Skip to content

Advertisement

  • Research article
  • Open Access
  • Open Peer Review

LC-MS based sphingolipidomic study on A549 human lung adenocarcinoma cell line and its taxol-resistant strain

BMC Cancer201818:799

https://doi.org/10.1186/s12885-018-4714-x

  • Received: 17 August 2017
  • Accepted: 1 August 2018
  • Published:
Open Peer Review reports

Abstract

Background

Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T).

Methods

A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis.

Results

Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 (p < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T.

Conclusions

This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, “inhibition of the de novo synthesis pathway” and “activation of glycosphingolipid pathway” may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer.

Keywords

  • A549 human lung adenocarcinoma cell line - Taxol-resistant - LC-MS - Sphingolipids - Ceramide

Background

Lung cancer has been the leading cause of cancer mortality, and adenocarcinoma is its most prevalent form [1]. Paclitaxel (taxol) is commonly used as part of combination chemotherapy for the treatment of non-small cell lung cancer including adenocarcinoma A549. However, resistance to natural product chemotherapy drugs still constitutes a huge problem of successful cancer treatment, and the efficiency of chemotherapy is weakened because of paclitaxel resistance [2]. Potential mechanisms have been reported including multidrug resistance, β-tubulin alterations, detoxifying of paclitaxel, and apoptosis related genetic changes [3]. Although the extensive efforts have been made for understanding the underlying mechanisms, they are still elusive.

It has been recognized that the dysregulated metabolic profile of cancer is linked to the chemoresistance [4]. Cancer cells reprogram their metabolism to satisfy the demands of malignant phenotype, which decrease drug-induced apoptosis, conferring therapeutic resistance [5]. Since cellular SPLs appear to play a significant role in relation to cancer, their dysregulated synthesis and metabolism in drug-resistant cancer cells have been systematically studied [6]. Most previous studies focus on the biological effect of a kind of specific SPL like Cer [7] and S1P [8] on A549 cancer cell line. The sphingolipid profiles for A549 have been preliminary explored by using MALDI-TOF-MS, only two Cers have been defined as markers out of all the 9 SPLs detected in A549 [9]. The whole sphingolipidome in either A549 or A549T remains largely unrevealed. Recently, a versatile sphingolipidomic approach for both qualitative and quantitative analysis of up to 10 subclasses of SPLs has been established in our group [10]. In this study, the integrated LC-MS approach was employed to investigate the taxol resistance mechanism of A549T from the viewpoint of sphingolipidomic.

Methods

Chemicals and materials

The LIPID MAPS internal standard cocktail (internal standards mixture II, 25 μM each of 9 compounds in ethanol, catalog LM-6005) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). It was composed of uncommon SPLs which include: 17-carbon chain length sphingoid base analogs C17-sphingosine [So (d17:1)], C17-sphinganine [Sa (d17:0)], C17-sphingosine-1-phosphate [S1P (d17:1)], C17-sphinganine-1-phosphate [Sa1P (d17:0)], the C12-fatty acid analogs of the more complex SPLs C12-Ceramide [Cer (d18:1/12:0)], C12-ceramide-1-phosphate [C1P (d18:1/12:0)], C12-sphingomyelin [SM (d18:1/12:0)], C12-glucosylceramide [GlcCer (d18:1/12:0)], and C12-lactosylceramide [LacCer (d18:1/12:0)].

Acetic acid (CH3COOH, MS grade), formic acid (HCOOH, MS grade), ammonium acetate (NH4OAc, ACS grade) and potassium hydroxide (KOH, ACS grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC grade chloroform (CHCl3), isopropanol (IPA), as well as methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Fetal Bovine Serum (FBS), Penicillin-Streptomycin (PS) were obtained from Gibco, New Zealand. Sodium dodecyl sulfate (SDS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were acquired from Acros, USA. Ultrapure water (18.2 MΩ) was supplied with a Milli-Q system (Millipore, MA, USA).

Cell culture and SPLs extraction

A549 human lung adenocarcinoma cell line (Cat.No. KG007) and its taxol-resistant strain (A549T, Cat.No. KG124) were obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). A549 was cultured in DMEM supplemented with 10% FBS and 1% PS in a humidified 5% CO2 atmosphere at 37 °C. A549T was cultured in RPMI 1640 medium supplemented with solution consisted of 10% FBS, 1% PS and 200 ng/mL taxol in a humidified 5% CO2 atmosphere at 37 °C. For lipid analysis, A549 and A549T cells were respectively seeded into 6-well plates at the density of 1.5 × 105 cells/well and incubated for 48 h. Lipids were extracted from the cells, when they were grown to 80% confluence. After rinsed twice by ice-cold PBS, the cells were scraped into a borosilicate glass tube, in which 0.5 mL of MeOH, 0.25 mL of CHCl3 and 10 μL of 2.5 μM internal standards cocktail were added. The extract procedure was carried out by incubation at 48 °C for 12 h after sonicated at ambient temperature for 30 s. After 75 μL of KOH in MeOH (1 M) was added, the mixture was placed into a shaking incubator at 37 °C for 2 h. Acetic acid was used to neutralize the mixture before the typical four-step extraction was carried out for the preparation of SPLs. Further details for extracting SPLs and sample preparation were the same as previously described [11]. MTT assay was employed to evaluate the sensitivity of A549 and A549T cells to taxol. The IC50s were 67.72 nM and 124.7 μM, respectively corresponding to A549 and A549T, showing almost 2000-fold difference in taxol sensitivity between these two cell lines.

LC-MS conditions

Sphingolipid analysis was performed by using our developed LC-MS method with minor optimization, just as described previously [10, 11]. Chromatographic separation was achieved by using an Agilent 1290 UHPLC system, and it was interfaced with an Agilent ultrahigh definition 6550 Q-TOF mass spectrometer and an Agilent 6460 triple-quadrupole mass spectrometer respectively for qualitative- and quantitative-analysis. The acquisition and data analysis were operated by using Agilent MassHunter Workstation Software.

Data analysis

Based on the Agilent Personal Compound Database and Library (PCDL) software and LIPID MAPS Lipidomics Gateway, a personal database has been established with the latest update of 32,622 SPLs until August 06 2016. The screening and identification of SPLs were carried out by searching against it.

In qualitative research, the sphingolipidomic approach was applied by analyzing QC samples equally pooled by A549 and A549T. In quantitative research, A549 cells (models, n = 10) and A549T cells (models, n = 10), as well as QC samples (n = 5), were analyzed in parallel. Multivariate statistical analysis, including principle component analysis (PCA) and partial least squares to latent structure-discriminant analysis (PLS-DA) methods, were performed to examine significant differences between A549 and A549T, using SIMCA-P+ software version 14.0 (Umetrics, Umea, Sweden). Variable Importance in the Project (VIP) value in PLS-DA model was used for selecting and identifying biomarkers. The altered SPL with a VIP value larger than 1.00 was considered as a biomarker.

