoxic effect of ALS towards to K562 cells. K562 cells were treated with ALS at concentrations ranging from 0.1 to 100 µM for 24 and 48 h and the cell viability was determined using MTT assay. (A) Chemical structure of ALS and (B). The effect of ALS on viability of K562 cells.
ously described [24-26]. Peptide SILAC ratio was calculated using MaxQuant version 1.2.0.13. The SILAC ratio was determined by averaging all peptide SILAC ratios from peptides identified of the same protein.
Autophagy Kit, respectively [28]. The apoptotic and autophagic cells were analyzed using flow cytometry.
Pathway and network analysis: The protein IDs were identified using Scaffold 4.3.2 from Proteome Software Inc. (Portland, OR) and the pathway and network were analyzed using Ingenuity Pathway Analysis (IPA) from QIAGEN (www.ingenuity.com, Redwood City, CA). The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc. ncifcrf.gov/) was also used to provide biological functional interpretation of the potential targets of ALS derived proteomics [27].
The cell lysate was subject to Western blotting assay. Visualization was performed using an enhanced chemiluminescence kit and the blots were analyzed using Image Lab 3.0 (Bio-Rad).
Cell cycle distribution analysis using flow cytometry The effect of ALS treatment on cell cycle distribution was determined by flow cytometry as previously described [28]. A total number of 1×104 cells was subject to cell cycle analysis using a flow cytometer (BD LSR II Analyzer, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Cellular apoptosis and autophagy analysis using flow cytometry The effect of ALS on apoptosis and autophagy of K562 cells was quantitated using PE Anexin V Apotosis Detction Kit and ENZO Cyto-ID® 2444
Western blotting analysis
Statistical analysis The data are presented as the mean ± standard deviation (SD). Comparisons of multiple groups were evaluated by one-way analysis of variance followed by Tukey’s multiple comparison procedure. P
Original Article A SILAC-based proteomics elicits the molecular interactome of alisertib (MLN8237) in human erythroleukemia K562 cells Li-Ping Shu1,2, Zhi-Wei Zhou2, Dan Zi1,2, Zhi-Xu He1, Shu-Feng Zhou2 Guizhou Provincial Key Laboratory for Regenerative Medicine, Tissue Engineering and Stem Cell Research Center, Laboratory Animal Center, Department of Immunology, Guiyang Medical University, Guiyang, Guizhou 550004, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL 33612, USA 1
Received September 16, 2015; Accepted November 4, 2015; Epub November 15, 2015; Published November 30, 2015 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. Keywords: Alisertib, human erythroleukemia cells, cell cycle, apoptosis, autophagy, SILAC
Introduction Myeloproliferative neoplasms are a group of clonal hematopoietic malignancies that include chronic myeloid leukemia (CML), polycythemia vera, essential thrombocythemia, and primary myelofibrosis, with a characteristics of excessive proliferation of myeloid/erythroid lineage cells [1, 2]. CML accounts for 10-15% among those neoplasms [3, 4]. Almost all CML patients have a chromosomal abnormality known as the Philadelphia chromosome producing an abnormal protein called BCR-ABL that signals the bone marrow to keep generating abnormal white blood cells [3, 4]. Currently, CML therapies include surgery, chemotherapy, radiother-
apy, immunotherapy, and target therapy [1]. Imatibnib, a tyrosine kinase inhibitor (TKI), is the first targeted drug approved by FDA in 2001 and has become the “Gold standard” treatment for CML, due to its activity to specifically inhibit BCR-ABL protein. Other targeted therapeutics also included dasatinib, nilotinib, bosutinib, and ponatinib. Although there is great advances been made in the treatment of CML, many patients still develops resistance to TKI (e.g. imatinib) treatment mainly due to the mutations in ABL kinase. The drug resistance substantially compromises the clinical therapeutic outcome in CML treatment. Therefore, it is imperative to develop more efficacious and safe drug for the treatment of CML.
Proteomic responses to alisertib in K562 cells Aurora kinase A (AURKA), a member of a family of serine-threonine kinases, regulates mitosis [5]. The role of AURKA in the pathogenesis of cancer has been attracted increasing attention and AURKA has been proposed to be a therapeutic target in cancer treatment [6, 7]. Currently, the AURKA inhibitor alisertib (MLN8237, ALS, Figure 1A) is being tested in various Phase I and Phase II clinical trials for advanced solid tumors and hematologic malignancies [8-13]. ALS selectively inhibits AURKA and has been shown in preclinical studies to induce cell cycle arrest, polyploidy, and mitotic catastrophe in various types of tumour cells and induce tumour regression [14-16]. Notably, it has been reported that aberrant activity and expression of AURKA has been implicated in the pathogenesis of leukemia and that AURKA may function as a target for leukemia targeted therapy [1720]. In particular, it has been shown that ALS was active in resistant CML and significantly increased the efficacy of nilotinib [21]. However, the molecular interactome of ALS in CML treatment has not been investigated yet.
FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN). Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad Inc. (Hercules, CA). The proteomic quantitation kit, ionic detergent compatibility reagent (IDCR), Pierce BCA protein assay kit, and Western blotting substrate were obtained from Thermo Scientific Inc. (Hudson, NH). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and the other primary antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA).
Due to the lack of comprehensive and global understanding on the proteomic responses to ALS in the treatment of CML, it is challengable to evaluate the anticancer effect of ALS and to explore the underlying mechanism for its cancer cell killing effect. It therefore needs a practical approach to unveil the full spectrum of molecular targets of ALS in CML treatment. Stable-isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach to uncover the global proteomic responses to drug treatment and other interventions [22-24]. Particularly, it can be used to systemically and quantitatively evaluate and explore the target network of drugs, assess drug toxicity, and identify new biomarkers for the diagnosis and treatment of important diseases, including cancer [23-25]. In this regard, we evaluated the proteomic responses and validated the molecular targets of ALS in K562 cells using a combination of proteomic and functional approaches, with a focus on the effect of ALS on cell cycle progression, apoptosis, and autophagy.
For proteomic analysis, K562 cells were cultured in DMEM/F12 for SILAC with (heavy) or without (light) stable isotope labeled amino acids (13C6 L-lysine and 13C6 15N4 L-arginine) and 10% dialyzed FBS. Cells were cultured in SILAC medium for six cell doubling times to achieve a high level (>98%) of labeled amino acid incorporation. Then, the cells were grown in “light” media were treated with 0.05% DMSO for 24 h to function as the negative control; cells grown in “heavy” media were treated with predetermined ALS for 24 h. All the experiments were performed three times independently.
Materials and methods Chemicals and reagents ALS and all cell culture required materials were purchased from Sigma-Aldrich (St. Louis, MO). 2443
Cell line and cell culture The human erythroleukemia cell line K562 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/F12 medium supplemented with 10% heat-inactivated FBS. The cells were maintained at 37°C in a 5% CO2/95% air humidified incubator. ALS was dissolved in DMSO and the final concentration of DMSO was at 0.05% (v/v).
Proteomic response to ALS treatment analyzed by SILAC-based approach Digestion and desalting SILAC protein samples: Prior to the quantitative proteomic analysis, the protein samples were subject to digestion and desalting which were performed using SILACbased approach as previously described [2426]. The desalted samples were concentrated and resuspended in 0.1% formic acid prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS and statistical analysis: The concentrated samples (5 μL) were subject to the hybrid linear ion trap-Orbitrap (LTQ Orbitrap XL, Thermo Scientific Inc., Hudson, NH) as previAm J Transl Res 2015;7(11):2442-2461
Proteomic responses to alisertib in K562 cells
Figure 1. Chemical structure of ALS and the cytotoxic effect of ALS towards to K562 cells. K562 cells were treated with ALS at concentrations ranging from 0.1 to 100 µM for 24 and 48 h and the cell viability was determined using MTT assay. (A) Chemical structure of ALS and (B). The effect of ALS on viability of K562 cells.
ously described [24-26]. Peptide SILAC ratio was calculated using MaxQuant version 1.2.0.13. The SILAC ratio was determined by averaging all peptide SILAC ratios from peptides identified of the same protein.
Autophagy Kit, respectively [28]. The apoptotic and autophagic cells were analyzed using flow cytometry.
Pathway and network analysis: The protein IDs were identified using Scaffold 4.3.2 from Proteome Software Inc. (Portland, OR) and the pathway and network were analyzed using Ingenuity Pathway Analysis (IPA) from QIAGEN (www.ingenuity.com, Redwood City, CA). The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc. ncifcrf.gov/) was also used to provide biological functional interpretation of the potential targets of ALS derived proteomics [27].
The cell lysate was subject to Western blotting assay. Visualization was performed using an enhanced chemiluminescence kit and the blots were analyzed using Image Lab 3.0 (Bio-Rad).
Cell cycle distribution analysis using flow cytometry The effect of ALS treatment on cell cycle distribution was determined by flow cytometry as previously described [28]. A total number of 1×104 cells was subject to cell cycle analysis using a flow cytometer (BD LSR II Analyzer, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Cellular apoptosis and autophagy analysis using flow cytometry The effect of ALS on apoptosis and autophagy of K562 cells was quantitated using PE Anexin V Apotosis Detction Kit and ENZO Cyto-ID® 2444
Western blotting analysis
Statistical analysis The data are presented as the mean ± standard deviation (SD). Comparisons of multiple groups were evaluated by one-way analysis of variance followed by Tukey’s multiple comparison procedure. P
