Mathilde Bayet-Robert

Phase I dose escalation trial of docetaxel plus curcumin in patients with advanced and metastatic breast cancer

Background. Since the improvement of chemotherapy with safe molecules is needed for better efficacy without supplementary toxicity, we investigated the feasibility and tolerability of the combination of docetaxel and curcumin, a polyphenolic derivative extracted from Curcuma longa Patients and methods. Patients with advanced or metastatic breast cancer were eligible. Docetaxel (100 mg/m2) was administered as a 1-hour i.v. infusion every 3 weeks on d1 for 6 cycles. Curcumin was orally given from 500 mg/d for 7 consecutive days by cycle (from d-4 to d+2) and escalated until a dose-limiting toxicity should occur. The primary endpoint of this study was to determine the maximal tolerated dose of the combination of dose-escalating curcumin and standard dose of docetaxel chemotherapy in advanced and metastatic breast cancer patients. Secondary objectives included toxicity, safety, vascular endothelial growth factor and tumor markers measurements, and assessment of objective and clinical responses to the combination therapy. Results. Fourteen patients were accrued in this open-label phase I trial. At the last dose level of curcumin, 3 dose-limiting toxicities were observed and 2 out of 3 patients at this dose level refused to continue treatment, leading us to define the maximal tolerated dose of curcumin at 8000 mg/d. Eight patients out of 14 had measurable lesions according to RECIST criteria, with 5 PR and 3 SD. Some improvements as biological and clinical responses were observed in most patients. Conclusion. The recommended dose of curcumin is 6000 mg/d for 7 consecutive days every 3 weeks in combination with a standard dose of docetaxel. From the encouraging efficacy results, a comparative phase II trial of this regimen plus docetaxel versus docetaxel alone is ongoing in advanced and metastatic breast cancer patients.

Quantitative two-dimensional HRMAS 1H-NMR spectroscopy-based metabolite profiling of human cancer cell lines and response to chemotherapy.

NMR spectroscopy‐based metabolomics still needs development in quantification procedures. A method was designed for quantitative two‐dimensional high resolution magic angle spinning (HRMAS) proton‐NMR spectroscopy‐based metabolite profiling of intact cells. It uses referencing of metabolite‐related NMR signals to protein‐related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two‐dimensionally visible metabolites and combination of one‐ and two‐dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping. Magn Reson Med 63:1172–1183, 2010.

The New Combination Docetaxel, Prednisone and Curcumin in Patients with Castration-Resistant Prostate Cancer: A Pilot Phase II Study

Objectives: Favorable phase I results justified this pilot phase II study to assess the efficacy of docetaxel/curcumin in patients with chemotherapy-naive metastatic castration-resistant prostate cancer (CRPC). Methods: Thirty patients with progressing CRPC and a rising prostate-specific antigen (PSA) received docetaxel/prednisone in standard conditions for 6 cycles in combination with per os curcumin, 6,000 mg/day (day -4 to day +2 of docetaxel). The co-primary endpoint was the overall response rate determined by PSA and target assessments. An ancillary study assessed the seric values of chromogranin A (CgA) and neuron-specific enolase (NSE). Results: Twenty-six patients received the scheduled treatment, 2 progressed and 2 died before the end of treatment. A PSA response was observed in 59% of patients (14% of PSA normalization) and achieved within the first three cycles for 88% of responders. Partial response was reached for 40% of evaluable patients. The regimen was well tolerated, and no adverse event was attributed to curcumin. Twenty patients were 100% curcumin compliant. The PSA level and objective response rate were not correlated with the serum values of CgA and NSE. Conclusion: This study produced additional data on curcumin as a treatment for cancer, with a high response rate, good tolerability and patient acceptability, justifying the interest to conduct a randomized trial.

