Response of Cisplatin Resistant Skov-3 Cells to [Pt(O,O'-Acac)(γ-Acac)(DMS)] Treatment Revealed by a Metabolomic ¹H-NMR Study

Abstract

The novel [Pt(O,O'-acac)(γ-acac)(DMS)], Ptac2S, Pt(II) complex has recently gained increasing attention as a potential anticancer agent for its pharmacological activity shown in different tumor cell lines, studied both in vitro and in vivo. The mechanism of action of Ptac2S, operating on non-genomic targets, is known to be very different from that of cis-[PtCl₂(NH₃)₂], cisplatin, targeting nucleic acids. In this work, we evaluated the cytotoxicity of Ptac2S on the cisplatin resistant Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells, by the MTT assay. A ¹H-NMR metabolomic approach coupled with multivariate statistical analysis was used for the first time for Ptac2S to figure out the biological mechanisms of action of the complex. The metabolic variations of intracellular metabolites and the composition of the corresponding extracellular culture media were compared to those of cisplatin (cells were treated at the IC₅₀ doses of both drugs). The reported comparative metabolomic analysis revealed a very different metabolic profile between Ptac2S and cisplatin treated samples, thus confirming the different mechanism of action of Ptac2S also in the Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells line. In particular, higher levels of pyruvate were observed in Ptac2S treated, with respect to cisplatin treated, cells (in both aqueous and culture media). In addition, a very different lipid expression resulted after the exposure to the two drugs (Ptac2S and cisplatin). These results suggest a possible explanation for the Ptac2S ability to circumvent cisplatin resistance in SKOV-3 cells.

Keywords: 1H-NMR metabolomics; Epithelial Ovarian Carcinoma; Ptac2S; SKOV-3 cells; [Pt(O,O′-acac)(γ-acac)(DMS)]; cisplatin; platinum based drugs.

Figure 1

Chemical structure of [Pt(O,O′-acac)(γ-acac)(DMS)], Ptac2S.

Figure 2

Evaluation of the cytotoxicity of Ptac2S on SKOV-3 cells in comparison with cisplatin. The SKOV-3 cells were treated with increasing concentrations of Ptac2S or cisplatin by determining the viable cell number after 12 h (a), 24 h (b), 48 h (c). Panel (d) shows the IC₅₀ of Ptac2S and cisplatin after 24 h.

Figure 3

PCA score scatter plot of the 600 MHz ¹H-CPMG-NMR spectra (Pareto scaled) obtained from aqueous extracts of Ptac2S and cisplatin treated SKOV-3 cells in comparison with the untreated control samples. The indicated labels are referred to the culture times. (a) PCA t[1]/t[2] (four components give R²X = 0.862, Q² = 0.79). (b) PCA t[1]/t[2] scores scatter plot (two components give R²X = 0.865, Q² = 0.754).

Figure 4

OPLS-DA score plot (left panel) and corresponding coefficient plot (right panel) of the 600 MHz ¹H-CPMG-NMR spectra (Pareto scaled) of SKOV-3 cells aqueous extracts obtained from different pairwise groups (controls, Ptac2S and cisplatin) at 6 h. (a) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive + 1 orthogonal, give R²X = 0.911, R²Y = 0.999, Q² = 0.988). (b) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive + 1 orthogonal give R²X = 0.886, R²Y = 0.999, Q² = 0.984). (c) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive + 1 orthogonal give R²X = 0.875, R²Y = 0.995, Q² = 0.984).

Figure 5

OPLS-DA score plot (left panel) and corresponding coefficient plot (right panel) of the 600 MHz ¹H-CPMG-NMR spectra (Pareto scaled) of SKOV-3 cells aqueous extracts obtained from different pairwise groups (controls, Ptac2S, and cisplatin) at 24 h. (a) OPLS-DA t[1]/t[2] scores scatter plot (three components, 1 predictive + 2 orthogonal give R²X = 0.926, R²Y = 1, Q² = 0.998). (b) OPLS-DA t[1]/t[2] scores scatter plot (three components, 1 predictive + 2 orthogonal give R²X = 0.931, R²Y = 1, Q² = 0.999). (c) OPLS-DA t[1]/t[2] scores scatter plot (three components, 1 predictive + 2 orthogonal give R²X = 0.745, R²Y = 0.998, Q² = 0.899).