Results

Comprehensive profiling of sphingolipids in A549 and A549T cells

QC samples were analyzed repeatedly to achieve comprehensive profiling of SPLs in A549 and A549T. In various subclasses of SPLs, the [M + H]+ ions exhibits highest intensities in positive ion mode. Totally 114 SPLs have been identified in the QC samples, among which Cer (d18:2/26:2), DHCer (d18:0/24:2), phytosphingosine (PTSo) t19:2, and PTSo t16:1 were new SPLs. Notably, 4 pairs of isobaric species (A1-A4 vs a1-a4) and 21 pairs of isomeric species (B1-B21 vs b1-b21) were clearly distinguished in this study. Respective qualitative test of A549 and A549T revealed that they share all the same species of SPLs. The full identification result was listed in Table 1.
Table 1

Identification and quantification of SPLs in A549/A549T cells by using UHPLC-Q-TOF and UHPLC-QQQ MS

Class

Name

[M + H]+ m/z

tR (min)

Molecular Formula

Measured Mass

Calculated Mass

Error (ppm)

MS/MS Fragments (m/z)

MRM transitions

SM

d18:1/26:0 [B1]

843.7314

18.483

C49 H99 N2 O6 P

842.7240

842.7241

−0.12

264.2674, 184.0730

843.7

184.1

d18:1/26:1

841.7096

17.136

C49 H97 N2 O6 P

840.7026

840.7084

−6.90

264.2682, 184.0739

841.7

184.1

d18:1/25:0 [B2]

829.7154

17.702

C48 H97 N2 O6 P

828.7080

828.7084

−0.48

264.2683, 184.0732

829.7

184.1

d18:1/25:1 [B3]

827.6993

16.288

C48 H95 N2 O6 P

826.6915

826.6928

−1.57

264.2698, 184.0722

827.7

184.1

d18:1/24:0 [B4]

815.6992

17.020

C47 H95 N2 O6 P

814.6925

814.6928

−0.37

264.2697, 184.0732

815.7

184.1

d18:1/24:1 [B5]

813.6841

15.507

C47 H93 N2 O6 P

812.6769

812.6771

−0.25

264.2685, 184.0739

813.7

184.1

d18:1/24:2

811.6680

14.958

C47 H91 N2 O6 P

810.6607

810.6615

−0.99

264.2688, 184.0732

811.7

184.1

d18:1/24:3 [B6]

809.6526

14.243

C47 H89 N2 O6 P

808.6459

808.6458

0.12

264.2627, 184.0725

809.7

184.1

d18:1/23:0 [B7]

801.6839

16.371

C46 H93 N2 O6 P

800.6769

800.6771

−0.25

264.2645, 184.0727

801.7

184.1

d18:1/23:1 [B8]

799.6683

15.241

C46 H91 N2 O6 P

798.6608

798.6615

−0.88

264.2654, 184.0721

799.7

184.1

d18:1/23:2

797.6520

14.326

C46 H89 N2 O6 P

796.6443

796.6458

−1.88

264.2398, 184.0727

797.7

184.1

d18:1/22:0 [B9]

787.6679

15.723

C45 H91 N2 O6 P

786.6608

786.6615

−0.89

264.2665, 184.0730

787.7

184.1

d18:1/22:1 [B10]

785.6524

14.642

C45 H89 N2 O6 P

784.6452

784.6458

−0.76

264.2606, 184.0730

785.7

184.1

d18:1/22:2

783.6362

13.728

C45 H87 N2 O6 P

782.6291

782.6302

−1.41

264.2636, 184.0727

783.6

184.1

d18:1/21:0

773.6528

15.108

C44 H89 N2 O6 P

772.6455

772.6458

−0.39

264.2651, 184.0721

773.7

184.1

d18:1/21:1 [B11]

771.6361

14.010

C44 H87 N2 O6 P

770.6286

770.6302

−2.08

264.2624, 184.0732

771.6

184.1

d18:1/20:0

759.6368

14.459

C43 H87 N2 O6 P

758.6295

758.6302

−0.92

264.2658, 184.0730

759.6

184.1

d18:1/20:1 [B12]

757.6201

13.428

C43 H85 N2 O6 P

756.6128

756.6145

−2.25

264.2653, 184.0735

757.6

184.1

d18:1/19:0

745.6207

13.811

C42 H85 N2 O6 P

744.6134

744.6145

−1.48

264.2677, 184.0725

745.6

184.1

d18:1/18:0

731.6054

13.195

C41 H83 N2 O6 P

730.5982

730.5989

−0.96

264.2665, 184.0727

731.6

184.1

d18:1/18:1 [B13]

729.5869

12.081

C41 H81 N2 O6 P

728.5786

728.5832

−8.23

264.2659, 184.0732

729.6

184.1

d18:1/17:0

717.5896

12.613

C40 H81 N2 O6 P

716.5824

716.5832

−1.12

264.2677, 184.0726

717.6

184.1

d18:1/16:0

703.5737

12.081

C39 H79 N2 O6 P

702.5666

702.5676

−1.42

264.2680, 184.0748

703.6

184.1

d18:1/16:1 [B14]

701.5583

11.366

C39 H77 N2 O6 P

700.5511

700.5519

−1.14

264.2685, 184.0730

701.6

184.1

d18:1/15:0

689.5585

11.616

C38 H77 N2 O6 P

688.5512

688.5519

−1.02

264.2627, 184.0726

689.6

184.1

d18:1/15:1 [B15]

687.5420

10.752

C38 H75 N2 O6 P

686.5352

686.5363

−1.60

264.2629, 184.0720

687.5

184.1

d18:1/14:0

675.5427

11.117

C37 H75 N2 O6 P

674.5355

674.5363

−1.19

264.2686, 184.0734

675.5

184.1

d18:2/25:0 [b3]

827.6988

16.504

C48 H95 N2 O6 P

826.6896

826.6928

−3.87

262.2524, 184.0729

  

d18:2/24:0 [b5]

813.6763

15.873

C47 H93 N2 O6 P

812.6693

812.6771

−9.60

262.2542, 184.0726

  

d18:2/24:2 [b6]

809.6503

15.723

C47 H89 N2 O6 P

808.6457

808.6458

−0.12

262.2554, 184.0731

  

d18:2/24:3

807.6343

14.659

C47 H87 N2 O6 P

806.6272

806.6302

−3.71

184.0725

  

d18:2/23:0 [b8]

799.6684

15.474

C46 H91 N2 O6 P

798.6606

798.6615

−1.13

184.0732

799.7

184.1

d18:2/22:0 [b10]

785.6523

14.808

C45 H89 N2 O6 P

784.6451

784.6458

−0.89

262.2440, 184.0732

785.7

184.1

d18:2/21:0 [b11]

771.6357

14.193

C44 H87 N2 O6 P

770.6272

770.6302

−3.89

184.0722

  

d18:2/20:0 [b12]

757.6200

13.545

C43 H85 N2 O6 P

756.6128

756.6145

−2.25

262.2451, 184.0725

757.6

184.1

d18:2/18:0 [b13]

729.5896

12.347

C41 H81 N2 O6 P

728.5822

728.5832

−1.37

262.2554, 184.0728

  

d18:2/16:0 [b14]

701.5582

11.382

C39 H77 N2 O6 P

700.5510

700.5519

−1.28

262.2504, 184.0728

  

d18:2/15:0 [b15]

