Fluorescent Probes for Live Cell Thiol Detection.

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols' concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

2-(2-Cholesteroxyethoxyl)ethyl 3'-S-glutathionylpropionate and its self-assembled micelles for brain delivery: Design, synthesis and evaluation.

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.

Design, Synthesis, and Characterization of Bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c][1,2,5]oxadiazol-5-yl)sulfane for Nonprotein Thiol Imaging in Lysosomes in Live Cells.

Thiols are critical to cellular structures and functions. Disturbance of cellular thiols has been found to affect cell functions and cause various diseases. Intracellularly, thiols were found unevenly distributed in subcellular organelles. Probes capable of detecting subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. The subcellular organelle lysosome is the place where unwanted macromolecules are removed through degradation by hydrolytic enzymes. The degradation also serves as a regulation of a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cellular homeostasis. Thiols are found to be involved in the lysosomal degradation process. A probe that can detect lysosomal thiols in live cells will be a valuable tool in unveiling the roles of thiols in lysosomes. We would like to report bis(7-(N-(2-morpholinoethyl)sulfamoyl)benzo[c][1,2,5]-oxadiazol-5-yl)sulfane (BISMORX) as a thiol specific fluorogenic agent for live cell nonprotein thiol (NPSH) imaging in lysosomes through fluorescence microscopy. BISMORX itself shows no fluorescence and reacts readily with a NPSH to form a fluorescent thiol adduct with excitation and emission wavelengths of 380 and 540 nm, respectively. BISMORX does not react with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH2, and -COOH. No reaction was observed either when BISMORX was mixed with protein thiols. BISMORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. A colocalization experiment with LysoTracker Red DND-99 confirmed that the thiols imaged by BISMORX were indeed lysosomal thiols.

A simple method to discriminate Guangchenpi and Chenpi by high-performance thin-layer chromatography and high-performance liquid chromatography based on analysis of dimethyl anthranilate.

Citri Reticulatae Pericarpium (CRP), the dried pericarp of Citrus reticulata Blanco, can be divided into "Guangchenpi" (GCP, the dried pericarps derived from Citrus reticulata 'Chachi') and "Chenpi" (CP, the dried pericarps derived from other cultivars of Citrus reticulata Blanco). To discriminate between GCP and CP, a simple and reliable high-performance thin-layer chromatography (HPTLC) method was firstly developed to analyze the volatile compound dimethyl anthranilate, and a high-performance liquid chromatography (HPLC) method was established to simultaneously quantify dimethyl anthranilate and three predominant flavonoids (hesperidin, nobiletin and tangeretin) in CRP samples. Both the HPTLC analysis and HPLC-orthogonal partial least squares discrimination analysis (OPLS-DA) indicated that GCP can be effectively distinguished from CP based on analysis of dimethyl anthranilate. Our results indicated that dimethyl anthranilate can be used as a marker compound for discrimination of GCP and CP. This work provided a convenient approach which might be applied for quality evaluation of CRP.

Thiol-specific fluorogenic agent for live cell non-protein thiol imaging in lysosomes.

Thiol molecules play a significant role in cellular structures and functions. These molecules are distributed in cells unevenly at the subcellular level. Disturbance of cellular thiols has been associated with various diseases and disorders. Probes that are able to detect subcellular thiol density in live cells are valuable tools in determining thiols' roles at the subcellular level. Lysosomes are a subcellular organelle involved in the degradation of macromolecules through the action of proteolytic enzymes. The degradation not only serves as a way to dispose of unwanted macromolecules but also a way to regulate a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cell homeostasis. A probe that can detect lysosomal thiols in live cells will be useful in unveiling the roles of thiols in lysosomes. Currently, limited probes are available to detect lysosomal thiols in live cells. We would like to report 4,4'-{[7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl)]bis(oxy))bis(naphthalene-2,7-disulfonicacid) (TBONES) as a thiol-specific fluorogenic agent for lysosomal thiol imaging in live cells through fluorescence microscopy. TBONES exhibits no fluorescence and readily reacts with non-protein thiols to form fluorescent thiol adducts with λex = 400 nm and λem = 540 nm. No reaction was observed when TBONES was mixed with compounds containing nucleophilic functional groups other than thiols such as -OH, -NH2, and -COOH. No reaction was observed either when TBONES was mixed with protein thiols. When incubated with cells, TBONES selectively and effectively imaged lysosomal thiols in live cells. Imaging of lysosomal thiols was confirmed by a co-localization experiment with LysoTracker™ Blue DND-22.

