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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2015 Nov 15;7(11):2442–2461.

A SILAC-based proteomics elicits the molecular interactome of alisertib (MLN8237) in human erythroleukemia K562 cells

Li-Ping Shu 1,2, Zhi-Wei Zhou 2, Dan Zi 1,2, Zhi-Xu He 1, Shu-Feng Zhou 2
PMCID: PMC4697722  PMID: 26807190

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, radiotherapy, 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.

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 [17-20]. 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.

Figure 1.

Figure 1

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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.

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.

Materials and methods

Chemicals and reagents

ALS and all cell culture required materials were purchased from Sigma-Aldrich (St. Louis, MO). 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).

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).

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.

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 SILAC-based approach as previously described [24-26]. 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 previously 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.

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].

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® Autophagy Kit, respectively [28]. The apoptotic and autophagic cells were analyzed using flow cytometry.

Western blotting analysis

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).

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<0.05 was considered to be statistically significant. Assays were performed at least three times independently.

Results

ALS inhibits proliferation and AURKA phosphorylation of K562 cells

We first tested the effect of ALS on the viability of K562 cells using MTT assay. The results showed that ALS markedly inhibited the proliferation of K562 cells (Figure 1B). The percentage of viability was 75.4%, 78.7%, 92.9%, 68.6%, 56.4%, and 17.7% for 24 h treatment and 63.0%, 52.9%, 58.7%, 46.9%, 15.4%, and 4.5% for 48 h treatment, when cells were treated with ALS at 0.1, 1, 5, 25, 50, and 100 µM, respectively. The IC50 values were 45.49 µM and 7.37 µM for 24 and 48 h incubation, respectively (Figure 1B). Taken together, these data suggest that ALS inhibits the growth of K562 cells.

Moreover, the effect of ALS on the phosphorylation of AURKA was determined using Westering botting assay. As shown in Figure 1A and 1B, ALS markedly inhibited the phosphorylation of AURKA at Thr288. Compared to the control, there was a 18.0%, 79.3%, and 55.9% reduction in the level of p-AURKA, whereas there was a 1.4-, 1.6-, and 1.5-fold increase in the level of AURKA when treated with ALS at 0.2, 1, and 5 µM for 24 h, respectively. Consequently, there was a remarkable decrease in the ratio of p-AURKA/AURKA, with a 13.8%, 67.2%, and 44.3% decrease (Figure 2A and 2B). Taken together, ALS inflicts a substantial impact on cellular processes of K562 cells. Following the test of effect of ALS on cell viability and phosphorylation of AURKA, we performed quantitative proteomics to evaluate the proteomic responses to ALS in K562 cells.

Figure 2.

Figure 2

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ALS inhibits the phosphorylation of AURKA in K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the phosphorylation level of AURKA was determined by Western blotting assay. A. Representative blots of p-AURKA, AURKA, and β-actin. B. Relative level of p-AURKA, AURKA, and ratio of p-AURKA/AURKA. β-actin functions as the internal control. Data are expressed as mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.

Overview of proteomic responses to ALS treatment in K562 cells

To determine the proteomic responses to ALS in K562 cells, we conducted SILAC-based proteomics. There were 1,541 protein molecules which were identified as the potential molecular targets, of which 570 proteins’ expression level were increased and 971 proteins’ level were decreased (Table S1). Subsequently, these identified proteins were subject to IPA pathway analysis, showing that 299 signaling pathways and 459 cellular functional proteins directly responded to ALS treatment in K562 cells (Tables S2 and S3). These functional proteins were involved in a number of important cellular processes, such as cellular growth and proliferation, protein synthesis, RNA post-transcription modification, cell death and survival, and post-translational modification. The IPA analysis showed that the top five targeted signaling pathways were EIF2 signaling pathway, eIF4 and p70S6K signaling pathway, protein ubiquitination pathway, mTOR signaling pathway, and mitochondrial dysfunction signaling pathway (Table S2) and the top five proteins with increased expression level were DCTN2, NAP1L1, RPLP0, RPL15, and SNW1 (Table S3). In aggregate, ALS modulates various critical singling pathways and molecular proteins in K542 cells, which, eventually, lead to cell proliferation inhibition and cancer cell death.

ALS modulates networked signaling pathways in K562 cells

Further data mining using IPA and KEGG pathway analysis was carried out to determine whether identified proteins could be mapped to a specific functional network (Table S4). The network analysis from KEGG pathway analysis (Figure S1) and IPA (Figure S2) indicated the key functional proteins and signaling pathways that were involved in cellular growth and proliferation, protein synthesis, and cell survival and death. In Figure S1, the summarized pathways in cancer, including CML, showed the participation of PI3K/Akt, p53, and MAPK signaling pathways in the regulation of cell proliferation and cell death. In Figure S2, the networked signaling pathways revealed a crosstalk among EIF2 signaling pathway, eIF4 and p70S6K signaling pathway, protein ubiquitination pathway, mTOR signaling pathway, and mitochondrial dysfunction, etc. (Figures S3, S4, S5, S6, and S7). These functional proteins and signaling pathways are important in cell cycle regulation, cell survival, cell migration, cell metabolism, and cell autophagy.