687.5433

10.951

C38 H75 N2 O6 P

686.5355

686.5363

−1.16

262.2503, 184.0716

  

d18:1/12:0 [IS-1]

647.5116

10.402

C35 H71 N2 O6 P

646.5042

646.5050

−1.24

264.2699, 184.0732

647.5

184.1

DHSM

d18:0/26:0

845.7455

19.182

C49 H101 N2 O6 P

844.7382

844.7397

−1.78

266.2711, 184.0730

  

d18:0/26:1 [b1]

843.7271

17.702

C49 H99 N2 O6 P

842.7202

842.7241

−4.63

266.2787, 184.0727

  

d18:0/25:0

831.7297

18.317

C48 H99 N2 O6 P

830.7224

830.7241

−2.05

184.0731

  

d18:0/25:1 [b2]

829.7149

17.469

C48 H97 N2 O6 P

828.7071

828.7084

−1.57

266.2729, 184.0729

  

d18:0/24:0

817.7151

17.585

C47 H97 N2 O6 P

816.7081

816.7084

−0.37

266.2696, 184.0732

817.7

184.1

d18:0/24:1 [b4]

815.6992

16.405

C47 H95 N2 O6 P

814.6931

814.6928

0.37

266.2767, 184.0732

  

d18:0/23:0

803.6995

16.937

C46 H95 N2 O6 P

802.6921

802.6928

−0.87

184.0725

803.7

184.1

d18:0/23:1 [b7]

801.6842

15.756

C46 H93 N2 O6 P

800.6767

800.6771

−0.50

184.0728

801.7

184.1

d18:0/22:0

789.6838

16.272

C45 H93 N2 O6 P

788.6771

788.6771

0.00

184.0129

789.7

184.1

d18:0/22:1 [b9]

787.6684

15.141

C45 H91 N2 O6 P

786.6612

786.6615

−0.38

184.0731

  

d18:0/21:0

775.6672

15.640

C44 H91 N2 O6 P

774.6598

774.6615

−2.19

184.0728

  

d18:0/20:0

761.6528

14.958

C43 H89 N2 O6 P

760.6452

760.6458

−0.79

184.0728

761.7

184.1

d18:0/19:0

747.6355

14.326

C42 H87 N2 O6 P

746.6312

746.6302

1.34

184.0728

747.6

184.1

d18:0/18:0

733.6210

13.694

C41 H85 N2 O6 P

732.6140

732.6145

−0.68

184.0730

733.6

184.1

d18:0/17:0

719.6056

13.096

C40 H83 N2 O6 P

718.5982

718.5989

−0.97

266.2542, 184.0730

719.6

184.1

d18:0/16:0

705.5896

12.514

C39 H81 N2 O6 P

704.5823

704.5832

−1.28

184.0739

705.6

184.1

d18:0/15:0

691.5737

11.982

C38 H79 N2 O6 P

690.5666

690.5676

−1.45

184.0729

691.6

184.1

d18:0/14:0

677.5586

11.466

C37 H77 N2 O6 P

676.5512

676.5519

−1.03

266.2797, 184.0729

677.6

184.1

Cer

d18:1/26:0

678.6745

20.495

C44 H87 N O3

677.6679

677.6686

−1.03

264.2681

  

d18:1/25:0

664.6584

19.481

C43 H85 N O3

663.6526

663.6529

−0.45

264.2654

  

d18:1/24:0 [B16]

650.6436

18.566

C42 H83 N O3

649.6365

649.6373

−1.23

264.2652

650.6

264.3

d18:1/24:1 [B17]

648.6280

17.219

C42 H81 N O3

647.6208

647.6216

−1.24

264.2681

648.6

264.3

d18:1/23:0

636.6272

17.070

C41 H81 N O3

635.6251

635.6216

5.51

264.2681

636.6

264.3

d18:1/23:1

634.6120

16.588

C41 H79 N O3

633.6045

633.6060

−2.37

264.2685

634.6

264.3

d18:1/22:0 [B18]

622.6124

17.086

C40 H79 N O3

621.6050

621.6060

−1.61

264.2680

622.6

264.3

d18:1/22:1 [B19]

620.5964

15.956

C40 H77 N O3

619.5891

619.5903

−1.94

264.2681

620.6

264.3

d18:1/20:0

594.5805

15.706

C38 H75 N O3

593.5732

593.5747

−2.53

264.2686

594.6

264.3

d18:1/18:0

566.5497

14.376

C36 H71 N O3

565.5423

565.5434

−1.95

264.2673

566.5

264.3

d18:1/18:1

564.5302

13.478

C36 H69 N O3

563.5233

563.5278

0.53

264.2669

563.5

264.3

d18:1/17:0

574.5155

13.744

C35 H69 N O3

551.5259

551.5277

−3.26

264.2670

  

d18:1/16:0

538.5186

13.112

C34 H67 N O3

537.5114

537.5121

−1.30

264.2683

538.5

264.3

d18:1/16:1 [B20]

536.5028

12.298

C34 H65 N O3

535.4952

535.4964

−2.24

264.2681

536.5

264.3

d18:1/15:0

524.5026

12.564

C33 H65 N O3

523.4955

523.4964

−1.72

264.2659

524.5

264.3

d18:1/14:0

510.4868

11.982

C32 H63 N O3

509.4793

509.4808

−2.94

264.2696

  

d18:2/26:2

672.6258

18.566

C44 H81 N O3

671.6185

671.6216

−4.62

262.2530

672.6

262.3

d18:2/24:1

646.6121

16.288

C42 H79 N O3

645.6047

645.6060

−2.01

262.2528

646.6

262.3

d18:2/22:0 [b19]

620.5960

16.139

C40 H77 N O3

619.5886

619.5903

−2.74

262.2528

620.6

262.3

d18:2/16:0 [b20]

536.5029

12.554

C34 H65 N O3

535.4953

535.4964

−2.05

262.2532

  

d18:1/12:0 [IS-2]

482.4556

11.034

C30 H59 N O3

481.4479

481.4495

−3.32

264.2678

482.5

264.3

DHCer

d18:0/24:0

652.6587

19.198

C42 H85 N O 3

651.6513

651.6529

−2.46

266.2853

652.7

266.3

d18:0/24:1 [b16]

650.6434

17.735

C42 H83 N O3

649.6361

649.6373

−1.85

266.2837

  

d18:0/24:2 [b17]

648.6281

17.502

C42 H81 N O3

647.6209

647.6216

−1.08

266.2826

  

d18:0/22:0

624.6277

17.569

C40 H81 N O3

623.6202

623.6216

−1.12

266.2859

  

d18:0/22:1 [b18]

622.6111

16.438

C40 H79 N O3

621.6036

621.6060

−3.86

266.2779

  

d18:0/20:0

596.5956

16.222

C38 H77 N O3

595.5878

595.5903

−4.20

266.2811

  

d18:0/18:0

568.5650

14.858

C36 H73 N O3

567.5578

567.5590

−2.11

266.2844

  

d18:0/16:0

540.5343

13.561

C34 H69 N O3

539.5272

539.5277

−0.93

266.2822

  