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λex = 550 nm, λem = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

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.

In Vitro and In Vivo Tumor Growth Inhibition by Glutathione Disulfide Liposomes.

Glutathione disulfide (GSSG) is an endogenous peptide and the oxidized form of glutathione. The impacts of GSSG on cell function/dysfunction remain largely unexplored due to a lack of method to specifically increase intracellular GSSG. We recently developed GSSG liposomes that can specifically increase intracellular GSSG. The increase affected 3 of the 4 essential steps (cell detachment, migration, invasion, and adhesion) of cancer metastasis in vitro and, accordingly, produced a significant inhibition of cancer metastasis in vivo. In this investigation, the effect of GSSG liposomes on cancer growth was investigated with B16-F10 and NCI-H226 cells in vitro and with B16-F10 cells in C57BL/6 mice in vivo. Experiments were conducted to elucidate the effect on cell death through promotion of apoptosis and the effect on the cell cycle. The in vivo results with C57BL/6 mice implanted subcutaneously with B16-F10 cells showed that GSSG liposomes retarded tumor proliferation more effectively than that of dacarbazine, a chemotherapeutic drug for the treatment of melanoma. The GSSG liposomes by intravenous injection (GLS IV) and GSSG liposomes by intratumoral injection (GLS IT) showed a tumor proliferation retardation of 85% ± 5.7% and 90% ± 3.9%, respectively, compared with the phosphate-buffered saline (PBS) control group. The median survival rates for mice treated with PBS, blank liposomes, aqueous GSSG, dacarbazine, GLS IV, and GLS IT were 7, 7, 7.5, 7.75, 11.5, and 16.5 days, respectively. The effective antimetastatic and antigrowth activities warrant further investigation of the GSSG liposomes as a potentially effective therapeutic treatment for cancer.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism.

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc- cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24-ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24-ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.

In-vitro and in-vivo inhibition of melanoma growth and metastasis by the drug combination of celecoxib and dacarbazine.

Celecoxib has been found to be effective in cancer prevention and treatment. Its combination with other chemotherapeutic agents was reported to produce synergistic/additive effects on various cancers. Dacarbazine (DTIC) is one of the most commonly used drugs in the treatment of metastatic melanoma. This investigation aimed to determine the in-vitro and in-vivo effects of the drug combination of celecoxib and DTIC on melanoma growth and metastasis. Melanoma cells B16-F10 and SK-MEL-28, and female C57BL/6 mice were used for the study. Our in-vitro data showed that significant synergistic effects were obtained when celecoxib was used together with various concentrations of DTIC. A study with B16-F10 cells using flow cytometry analysis showed that the drug combination induced significantly more apoptosis than each drug used individually. Our in-vivo results showed that the drug combination was much more effective than each drug used alone for the inhibition of both melanoma growth and metastasis in the B16-F10+C57BL/6 mouse models. For melanoma growth, the median survival rates for phosphate-buffered saline (PBS) (control), celecoxib (30 mg/kg), DTIC-1 (10 mg/kg), DTIC-2 (positive control, 50 mg/kg), and the drug combination (DTIC 10 mg/kg+celecoxib 30 mg/kg) were 6, 6.5, 7.5, 7.5, and 9 days, respectively. For melanoma metastasis, the average number of metastatic tumors in murine lungs was 53.7±10.7, 31.8±18.6, 21.2±21.7, 7.0±9.0, and 0.8±2.0 for PBS, DTIC-1, celecoxib, the drug combination, and DTIC-2. Our results warrant further investigation of the combination as an effective treatment for melanoma patients.

Glutathione Disulfide Liposomes - a Research Tool for the Study of Glutathione Disulfide Associated Functions and Dysfunctions.

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1 ± 6.9 folds (n = 3) in 4 hours and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at -80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.

Evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine as an irreversible inhibitor of mammalian thioredoxin reductase1.

