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1.  Induction of Lung Cancer Cell Apoptosis through a p53 Pathway by [6]-Shogaol and Its Cysteine-Conjugated Metabolite M2 
Dietary chemoprevention of cancer offers the possibility to suppress or inhibit cancer growth before it develops into more advanced and lethal stages. To this end, identification of novel compounds and their mechanisms of action is constantly needed. In this study, we describe that a major component of dry ginger (Zingiber officinalis), [6]-shogaol (6S), can be quickly metabolized in A549 human lung cancer cell line. One of the resulting metabolites, the cysteine-conjugated 6S (M2), exhibits toxicity to cancer cells similar to the parent compound 6S, but is relatively less toxic toward normal cells than 6S. We further demonstrate that both compounds can cause cancer cell death by activating the mitochondrial apoptotic pathway. Our results show that the cancer cell toxicity is initiated by early modulation of glutathione (GSH) intracellular content. The subsequently generated oxidative stress activates a p53 pathway that ultimately leads to the release of mitochondria-associated apoptotic molecules such as cytochrome C, and cleaved caspases 3 and 9. In a xenograft nude mouse model, a dose of 30 mg/kg of 6S or M2 was able to significantly decrease tumor burden, without any associated toxicity to the animals. This effect was correlated with an induction of apoptosis and reduction of cell proliferation in the tumor tissues. Taken together, our results show that 6S metabolism is an integral part of its anticancer activities in vitro and in vivo. This allows us to characterize M2 as a novel compound with superior in vivo chemopreventive properties that targets similar anticancer mechanisms as 6S.
doi:10.1021/jf405573e
PMCID: PMC3983336  PMID: 24446736
[6]-shogaol; cysteine conjugated metabolite; lung cancer; apoptosis; xenograft
2.  Cysteine-conjugated metabolite of ginger component [6]-shogaol serves as a carrier of [6]-shogaol in cancer cells and in mice 
Chemical research in toxicology  2013;26(6):976-985.
Shogaols, a series of major constituents in dried ginger (Zingiber officinale), show high anti-cancer potencies. Previously, we reported that a major metabolite resulting from the mercapturic acid pathway, 5-cysteinyl-[6]-shogaol (M2), showed comparable growth inhibitory effects towards cancer cells to [6]-shogaol (6S). Here we probe the mechanism by which M2 exerts its bioactivity. We utilized a series of chemical stability tests in conjunction with bioassays to show that thiol-conjugates display chemopreventative potency by acting as carriers of active ginger component 6S. M2 chemical degradation to 6S was observed in an environment most resembling physiological conditions, with a pH of 7.4 at 37°C. The metabolic profiles of M2 in cancer cells HCT-116 and H-1299 resembled those of 6S, indicating that its biotransformation route was initiated by deconjugation. Further, the presence of excess glutathione significantly delayed 6S and M2 metabolism and counteracted cell death induced by 6S and M2, suggesting that increasing available free thiols exogenously both promoted formation of 5-glutathionyl-[6]-shogaol (M13) and inhibited the production of free 6S from M2 deconjugation, resulting in delayed 6S cell entry and bioactivity. Given the chemopreventative properties of M2 and our observations in vitro, we investigated its metabolism in mice. M2 and 6S showed similar metabolic profiles in mouse urine and fecal samples. Six new thiol-conjugated metabolites (M16–M21), together with previously reported ones, were identified by LC/MS. In particular, the increase of 5-N-acetylcystenyl-[6]-shogaol (M5) and its 3′-demethylated product (M16) abundance in mouse feces after treatment with M2 indicate that in addition to acting as a carrier of 6S, M2 is also directly acetylated to M5, which is further demethylated to M16 in vivo. In conclusion, cysteine-conjugated metabolite of [6]-shogaol M2 exerts its bioactivity by acting as a carrier of 6S in both cancer cells and in mice.
doi:10.1021/tx4001286
PMCID: PMC3767927  PMID: 23638641
ginger; [6]-shogaol; 5-cysteinyl-[6]-shogaol; carrier; metabolism; cancer cells; mice
3.  [10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos 
Journal of agricultural and food chemistry  2013;61(22):10.1021/jf401501s.
Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound towards pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos, and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and in humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MSn spectra and comparison to authentic standards that we synthesized. After 24 hours of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and in humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos.
doi:10.1021/jf401501s
PMCID: PMC3840088  PMID: 23701129
[10]-Gingerol; Metabolism; Hematopoiesis; Zebrafish embryos; Human urine
4.  Metabolism of Ginger Component [6]-Shogaol in Liver Microsomes from Mouse, Rat, Dog, Monkey, and Human 
Molecular nutrition & food research  2013;57(5):10.1002/mnfr.201200708.
Scope
There are limited data on the metabolism of [6]-shogaol, a major bioactive component of ginger. This study demonstrates metabolism of [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human.
Methods and results
The in vitro metabolism of [6]-shogaol was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with [6]-shogaol, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E, 4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than [6]-shogaol.
Conclusion
We conclude that [6]-shogaol is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning pre-clinical trials towards [6]-shogaol chemoprevention.
doi:10.1002/mnfr.201200708
PMCID: PMC3815528  PMID: 23322474
[6]-Shogaol; Ginger; Metabolism; Liver microsomes; Cancer
5.  A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells 
eLife  2014;3:e01630.
Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).
DOI: http://dx.doi.org/10.7554/eLife.01630.001
eLife digest
Cells are complex environments: at any one time, thousands of different genes act as molecular templates to produce messenger RNA (mRNA) molecules, which themselves are templates used to produce proteins. However, not all genes are active at all times inside all cells: as cells grow and divide as part of the cell division cycle, genes are switched on and off on a regular basis. Similarly, the patterns of mRNA and protein production are different in, say, immune and skin cells.
In recent years, the tools available for detecting mRNA molecules and proteins have become more powerful, allowing researchers to move beyond just measuring the total amounts of mRNA and protein in the cell to now measuring individual amounts of specific mRNA and protein molecules encoded by specific genes. However, it has been a challenge to make these measurements at different stages of the cell cycle. Most of the methods used to do this have involved artificially ‘arresting’ the cell cycle, which can lead to side effects that are difficult to account for.
Ly et al. have now overcome these problems using a combination of three methods to measure the levels of mRNA and protein molecules associated with over 6000 genes in human cancer cells derived from myeloid leukemia. Exploiting the fact that cells change size during the cell cycle, Ly et al. used a centrifugation technique to separate cells based on their size and, therefore, the stage of the cell cycle they were at, thus avoiding the need to arrest the cell cycle. An approach called RNA-Seq was then employed to measure the levels of the different mRNA molecules in the cells, and a device called a mass spectrometer was used to identify and measure the levels of many different proteins.
In addition to being able to follow the level of mRNA and protein production for a large number of genes throughout the cell division cycle, while also obtaining detailed information about how many of the proteins are modified, Ly et al. discovered that—contrary to expectations—low numbers of mRNA molecules were sometimes associated with high numbers of the corresponding protein, and vice versa. This work provides a better understanding of the complex relationship between the levels of an mRNA and its corresponding protein product, and also demonstrates how it may be possible to detect subtle but important differences between cell types and disease states, including different types of cancer.
DOI: http://dx.doi.org/10.7554/eLife.01630.002
doi:10.7554/eLife.01630
PMCID: PMC3936288  PMID: 24596151
proteomics; mass spectrometry; RNA-Seq; cell cycle; transcriptomics; human
6.  Characterization of thiol-conjugated metabolites of ginger components shogaols in mouse and human rrine and modulation of the glutathione levels in cancer cells by [6]-shogaol 
Molecular nutrition & food research  2013;57(3):10.1002/mnfr.201200679.
Scope
Shogaols, a series of major constituents in dried ginger with the most abundant being [6]-, [8]-, and [10]-shogaols, show much higher anti-cancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]-shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols and how shogaols are metabolized in humans.
Methods and results
We first investigate the metabolism of [10]-shogaol in mouse urine, and then investigate the biotransformation of shogaols in human urine. Our results show that eight major thiol-conjugated metabolites of [10]-shogaol were detected in mouse urine, while six major thiol-conjugated metabolites of [6]-shogaol, two thiol-conjugated metabolites of [8]-shogaol, and two thiol-conjugated metabolites of [10]-shogaol were detected in urine collected from human after drinking ginger tea, using liquid chromatography/electrospray ionization tandem mass spectrometry. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for [10]-shogaol in mice and for shogaols in human. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]-shogaol in human colon cancer cells HCT-116. Our results show [6]-shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time.