Biochemical disorders induced by cytotoxic marine natural products in breast cancer cells as revealed by proton NMR spectroscopy-based metabolomics

Marine plants and animals are sources of a huge number of pharmacologically active compounds, some of which exhibit antineoplastic activity of clinical relevance. However the mechanism of action of marine natural products (MNPs) is poorly understood. In this study, proton NMR spectroscopy-based metabolomics was applied to unravel biochemical disorders induced in human MCF7 breast cancer cells by 3 lead candidate anticancer MNPs: ascididemin (Asc), lamellarin-D (Lam-D), and kahalalide F (KF). Asc, Lam-D, and KF provoked a severe decrease in DNA content in MCF7 cells after 24-h treatment. Asc and Lam-D provoked apoptosis, whereas KF induced non-apoptotic cell death. Metabolite profiling revealed major biochemical disorders following treatment. The response of MCF7 tumor cells to Asc involved the accumulation of citrate (x17 the control level, P<0.001), testifying enzyme blockade in citrate metabolism, and the accumulation of gluconate (x9.8, P<0.005), a metabolite never reported at such concentration in tumor cells, probably testifying glycolysis shutdown. The response to Lam-D involved the accumulation of aspartate (x7.2, P<0.05), glutamate (x14.7, P<0.05), and lactate (x2.3, P<0.05), probably in relation with the targeting of the malate-aspartate shuttle, as discussed. The response to KF involved increased lipid accumulation (polyunsaturated fatty acids x9.8, P<0.05), and phospholipid and acetate derivative alterations. Altogether, this study demonstrates the potential of proton NMR spectroscopy-based metabolomics to help uncover metabolic targets and elucidate the mechanism of cytotoxicity of candidate antineoplastic MNPs.

A polyamine-deficient diet prevents oxaliplatin-induced acute cold and mechanical hypersensitivity in rats.

Background Oxaliplatin is an anticancer drug used for the treatment of advanced colorectal cancer, but it can also cause painful peripheral neuropathies. The pathophysiology of these neuropathies has not been yet fully elucidated, but may involve spinal N-methyl-D-aspartate (NMDA) receptors, particularly the NR2B subunit. As polyamines are positive modulators of NMDA-NR2B receptors and mainly originate from dietary intake, the modulation of polyamines intake could represent an interesting way to prevent/modulate neuropathic pain symptoms by opposing glutamate neurotransmission. Methods The effect of a polyamine deficient diet was investigated in an animal model of oxaliplatin-induced acute pain hypersensitivity using behavioral tests (mechanical and cold hypersensitivity). The involvement of spinal glutamate neurotransmission was monitored by using a proton nuclear magnetic resonance spectroscopy based metabolomic approach and by assessing the expression and phosphorylation of the NR2B subunit of the NMDA receptor. Results A 7-day polyamine deficient diet totally prevented oxaliplatin-induced acute cold hypersensitivity and mechanical allodynia. Oxaliplatin-induced pain hypersensitivity was not associated with an increase in NR2B subunit expression or phosphorylation, but with an increase of glutamate level in the spinal dorsal horn which was completely prevented by a polyamine deficient diet. As a validation that the oxaliplatin-induced hypersensitivity could be due to an increased activity of the spinal glutamate system, an intrathecal administration of the specific NR2B antagonist, ifenprodil, totally reversed oxaliplatin-induced mechanical and cold hypersensitivity. Conclusion A polyamine deficient diet could represent a promising and valuable nutritional therapy to prevent oxaliplatin-induced acute pain hypersensitivity.

Possible Benefits of Curcumin Regimen in Combination With Taxane Chemotherapy for Hormone-Refractory Prostate Cancer Treatment

Complementary and alternative therapies for neoplastic diseases treatment and prevention receive increasing attention from the medical community. Prostate cancer (PC) is the most frequently diagnosed malignancy and the second major cause of male death in industrialized countries. The chemopreventive properties and clinical safety of curcumin, a polyphenolic derivative, have already been established. However, curcumin regimen value in addition to conventional hormone refractory (HR) PC treatment remains largely unknown. This review article summarizes mechanisms by which curcumin may decrease HRPC aggressive proliferation and potentiate activity of taxane therapy. Our analysis suggests that curcumin alone has a therapeutic value in HRPC. In combination with a taxane agent, this compound may enhance cytotoxicity and retard PC cell resistance to taxane. As a consequence, a rationale is provided for considering the possible benefits of curcumin regimen in combination with taxane therapy in HRPC patients.