ALS regulates cell cycle progression

The proteomic data showed that ALS treatment led to a marked response in cell cycle with the involvement of signaling pathways in G2/M DNA damage checkpoint regulation and cyclins and cell cycle regulation (Table S2). The functional proteins involved in G2/M DNA damage checkpoint regulation included YWHAQ, YWHAG, and CDK1; and the functional protein involved in cyclins and cell cycle regulation included PPP2CB, HDAC1, and CDK1. It suggests that ALS possesses a regulatory effect on cell cycle progression in K562 cells.

ALS regulates EIF2 signaling pathways and ribosome network in K562 cells

ALS also induced a marked response with regard to the protein synthesis, which is a complex process that requires cooperation among a large number of polypeptides including ribosomal proteins, modification of enzymes, and ribosome-associated translation factors. ALS showed a potent effect on EIF2 signaling pathway (Figure S3) and ribosome network (Figure S4). The molecules involved in EIF2 signaling pathway included RPL27A, EIF2B4, MAPK1, RPS8, EIF4G1, EIF4E, EIF2A, RPL7, RPL7A, EIF3B, RPS20, RPS13, EIF3D, RPL23A, RPL31, RPL13, RPS24, RPL32, PABPC1, RPL4, RPS2, RPL17, RPL29, RPS10, EIF3J, RPS21, RPL9, RPLP0, RPS6, RPL15, EIF3F, RPS16, RPL28, EIF3L, RPL13A, and RPS14; and the molecules involved in ribosome network included 60S ribosomal subunit, AP-3, CALB1, CNBP, DDOST, DPP3, Fascin, GSR, IGF2BP1, IGF2BP2, LUC7L3, LYAR, NAT10, NF-κB (complex), OTUB1, PHF6, PUF60, RPL4, RPL7, RPL9, RPL13, RPL15, RPL17, RPL28, RPL29, RPL31, RPL32, RPL13A, RPL23A, RPL27A, RPL7A, SRSF11, TROVE2, UBE2, and ZC3H18.

ALS regulates cell death singals

As showed in Figure S1, ALS has been predicted to affect cell death by KEGG pathway analysis in K562 cells. The IPA results further showed that treatment of K562 cells regulated apoptosis signaling pathway (Table S2) and led to mitochondrial dysfunction (Figure S5). MAPK1, LMNA, and CDK1 responded to ALS treatment in apoptosis signaling pathway. ALS-induced mitochondrial dysfunction was one of the top signaling pathways responding to ALS treatment in K562 cells (Table S2 and Figure S5). The molecular proteins included ATP5J, NDUFV1, COX17, PRDX5, ATP5A1, ACO2, VDAC3, CYB5R3, UQCRB, NDUFB10, VDAC2, GSR, NDUFB9, NDUFS8 NDUFV2, ATP5B, COX7A2, NDUFA10, OGDH, and VDAC1. The data suggest that ALS may induce apoptosis of K562 through mitochondria-mediated pathway.

ALS regulates PI3K/Akt/mTOR, ERK/MAPK, and AMPK signaling pathways

Additionally, the proteomics and IPA data showed that there were marked alteration in signal transduction in response to ALS treatment in K562 cells. As shown in Table S2 and Figure S6, treatment of K562 cells with ALS resulted in a remarkable change in PI3K/Akt/mTOR, MAPK, and AMPK signaling pathways. The protein molecules included YWHAQ, PPP2CB, YWHAG, HSP90AA1, MAPK1, RPS2, RPS10, EIF3J, RPS8, FKBP1A, RPS21, EIF4G1, EIF4E, RPS6, PPP2CB, EIF3F, RPS16, EIF3B, RPS20, RPS13, EIF3D, EIF3L, RPS24, RPS14, and EIF4B in PI3K/Akt/mTOR signaling pathway (Table S2). The proteins included RAP1B, YWHAQ, PPP2CB, YWHAG, MAPK1, TLN1, EIF4E, and KSR1 which were involved in ERK/MAPK signaling pathway (Table S2). PPP2CB, SLC2A1, MAPK1, and PFKP were involved in AMPK signaling pathway (Table S2). Moreover, the IPA also showed that ALS regulated PTEN and eIF4 and p70S6K signaling pathways in K562 cells (Table S2 and Figure S7), which were closely orchestrated with PI3K/Akt/mTOR signaling pathway. The proteins included MAPK1, CDC42, and CSNK2B in PTEN signaling pathway (Table S2) and PABPC1, EIF2B4, MAPK1, RPS2, RPS10, EIF3J, RPS8, RPS21, EIF4G1, EIF4E, EIF2A, RPS6, PPP2CB, EIF3F, RPS16, EIF3B, RPS20, RPS13, EIF3D, EIF3L, RPS14, and RPS24 in eIF4 and p70S6K signaling pathway (Figure S7).

