Ming Chiu

Enhancement of curcumin oral absorption and pharmacokinetics of curcuminoids and curcumin metabolites in mice.

PurposeCurcumin has shown a variety of biological activity for various human diseases including cancer in preclinical setting. Its poor oral bioavailability poses significant pharmacological barriers to its clinical application. Here, we established a practical nano-emulsion curcumin (NEC) containing up to 20% curcumin (w/w) and conducted the pharmacokinetics of curcuminoids and curcumin metabolites in mice.MethodsThis high loading NEC was formulated based on the high solubility of curcumin in polyethylene glycols (PEGs) and the synergistic enhancement of curcumin absorption by PEGs and Cremophor EL. The pharmacokinetics of curcuminoids and curcumin metabolites was characterized in mice using a LC–MS/MS method, and the pharmacokinetic parameters were determined using WinNonlin computer software.ResultsA tenfold increase in the AUC0→24h and more than 40-fold increase in the Cmax in mice were observed after an oral dose of NEC compared with suspension curcumin in 1% methylcellulose. The plasma pharmacokinetics of its two natural congeners, demethoxycurcumin and bisdemethoxycurcumin, and three metabolites, tetrahydrocurcumin (THC), curcumin-O-glucuronide, and curcumin-O-sulfate, was characterized for the first time in mice after an oral dose of NEC.ConclusionThis oral absorption enhanced NEC may provide a practical formulation to conduct the correlative study of the PK of curcuminoids and their pharmacodynamics, e.g., hypomethylation activity in vivo.

Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia

Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment.

CCI-779 Inhibits Cell-Cycle G2/M Progression and Invasion of Castration Resistant Prostate Cancer via Attenuation of UBE2C Transcription and mRNA Stability

The cell-cycle G(2)-M phase gene UBE2C is overexpressed in various solid tumors including castration-resistant prostate cancer (CRPC). Our recent studies found UBE2C to be a CRPC-specific androgen receptor (AR) target gene that is necessary for CRPC growth, providing a potential novel target for therapeutic intervention. In this study, we showed that the G(1)-S cell-cycle inhibitor-779 (CCI-779), an mTOR inhibitor, inhibited UBE2C mRNA and protein expression in AR-positive CRPC cell models abl and C4-2B. Treatment with CCI-779 significantly decreased abl cell proliferation in vitro and in vivo through inhibition of cell-cycle progression of both G(2)-M and G(1)-S phases. In addition, exposure of abl and C4-2B cells to CCI-779 also decreased UBE2C-dependent cell invasion. The molecular mechanisms for CCI-779 inhibition of UBE2C gene expression involved a decreased binding of AR coactivators SRC1, SRC3, p300, and MED1 to the UBE2C enhancers, leading to a reduction in RNA polymerase II loading to the UBE2C promoter, and attenuation of UBE2C mRNA stability. Our data suggest that, in addition to its ability to block cell-cycle G(1) to S-phase transition, CCI-779 causes a cell-cycle G(2)-M accumulation and an inhibition of cell invasion through a novel UBE2C-dependent mechanism, which contributes to antitumor activities of CCI-779 in UBE2C overexpressed AR-positive CRPC.

Reactivation of RASSF1A in Breast Cancer Cells by Curcumin

Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.

A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma.

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin<THC<TMC<DMCHC in both matrices. Following an i.p. cassette dose, both TMC and DMCHC showed the prolonged elimination half life (1.0, 1.0 h, respectively vs 0.4h for curcumin) and an increased drug exposure as described by the area under the curve (0.64, 0.98 μM h, respectively vs 0.4 μM h for curcumin).

Abstract 2014: Modulation of DNA methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Curcumin, a natural product of curcuma longa and a safe and widely consumed spice, has been shown to exhibit strong in-vitro and in-vivo anti-tumor activity through the inhibition of NF-κB activity, increase of reactive oxygen species (ROS) levels and decrease of the global DNA methylation levels in leukemia cells. We investigated here the molecular mechanisms of down-regulation of DNA Methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities. Method: Leukemia cell lines MV4-11, Kasumi-1 and K562 cells and primary leukemia cells from patient with AML were treated with 1-30 μM of curcumin and MV4-11 xenografted nu/nu athymic mice was given i.p. curcumin at 100 mg/kg, and the transcription and protein levels of DNMT1, DNMT3a, DNMT3b, Sp1, p15INK4B and p21 were assessed by either RT-PCR or western blot. The inhibition of NF-κB was determined using Electrophoretic Mobility Shift Assays and the disruption of Sp1-NF-κB/DNMTs promoter complex was investigated by anti-body gel supershifts. The hypomethylating activity of curcumin on the promoter of p15INK4B in MV4-11 cells in-vitro was assessed by a LC-MS/MS method for regional DNA methylation. The anti-proliferative activity of curcumin and cell cycle distribution and apoptosis were evaluated using MTS assay and flow cytometry. Results: In addition to the pre-defined direct inhibition of the enzymatic activity of DNA methyltransferase 1 (DNMT1), curcumin disrupts Sp1/NF-κB complex on DNMT1, DNMT3a and DNMT3b promoter thereby decreasing the transactivation activity of these complexes and down-regulating their mRNA and protein expression levels in AML cells in vitro and in vivo. Consistent with these findings, we showed that promoter hypermethylation of silenced TSGs i.e., p15INK4B decreased while their expression was up-regulated. This results in the increase of protein function, as we showed that in association with p15INK4B re-expression, curcumin induced cell cycle arrest in the S-phase and increase in SubG1 cell fraction, thereby resulting in significant anti-leukemia activity. Conclusion: Different from nucleoside analogs (e.g., azanucleosides), curcumin is an effective S-phase independent DNA methylation inhibitor. Further evaluation of curcumin as a single agent and in combination with other epigenetic targeting compounds in human disease is warranted. Supported by NIH-NCI-R21 CA135478 [ZL], NIH-NCI-RO1 CA102031 [GM], and the BioMedical Mass Spectrometry Laboratory [KC & ZL]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2011-2014

