Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of ...flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21
and p27
and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.
Context: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few ...inhibitors of GRX-1 have been reported.
Objective: The objective of this paper is to report 2-acetylamino-3-4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanylpropionic acid (2-AAPA) as an inhibitor of human GRX-1.
Materials and methods: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry.
Results: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells.
Discussion: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors.
Conclusion: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.
This study examined the feasibility of an interprofessional high-fidelity pharmacology simulation and its impact on pharmacy and nursing students’ perceptions of interprofessionalism and pharmacology ...knowledge.
Pharmacy and nursing students participated in a pharmacology simulation using a high-fidelity patient simulator. Faculty-facilitated debriefing included discussion of the case and collaboration. To determine the impact of the activity on students’ perceptions of interprofessionalism and their ability to apply pharmacology knowledge, surveys were administered to students before and after the simulation. Attitudes Toward Health Care Teams scale (ATHCT) scores improved from 4.55 to 4.72 on a scale of 1–6 (p = 0.005). Almost all (over 90%) of the students stated their pharmacology knowledge and their ability to apply that knowledge improved following the simulation.
A simulation in pharmacology is feasible and favorably affected students’ interprofessionalism and pharmacology knowledge perceptions.
Pharmacology is a core science course required by multiple health professions in early program curricula, making it favorable for incorporation of interprofessional learning experiences. However, reports of high-fidelity interprofessional simulation in pharmacology courses are limited. This manuscript contributes to the literature in the field of interprofessional education by demonstrating that an interprofessional simulation in pharmacology is feasible and can favorably affect students’ perceptions of interprofessionalism. This manuscript provides an example of a pharmacology interprofessional simulation that faculty in other programs can use to build similar educational activities.
Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to ...increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC50 = 647 μm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanylpropionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm and 0.1 min–1, respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.
Depletion of the reduced form of glutathione (GSH) has been extensively studied for its effect on sensitizing cancer to radiation. However, little is known about the effects of thiol oxidative stress ...created through an increase in glutathione disulfide (GSSG) on cancer sensitivity to radiation. In this study, an increase in GSSG was effectively created using 2-acetylamino-3-4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanylpropionic acid (2-AAPA), an irreversible glutathione reductase (GR) inhibitor. Our results demonstrate that the GSSG increase significantly enhanced cancer sensitivity to X-ray irradiation in four human cancer cell lines (A431, MCF7, NCI-H226, and OVCAR-3). When cells were pretreated with 2-AAPA followed by X-ray irradiation, the IC
50 values for X-ray irradiation of A431, MCF7, NCI-H226, and OVCAR-3 cells were reduced, from 24.2
±
2.8, 42.5
±
3.0, 43.0
±
3.6, and 27.8
±
3.5 Gy to 6.75
±
0.9, 8.1
±
1.1, 6.75
±
1.0, and 12.1
±
1.7 Gy, respectively. The synergistic effects observed from the combination of X-rays plus 2-AAPA were comparable to those from the combination of X-rays plus buthionine sulfoximine, a reference compound known to increase cancer sensitivity to radiation. The synergistic effect was correlated with an increase in cell thiol oxidative stress, which was reflected by a five-to sixfold increase in GSSG and 25% increase in total disulfides. No change in GSH or total thiols was observed as a result of GR inhibition.
Microtubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G2/M phase. ...Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S-glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S-glutathionylation can be a novel approach for antimitotic agents.
2-Acetylamino-3-4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanylpropionic acid (2-AAPA) was used as a tool to induce microtubule S-glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S-transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S-glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S-glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S-glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines.
Our investigation demonstrated that cell morphology was changed and microtubules were S-glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G2/M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition.
The data from this investigation confirms that microtubule S-glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.
Cancer metastasis is the major cause of cancer mortality. Despite extensive research efforts, effective treatment for cancer metastasis is still lacking. Cancer metastasis involves 4 essential steps: ...cell detachment, migration, invasion, and adhesion. Detachment is the first and required step for metastasis. Glutathione disulfide (GSSG) is derived from the oxidation of glutathione (GSH), which is present in biological systems in millimolar concentration. Although GSSG is commercially available, the impact of GSSG on cell functions/dysfunctions has not been fully explored due to the fact that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed GSSG liposomes that effectively deliver GSSG to cells. Unexpectedly, cells treated with GSSG liposomes were resistant to detachment by trypsinization. This observation led to the investigation of the antimetastatic effect of GSSG liposomes. Our data demonstrate that GSSG liposomes at 1 mg/mL completely blocked cell detachment and migration, and significantly inhibited cancer cell invasion. Aqueous GSSG showed no such effect, confirming that the effects on cell detachment, migration, and invasion were caused by the intracellular delivery of GSSG. An in vivo experiment with a murine melanoma experimental metastasis model showed that GSSG liposomes prevented melanoma lung metastasis. The unique antimetastatic mechanism through the effects on detachment and migration, and effective in vitro and in vivo metastasis inhibition, warrants further investigation of the GSSG liposomes as a potential treatment for cancer metastasis.
Our results demonstrate the potential of COXP micelles for delivering therapeutic, preventative, or diagnostic agents to the brain.