PTCer

t18:0/14:0

528.4981

11.333

C32 H65 N O4

527.4908

527.4914

−1.14

514.4823, 264.2687

  

HexCer

d18:1/26:0

840.7273

18.467

C50 H97 N O8

839.7189

839.7214

− 2.98

264.2685

840.7

264.3

d18:1/24:0

812.6974

17.020

C48 H93 N O8

811.6893

811.6901

−0.99

264.2685

812.7

264.3

d18:1/24:1

810.6809

16.654

C48 H91 N O8

809.6732

809.6745

−1.61

264.2677

810.7

264.3

d18:1/23:0

798.6804

16.388

C47 H91 N O8

797.6725

797.6745

−2.51

264.2676

798.7

264.3

d18:1/22:0

784.6656

15.740

C46 H89 N O8

783.6579

783.6588

−1.15

264.2689

784.7

264.3

d18:1/16:0

700.5717

12.115

C40 H77 N O8

699.5621

699.5649

−4.00

264.2689

700.6

264.3

d18:1/12:0 [IS-3]

644.5101

10.435

C36 H69 N O8

643.5007

643.5023

−2.49

264.2684

644.5

264.3

LacCer

d18:1/24:0

974.7508

16.388

C54 H103 N O13

973.7432

973.7429

0.31

264.2672

974.7

264.3

d18:1/24:1

972.7329

15.257

C54 H101 N O13

971.7253

971.7273

−2.06

264.2650

972.7

264.3

d18:1/22:0

946.7190

15.124

C52 H99 N O13

945.7112

945.7116

−0.42

264.2679

946.7

264.3

d18:1/20:0

918.6866

13.894

C50 H95 N O13

917.6780

917.6803

−2.51

264.2688

  

d18:1/18:0

890.6552

12.713

C48 H91 N O13

889.6469

889.6490

−2.36

264.2696

890.7

264.3

d18:1/16:0

862.6250

11.682

C46 H87 N O13

861.6175

861.6177

−0.23

264.2687

862.6

264.3

d18:1/12:0 [IS-4]

806.5623

10.219

C42 H79 N O13

805.5550

805.5551

−0.13

264.2683

806.7

264.3

Sa

d19:0 [A1]

316.3202

6.532

C19 H41 N O2

315.3135

315.3137

−0.63

298.3106, 272.2906

274.3

256.3

d18:0 [B21] [A2]

302.3050

6.993

C18 H39 N O2

301.2972

301.2981

−2.98

284.2921

302.3

284.3

d16:0

274.2739

4.881

C16 H35 N O2

273.2666

273.2668

0.73

256.2627

274.3

256.3

PTSa

t17:0

304.2874

5.468

C17 H37 N O3

303.2782

303.2773

2.68

286.2751

  

d17:0 [A3] [IS-5]

288.2901

6.632

C17 H37 N O2

287.2829

287.2824

1.65

270.2794

288.3

270.3

So

d19:1

314.3051

10.668

C19 H39 N O2

313.2978

313.2981

−0.96

296.3320

  

d18:1

300.2896

6.760

C18 H37 N O2

299.2822

299.2824

−0.67

282.2787, 264.2689

300.3

282.3

d17:1 [A4]

286.2737

6.444

C17 H35 N O2

285.2659

285.2668

−3.15

270.2783

  

d16:1

272.2582

5.264

C16 H33 N O2

271.2505

271.2511

−2.36

254.2833

272.3

254.3

d15:1

258.2425

6.711

C15 H31 N O2

257.2347

257.2355

−3.11

240.2319

  

d15:2

256.2270

5.064

C15 H29 N O2

255.2192

255.2198

−2.35

238.2214

  

PTSo

t19:1

330.3003

8.041

C19 H39 N O3

329.2954

329.2930

7.29

312.3278

  

t19:2

328.2846

6.245

C19 H37 N O3

327.2771

327.2773

−0.61

310.2996

  

t18:1 [a1]

316.2850

6.874

C18 H37 N O3

315.2739

315.2773

10.8

298.2739, 280.2632, 262.2522

316.3

298.3

t17:1 [a2]

302.2687

7.176

C17 H35 N O3

301.2618

301.2617

0.33

284.2921, 266.2838

302.3

284.3

t16:1 [a3]

288.2537

5.663

C16 H33 N O3

287.2459

287.2460

−0.35

270.2786

  

d17:1 [a4] [IS-6]

286.3106

6.558

C18 H39 N O

285.3034

285.3032

0.74

268.2643

286.3

268.3

SBA

Enigmol [b21] [A2]

302.3052

5.081

C18 H39 N O2

301.2974

301.2981

−2.32

284.2930, 266.2090

  

SBA

Xestoaminol C

230.2477

5.131

C14 H31 N O

229.2404

229.2406

−0.87

212.2355

  

C1P

d18:1/12:0 [IS-7]

562.4223

10.006

C30 H60 N O6 P

561.4149

561.4158

−1.72

264.2688

562.5

264.3

Sa1P

d17:0 [IS-8]

368.2574

6.774

C17 H38 N O5 P

367.2504

367.2488

4.57

 

368.3

270.3

So1P

d17:1 [IS-9]

366.2406

6.558

C17 H36 N O5 P

365.2331

365.2331

0.03

250.2510

366.2

250.3

The sphingolipids are classified according to “lipid classification system” (http://www.lipidmaps.org/)

SM sphingomyelin, DHSM dihydrosphingomyelin, Cer Ceramide, DHCer dihydroceramide, PTCer phytoceramides, HexCer hexosylceramide, LacCer lactosylceramide, Sa sphinganine, PTSa phytosphinganine, So sphingosine, PTSo phytosphingosine, SBA sphingoid base analog, C1P ceramide-1-phosphate, Sa1P sphinganine-1-phosphate, So1P sphingosine-1-phosphate

[A1-A4 vs a1-a4] 4 pairs of isomeric sphingolipids; [B1-B21 vs b1-b21], 21 pairs of isomeric sphingolipids; [IS], internal standard

Interpretation of high resolution MS and MS/MS spectra of each identified ion, as well as searching against the latest database, allowed for the accurate identification of SPLs. For instance, isobaric lipids could be differentiated by the high-resolution mass spectrometry-based approaches. Two peaks yield m/z 316 ions, with accurate mass acquired by Q-TOF, m/z 316.3202 at 6.532 min and m/z 316.2850 at 6.874 min correspond to [C19H41NO2 + H]+ and [C18H37NO3 + H]+ respectively, facilitating assignment of sphinganine (Sa) d19:0 and phytosphingosine (PTSo) t18:1. Further fragmentation in MS/MS confirmed the identification, a consecutive loss of 3 hydroxy groups can be observed in the latter case, which is the characteristic cleavage of PTSo (Fig. 1).
Fig. 1
Fig. 1

Differentiation of isobaric SPLs by high resolution mass spectrometry. Accurate mass and isotope distribution can distinguish two m/z 316 compounds, corresponding to d19:0 sphinganine (C19H41NO2) and t18:1 phytosphingosine (C18H37NO3), respectively. Typical ion fragments in MS/MS confirmed the identification

