Abstract: Curcuminoids have proven efficacy in breast cancer prevention [1]. In addition, there is growing evidence that these polyphenolic compounds may be relevant in cancer therapy [2]. However, the mechanisms for curcuminoid cytotoxicity remain partially unknown. Prooxydant and proapoptotic activities could participate to cytotoxicity of curcuminoids at high dose [3]. Functional genomics include several tools increasingly applied in pharmacology, like DNA and RNA microarrays, and proteomics. It was recently shown that a novel technology, NMR spectroscopy-based metabolomics, was able to unravel metabolic pathways of the response to anticancer drugs [4,5]. We applied this technology to investigate metabolic features of the response of MCF7 human breast carcinoma cells to curcuminoids at high dose, and focused on the glutathione metabolism subset of our data.
MCF7 cells were exposed to 10 µg/mL curcuminoids, and a kinetics of the response was recorded at 6, 16, 24, 48, 72 and 96h. Cytotoxicity assay was performed using Hoescht 33342. A recently developed high resolution magic angle spinning proton-NMR spectroscopy-based metabolomics technique was used to obtain a quantitative metabolite profiling of about thirty metabolites, 9 of which, belonging to the glutathione metabolism subset, were here scrutinized, in intact treated and untreated MCF7 cells.
The decrease in MCF7 cell survival after treatment was continuous (96% at 6h, 61% at 24h (p<0.01), 14% at 48h (p<0.01), and 4% at 72h (p<0.01), although the time response of glutathione subset metabolites could be described as biphasic (early and delayed phases). The early phase (6h-24h) involved an acute increase of creatine (tCr) by +149% at 6h (p<0.05), followed by the decrease of homocysteine (Hcy) by -95% at 16h (p<0.05) and the increase of total glutathione (GSx) by +119% at 24h (p<0.05). The delayed phase, between 48h and 96h, was associated with the progressive return to baseline of GSx from +106% at 48h (p<0.05) to +24% at 96h (p=0.09) and, after a transient return to baseline, the decrease of Hcy by -91% at 96h (p<0.05).
The bell-shaped GSx variation and coordinated metabolite variation reveal a redox-related regulation of transsulfuration at the transcriptional and enzyme levels. The enzyme cystathionine beta-synthase activity is well known to be activated by reactive oxygen species (ROS) [6]. Also such regulation of GSx levels may result from combined involvement of ROS production and nitric oxide synthase induction [7].
In conclusion, metabolomics supports the implication of prooxidant effects in the mechanism of high dose curcuminoid cytoxicity in breast carcinoma cells.
