Table 1.
Proton chemical shift of signals used for metabolite quantification.
Figure 1.
Dose-dependent response to CUR.
A- Cellular DNA content in attached MCF7 (black bars) and MDA-MB-231 (white bars) breast cancer cells exposed to increasing concentrations (0.5, 2.5, 10, 25, and 50 mg.l−1) of CUR for 24 h, measured using the Hoechst fluorescence intensity assay. *, P<0.05, **, P<0.01, CUR vs. CTL, Mann-Whitney test. B- DNA fragmentation expressed in tail DNA in MCF7 tumor cells exposed to increasing concentrations (0.5, 2.5, 10, 25, and 50 mg.l−1) of CUR for 24 h, measured using the alkaline Comet assay. *, P<0.05, CUR vs. CTL, Mann-Whitney test. C- Total glutathione S-transferase (GST) activity in MCF7 tumor cells exposed to increasing concentrations (0.5, 2.5, 10, 25, and 50 mg.l−1) of CUR for 24 h, measured using the Cayman Chemical Company assay kit. **, P<0.01, CUR vs. CTL, Mann-Whitney test. D- Set of typical 1D HRMAS 1H-NMR spectra of MCF7 breast cancer cells in the 0.5–4.5 ppm spectral range. From bottom to top, cells exposed to 0, 0.5, 2.5, 10, 25, and 50 mg.l−1 CUR. Thin arrows, signals from glutathione (GSx) at positions 2.25, 2.55, 2.98, and 3.78 ppm. Grey arrowheads, signal from total free fatty acids (tFA) at positions 1.30 and 0.89 ppm. E- Set of typical 1D HRMAS 1H-NMR spectra of MDA-MB-231 breast cancer cells. From bottom to top, cells exposed to 0, 10, and 50 mg.l−1 CUR. Arrows, as in Fig. 1D. F- Two-dimensional HRMAS 1H-NMR spectra of intact MCF7 tumor cells, here from the 25 mg.l−1 CUR group. Thin arrows, cross-peaks of glutathione (GSx) at positions 2.25×2.55, 2.25×3.78, 2.55×3.78, and 2.98×4.55 ppm×ppm. Square, area containing gluconate (Gna) signals. G- Square area magnified from the spectrum in Fig. 1F. Arrowheads, cross-peaks from Gna at positions 4.13×4.02, 4.02×3.76, and 3.76×3.66 ppm×ppm. Gna was quantified from the first 2 cross-peaks.
Figure 2.
Co-mapping of dose-related metabolite changes with metabolic schemes.
A- Glutathione cycle and metabolism. Abbreviations: GS-X, conjugated GSH; GSSG, oxidized GSH; GS-L, lactoyl-glutathione; Glu-Cys, glutamylcysteine; Cys, cysteine; Met, methionine; Ser, serine; GST, glutathione S-transferase; GSR, glutathione S-reductase; GLO1, glyoxalase-1; GS, glutathione synthetase; GCL, glutamate-cysteine ligase; OPase, oxoprolinase, CBS, cystathionine beta-synthase. Other metabolite abbreviations, as in Table 1. Italicized, enzymes; Grey, undetected/unmeasured metabolites; #, P<0.05; *, P<0.025; $, P<0.01, Mann-Whitney test. B- Glycolysis and lipid metabolism. Abbreviations: PtE, phosphatidylethanolamine; Glc, glucose; DAG, diacylglycerol; Pyr, pyruvate; ACoA, acetyl-CoA; PG, prostaglandin; CHK, choline kinase; CCT, choline cytidylyltransferase; PDH, pyruvate dehydrogenase; PLA2, phospholipase A2; FASN, fatty acid synthase; COX-2, cyclooxygenase-2. Other metabolite abbreviations, as in Table 1. #, P<0.05; *, P<0.025; $, P<0.01, Mann-Whitney test.
Table 2.
CUR dose-related data in MCF7 cells (fold variation vs. 24 h controls).
Figure 3.
Multivariate analysis of CUR dose-related metabolite data.