Taken together, the data showed that ALS may exert its anticancer effect via the regulation of the multiple functions proteins and signaling pathways in K562 cells. Subsequentially, we verified the effect of ALS on cell cycle distribution and programmed cell death and explored the potential mechanisms in K562 cells.

Verification of reguatory effects and molecular targets of ALS in K562 cells

Our above quantitative proteomic studies have shown that ALS modulated a number of functional proteins and related signaling pathways in cell proliferation, cell invasion and migration, and cell survival and death. In order to verify these effects, we tested how ALS affected the cell cycle distribution, apoptosis, and autophagy in K562 cells.

ALS induces cell cycle arrest in G2/M phase in K562 cells

First, the cell cycle distribution was determined in K562 cells using flow cytometry. As shown in Figure 3A and 3B, there was a marked alteration in the cell population in cell cycle distribution. Compared to the control cells (9.6%), the percentage of cell population in G2/M phase was 68.0%, 69.0% and 61.9% when cells were treated with ALS at 0.2, 1, and 5 µM for 24 h, with a 7.1-, 7.2-, and 6.4-fold rise in the number of cells arrested in G2/M phase, respectively (P<0.0001; Figure 3B). In contrast, there was a remarkable decrease in the number of cells in G1 and S phases in K562 cells when treated with ALS at 0.2, 1, and 5 µM for 24 h (P<0.01 or 0.0001; Figure 3B). Taken together, the results show that ALS can regulate the cell cycle distribution, contributing to its anticancer effect in K562 cells. Moreover, the inducing effect of ALS on cell cycle arrest further verifies the regulatory activities of ALS on cell proliferation determined by proteomics.

Figure 3.

Figure 3

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ALS induces cell cycle arrest at G2/M phase in K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the cell samples were subject to flow cytometry. (A) Representative plots of cell cycle distribution of K562 cells and (B). The population of K562 cells at G1, S, and G2/M phases. Data are expression as mean ± SD of three independent experiments. **P<0.01; and ****P<0.0001 by one-way ANOVA.

ALS regulates cell cycle regulators of K562 cells

Following the observations on the cell cycle arrest, we examined the effect of ALS on the expression of several key cell cycle regulators in K562 cells. As shown in Figure 4A and 4B, ALS treatment exhibited a marked regulating effect on the expression of PLK-1, CDK1/CDC2, cyclin B1, p21 Waf1/Cip1, p27 Kip1, and p53. There was a marked decrease in the level of PLK-1, CDK1/CDC2, and cyclin B1 that are the positive regulators of cell cycle progression, whereas there was a remarkable increase in the level of p21 Waf1/Cip1 and p27 Kip1 that exhibit inhibitory effect on cell cycle progression (Figure 4A and 4B). Compared to the control cells, 5 µM ALS decreased 23.9% in the level of PLK-1 in K562 cells (P<0.01; Figure 4A and 4B). Treatment of K562 cells with ALS concentration-dependently reduced the expression level of CDK1/CDC2 and cyclin B1 (Figure 4A and 4B). There was a 24.8% and 28.9% reduction in the level of CDK1/CDC2 and 21.1% and 43.8% decline in the level of cyclin B1 when K562 cells were treated with ALS at 1 and 5 µM for 24 h, respectively (P<0.01, 0.001 or 0.0001; Figure 4A and 4B). On the other hand, treatment of K562 cells with 1 µM ALS led to a 1.5-fold increase in the level of p21 Waf1/Cip1, compared to the control cells (P<0.05; Figure 4A and 4B). Moreover, there was a 1.3-, 1.3-, and 1.4-fold elevation in the level of p27 Kip1 when treated with ALS at 0.2, 1, and 5 µM for 24 h, respectively (P<0.05; Figure 4A and 4B). ALS treatment did not significantly altered the expression of p53 in K562 cells. Taken together, the results suggest that the cell cycle arresting effect of ALS may be attributed to the regulating effect on the key cell cycle modulators in K562 cells, which also further verifies the proteomic data showing the effect of ALS on cell proliferation and cell cycle distribution.

Figure 4.

Figure 4

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ALS alters the expression of key cell cycle regulators in K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the expression level of PLK-1, CDK1/CDC2, cyclin B1, p21 Waf1/Cip1, p27 Kip1, and p53 was determined by Western blotting assay. A. Representative blots of PLK-1, CDK1/CDC2, cyclin B1, p21 Waf1/Cip1, p27 Kip1, and p53. B. Relative level of PLK-1, CDK1/CDC2, cyclin B1, p21 Waf1/Cip1, p27 Kip1, and p53. β-actin functions as the internal control. Data are expressed as mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001 by one-way ANOVA.