CONTEXT: Thioredoxin reductase (TrxR) is up-regulated in a number of human malignant cells and becomes a promising target for anticancer drug development. OBJECTIVE: To evaluate N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC), a potent anticancer agent against melanoma, as an inhibitor of mammalian TrxR1. MATERIAL AND METHODS: The mechanism of inhibition against TrxR1 was investigated using substrate protection, dialysis and liquid chromatography-tandem mass spectrometry. RESULTS: NACC inhibits TrxR1 in a time and concentration dependent manner. The K(I) and k(inact) of NACC against TrxR1 were determined to be 80 µM and 0.178 min(-1), respectively. The inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis demonstrated that the selenocysteine rather than cysteine residue at the active site was p-chlorophenyl carbamoylated by NACC. Inhibition of intracellular TrxR by NACC in cultured melanoma cells was observed. DISCUSSION AND CONCLUSION: NACC which irreversibly inhibits TrxR1 by forming a covalent bond with selenocysteine can be an effective tool in the study of TrxR1.

Cancer metastases: challenges and opportunities.

Cancer metastasis is the major cause of cancer morbidity and mortality, and accounts for about 90% of cancer deaths. Although cancer survival rate has been significantly improved over the years, the improvement is primarily due to early diagnosis and cancer growth inhibition. Limited progress has been made in the treatment of cancer metastasis due to various factors. Current treatments for cancer metastasis are mainly chemotherapy and radiotherapy, though the new generation anti-cancer drugs (predominantly neutralizing antibodies for growth factors and small molecule kinase inhibitors) do have the effects on cancer metastasis in addition to their effects on cancer growth. Cancer metastasis begins with detachment of metastatic cells from the primary tumor, travel of the cells to different sites through blood/lymphatic vessels, settlement and growth of the cells at a distal site. During the process, metastatic cells go through detachment, migration, invasion and adhesion. These four essential, metastatic steps are inter-related and affected by multi-biochemical events and parameters. Additionally, it is known that tumor microenvironment (such as extracellular matrix structure, growth factors, chemokines, matrix metalloproteinases) plays a significant role in cancer metastasis. The biochemical events and parameters involved in the metastatic process and tumor microenvironment have been targeted or can be potential targets for metastasis prevention and inhibition. This review provides an overview of these metastasis essential steps, related biochemical factors, and targets for intervention.

Rapid and thiol-specific high-throughput assay for simultaneous relative quantification of total thiols, protein thiols, and nonprotein thiols in cells.

Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λ(ex) = 430 nm and λ(em) = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1.

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)phenylthiocarbamoylsulfanyl]propionic 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.

Benzofurazan sulfides for thiol imaging and quantification in live cells through fluorescence microscopy.

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a-1e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH(2), -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 and 520 nm as the excitation and emission wavelengths, respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an high-pressure liquid chromatography/ultraviolet (HPLC/UV) total thiol assay method. The reagents and method will be of a great value to thiol redox-related research.

Microtubule S-glutathionylation as a potential approach for antimitotic agents.

BACKGROUND: 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. METHODS: 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic 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. RESULTS: 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. CONCLUSIONS: 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.

Structure-skin permeability relationship of dendrimers.

PURPOSE: To investigate skin penetration of poly (amidoamine) (PAMAM) dendrimers as a function of surface charge and molecular weight in presence and absence of iontophoresis. METHODS: Dendrimers were labeled with fluoroisothiocynate (FITC); skin penetration of dendrimers was studied using excised porcine skin in-vitro. Skin penetration of FITC-labeled dendrimers was quantified using confocal laser scanning microscope (CLSM). G2-G6 NH(2), G3.5-COOH and G4-OH dendrimers were used. RESULTS: Cationic dendrimers showed higher skin penetration than neutral and anionic dendrimers. Skin penetration of cationic dendrimer increased linearly with increase in treatment time. Iontophoresis enhanced skin penetration of cationic and neutral dendrimers. Increase in current strength and current duration increased skin transport of dendrimers. Passive and iontophoretic skin penetration of cationic dendrimers was inversely related to their molecular weight. Dendrimer penetrated the skin through intercellular lipids and hair follicles. With iontophoresis, dendrimer was also found in localized skin regions. CONCLUSIONS: The study demonstrates that the physicochemical properties of dendrimers influence their skin transport. Findings can be used to design dendrimer-based nanocarriers for drug delivery to skin.