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•Blood brain barrier (BBB) is the major cause of ...treatment failure for most brain diseases.•The BBB is enriched with glutathione (GSH) transporters.•COXP was designed by connecting a GSH to a cholesterol through a short chain.•COXP is a GSH transporter-based brain targeting agent.•COXP self-assembles to form brain targeting micelles (COXP micelles).•COXP micelles delivered DiR to a mouse brain 20 folds higher than DiR alone.•GSH further increased COXP micelles’ brain-targeting in mice by more than one-fold.
The blood–brain barrier (BBB) is a barrier that prevents almost all large and most small exogenous molecules from reaching the brain. The barrier is the major cause of treatment failure for most brain diseases. Extensive efforts have been made to facilitate drug molecules to cross the BBB. One of the approaches is to employ an endogenous ligand or ligand analogue that can enter the brain through its transporter or receptor at the BBB as a brain-targeting agent.
Glutathione (GSH) transporters are richly expressed at the BBB with limited presence in other tissues except kidneys. 2-(2-Cholesteroxyethoxyl)ethyl 3′-S-glutathionylpropionate (COXP), formed by connecting GSH with cholesterol through a linker, was designed as a GSH transporter-mediated brain targeting molecule. The amphiphilic nature of COXP enables the molecule to self-assemble to form micelles with a CMC value of 3.9 μM. By using DiR as a fluorescence tracking agent and the whole-body fluorescence imaging technique, the brain distribution of DiR delivered by COXP micelles in mice was 20 folds higher when compared with free DiR. Interestingly, the brain targeting effect was further enhanced by co-administration of GSH. The low CMC value and effective brain targeting make COXP micelles a promising drug delivery system to the brain.
This work presents an assay for total thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of thiols with ...5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH–DTNB adduct (GSH–TNB). By using 326
nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH–TNB adduct was determined to be 15 and 7.5
pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.4
±
1.2% (
n
=
3), 98.1
±
5.0% (
n
=
3), 95.6
±
0.9% (
n
=
3), and 96.6
±
2.3% (
n
=
3) respectively. The recovery yield for GSH–TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides was 99.0
±
0.3% (
n
=
3), 95.1
±
4.9% (
n
=
3), 96.8
±
0.6% (
n
=
3), and 95.1
±
2.9% (
n
=
3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (
n
=
6) for non-protein thiols, 3.9% (
n
=
6) for protein thiols, 3.6% (
n
=
6) for non-protein disulfides and 4.6% (
n
=
6) for protein disulfides. The reproducibility for GSH–TNB was determined to be 1.6% (
n
=
6) for non-protein thiols and 2.6% (
n
=
6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH
4 reduction with that after the reduction, this method can provide information associated with thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for thiol and disulfide quantification and will be a useful analytical method in the study of thiol redox state and thiol glutathionylation.
An in vitro study was conducted to determine effects of reducing pH of porcine in vitro fermentation medium on compositions of glucosinolate degradation products and porcine in vitro fermentation ...characteristics for solvent-extracted canola meal (SECM) and cold-pressed canola cake (CPCC). Two canola co-products were subjected to porcine in vitro fermentation for 72 h. Accumulated gas production during microbial fermentation was recorded and modeled to estimate kinetics of gas production. Glucosinolate degradation products after microbial fermentation were determined and fermentation medium pH after incubation was recorded. Total and individual volatile fatty acids (VFA) concentrations per unit weight of dry matter (DM) of feedstuffs were determined. On DM basis, SECM and CPCC contained 6.15 and 11.1 µmol/g of glucosinolates, respectively. Goitrin concentration for CPCC was lower (P < 0.05) than that for SECM. Isothiocyanate and indole-3-acetonitrile concentrations for CPCC were lower (P < 0.05) than those for SECM, whereas thiocyanate concentration for CPCC was greater (P < 0.05) than that for SECM. A reduction in fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) goitrin and indole-3-acetonitrile concentrations and decreased (P < 0.05) isothiocyanates concentration for SECM, but did not affect concentration of these 2 glucosinolate degradation products for CPCC. Fermentation medium pH after in vitro fermentation for SECM tended to be greater (P = 0.081) than that for CPCC. Canola co-product type and fermentation medium pH did not interact on gas production parameters. However, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased (P < 0.05) lag time and reduced (P < 0.05) fractional rate of degradation and total gas production. Canola co-product type and fermentation medium pH did not interact on total or individual VFA production per gram of DM of feedstuff fermented. However, reducing fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) total VFA and acetate productions, and tended to reduce (P = 0.083) branched-chain VFA production SECM and CPCC. In conclusion, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased production of goitrin and indole-3-acetonitriles from SECM-derived glucosinolates, but did not affect production of thiocyanate from SECM-derived glucosinolates. Glucosinolates in CPCC were less affected by the fermentation medium pH used in the current study. It appears that there are other factors other than pH that affect the degradation of canola-derived glucosinolates by microorganisms from hindgut of pigs.
•Glucosinolates in canola cake were less degradable than those in canola meal.•Reduction in fermentation medium pH increased nitrile production from canola meal.•Reduction in fermentation medium pH increased goitrin production from canola meal.•Reduction in fermentation medium pH unaffected nitrile production from canola cake.•Reduction in fermentation medium pH unaffected goitrin production from canola cake.
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