A more realistic interference in the identification of SPLs is the isomeric species that have same number of atoms of each element, thus MS/MS fragment data with the assistance of optimized separation are essential for distinguishing the isomers. Take SM (d18:1/22:1) and SM (d18:2/22:0) as example, there are 2 peaks corresponding to m/z 785.65 in extracted ion chromatogram of TOF MS. In accurate MS/MS data acquired by Q-TOF, two characteristic fragments (264.3 & 262.3) respectively corresponding to the sphingoid base chain of SM (d18:1/22:1) and SM (d18:2/22:0) were observed (Fig. 2). The targeted ion pairs as well as complete chromatographic separation make the accurate MRM quantification of isomers possible.
Fig. 2
Fig. 2

Differentiation of isomeric SPLs by accurate MS/MS. The extracted ion chromatogram of m/z 785.6423 at 5 ppm mass accuracy showed two peaks at 14.642 and 14.808 min. Targeted MS/MS of m/z 785.64 at respective time points gave distinct product ions corresponding to backbone of SM (d18:1/22:1) (m/z 264.3) and SM (d18:2/22:0) (m/z 262.3), providing evidence for the identification of these two species

Ceramides are prone to fragment into product ions corresponding to the sphingoid base backbone (e.g. m/z 262.25, 264.27, 266.28). In A549 QC samples, 29 Cers, including 20 dehydroceramides, 8 dihydroceramides (DHCers) and 1 phytoceramide (PTCer), were identified by comparing the MS information and retention time with those of SPLs in our previous study [10, 11]. Most Cers detected in the samples were with a d18:1 sphingoid backbone and the carbon number of N-acyl side chain varied from 14 to 26. A new dihydroceramide DHCer (d18:0/24:2), and Cer (d18:2/26:2), a dehydroceramide with high degree of unsaturation and long N-acyl chain, have been characterized for the first time to the best of our knowledge.

SM is the most multitudinous subclass of SPLs in A549 and A549T. Based on the exact mass in TOF MS and characteristic product ions obtained by Q-TOF MS/MS, a total of 56 SMs, including 38 dehydrosphingomyelins and 18 dihydrosphingomyelins (DHSMs), were unambiguously identified. All these SMs were characterized with a C18 sphingoid base chain, among which d18:1 type takes the largest proportion. In the N-acyl side chain, the number of carbon ranged between 14 and 26, with an unsaturation degree up to 5. Notably, all the DHSMs with 21 or less carbons in the N-acyl chain are fully saturated, while the others (with more than 21 carbons in the N-acyl chain) can be detected together with their corresponding de-hydrogen form. Three highly unsaturated SMs (total unsaturation degree no less than 4) including SM (d18:1/24:3), SM (d18:2/24:2) and SM (d18:2/24:3), have been detected in the QC sample of A549 & A549T cells.

Hexose-linked glycoceramide including galactosylceramide (GalCer) and glucosylceramide (GluCer) were represented as HexCer. All the 6 HexCers and 6 LacCers were found with d18:1 sphingoid base backbone. Only one HexCer with N-acyl chain in odd carbon number, HexCer (d18:1/23:0) was identified in A549. Notably, among all the HexCers and LacCers, only d18:1/24:1 species were identified as glycoceramides with unsaturated N-acyl fatty chain.

Seventeen sphingoid bases as well as the analogs were also successfully identified. The carbon number ranging from 14 to 19 and the degree of unsaturation falls between 0 and 2. Two PTSo with 3 hydroxyl groups, PTSo t19:2 and PTSo t16:1, have been discovered for the first time.

Quantitation of sphingolipids in A549 and A549T cells

MRM mode of UHPLC-QQQ MS could provide accurate and sensitive approach under a wide range for quantitative analysis of SPLs. As the accuracy of triple-quadruple is about 0.1 Da, the quantification of SPLs cannot be accurately achieved merely with a QQQ analyzer, especially when suffering the isotopic interferences. Every unsaturated SPL could be recognized as an isotope of another one with the same characteristic backbone but less degree of unsaturation. For instance, if the LC separation is incomplete, the content of Cer (d18:1/24:0) will be artificially high due to the interference of Cer (d18:1/24:1) (Fig. 3). In this study, based on UHPLC complete separation and Q-TOF comprehensive profiling, accurate quantification was accomplished by eliminating the isotopic interference. By using the UHPLC-QQQ MS method with the optimized MRM parameters, a total of 75 species out of 114 identified SPLs were quantified in A549 and A549T cells, respectively. The amounts of these SPLs were quantified by comparing with the foregoing mentioned ISs.
Fig. 3
Fig. 3

Differentiation of isotopic SPLs by accurate MS together with complete separation. Cer (d18:1/24:1) (tR = 17.219 min) yields precursor ions at m/z 648.6280 and m/z 650.6343, the latter one is the [M + 2] isotopic ion which will interfere with the precursor ion of Cer (d18:1/24:0) (tR = 18.566 min) at m/z 650.6436. The mass differentiation cannot be distinguished by QQQ. If the two peaks cannot be completely separated by LC, the quantification result of Cer (d18:1/24:0) will be artificially high

The quantitative results indicated that SMs account for the majority of all the SPLs in A549 and A549T, among which SMs with C16/C18/C22/C24 N-acyl side chain took the largest proportion of the total content. SMs with d18:1 sphingoid backbone are the most dominant species, which take 27 out of all the 41 quantified SMs (Fig. 4). For some SMs with high unsaturation degree or long N-acyl chain, the content is extremely low which cannot reach the limit of quantitation (LOQ). Figure 5 shows quantification data of 17 Cers. In general, the amounts of various Cers are significantly higher in A549 rather than those in A549T. Similar to SM, d18:1 Cers with C16/C18/C22/C24 N-acyl side chain showed relative high levels in both A549 and A549T, which take most proportion of Cer. LacCers and HexCers were only found with d18:1 sphingoid base backbone. All the 6 LacCers showed higher intensity in A549T than that in A549. But HexCer showed a species-dependent trend, HexCer d18:1/16:0, HexCer d18:1/22:0 and HexCer d18:1/23:0 increased in A549T, while HexCer d18:1/24:0, HexCer d18:1/24:1 and HexCer d18:1/26:0 decreased (Fig. 6). The overall content of sphingoid bases was similar in both cell types, Sa d16:0 was found with the highest intensity (Fig. 7). The relative abundance of each SPL varied greatly, but SPLs with N-acyl chain length of C16 and C24, respectively, are the most abundant species within each subclass.
Fig. 4
Fig. 4

Content of SM and DHSM in A549 and A549T. The X and Z axis represent the compose of fatty acid acyl chain and backbone chain, respectively. Comparisons were performed by the non-parametric Mann-Whitney test. Most SMs and DHSMs showed statistical significance between A549 and A549T (P < 0.0001), except for SM (d18:1/16:1) and SM (d18:2/23:0) (P > 0.05)

