source: https://doi.org/10.48350/156975 | downloaded: 31.1.2022
Investigating differences in lung cancer cells with induced and de-induced cisplatin resistance by 1 H HR-MAS NMR metabolomics
Martina Vermathen
a, Hendrik von Tengg-Kobligk
b,c, Martin Nils Hungerbühler
b,c, Christoph Kempf
b,c, Peter Vermathen
b,c, Nico Ruprecht
b,caDept. Chemistry, Biochemistry & Pharm. Sciences, University of Bern, Switzerland, bUniversity Inst. Diagnostic, Interventional & Pediatric Radiology, Inselspital, University of Bern, Switzerland,cDept. BioMedical Research, University of Bern, Switzerland
(D-)A24cisPt0.5 (D-)A24cisPt2.0 (D-)A24cisPt4.0 A24cisPt8.0 A24-0a A24-0b A24cisPt0.5
(D-)A24cisPt8.0 A24cisPt2.0 A24cisPt4.0
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -0.6
-0.4 -0.2 0 0.2 0.4 0.6
Scores on PC 1 (45.54%)
Scores on PC 2 (21.10%)
Resistance
PCA
(D-)A24cisPt0.5 (D-)A24cisPt2.0
(D-)A24cisPt4.0 A24cisPt8.0
A24-0a A24-0b
A24cisPt0.5 (D-)A24cisPt8.0
A24cisPt2.0
A24cisPt4.0
Batch
# 1223
INTRODUCTION
Cisplatin (cisPt)-resistance poses a major clinical problem in the treatment of non-small cell lung cancer (NSCLC).1, 2However, the mechanisms accounting for metabolic adaptations in cisPt-resistant cells are not well understood. In cultured NSCLC cells with induced cisPt resistance a long-term resistance is retained after de-induction.3High resolution magic angle spinning (HR-MAS) NMR spectroscopy allows to metabolically characterize biological samples like cells or tissue.4-6
A24 0.5 μM 2.0 μM 4.0 μM 8.0 μM
cisplatin (cisPt) concentration in medium
A24 D-Pt2.0
A24 D-Pt0.5 A24 D-Pt4.0 A24 D-Pt8.0
A24-0a
A24 Pt0.5 A24 Pt2 A24 Pt4 A24 Pt8
A24-0b
METHODS
cisPt-resistant cell lines
Generated from cisPt-sensitive WT-lung adenocarcinoma cells (A24)3
Exposure to stepwise increasing concentrations of cisPt in the culture medium ranging from 0.5μM to 8μM (Fig.1)
For de-induction cells branched off and grown in the absence of cisPt
A24 cells/sublines cultured (RPMI 1640) 2 batches (a,b), 2x15 samples
B0
= 54.74°
1H HR-MAS NMR 500 MHz, 3kHz MAS, T=276K 1D-PROJECT7, TE=400 ms, ns=512 water-presaturation Sample preparation for HR-MAS NMR:
5x106cells Lysed, heated 70°C (20 min)
2 x 15 Fig. 2: 1D PROJECT spectrum (excerpt for 0.5-3.5 ppm spectral region)
More than 50 metabolites were assigned including:
Amino acids, peptides, organic acids, amines, choline containing compounds, nucleobases, nucleosides, nucleotides, phosphate sugars and lipids
AIMS
Systematically investigate metabolic alterations in cultured NSCLC- cells with incremented induced and de-induced cisPt-resistance
Apply HR-MAS NMR-based metabolomics to study the metabolome using cisPt-sensitive NSCLC-cells (A240286S, A24) as controls
Identify A24 cell metabolites and potential markers of cisPt-resistance
Address the question if the maintenance of cisPt-resistance in de- induced NSCLC cells is also reflected in the metabolic profile
RESULTS
RESULTS
CONCLUSIONS
-0.6 -0.4 -0.2 0 0.2 0.4 0.6