Conclusion
Shogaols are metabolized extensively in mouse and human to form thiol-conjugated metabolites and GSH might play an important role in the cancer preventative activity of ginger.
doi:10.1002/mnfr.201200679
PMCID: PMC3817846  PMID: 23322393
Shogaols; Thiol-conjugated metabolites; Human urine; Glutathione
7.  6-Gingerdiols as the Major Metabolites of 6-Gingerol in Cancer Cells and in Mice and Their Cytotoxic Effects on Human Cancer Cells 
6-Gingerol, a major pungent component of ginger (Zingiber officinale Roscoe, Zingiberaceae), has been reported to have anti-tumor activities. However, the metabolic fate of 6-gingerol and the contribution of its metabolites to the observed activities are still unclear. In the present study, we investigated the biotransformation of 6-gingerol in different cancer cells and in mice, purified and identified the major metabolites from human lung cancer cells, and determined the effects of the major metabolites on the proliferation of human cancer cells. Our results show that 6-gingerol is extensively metabolized in H-1299 human lung cancer cells, CL-13 mouse lung cancer cells, HCT-116 and HT-29 human colon cancer cells, and in mice. The two major metabolites in H-1299 cells were purified and identified as (3R,5S)-6-gingerdiol (M1) and (3S,5S)-6-gingerdiol (M2) based on the analysis of their 1D and 2D NMR data. Both metabolites induced cytotoxicity in cancer cells after 24 hours, with M1 having a comparable effect to 6-gingerol in H-1299 cells.
doi:10.1021/jf303879b
PMCID: PMC3649839  PMID: 23066935
Ginger; 6-Gingerol; 6-Gingerdiol; Metabolite; Cancer
8.  Metabolites of Ginger Component [6]-Shogaol Remain Bioactive in Cancer Cells and Have Low Toxicity in Normal Cells: Chemical Synthesis and Biological Evaluation 
PLoS ONE  2013;8(1):e54677.
Our previous study found that [6]-shogaol, a major bioactive component in ginger, is extensively metabolized in cancer cells and in mice. It is unclear whether these metabolites retain bioactivity. The aim of the current study is to synthesize the major metabolites of [6]-shogaol and evaluate their inhibition of growth and induction of apoptosis in human cancer cells. Twelve metabolites of [6]-shogaol (M1, M2, and M4–M13) were successfully synthesized using simple and easily accessible chemical methods. Growth inhibition assays showed that most metabolites of [6]-shogaol had measurable activities against human cancer cells HCT-116 and H-1299. In particular, metabolite M2 greatly retained the biological activities of [6]-shogaol, with an IC50 of 24.43 µM in HCT-116 human colon cancer cells and an IC50 of 25.82 µM in H-1299 human lung cancer cells. Also exhibiting a relatively high potency was thiol-conjugate M13, with IC50 values of 45.47 and 47.77 µM toward HCT-116 and H-1299 cells, respectively. The toxicity evaluation of the synthetic metabolites (M1, M2, and M4–M13) against human normal fibroblast colon cells CCD-18Co and human normal lung cells IMR-90 demonstrated a detoxifying metabolic biotransformation of [6]-shogaol. The most active metabolite M2 had almost no toxicity to CCD-18Co and IMR-90 normal cells with IC50s of 99.18 and 98.30 µM, respectively. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay indicated that apoptosis was triggered by metabolites M2, M13, and its two diastereomers M13-1 and M13-2. There was no significant difference between the apoptotic effect of [6]-shogaol and the effect of M2 and M13 after 6 hour treatment.
doi:10.1371/journal.pone.0054677
PMCID: PMC3559867  PMID: 23382939
9.  Synthesis and proteasome inhibition of lithocholic acid derivatives 
A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC50 = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are non-competitive inhibitors of the proteasome.
doi:10.1016/j.bmcl.2011.02.041
PMCID: PMC3072167  PMID: 21388808
Lithocholic acid; proteasome; proteasome inhibitor

Results 1-9 (9)