ALS promotes the expression of DCTN2, NAP1L1, RPLP0, and RPL15 in K562 cells

As shown in the proteomic data, treatment of K562 cells with ALS dramatically altered the expression of DCTN2, NAP1L1, RPLP0, and RPL15. DCTN2 modulates cytoplasmic dynein binding to an organelle, plays a role in prometaphase chromosome alignment and spindle organization during mitosis, and is involved in anchoring microtubules to centrosomes [29]. NAP1L1 participates in DNA replication and may play a role in modulating chromatin formation and contribute to the regulation of cell proliferation [30,31]. RPLP0 and RPL15 are ribosomal proteins involved in protein synthesis [32,33]. The results showed a promoting effect of ALS on the expression of DCTN2, NAP1L1, RPLP0, and RPL15 in K562 cells (Figure 5A and 5B). Compared to the control cells, there was a 1.7-fold increase in the level of DCTN2 when K562 cells were treated with 1 µM ALS for 24 h (P<0.01; Figure 5A and 5B). There was a concentration-dependent elevation in the level of two ribosomal proteins, RPLP0 and RPL15 in K562 cells (Figure 5A and 5B). In comparison to the control cells, there was a 2.7- and 2.8-fold increase in the level of RPLP0 and 1.2- and 1.3-fold rise in the level of RPL15 when cells were treated with ALS at 1 and 5 µM, respectively (P<0.05; Figure 5A and 5B). Although there was no significant increase in the level of NAP1L1, there was a 1.1-, 1.1-, and 1.2-fold increase in the level of NAP1L1 when treated with ALS at 0.2, 1, and 5 µM, respectively (P>0.05; Figure 5A and 5B). Taken together, the results show that ALS exerts a potent effect on protein synthesis and cell proliferation, which may contribute to the cell cycle arresting and cancer cell killing effect in K562 cells.

Figure 5.

Figure 5

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ALS alters the expression of critical proteins in ribosome in K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the expression level of DCTN2, NAP1L1, RPLP0, and RPL15 was determined by Western blotting assay. A. Representative blots of DCTN2, NAP1L1, RPLP0, and RPL15. B. Relative level of DCTN2, NAP1L1, RPLP0, and RPL15. β-actin functions as the internal control. Data are expressed as mean ± SD of three independent experiments. *P<0.05 and **P<0.01 by one-way ANOVA.

ALS induces apoptosis of K562 cells

We further validated apoptosis-inducing effect in K562 cells using flow cytometry and Western blotting assay. As shown in Figure 6A and 6B, ALS induced apoptosis of K562 cells in a concentration-dependent manner. In comparison to the control cells, there was a 1.4-, 1.5-, and 1.7-fold increase in apoptotic K562 cells when treated with ALS at 0.2, 1, and 5 µM, respectively (Figure 6A and 6B). Furthermore, the expression of pro-apoptotic and anti-apoptotic proteins were examined. As shown in Figure 6C and Figure S8, treatment of K562 cells markedly increased the level of Bax, while decreasing the level of Bcl-xl and Bcl-2. In comparison to the control cells, there was a 2.1- and 2.3-fold elevation in the level of Bax when treated with 1 and 5 µM ALS, respectively (P<0.01; Figure 6C and Figure S8); whereas there was a 30.6% and 55.3% reduction in the level of Bcl-2 and 45.6% and 25.6% decline in Bcl-xl when treated with ALS at 1 and 5 µM, respectively (P<0.05 or 0.01; Figure 6C and Figure S8). The concentration-dependent increase in the level of Bax and the decrease in the level of Bcl-2 consequently resulted in a remarkable increase in the ratio of Bax/Bcl-2, which impaired the mitochondrial function in K562 cells. Indeed, treatment of K562 cells induced an impairment in mitochondrial membrane potential, evident from the release of cytochrome c (Figure 6C). There was a 1.9-fold increase the level of cytosolic cytochrome c compared to the control cells, when treated with 5 µM ALS (P<0.05; Figure S8). Increased level of cytosolic cytochrome c triggers the activation of caspase cascade. Compared to the control cells, treatment of ALS at 0.2, 1, and 5 µM resulted in a 1.8-, 1.9-, and 1.9-fold rise in the level of cleaved caspase 9 (P<0.0001; Figure S8). Also, incubation of 5 µM ALS led to a 1.6-fold increase in the level of caspase 3 (P<0.01; Figure S8). Furthermore, there was a 2.4- and 2.5-fold increase in the level of cleaved PARP (P<0.01; Figure S8). Additionally, ALS also up-regulated the negative regulator of Bcl-2 family, PUMA, in K562 cells. Treatment of cells with ALS at 0.2, 1, and 5 µM resulted in 1.6-, 1.6-, and 1.6-fold increase in the level of PUMA compared to the control cells, respectively (P<0.05; Figure S8). Taken together, the results indicate that ALS exhibits a pro-apoptotic effect in K562 cells.

Figure 6.