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Scores on LV 1 (20.94%)
Scores on LV 2 (46.11%)
oPLS
A24cisPt8.0 A24-0a A24cisPt2.0
Batch
Resistance B
A24-0b
(D-)A24cisPt0.5 A24cisPt0.5
A24cisPt4.0 (D-)A24cisPt4.0
(D-)A24cisPt8.0 (D-)A24cisPt2.0 A
C
0 1 2 3 4 5 6 7 8
1
0 1 2 3 4 5 6 7 8 9
Y Measured
Y CV Predicted
50 100 150 200 250 300
- - - -
0.4 0.3 0.2 0.1 0 0.1 0.2 0.3
Bucket
LV 1 (Component) (20.94%)
GPC Ile
Phe Met
Lac, Lip AMP
NA UTP
GSHUNGlc UNGal Lac
GSH
GPC Tau
Pro Tau
Tyr GSH GSH Asnb-Ala, GSH GSH
Lac
Ino Hxn NAD+
Ura InoCyd
Cyd, UrdUraIno Ino
PC Cho
Cre PC
Gly Cho
PC b-Ala
Cre Cit Glu
Glu Val
Met Ala Lip-CH3 Lip-(CH2)n
GSH
Lip-(CH2)n
Data analysis:
Spectra subdivided into 309 individually sized buckets
Probabalistic quotient normalization; mean centering, pareto scaling
Principal Component Ananlysis (PCA) and orthogonal Partial least squares analysis (o-PLS) unsing PLS-Toolbox (Eigenvector)
Fig. 3A: Unsupervised PCA on all samples (30 x 309) demonstrates:
Close clustering of replicates
Scores along PC-2 correlate with increasing cisPt resistance
De-induced samples are close to their induced counterparts
Clear separation of the two batches along PC-1 (possibly due to passage and media differences)
cisPt resistance is reflected in metabolic alterations
GSH and Tau may serve as biomarkers with elevated levels in cisPt resistant cells
GSH and Tau may function as reactive oxygen species scavenger and for cellular defense (anitapoptotic)8,9
Developed cisPt-resistance is an adaptation that is maintained even after cisPt removal and indicates a metabolic long-term memory of the cells
Fig. 1
Fig. 3 A - D
Glu
PC Cre
Tau Tau Cho
GSH
Lys Met Val
Cys Cys
-Ala GSH
Suc Pro Pro Pro
Asn Cit Cit Tyr
Tyr Phe Phe
-Ala
Gln HT HT
EtOH ValIle Leu
Ile Lip Lip
Lac Thr Ala
Lys LysLeu Ac Glu
Ile Val Ile
Ile
Lys Met
GSH Pro
UNGal UNGlc
Gln
Fig. 2
D
Fig. 3B: oPLS on all samples (30 x 309) demonstrates:
Close clustering of replicates
Scores along LV-1 correlate with increasing cisPt resistance
De-induced samples are metabolically similar to their induced counterparts
Batches separate along LV-2
Fig. 3C: PLS model prediction performs very well
For all samples, the predicted resistance is close to the measured one
Fig. 3D: Loading plot for oPLS LV-1
Glutathione (GSH) and taurine (Tau) increased in cisPt-resistant cells
Creatine (Cr) and Phosphocholine (PC) appear reduced References: 1: Fennell, D.A. et al. Cancer Treat. Rev. 2016, 44, 42–50. 2: Chen, S.H. et al. Int. J. Mol. Sci. 2019, 20, 4136. 3: Ruprecht, N. et al. Pharmaceuticals 2020, 13, 109. 4: Lindon, J.C. et al. Prog Nucl Mag Res Sp. 2009; 55:
79–100. 5: Vermathen, M. et al. PLoS ONE 2015, 10(5): e0128478. 6: Primasovà, H. et al. Metabolites 2019, 9, 146. 7: Aguilar J.A. et al. Chem. Commun. 2012; 48:811-813. 8: Brozovic A. et al. Critical Reviews in Toxicology, 2010; 40(4): 347–359. 9: Sørensen B.H. et al. Am J Physiol Cell Physiol 2014; 307: C1071–C1080.