Drimane-type sesquiterpene coumarins from Ferula gummosa fruits enhance doxorubicin uptake in doxorubicin-resistant human breast cancer cell line

Multidrug resistance (MDR) is the main cause of failure in the chemotherapy of cancer patients. The present study aimed to evaluate the effects of sesquiterpene coumarins of Ferula gummosa fruits on P-glycoprotein (P-gp)-mediated MDR. Drimane-type sesquiterpene coumarins from the fruits of F. gummosa were extracted with dichloromethane and subjected to column chromatography. The effects of the isolated compounds on P-gp-mediated MDR were evaluated in the breast cancer cell line MCF-7 which shows high resistance to doxoribicin (MCF-7/Dox). Phytochemical investigation of dichloromethane extract of F. gummosa fruits resulted in three sesquiterpene coumarins including conferone (1), mogoltacin (2), and feselol (3). The structures of these compounds were confirmed by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. Exposure of cells to conferone, mogoltacin, feselol, and verapamil (positive control) enhanced doxorubicin uptake by MCF-7/Dox cells. This effect was dose dependent, but varied with the structure of the chemical. At 25 μM, all the tested sesquiterpene coumarins restored at least 50% of the reference uptake (uptake by sensitive cells); but at 10 μM, their potency varied where conferone showed the highest potency and feselol showed the lowest potency. Conferone, mogoltacin, and feselol from F. gummosa suppress P-gp-mediated drug efflux in highly resistant human breast cancer cells.

Abstract 3653: Cytotoxicity of curcumin combined with docetaxel at low or high dose implicates the antioxidant response and the glutathione cycle in MCF7 tumor cells: A metabolomics analysis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Curcumin (CCM) has antioxidant and chemopreventive properties at low dose. However, at high dose, it exhibits prooxidant activity. Recently, we showed using pharmacometabolomics that MCF7 cells response to docetaxel (DTX) involved, at low dose (1 nM), blockade of glutathione (GSH) biosynthesis, whereas, at high dose (5 µM), extensive GSH consumption. Cytotoxicity was significant even at low dose DTX. Here, we investigated whether CCM at 10 mg/l, a dose stimulating GSH synthesis, may interfere with GSH metabolism, reactive oxygen species (ROS) production, and cytotoxicity when combined to DTX at low and high doses. MCF7 cells were exposed for 72h to DTX at 1 nM and 5 µM in combination with 10 mg/l CCM as a 24h-pretreatment (PRE), cotreatment (CO) and 24h- and 48h-posttreatment (POST24 and POST48). Cytotoxicity was assessed from DNA content (Hoescht 33342), and ROS production from DNA strand breaks (Comet assay). Metabolomics analysis used high resolution magic angle spinning 1H-NMR spectroscopy, 2D spectral acquisitions and a new 2D-based quantification procedure, enabling to measure 32 metabolites. We focused on the variations of 13 metabolites from the GSH metabolism. Overall cytotoxicity of combinations increased according to POST48<POST24<CO<PRE (all, P<0.05), and ROS production was increased in PRE and CO (both, P<0.05), at low and high dose DTX. Metabolomics analysis of PRE at low and high dose DTX involved the preservation of GSH levels but an accumulation of 5-oxoproline (OP, X3.85 at 1 nM and X5.90 at 5 µM, both P<0.05), suggesting a blockade at oxoprolinase (OPase), step of the GSH cycle. Metabolomics analysis of CO at low dose DTX showed an increase in GSH (X1.25, P<0.05), together with starvation of most GSH precursors: glycine (Gly, X0.26, P<0.05) and homocysteine (Hcy, X0.24, P<0.05)). At high dose DTX, the GSH level was unchanged, but OP was increased (X4.26, P<0.05). Metabolomics analysis of POST24 at low dose DTX involved an increase in GSH (X1.22, P<0.05) suggesting increased biosynthesis of GSH but at the expense of taurine (X0.44, P=NS). At high dose DTX, GSH level was maintained, but associated with an increase in glutamate (Glu, X1.73, P<0.05). Metabolomics analysis of POST48 at low dose DTX showed an increase in GSH (X1.21, P<0.05) without signs of blockade of transsulfuration (Hcy X0.80, P=NS). At high dose DTX, the GSH level was maintained with mild signs of precursor starvation (Gly, X0.27, P<0.05), and decreased Glu derivatives (P<0.05), suggesting increased GSH biosynthesis and consumption. In all combinations, CCM at 10 mg/l generated an antioxidant response in MCF7 cells, with preserved or increased GSH levels. However, the underlying biochemical mechanism was different. Maximal cytotoxicity was obtained in PRE conditions involving ROS production, probable OPase blockade with the accumulation of biomarker OP. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3653.