Abstract 4426: Metabolism and tissue distribution of curcumin in mouse GI tract.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: Curcuminoids have shown pronounced preventive activities on gastrointestinal (GI) cancers in preclinical settings and have now advanced to clinical evaluation. However, their tissue distribution and metabolism in the GI tissues remain largely unknown. The objective of this study is to establish an LC-MS/MS method to profile curcumin (CUR), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and CUR Metabolites: CUR O-glucuronide (COG), CUR O-sulfate (COS), and tetrahydrocurcumin (THC) in plasma, and to study their disposition and kinetics in the GI tissues and lumens. Methods: The following ion transitions at m/z: 367.4/149.1, 337.3/216.9, 307.5/186.8, 543.7/216.9, 447.4/216.9, and 371.2/235.1, and 301.5/163.9 were used for monitoring CUR, DMC, BDMC, COG, COS, THC and hesperetin (internal standard), respectively, on an API mass spectrometer. Nano-emulsion CUR (50 mg/kg) was orally administered to CD2F1 mice. Plasma, stomach tissues (ST) and lumens (SL), intestine tissue (IT) and lumens (IL), and colon tissue (CT) and feces (F) were collected up to 6 h after the dose and analyzed using an LC-MS/MS method. Results: CUR and its metabolites were simultaneously quantified in mouse plasma, GI tissue homogenates, and GI lumens using the LC-MS/MS method with a linear range of 2 to 1000 ng/mL. COG was found to be the most abundant circulating CUR metabolite with a peak concentration of 3.99 μM in plasma at 30 min, which is approximately 8 and 36 fold higher than that of CUR and COS, respectively. In contrast, CUR was the most abundant transient species in ST, IT and CT, and reached pharmacological effective peak concentrations (μM) of 118, 55, and 142 in ST, IT and CT at 20, 60 and 30 min, respectively. The concentrations of CUR were 8261, 923 and 9.74 μM in SL, IL and F at 30, 60 and 30 min, respectively. COG and COS were two main metabolites of CUR with relative lower peak concentrations of 6.46 and 8.79 μM in IT and 30.4 and 80.1 μM in IL at 60 min, respectively, while COG and COS concentrations were much lower in ST, CT and F, and they were not detectable in SL. Notably, THC was the main metabolite of CUR with peak concentrations of 24 and 1.89 μM in ST and CT at 10 and 360 min, respectively, while no THC was detected in SL, IT, IL and F. In addition, DMC, BDMC, and THC, and their glucuronide and sulfate adducts were also profiled using these ion transition channels. Conclusions: The LC-MS/MS method provides a critical analytic method to study the metabolism, tissue distribution, and pharmacokinetics (PK) of CUR in mice. These results demonstrated that orally ingested CUR could deliver pharmacological effective level of CUR to GI tissues and lumens, which is completely different from that in plasma. Therefore, further investigation of curcuminoids as a preventive or therapeutic agent for GI cancers is warranted. Supported by R21CA135478 (Liu), CA159077-01 (Liu & Xu) and Biomedical Mass Spectrometric Laboratory (Chan & Liu). Citation Format: Yu Cao, Ming Chiu, Kenneth K. Chan, Ronald Xu, Zhongfa Liu. Metabolism and tissue distribution of curcumin in mouse GI tract. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2013-4426

Abstract 5494: A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell culture medium and mouse plasma and their pharmacokinetics in mice

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis (3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. Purpose: A sensitive and rapid LC-MS/MS method for simultaneous determination of curcumin and its metabolite THC and analogs TMC and DMCHC in RPMI cell culture medium and mouse plasma has been developed and validated and their stability in these matrices and their pharmacokinetics in mice were evaluated. Method: A triple quadrupole mass spectrometer with electro-spray (ESI) ionization was used for quantification. Spiked curcuminoids and the internal standard, hesperetin were extracted from mouse plasma with ethyl acetate, and the components were separated on a BetaBasic C8 column. The ion transitions at m/z 369→177, m/z 373→137, m/z 425→191, m/z 465→191 and m/z 303→153 for curcumin. THC, TMC, DMHC, and hesperetin, respectively, were monitored for their quantification. These curcuminoids were incubated in mouse plasma or cell culture at different temperatures and aliquots were removed at different times up to 24 hrs. Cassette dose of curcumin and TMC (10 mg/kg each) was intraperitoneally administered to CD1F2 mice and timed plasma samples were collected up to 8 hr. Curcuminoids in these samples were quantified using this method. Result: The lower limit of quantifications for all four curcuminoids was found to be 1 ng/mL in both mouse plasma and cell culture medium. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85% to 115%. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, IHC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability are in the order of curcumin Conclusion: A sensitive LC-MS/MS method for simultaneous quantification of these curcumionids has been established. TMC and DMCHC were more stable than curcumin in cell culture medium and mouse plasma. Further pharmacokinetic study is ongoing. Supported by NIH/NCI R21 CA135478, and BioMedical Mass Spectrometric Laboratory. 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 5494.