1H HR-MAS NMR Based Metabolic Profiling of Cells in Response to Treatment with a Hexacationic Ruthenium Metallaprism as Potential Anticancer Drug

Abstract

(1)H high resolution magic angle spinning (HR-MAS) NMR spectroscopy was applied in combination with multivariate statistical analyses to study the metabolic response of whole cells to the treatment with a hexacationic ruthenium metallaprism [1](6+) as potential anticancer drug. Human ovarian cancer cells (A2780), the corresponding cisplatin resistant cells (A2780cisR), and human embryonic kidney cells (HEK-293) were each incubated for 24 h and 72 h with [1](6+) and compared to untreated cells. Different responses were obtained depending on the cell type and incubation time. Most pronounced changes were found for lipids, choline containing compounds, glutamate and glutathione, nucleotide sugars, lactate, and some amino acids. Possible contributions of these metabolites to physiologic processes are discussed. The time-dependent metabolic response patterns suggest that A2780 cells on one hand and HEK-293 cells and A2780cisR cells on the other hand may follow different cell death pathways and exist in different temporal stages thereof.

Fig 1. Ruthenium metallaprism [1]⁶⁺.

Structure of the metallaprism [1]⁶⁺ used for cell incubation [(p-cymene)₆Ru₆(tpt)₂(dhnq)₃]⁶⁺, isolated as its triflate salt.

Fig 2. ¹H HR-MAS NMR spectrum of cells.

¹H HR-MAS-cpmg spectrum of a cell suspension (A2780) in PBS with resonance assignments according to Table 1. A: aliphatic region (0.5–4.5 ppm), B: aromatic region (5–9 ppm, scaled up ~*4).

Fig 3. PCA scores plot for all control samples.

PCA scores plot (PC 1—PC 3) for all cell samples A2780, A2780cisR and HEK-293 at incubation times of 24h and 72h.

Fig 4. ¹H HR-MAS NMR spectral region of phosphate sugars.

Spectral region 5–6.3 ppm for averaged ¹H HR-MAS spectra of control cell samples A2780, A2780cisR and HEK-293 at incubation times of 24h and 72h. Region with resonances of phosphate sugars Glc1P, UNGlc, UNGal, and UGlcA is highlighted.

Fig 5. PLS-DA of control and drug-treated cells.

PLS-DA scores plot (LV 1—LV 2) comparing control (blue) and drug treated (red) cell samples for the 3 different cell types A2780, A2780cisR and HEK-293 at incubation times of 24h and 72h. ∙∙∙∙∙∙∙∙∙ 95% Confidence level

Fig 6. PLS-loadings (24 h).

PLS-loadings of the first PLS component (LV 1) for the 3 cell lines A2780, A2780cisR and HEK-293 and 24h incubation time. Buckets are assigned to metabolites. Strong lipid contributions (and Lac for A2780cisR) are highlighted according to their sign (blue: < -0.1, red: > 0.1).

Fig 7. PLS-loadings (72 h).

PLS-loadings of the first PLS component (LV 1) for the 3 cell lines A2780, A2780cisR and HEK-293 and 72h incubation time. Buckets are assigned to metabolites. Strong lipid contributions are highlighted according to their sign (blue: < -0.1, red: > 0.1).

Fig 8. ¹H HR-MAS NMR mean spectra.

PQN-normalized mean 1D noesy spectra for control (blue) and drug treated (red) cell samples for spectral regions of (A) saturated lipids and (B) choline containing compounds.

Fig 9. Relative changes for lipids.

Relative differences (control—drug) of bucket integrals (means, PQN-normalized) with SE for saturated and unsaturated lipid resonances. (A) 24h and (B) 72h incubation time. * p<0.05; ** p<0.01 (corrected for multiple comparisons).

Fig 10. Relative changes for choline containing compounds.

Relative differences (control—drug) of bucket integrals (means, PQN-normalized) with SE for choline containing compound resonances. (A) 24h and (B) 72h incubation time. * p<0.05; ** p<0.01 (corrected for multiple comparisons).

Fig 11. Relative changes for selected metabolites.

Relative differences (control—drug) of bucket integrals (means, PQN-normalized) with SE for selected metabolite resonances. (A) 24h and (B) 72h incubation time. * p<0.05; ** p<0.01 (corrected for multiple comparisons).

Fig 12. Integrals of metabolites from spectral region 5–9 ppm.

Normalized mean integrals (± SD) of selected spectral regions from control and drug-treated A2780, A2780cisR and HEK-293 cell spectra (cpmg): α-Glucose-1-phosphate (Glc1P), UDP-N-acetylglucosamine (UNGlc), UDP-N-acetylgalactosamine (UNGal), UDP-glucuronic acid (UGlcA), and UDP/UTP. (A): 24h, (B): 72h incubation time. * p<0.05; ** p<0.01 (corrected for multiple comparisons).