Figure 6

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ALS induces apoptosis of K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the cell samples were subject to flow cytometry and the cell lysates were subject to Western blotting assay. (A) Representative flow cytometric plots of apoptotic K562 cells; (B) Bar graphs showing the percentage of apoptosis of K562 cells and (C). Representative blots of Bcl-xl, Bcl-2, Bax, PUMA, cytochrome c, cleaved caspase 9, cleaved caspase 3, and cleaved PARP. Data are expression as mean ± SD of three independent experiments. *P<0.05 and **P<0.01 by one-way ANOVA.

ALS induces autophagy of K562 cells

Following the findings on the apoptosis-inducing effect of ALS in K562 cells, the effect of ALS on autophagy of K562 cells was also examined. As shown in Figure 7A, exposure of K562 cells to ALS concentration-dependently increased the autophagy of K562 cells. There was a 1.2-, 1.6-, and 2.2-fold elevation in the autophagic level of K562 cells when treated with ALS at 0.2, 1, and 5 µM, respectively (Figure 7B). Furthermore, the effect of ALS on the autophagy-related signaling pathways was examined (Figure 7C, Figures S9 and S10).

Figure 7.

Figure 7

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ALS induces autophagy of K562 cells. K562 cells were treated with ALS at 0.2, 1, and 5 µM for 24 h and the cell samples were subject to flow cytometry. (A) Representative flow cytometric plots of autophagic K562 cells and (B). Bar graphs showing the percentage of autophagy of K562 cells. (C) Representative blots of phosphorylated PI3K, Akt, mTOR, AMPK, and p38 MAPK, and the expression of PI3K, PTEN, Akt, mTOR, AMPK, p38 MAPK, beclin 1, LC3-I, and LC3-II. Data are expression as mean ± SD of three independent experiments. **P<0.01 by one-way ANOVA.

We further examined the phosphorylation level of PI3K at Tyr458, AMPK at Thr172, and p38 MAPK at Thr180/Tyr182, which are upstream regulators of Akt/mTOR pathway with important role in the regulation of cell proliferation and death [34,35]. ALS significantly inhibited the phosphorylation of PI3K at Tyr458 in K562 cells compared to the control cells (Figure 7C). Exposure of K562 cells to 5 μM ALS for 24 h decreased the phosphorylation level of PI3K at Tyr458 53.3% (P<0.05; Figure S9). However, incubation of K562 cells with ALS did not significantly affect the expression of total PI3K (P>0.05; Figure S9). The ratio of p-PI3K/PI3K was concentration-dependently decreased by ALS in K562 cells. Compared to the control cells, the p-PI3K/PI3K ratio was decreased 60.5%, when treated with 5 μM ALS (P<0.05; Figure S9).

AMPK plays a crucial role in the regulation of energy homeostasis, cell survival, and cell death [36]. In the present study, ALS exhibited a promoting effect on the phosphorylation of AMPK at Thr172 in K562 cells (Figure 7C and Figure S10). In comparison to the control cells, there was a 2.7-fold increase in the phosphorylation level of AMPK at Thr172 in K562 cells when treated with 5 μM ALS for 24 h (P<0.01; Figure S10). However, there was no significant change in the expression of total AMPK compared to the control cells. Of note, with increasing concentration of ALS, an increased ratio of p-AMPK/AMPK was observed in K562 cells. Compared to the control cells, the p-AMPK/AMPK ratio was increased 1.9- and 2.2-fold when treated with ALS at 0.2 and 5 μM, respectively (P<0.05 or 0.01; Figure S10).

p38 MAPK exerts a dual role in the regulation of cell death, and it can either promote cell survival or cell death depending not only on the type of stimulus but also in a cell type specific manner [37]. In contrast to the promoting effect on AMPK phosphorylation, we observed an inhibitory effect of ALS on the activation of p38 MAPK at Thr180/Tyr182 in K562 cells (Figure 7 and Figure S10). In comparison to the control cells, there was a 30.3%, 23.9%, and 7.5% reduction in the phosphorylation of p38 MAPK at Thr180/Tyr182 in K562 cells when treated with0.2, 1, and 5 μM of ALS for 24 h, respectively (Figure S10). Exposure of K562 cells to ALS increased the expression level of total p38 MAPK (P>0.05; Figure S10). Notably, a decreased ratio of p-p38 MAPK/p38 MAPK was observed in both cell lines with increasing concentration of ALS. In comparison to the control cells, the ratio of p-p38 MAPK/p38 MAPK was decreased 33.8%, 37.6%, and 21.8% when K562 cells were treated with ALS at 0.2, 1, and 5 μM, respectively (Figure S10). These findings demonstrate that ALS inhibited the phosphorylation of PI3K Tyr458 and p38 MAPK Thr180/Tyr182 but enhanced the phosphorylation of AMPK Thr172 in K562 cells, contributing to the increase in autophagy flux.