A- PLS-DA of metabolite data obtained in MCF7 and MDA-MB-231 cells types responding to 10 mg.l−1 CUR. Comapping of metabolites and 3 factors (cell type: MCF7 and MDA-MB-231) and common response to CUR at 10 mg.l−1 (CUR10). Metabolite abbreviations, as in Table 1. B- PLS-DA of metabolite data obtained in MCF7 and MDA-MB-231 cells types responding to 50 mg.l−1 CUR. Comapping of metabolites and 3 factors (cell type: MCF7 and MDA-MB-231) and common response to CUR at 50 mg.l−1 (CUR50). Metabolite abbreviations, as in Table 1. C- PCA of CUR dose related data. Each score corresponds to an individual measurement and is labeled CTL or the corresponding CUR dose level. F1 and F2, first 2 principal components with the percentage of total variance of data they account for. Two elliptic clouds separate, to the left, the set of CTL and low dose CUR (0.5, 2.5, and 10 mg.l−1) samples, and, to the right, the set of high dose CUR (25 and 50 mg.l−1) samples. Grey arrow, trajectory between low and high dose clouds. D- Loading plot of PCA. Metabolite abbreviations, as in Table 1. E- Partial correlation of CUR dose-related data with available covariables (DNA content, tail DNA and GST activity). Red, positive correlation; green, negative correlation; left, color scale of correlations, between -1 and +1. F- PLS-DA of metabolite data, after fitting to a hormetic model, showing the VIP plot of the 5 most discriminant metabolites with hormetic behaviour (left). Corresponding loading plot (right). Metabolite abbreviations, as in Table 1.
Figure 4.
Duration of exposure-related response to CUR.
Cells were treated with 10 mg.l−1 CUR and followed for 96 h. A- Cellular DNA content in attached MCF7 tumor cells relative to time-matched CTL. DNA content was measured using the Hoechst fluorescence intensity assay. *, P<0.05, **, P<0.01 CUR vs. CTL, Mann-Whitney test. B- Tail DNA relative to time-matched CTL. DNA. Tail DNA was assessed using the alkaline Comet assay. *, P<0.05, CUR vs. CTL, Mann-Whitney test. C- Total GST activity relative to time-matched CTL. GST activity was measured using the Cayman Chemical Company assay kit. **, P<0.01, CUR vs. CTL, Mann-Whitney test. D- Metabolite variations relative to time-matched CTL. #, P<0.05; *, P<0.025; $, P<0.01, CUR vs. CTL, Mann-Whitney test. E- PCA of duration of exposure-related data. The scores plot is displayed with individual scores labeled by their duration of exposure, between 24 h and 96 h, with CTL data also displayed. F1 and F2, first 2 principal components with the percentage of total variance of data they account for. Grey arrows, duration of exposure-related trajectory. F- Corresponding loading plot of PCA of duration of exposure-related data. Metabolite abbreviations, as in Table 1.
Figure 5.
Response of MCF7 cells to co-treatment with CUR and DTX.
A- Protocol of cotreatment. Thin arrows, onset of DTX treatment at either low or high dose. White arrow, onset of CUR treatment (10 mg.l−1). Right, abbreviations designating each condition: CO, cotreatment; L or H, low dose DTX or high dose DTX; 24–96 h, duration of exposure to CUR at the time of measurement. B- Cellular DNA content in attached MCF7 tumor cells relative to time-matched CTL in combinations with low dose DTX (DTX-L). DNA content was measured using the Hoechst fluorescence intensity assay. C- Cellular DNA content in attached MCF7 tumor cells relative to time-matched CTL in combinations with high dose DTX (DTX-H). D- Tail DNA for cotreatment with low dose DTX (DTX-L) relative to DTX-L. Tail DNA was assessed using the alkaline Comet assay. E- Tail DNA for cotreatment with high dose DTX (DTX-H) relative to DTX-H. F- Metabolite variations as a function of duration of cotreatment with low dose DTX (DTX-L). Abbreviations, as in Table 1. #, P<0.05; *, P<0.025; $, P<0.01, cotreatment vs. DTX-L, Mann-Whitney test. G- Metabolite variations as a function of duration of cotreatment with high dose DTX (DTX-H). Abbreviations, as in Table 1. #, P<0.05; *, P<0.025; $, P<0.01, cotreatment vs. DTX-H, Mann-Whitney test.
Figure 6.
Multivariate analysis of cotreatment-related metabolite data.
A- PCA of data related to cotreatment with curcumin and DTX-L. Each score corresponds to an individual measurement and is labeled as in Fig. 5A. F1 and F2, first 2 principal components with the percentage of total variance of data they account for. Cloud contours are displayed for DTX-L, CO-L-24 h, the set of CO-L-48 h and CO-L-72 h, and CO-L-96 h. Arrows, duration of exposure-related trajectory. B- Corresponding loading plot. Metabolite abbreviations, as in Table 1. C- PCA of data related to cotreatment with curcumin and DTX-H. Each score corresponds to an individual measurement and is labeled as in Fig. 5A. F1 and F2, first 2 principal components with the percentage of total variance of data they account for. Cloud contours are displayed for CO-H-24 h, the set of DTX-H, CO-H-48 h and CO-H-72 h, and CO-H-96 h. Grey arrows, duration of exposure-related trajectory. D- Corresponding loading plot. Metabolite abbreviations, as in Table 1.