Figure 6

Relevant discriminant metabolites comparison of SKOV-3 cells aqueous extracts obtained from different pairwise groups (controls, Ptac2S, and cisplatin). The values of −Log2(FC) and the p-values are provided (Student’s t test, p-value < 0.05). (a,b) Metabolites with −Log2 (FC) negative values have lower concentration with respect to those of the 6 h (left panel) and 24 h (right panel) of the control, while −Log2 (FC) positive values have higher concentration with respect to those of the 6 h (left panel) and 24 h (right panel) of the control. (c) Metabolites with −Log2 (FC) negative values have lower concentration with respect to those of the 6 h (left panel) and 24 h (right panel) of cisplatin, while −Log2 (FC) positive values have higher concentration with respect to those of the 6 h (left panel) and 24 h (right panel) of cisplatin.

Figure 7

PCA score scatter plots of the 600 MHz ¹H-ZG-NMR spectra (Pareto scaled) obtained from lipophilic extracts of Ptac2S and cisplatin treated SKOV-3 cells in comparison with the untreated control samples. The indicated labels are referred to the culture times. (a) PCA t[1]/t[2] (four components give R²X = 0.905, Q² = 0.82). (b) PCA t[1]/t[2] (two components give R²X = 0.854, Q² = 0.771).

Figure 8

OPLS-DA score plot (left panel) and corresponding coefficient plot (right panel) of the 600 MHz ¹H-ZG-NMR spectra (Pareto scaled) of SKOV-3 cells lipophilic extracts obtained from different pairwise groups (controls, Ptac2S, and cisplatin) at 24 h. (a) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive + 1 orthogonal give R²X = 0.887, R²Y = 0.986, Q² = 0.944. (b) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive +1 orthogonal give R²X = 0.784, R²Y = 0.985, Q² = 0.936). (c) OPLS-DA t[1]/t[2] scores scatter plot (two components, 1 predictive + 1 orthogonal give R²X = 0.893, R²Y = 0.996, Q² = 0.932).

Figure 9

PCA score scatter plot of the 600 MHz ¹H-CPMG-NMR spectra (Pareto scaled) obtained from recovered culture media of Ptac2S and cisplatin treated SKOV-3 cells in comparison with the untreated control samples. The indicated labels are referred to the culture times. (a) PCA t[1]/t[2] (four components give R²X = 0.969, Q² = 0.936). (b) PCA t[1]/t[2] (two components give R²X = 0.857, Q² = 0.678).

Figure 10

OPLS-DA score scatter plot (left panel) and corresponding coefficient plot (right panel) of the 600 MHz ¹H-CPMG-NMR spectra (Pareto scaled) of SKOV-3 cells culture media obtained from different pairwise groups (controls, Ptac2S, and cisplatin) at 24 h. (a) OPLS-DA t[1]/t[2] (two components, 1 predictive + 1 orthogonal, give R²X = 0.944, R²Y = 0.995, Q² = 0.99. (b) OPLS-DA t[1]/t[2] (two components, 1 predictive + 1 orthogonal, give R²X = 0.913, R²Y = 0.989, Q² = 0.957). (c) OPLS-DA t[1]/t[2] (two components, 1 predictive + 1 orthogonal, give R²X = 0.792, R²Y = 0.988, Q² = 0.871).

Figure 11

Relevant discriminant metabolites comparison of the culture media of treated, with either Ptac2S, cisplatin, and untreated (controls) SKOV-3 cells. The values of −Log₂(FC) and the p-values < 0.05 are provided (Student’s t-test). (a–b) Metabolites with −Log₂ (FC) negative values have a lower concentration compared to those of the 24 h control, while −Log₂ (FC) positive values have higher concentration compared to those of the 24 h control. (c) Metabolites with −Log₂ (FC) negative values have a lower concentration compared to those of the 24 h cisplatin group, while −Log₂ (FC) positive values have a higher concentration compared to those of the 24 h cisplatin group.

Figure 12

Schematic representation of main metabolic pathways altered at 24 h after Ptac2S (blue) and cisplatin (red) treatment of SKOV-3 cells. Comparison with the 24 h of the controls. Positive (+) and negative (−) signs indicate positive and negative correlations in the concentration, respectively.