Fig. 5
Fig. 5

Content of Cer and DHCer in A549 and A549T. The X and Z axis represent the compose of fatty acid acyl chain and backbone chain, respectively. Comparisons were performed by the non-parametric Mann-Whitney test. All Cers and DHCers showed statistical significance between A549 and A549T (P < 0.0001)

Fig. 6
Fig. 6

Content of HexCer and LacCer in A549 and A549T. The X axis represents the compose of fatty acid acyl chain of the d18:1 HexCer and d18:1 LacCer. Comparisons were performed by the non-parametric Mann-Whitney test. All HexCers and LacCers showed statistical significance between A549 and A549T (P < 0.0001), except for HexCer (d18:1/22:0) (P < 0.01) and HexCer (d18:1/23:0) (P < 0.001)

Fig. 7
Fig. 7

Content of sphingoid base in A549 and A549T. Comparisons were performed by the non-parametric Mann-Whitney test. All sphingoid bases showed statistical significance between A549 and A549T (P < 0.0001), except for Sa (d16:0) (P < 0.01), So (d16:1) (P > 0.05) and So (t18:1) (P > 0.05)

PCA was used for the overview of SPL dataset and the spotting of outliers, and thereby pick out trends of grouping or separation. It was performed to visualize general clustering among A549, A549T and QC groups [R2X (cum) = 0.874, Q2 (cum) = 0.845; Fig. 8a]. Supervised PLS-DA was used to further study the differences between A549 and A549T and to select potential biomarkers. In PLS-DA, the result of model showed the performance statistics of R2X (cum) = 0.880, R2Y (cum) = 0.999 with an excellent prediction parameter Q2 (cum) = 0.998, and the score plot showed good visual separation between A549 and A549T groups as well (Fig. 8b). A total of 57 potential biomarkers were identified according to scattering-plot and the VIP value (Table 2), among which most of them are SM and Cers. SM (d18:0/18:0) showed the largest decline in A549T, the content in decreased from 17.0 to 0.10 pmol/(5 × 105cells), that markedly contributes to the classification.
Fig. 8
Fig. 8

Principal component analysis and partial least squares discriminant analysis projecting scatter plots. a PCA [R2X (cum) = 0.874, Q2 (cum) = 0.845] score plots based on the content of SPLs obtained from A549 (red, diamond), A549T (blue, triangle), and QC (green, circle) groups; b PLS-DA [R2X (cum) = 0.880, R2Y (cum) = 0.999, Q2 (cum) = 0.998] score plots based on the content of SPLs obtained from A549 (red, diamond) and A549T (blue, triangle) groups

Table 2

Quantification of SPLs (VIP > 1) in A549 and A549T

SPLs

Content (pmol/5*105 cells)

ChangeA549T vs A549

p value

VIP

A549 (n = 20)

A549T (n = 20)

SM (d18:2/20:0)

1.96 ± 0.16

0.53 ± 0.07

< 0.001

1.07408

SM (d18:1/26:1)

19.8 ± 1.22

0.34 ± 0.04

< 0.001

1.08696

SM (d18:1/26:0)

5.67 ± 0.31

0.20 ± 0.02

< 0.001

1.08775

SM (d18:1/25:1)

9.28 ± 0.43

0.45 ± 0.05

< 0.001

1.08860

SM (d18:1/25:0)

4.09 ± 0.23

0.42 ± 0.04

< 0.001

1.08699

SM (d18:1/24:3)

3.34 ± 0.26

0.47 ± 0.07

< 0.001

1.08120

SM (d18:1/24:2)

32.5 ± 1.55

8.99 ± 0.87

< 0.001

1.08501

SM (d18:1/24:1)

542 ± 23.8

37.9 ± 2.59

< 0.001

1.08879

SM (d18:1/24:0)

298 ± 13.0

41.5 ± 3.16

< 0.001

1.08829

SM (d18:1/23:2)

1.12 ± 0.11

0.25 ± 0.04

< 0.001

1.06488

SM (d18:1/23:1)

21.6 ± 1.08

2.58 ± 0.30

< 0.001

1.08745

SM (d18:1/23:0)

26.4 ± 1.26

6.57 ± 0.51

< 0.001

1.08618

SM (d18:1/22:2)

1.25 ± 0.12

0.15 ± 0.02

< 0.001

1.07067

SM (d18:1/22:1)

16.4 ± 0.81

2.07 ± 0.18

< 0.001

1.08760

SM (d18:1/22:0)

142 ± 6.17

26.9 ± 1.78

< 0.001

1.08783

SM (d18:1/21:1)

0.82 ± 0.07

0.15 ± 0.02

< 0.001

1.07308

SM (d18:1/21:0)

4.66 ± 0.25

1.39 ± 0.12

< 0.001

1.08216

SM (d18:1/20:1)

0.87 ± 0.13

0.06 ± 0.00

< 0.001

1.06102

SM (d18:1/20:0)

17.6 ± 0.86

3.08 ± 0.31

< 0.001

1.08689

SM (d18:1/19:0)

1.94 ± 0.20

0.21 ± 0.03

< 0.001

1.07588

SM (d18:1/18:1)

39.0 ± 1.99

8.13 ± 0.58

< 0.001

1.08637

SM (d18:1/18:0)

69.5 ± 2.72

9.07 ± 0.54

< 0.001

1.08896

SM (d18:1/17:0)

14.1 ± 0.72

4.76 ± 0.39

< 0.001

1.08307

SM (d18:1/16:0)

982 ± 38.5

629 ± 23.5

< 0.001

1.06108

SM (d18:1/14:0)

27.3 ± 1.04

17.2 ± 0.65

< 0.001

1.06732

SM (d18:0/24:0)

15.3 ± 0.78

0.11 ± 0.02

< 0.001

1.08843

SM (d18:0/23:0)

2.45 ± 0.15

0.02 ± 0.00

< 0.001

1.08694

SM (d18:0/22:0)

23.3 ± 1.05

0.23 ± 0.04

< 0.001

1.08900

SM (d18:0/20:0)

4.59 ± 0.24

0.08 ± 0.01

< 0.001

1.08808

SM (d18:0/19:0)

0.49 ± 0.07

0.01 ± 0.00

< 0.001

1.05519

SM (d18:0/18:0)

17.0 ± 0.55

0.10 ± 0.03

< 0.001

1.09013

SM (d18:0/17:0)

0.65 ± 0.07

0.06 ± 0.01

< 0.001

1.07030

SM (d18:0/16:0)

545 ± 28.7

12.3 ± 0.94

< 0.001

1.08809

SM (d18:0/15:0)

1.74 ± 0.16

0.10 ± 0.02

< 0.001

1.08041

SM (d18:0/14:0)

5.83 ± 0.40

0.28 ± 0.03

< 0.001

1.08565

Cer (d18:2/24:1)

1.01 ± 0.21

0.03 ± 0.01

< 0.001

1.04397

Cer (d18:1/24:1)

46.1 ± 5.83

3.60 ± 0.44

< 0.001

1.07110

Cer (d18:1/24:0)

42.0 ± 2.08

9.83 ± 0.41

< 0.001

1.08653

Cer (d18:1/23:1)

1.37 ± 0.22

0.06 ± 0.01

< 0.001

1.06057

Cer (d18:1/23:0)