We also examined the regulatory effect of ALS on the phosphorylation of Akt at Ser473 and mTOR at Ser2448 and the expression of PTEN in K562 cells (Figure S10). In comparison to the control cells, the phosphorylation level of Akt at Ser473 was decreased 45.4% and 18.4% in K562 cells with the treatment of ALS at 1 and 5 μM for 24 h, respectively (Figure S10). Notably, there was a significant alteration in the expression of Akt in K562 cells and there was a 1.7-, 2.1-, and 2.1-fold increase in the level of Akt when K562 cells were treated with ALS at 0.2, 1, and 5 µM, compared to the control cells, respectively (P<0.05 or 0.01; Figure S10). Consequently, the ratio of p-Akt/Akt was significantly decreased in K562 cells treated with ALS. In K562 cells, the ratio of p-Akt/Akt was decreased 30.2%, 73.8%, and 59.5% when cells were treated with ALS at 0.2, 1, and 5 μM for 24 h, respectively (Figure S10).

In addition, the expression level of PTEN which is the negative regulator of PI3K/Akt signaling pathway, was significantly increased when K562 cells were treated with 0.2 and 1 μM ALS for 24 h (P<0.01; Figure 7 and Figure S10). And exposure of K562 cells to 5 μM ALS resulted in a 57.5% decrease in the phosphorylation level of mTOR at Ser2448 (P<0.001; Figure S9). There was no significant change in the expression of total mTOR in K562 cells when treated with ALS for 24 h. However, a decreased ratio of p-mTOR/mTOR was observed in K562 cells when treated with ALS. In K562 cells, the ratio of p-mTOR/mTOR was decreased 56.3% when treated with 5 μM PLB (P<0.05; Figure S9).

Additionally, we examined the effect of PLB on the expression level of beclin 1 and LC3-I/II. Treatment of K562 cells with ALS for 24 h concentration-dependently increased the expression of beclin 1 (Figure 7 and Figure S10). There was a 1.4 and 1.5-fold increase of beclin 1 in K562 cells when treated with 1 and 5 μM ALS for 24 h (P<0.01; Figure S10). The results showed two bands of LC3-I and II in K562 cells (Figure 7). Compared to the control cells, there was a 1.4-fold increase in the LC3-II level in K562 cells treated with 5 μM ALS for 24 h (P<0.001; Figure S10). In addition, treatment of K562 cells with ALS decreased the expression of LC3-I, although which was not significantly different. The ratio of LC3-II/LC3-I was remarkably increased 1.3-, 1.3-, and 1.3-fold in K562 cells with treatment of ALS at 0.2, 1, and 5 μM, respectively (P<0.05 or 0.01; Figure S10). These findings indicate that ALS exhibited a strong autophagy-inducing effect on K562 cells via inhibition of the PI3K/Akt/mTOR pathway.

ALS induces apoptosis of K562 cells involving PI3K/Akt/mTOR and p38 MAPK signaling pathways

To further confirm the role of PI3K/Akt/mTOR and p38 MAPK signaling pathways in ALS-induced apoptosis in K562 cells, we employed the specific chemical inhibitors of mTOR (0.5 µM rapamycin), PI3K (10 µM wortmannin), Akt (1 µM MK-2206), and p38 MAPK (10 µM SB202190) to examine the apoptosis of K562 cells in the presence and absence of 5 µM ALS using flow cytometry. In the absence of ALS, incubation of 0.5 µM rapamycin, 10 µM wortmannin, 1 µM MK-2206, and 10 µM SB202190 resulted in an increase in the apoptosis of K562 cells (Figure 8A and 8B). In compared to the control cells (DMSO), there were 2.0- and 1.5-flod elevation in the percentage of apoptotic K562 cells when treated with 10 µM wortmannin and 10 µM SB202190, respectively (P<0.05 or 0.001; Figure 8A and 8B). Moreover, although there was no significant inducing effect of 0.5 µM rapamycin and 1 µM MK-2206 on the apoptosis of K562 cells, there was a 1.2- and 1.3-fold increase in the percentage of apoptotic K562 cells compared to DMSO-treated cells, respectively (P>0.05; Figure 8A and 8B). Notably, co-incubation of K562 cells with 0.5 µM rapamycin, 10 µM wortmannin, 1 µM MK-2206, or 10 µM SB202190 and 5 µM ALS remarkably enhanced ALS-induced apoptosis (Figure 8A and 8B). Compared to DMSO-treated cells, ALS induced a 1.6-fold increase in the percentage of apoptotic K562 cells, respectively (P<0.001; Figure 8A and 8B). In comparison to ALS-treated cells, there was a 1.2-, 1.6-, 1.2-, and 1.4-fold elevation in the percentage of apoptotic K562 cells when co-incubated with rapamycin and ALS, wortmannin and ALS, MK-2206 and ALS, or SB202190 and ALS, respectively (P<0.05 or 0.001; Figure 8A and 8B). On the other hand, compared to the mono-treatment of cell with 0.5 µM rapamycin, 10 µM wortmannin, 1 µM MK-2206, or 10 µM SB202190, there was a marked increase in the percentage of apoptotic K562 cells with the co-incubation with rapamycin and ALS, wortmannin and ALS, MK-2206 and ALS, or SB202190 and ALS (P<0.001; Figure 8A and 8B). Taken together, the results further demonstrate the involvement of PI3K/Akt/mTOR and p38 MAPK signaling pathways in ALS-induced apoptosis in K562 cells.