Cytotoxic doses of curcuminoids provoke a down-regulation of phospholipid biosynthesis – a focused metabolomic profiling.

Abstract #6029 Background: Curcuminoids of which curcumin have proved efficacy in breast cancer prevention [Aggarwal et al, Anticancer Res (2003);23(1A):363]. In addition, there is growing evidence that these compounds may treat cancer [Karunagaran et al, Curr Cancer Drug Targets (2005);5(2):117]. However, the mechanisms of cytotoxicity of curcuminoids remain partially unknown. Functional genomics include several tools increasingly applied in pharmacology, like DNA and RNA microarrays, and proteomics. It was recently shown that a novel post-genomics technology, NMR spectroscopy-based metabolomics, was able to unravel metabolic pathways of the response to anticancer drugs [Morvan et al, Cancer Res. (2007);67(5):2150 ; Bayet-Robert et al, AACR Proceedings (2008);abstract 3938]. We applied this technique to investigate the metabolic features of the response of MCF7 human breast carcinoma cells to curcuminoids. We here report the analysis of the phospholipid metabolism subset of our data.
 Material and Methods: MCF7 cells were exposed to 10 µg/mL curcuminoids and followed at 12, 36 and 72 h intervals of time. Cytotoxicity assay was performed using Hoescht 33342. A novel high resolution magic angle spinning 1H-NMR spectroscopy-based metabolomics technique was used to obtain a quantitative metabolite profiling of about thirty metabolites, among which 8 phospholipid metabolism derivatives, in intact treated and untreated MCF7 cells.
 Results: A 72 h-exposure to 10 µg/mL curcuminoids decreased MCF7 cell survival to 4% (p=0.0039). Polyunsaturated fatty acids increased by +120% at 36 h and +62% at 72 h (both p=NS, Mann-Whitney test), phosphoethanolamine by +24% at 36 h (p=NS) and +120% at 72 h (p=0.0062), cytidinediphosphocholine (CDP-Cho) by +22% at 12 h, +132% at 36 h (both p=NS), and +547% at 72 h (p=0.0062). There was a decrease of phosphatidylcholine (PtdCho) by -30% at 36 h (p=NS), and -42% at 72 h (p=NS). Glycerophosphoethanolamine decreased by -28% at 12 h (p=0.0446) and glycerophosphocholine by -53% (p=0.0106). The PtdCho/CDP-Cho ratio decreased from +93% to +8% between 12 and 72 h, which indicates a blockade of PtdCho biosynthesis.
 Discussion and Conclusion: Our results are consistent with a rapidly progressive down-regulation of cholinephosphotransferase activity in response to curcuminoids, a biochemical mechanism closely linked to apoptosis [Miquel et al, J Biol Chem. (1998);273(40):26179 ; Cui et al, Biochim Biophys Acta. (2002);1585(2-3):87]. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6029.