2.54 ± 0.27

0.53 ± 0.09

< 0.001

1.06941

Cer (d18:1/22:1)

1.20 ± 0.27

0.04 ± 0.01

< 0.001

1.03542

Cer (d18:1/22:0)

14.4 ± 0.94

1.90 ± 0.39

< 0.001

1.08443

Cer (d18:1/20:0)

1.69 ± 0.33

0.07 ± 0.02

< 0.001

1.04721

Cer (d18:1/18:1)

20.1 ± 1.33

4.03 ± 0.49

< 0.001

1.08187

Cer (d18:1/18:0)

8.03 ± 0.72

0.26 ± 0.04

< 0.001

1.08142

Cer (d18:1/16:0)

52.0 ± 3.59

10.7 ± 0.98

< 0.001

1.08243

Cer (d18:1/15:0)

3.14 ± 0.46

1.34 ± 0.30

< 0.001

1.00100

Cer (d18:0/24:0)

1.44 ± 0.11

0.34 ± 0.04

< 0.001

1.07587

Cer (d18:0/16:0)

3.46 ± 0.25

0.07 ± 0.01

< 0.001

1.05883

HexCer (d18:1/26:0)

0.41 ± 0.10

0.04 ± 0.02

< 0.001

1.00780

HexCer (d18:1/24:1)

9.25 ± 0.97

0.02 ± 0.00

< 0.001

1.07959

HexCer (d18:1/16:0)

5.06 ± 0.89

21.6 ± 2.10

< 0.001

1.07118

LacCer (d18:1/24:1)

0.22 ± 0.05

19.9 ± 2.17

< 0.001

1.07014

LacCer (d18:1/24:0)

0.37 ± 0.03

56.4 ± 3.16

< 0.001

1.07766

LacCer (d18:1/22:0)

0.06 ± 0.01

21.6 ± 2.85

< 0.001

1.07258

LacCer (d18:1/16:0)

1.15 ± 0.55

115 ± 11.6

< 0.001

1.08013

So (t17:1)

2.21 ± 0.17

0.62 ± 0.08

< 0.001

1.07483

Discussion

Using the sphingolipidomic approach, we obtained the detailed sphingolipid profiles for human lung adenocarcinoma cell A549 and its taxol resistant strain A549T, and then performed quantification. We found A549 and A549T share all the same species of SPLs, among which SM (dehydrosphingomyelin and DHSM), Cer (dehydroceramide, DHCer and PTCer), HexCer, LacCer, and sphingoid base were identified as the major SPLs. In contrast to normal A549, decreasing levels of Cer and SM concomitant with increasing of glycosphingolipids represent the main SPL metabolic profile of A549T. Totally 35 SMs, 14 Cers, 3 HexCers, 4 LacCers, and 1 sphingosine are recognized as metabolic pathway related biomarkers.

Cer is the basic SPL structural unit which balances cell growth and death by inducing apoptosis [12], and its definite efficacy in promoting apoptosis in A549 cells has been well studied [7]. It is noteworthy that Cers can be classified into SM-hydrolyzed and de novo-synthesized. The former is well known as triggering apoptotic death signaling in many cell types, while the specific role of the latter one seems important to tumor survival [13]. In human ovarian carcinoma cell line CABA I, anti-cancer drugs including taxol have been reported to activate SMase to generate Cer, which acts as a second messenger in triggering apoptosis [14]. While in lung carcinoma cells, the tumor tissues produce large amounts of both dihydroceramide and ceramide through the de novo synthesis pathway, but not through SM hydrolysis [13]. More relevantly, treatment of A549 cells with gemcitabine was demonstrated to increase Cer levels via the activation of de novo synthesis [15]. In the case of study of A549T in this paper, both Cer and SM levels were much lower than their levels in taxol-sensitive A549 cells, which indicates that the decrease of Cer may not attribute to “activating the SM pathway” as our previous study in A2780T [11]. Furthermore, DHCer was decreasing accompanied with Cer, which revealed the mechanism of taxol-resistance in A549T could be explained as “inhibiting the de novo synthesis pathway”. (Fig. 9).
Fig. 9
Fig. 9

Biosynthesis and metabolism pathway of sphingolipids

Both Cer and its catabolite sphingosine as negative regulators of cell proliferation could promote apoptosis, and the role of sphingosine as a messenger of apoptosis is of importance [16]. In small cell lung cancer (SCLC), multidrug-resistance-associated protein (MRP) contributes to the drug resistance, and pro-apoptotic SPLs (Cer and sphingosine) could further induce apoptosis overcome or bypass MRP-mediated drug resistance [17]. In non-small cell lung cancer (NSCLC) including A549, sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling which may contribute to the apoptosis resistance [18]. Inhibition of SphK2 can enhance the apoptosis of NSCLC cells, and it will certainly result in an increase of the substrate sphingosine. In A549T, all sphingosines showed consistent trend of decrease comparing to A549. It’s known that sphingosine in mammalian cells is not synthesized de novo but it is generated from ceramides by ceramidases [19]. Thus, we can deduce that in A549T the concomitant decrease of sphingosine and Cer may be the result of activation of SphK2, which leads to the inhibition of apoptosis in taxol resistant strain.

Besides Cer and SM, glycosphingolipids including HexCers (GalCers & GluCers) and LacCers account for a large proportion of biomarkers in A549T. It has been observed that glucosylceramide synthase is up-regulated after drug intervention and suggests that glycolipids may be involved in chemotherapy resistance [2]. For decades, GluCer has been found to increase in the resistant cancer cells [20], suggesting that glycosylation plays an important role in evading Cer induced apoptosis. Glycosphingolipids have recently been reported as transactivating multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents accumulation of ceramide and stimulates drug efflux [21]. Specifically, GalCer and LacCer were characteristically increased in taxol-resistant human ovarian carcinoma-derived KF28TX cells [22]. Moreover, GalCer was demonstrated to be the apoptosis protector, and its upregulation was also thought to attenuate the Cer-mediated apoptotic signals [23]. Our findings revealed a significant overall increase of glycosphingolipids in A549T, among which all LacCers showed a consistent tendency of increase, while HexCers (including GalCer and GluCer) showed a species-dependent trend. It should be noted that GluCer could be converted into LacCer under the influence of lactosylceramide synthase. Therefore the decreased species of HexCer might be GluCer. For instance, the decrease of HexCer (d18:1/24:1) resulted in the concomitant increase of LacCer (d18:1/24:1).

Conclusions

Evidences suggest that tumor microenvironment including the sphingolipidome plays an important role in cancer drug resistance. So far to our knowledge, there is no sphingolipidomic study on taxol-resistant A549 human adenocarcinoma cell line. Based on the comprehensive identification and accurate quantification of SPLs, decreasing of Cer, SM and sphingosine concomitant with increasing of HexCer and LacCer have been characterized as the metabolic profile of A549T. It indicated that “inhibition of the de novo synthesis pathway” and “activation of glycosphingolipid pathway” played the dominant role for taxol-resistance, and the key enzymes related to the pathways may have been altered. These results provide evidence to unravel the mechanism of taxol resistance in A549T. The distinctive phenotype could facilitate clinical diagnosis of taxol-resistant adenocarcinoma and provide insights into targets for the development of new drug against taxol resistance.