Figure 8.

Figure 8

Open in a new tab

ALS-induced apoptosis with the involvement of mTOR/Akt and p38 MAPK signaling pathways in K562 cells. K562 cells were treated with ALS in the presence or absence of rapamycin, wortmannin, MK-2206, and SB202190 for 24 h and the cell samples were subject to flow cytometry. (A) Representative plots of apoptotic K562 cells and (B). Bar graphs showing the percentage of apoptosis of K562 cells. Data are expression as mean ± SD of three independent experiments. *P<0.05 and ***P<0.001 by one-way ANOVA.

Discussion

Current CML therapies often fail mainly due to the drug resistance, which requires and spurs the development of new therapeutics with novel targets. As stated above, identification of the molecular targets of ALS is critical for CML therapy. With the application of SILAC-based proteomic approach, the present study has evaluated the proteomic responses to ALS treatment in K562 cells, including numerous functional proteins and related signaling pathways. The subsequential validating assays indicated that ALS-regulated proteins and signaling pathways were involved in cell cycle distribution, apoptosis, and autophagy with the participation of PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways in K562 cells.

The SILAC-based proteomics possesses the capability of quantitatively and comprehensively evaluating the effect of a given compound and identify its potential molecular targets and related signaling pathways in vitro or in vivo [38-40]. Our previous studies have unveiled the molecular interactome of plumbagin and 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in several cancer cell lines [41,42], and explored the potential molecular targets and possible mechanisms for the anticancer effects. In the present study, we employed SILAC-based proteomic approach to evaluate the cellular proteomic responses to ALS treatment in K562 cells, showing that ALS regulated a number of functional proteins and signaling pathways involved in cell cycle progression, apoptosis, and autophagy in K562 cells, such as DCTN2, NAP1L1, RPLP0, RPL15, PI3K/Akt/mTOR, PTEN, ERK/MAKPK, and AMPK and their related signaling pathways. The proteomics results suggest that ALS may target these signaling molecules to elicit its anticancer effects in the treatment of CML. Notably, we further validated the proteomic responses to ALS treatment in K562 cells.

With increasing preclininal and clinical studies focusing on the role of the Aurora kinases in the formation and the treatment of tumours, AURKA becomes an important therapeutic target in cancer therapy [43-45]. Accumulating evidence shows that abberation in the activity and expression of AURKA leads to tumor development and progression [46]. Recent reports have shown that AURKA can induce drug resistance and regulate several key signaling pathways related to cell cycle progression, cell migration and invision, and programmed cell death in cancer cells [46-48], suggesting a pivotal role in cancer cell signaling [49]. Our recent studies showed that inhibition of AURKA/B led to a marked cancer cell death [28,50-53]. In the present study, we found that ALS significantly inhibited the phosphorylation of ARUKA and induced cell death in K562 cells, suggesting an anticancer potential in CML treatment.

Due to the central role of AURKA in mitosis, we examined the effect of ALS on cell cycle distribution and found that ALS dramatically arrested K562 cells in G2/M phase, which also verified the proteomic data. Based on the proteomic and flow cytometric data, we speculated that the possible mechanism of ALS on G2/M arrest in K562 cells might involve a number of key regulators, such as PLK-1, p21 Waf1/Cip1, p53, cyclins and cyclin-dependent kinases. In our study, the findings of proteomics and Western blotting assays clearly showed a potent regulatory effect of ALS on the expression of PLK-1, p21 Waf1/Cip1, p53, cyclins, and CDKs. Notably, cell cycle progression is tightly regulated by cyclins and CDKs [54]. p21 Waf1/Cip1, a cyclin-dependent kinase inhibitor, is regulated by p53. It binds to CDC2-cyclin B1 complex, inducing cell cycle arrest [55]. The CDC2 and cyclin B1 complex plays a major role in the entry of cells into mitosis, because cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. Thus, taken the proteomic and validating results into consideration, ALS-induced cell cycle arrest may be through the regulation of key modulators controlling the G2/M check point in K562 cells.