Abbreviations

A549T: 

Taxol-resistant strain of A549 cell

C1P: 

Ceramide-1-phosphate

Cer: 

Ceramide

DHCer: 

Dihydroceramide

DHSM: 

Dihydrosphingomyelin

HexCer: 

Hexosylceramide

LacCer: 

Lactosylceramide

QC: 

Quality control

SM: 

Sphingomyelin

SPL: 

Sphingolipid

Declarations

Funding

This work was financially supported by Tertiary Education Services Office, Macau Special Administrative Region (GAES-17-001-SKL to Z.-H. Jiang); Macao Science and Technology Development Fund, Macau Special Administrative Region (015/2017/AFJ to Z.-H. Jiang and 023/2016/AFJ to J.-R Wang); and in part by Ph.D. start-up fund of Gannan Medical University (No.201304 granted to H. Huang). The funding bodies have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

The datasets used and analyzed during the current study were available from the corresponding author(s) on reasonable request.

Authors’ contributions

ZHJ and JRW conceived the research; HH drafted the manuscript, ZHJ, JRW and LFY reviewed and revised it critically; TTT and LFY performed the cell experiments; HH, CCY and MJN carried out the sample preparation, LC-MS analysis and data interpretation; HH and TTT designed the figures. All authors read and approved the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China
(2)
College of Pharmacy, Gannan Medical University, Ganzhou, 341000, China
(3)
International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China

References

  1. Bender E. Epidemiology: the dominant malignancy. Nature. 2014;513(7517):S2–3.View ArticlePubMedGoogle Scholar
  2. Gouaze V, Yu JY, Bleicher RJ, Han TY, Liu YY, Wang H, Gottesman MM, Bitterman A, Giuliano AE, Cabot MC. Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy. Mol Cancer Ther. 2004;3(5):633–9.PubMedGoogle Scholar
  3. Yusuf RZ, Duan Z, Lamendola DE, Penson RT, Seiden MV. Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation. Curr Cancer Drug Targets. 2003;3(1):1–19.View ArticlePubMedGoogle Scholar
  4. Wang JB, Erickson JW, Fuji R, Ramachandran S, Gao P, Dinavahi R, Wilson KF, Ambrosio AL, Dias SM, Dang CV, et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell. 2010;18(3):207–19.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Zhao Y, Butler EB, Tan M. Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis. 2013;4:e532.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Giussani P, Tringali C, Riboni L, Viani P, Venerando B. Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance. Int J Mol Sci. 2014;15(3):4356–92.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Kurinna SM, Tsao CC, Nica AF, Jiffar T, Ruvolo PP. Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase. Cancer Res. 2004;64(21):7852–6.View ArticlePubMedGoogle Scholar
  8. Pettus BJ, Bielawska A, Subramanian P, Wijesinghe DS, Maceyka M, Leslie CC, Evans JH, Freiberg J, Roddy P, Hannun YA, et al. Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2. J Biol Chem. 2004;279(12):11320–6.View ArticlePubMedGoogle Scholar
  9. Yu Y, Sun G, Liu G, Wang Y, Shao Z, Chen Z, Yang J. Effects of mycoplasma pneumoniae infection on sphingolipid metabolism in human lung carcinoma A549 cells. Microb Pathog. 2009;46(2):63–72.View ArticlePubMedGoogle Scholar
  10. Wang JR, Zhang H, Yau LF, Mi JN, Lee S, Lee KC, Hu P, Liu L, Jiang ZH. Improved sphingolipidomic approach based on ultra-high performance liquid chromatography and multiple mass spectrometries with application to cellular neurotoxicity. Anal Chem. 2014;86(12):5688–96.View ArticlePubMedGoogle Scholar
  11. Huang H, Tong TT, Yau LF, Chen CY, Mi JN, Wang JR, Jiang ZH. LC-MS based Sphingolipidomic study on A2780 human ovarian Cancer cell line and its Taxol-resistant strain. Sci Rep. 2016;6:34684.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Ravid T, Tsaba A, Gee P, Rasooly R, Medina EA, Goldkorn T. Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol. 2003;284(6):L1082–92.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Koyanagi S, Kuga M, Soeda S, Hosoda Y, Yokomatsu T, Takechi H, Akiyama T, Shibuya S, Shimeno H. Elevation of de novo ceramide synthesis in tumor masses and the role of microsomal dihydroceramide synthase. Int J Cancer. 2003;105(1):1–6.View ArticlePubMedGoogle Scholar
  14. Prinetti A, Millimaggi D, D'Ascenzo S, Clarkson M, Bettiga A, Chigorno V, Sonnino S, Pavan A, Dolo V. Lack of ceramide generation and altered sphingolipid composition are associated with drug resistance in human ovarian carcinoma cells. Biochem J. 2006;395(2):311–8.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Chalfant CE, Rathman K, Pinkerman RL, Wood RE, Obeid LM, Ogretmen B, Hannun YA. De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1. J Biol Chem. 2002;277(15):12587–95.View ArticlePubMedGoogle Scholar
  16. Cuvillier O. Sphingosine in apoptosis signaling. Biochim Biophys Acta. 2002;1585(2–3):153–62.View ArticlePubMedGoogle Scholar
  17. Khodadadian M, Leroux ME, Auzenne E, Ghosh SC, Farquhar D, Evans R, Spohn W, Zou Y, Klostergaard J. MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro. Lung Cancer. 2009;66(1):48–57.View ArticlePubMedGoogle Scholar
  18. Yang J, Yang C, Zhang S, Mei Z, Shi M, Sun S, Shi L, Wang Z, Wang Y, Li Z, et al. ABC294640, a sphingosine kinase 2 inhibitor, enhances the antitumor effects of TRAIL in non-small cell lung cancer. Cancer Biol Ther. 2015;16(8):1194–204.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Mao C, Obeid LM. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim Biophys Acta. 2008;1781(9):424–34.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Nicholson KM, Quinn DM, Kellett GL, Warr JR. Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase. Br J Cancer. 1999;81(3):423–30.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Tyler A, Johansson A, Karlsson T, Gudey SK, Brannstrom T, Grankvist K, Behnam-Motlagh P. Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells. Exp Cell Res. 2015;336(1):23–32.View ArticlePubMedGoogle Scholar
  22. Kiguchi K, Iwamori Y, Suzuki N, Kobayashi Y, Ishizuka B, Ishiwata I, Kita T, Kikuchi Y, Iwamori M. Characteristic expression of globotriaosyl ceramide in human ovarian carcinoma-derived cells with anticancer drug resistance. Cancer Sci. 2006;97(12):1321–6.View ArticlePubMedGoogle Scholar
  23. Grazide S, Terrisse AD, Lerouge S, Laurent G, Jaffrezou JP. Cytoprotective effect of glucosylceramide synthase inhibition against daunorubicin-induced apoptosis in human leukemic cell lines. J Biol Chem. 2004;279(18):18256–61.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2018

Advertisement