Notably, the proteomic data showed that ALS regulated ribosomal signal and dramatically altered the expression of DCTN2, NAP1L1, RPLP0, and RPL15 in K562 cells, which have important roles in protein synthesis and cell division. Human DCTN2 encodes a 50 kD subunit of dynactin, a macromolecular complex consisting of 10-11 subunits ranging in size from 22 to 150 kD. DCTN2 is involved in a diverse array of cellular functions, including endoplasmic reticulum to Golgi transport, the centripetal movement of lysosomes and endosomes, spindle formation, chromosome movement, nuclear positioning, and axonogenesis [29]. Moreover, NAP1L1 participates in DNA replication and may play a role in modulating chromatin formation and contribute to the regulation of cell proliferation [30,31]; RPLP0 and RPL15 are ribosomal proteins involved in protein synthesis [32,33]. Thus, we tested the expression level of DCTN2, NAP1L1, RPLP0, and RPL15 in K562 cells when treated with ALS. The findings showed that ALS exhibited a potent promoting effect on the expression of DCTN2, NAP1L1, RPLP0, and RPL15, which may provide further explanation on the cell cycle arresting effect of ALS on K562 cells.

In the present study, the proteomic study also showed that ALS regulated mitochondrial function and cell death. Disruption of mitochondrial function and the resultant cytochrome c release initiate apoptosis process, with the latter being activated caspase cascade [56,57]. Also, pro-apoptotic members of the Bcl-2 family but antagonized by anti-apoptotic members of this family were highly involved in apoptosis [56,57]. Anti-apoptotic members of Bcl-2 is suppressed by post-translational modification and/or by increased expression of PUMA, an essential regulator of p53-mediated cell apoptosis [58]. Cytochrome c released from mitochondria to cytosol induces that activation of caspase 9, subsequently activating caspase 3 [59]. In our study, the finding showed that cytosolic level of cytochrome c was significantly increased and that caspase cascade was markedly activated in response to ALS treatment, which contributes to ALS-induced apoptosis of K562 cells. Intriguingly, the specific chemical inhibitors of mTOR (rapamycin), PI3K (wortmannin), Akt (MK-2206), and p38 MAPK (SB202190) enhanced ALS-induced apoptosis of K562 cells, indicating the involvement of PI3K/AKT/mTOR, MAPK, and AMPK signaling pathways in ALS-induced apoptosis. Furthermore, the proteomic results showed that ALS exhibited a modulating effect on PI3K/Akt/mTOR, ERK/MAPK, and AMPK signaling pathways in K562 cells, which play critical role in regulation of cellular process, including autophagy. Autophagy (also known as type II programmed cell death) is extremely important for a variety of human diseases, especially cancers. It affects various stages of initiation and progression of cancer with the participation of overlapped signaling pathways of autophagy and carcinogenesis [35,60,61]. Accumulating evidence shows that the PI3K/Akt/mTOR, MAPK, and AMPK signaling pathways have been regarded to be the key regulators of a series of cell processes as they can be deregulated by various genetic and epigenetic mechanisms, in a wide range of cancer cells [60,62]. PI3K activates the serine/threonine kinase Akt, which in turn through a cascade of regulators results in the phosphorylation and activation of the serine/threonine kinase mTOR, activated mTORC1 inhibits autophagy by direct phosphorylation of Atg13 and ULK1 at Ser757 [34,35,63,64]. Also, p38 MAPK and AMPK signals were orchestrated with autophagy process [60]. In the present study, ALS induced autophagy in K562 cells as indicated by flow cytometric data and the increase in the expression of beclin 1 and the ratio of LC3-II over LC3-I. Of note, the PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways were altered in response to ALS treatment. Taken together, out findings indicate that PI3K/AKT/mTOR, MAPK, and AMPK signaling pathways contribute to ALS-induced programmed cell death in K562 cells.

In summary, the quantitative SILAC-based proteomic approach showed that ALS inhibited cell proliferation, induced cell cycle arrest, activated mitochondria-dependent apoptotic pathway and induced autophagy in human K562 cells involving a number of key functional proteins and related molecular signaling pathways, such as PI3K/Akt/mTOR, MAPK, and AMPK signaling pathways. This study may provide a clue to fully identify the molecular targets and elucidate the underlying mechanism of ALS in the treatment of CML, resulting in an improved therapeutic effect and reduced side effect in clinical settings.

Acknowledgements

The authors appreciate the financial support from the Startup Fund of the College of Pharmacy, University of South Florida, Tampa, Florida 33612, USA. Dr. Zhi-Wei Zhou is a holder of a postdoctoral scholarship from College of Pharmacy, University of South Florida, Tampa, Florida 33612, USA.

Disclosure of conflict of interest

None.

Authors’ contribution

Participated in research design: Li-Ping Shu, Zhi-Wei Zhou, Dan Zi, Zhi-Xu He, and Shu-Feng Zhou. Conducted experiments: Li-Ping Shu and Zhi-Wei Zhou. Contributed new reagents or analytic tools: Zhi-Xu He and Shu-Feng Zhou. Performed data analysis: Li-Ping Shu, Zhi-Wei Zhou and Dan Zi. Wrote or contributed to the writing of the manuscript: Li-Ping Shu, Zhi-Wei Zhou, Zhi-Xu He and Shu-Feng Zhou.

Supporting Information

ajtr0007-2442-f9.pdf (3.9MB, pdf)

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