Biehuoy Shieh

Potent activity of indolequinones against human pancreatic cancer: identification of thioredoxin reductase as a potential target.

The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cell-free systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.

Aldose reductase expression as a risk factor for cataract

Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the lens to disturbances in signaling through the ERK and JNK pathways and thereby alter the balance of cell growth and apoptosis that is critical to lens transparency and homeostasis.

Suppression of transcription factor early growth response 1 reduces herpes simplex virus lethality in mice

Herpes simplex virus type 1 (HSV-1) infection is the most common cause of sporadic, fatal encephalitis, but current understanding of how the virus interacts with cellular factors to regulate disease progression is limited. Here, we show that HSV-1 infection induced the expression of the cellular transcription factor early growth response 1 (Egr-1) in a human neuronal cell line. Egr-1 increased viral replication by activating promoters of viral productive cycle genes through binding to its corresponding sequences in the viral promoters. Mouse studies confirmed that Egr-1 expression was enhanced in HSV-1-infected brains and that Egr-1 functions to promote viral replication in embryonic fibroblasts. Furthermore, Egr-1 deficiency or knockdown of Egr-1 by a DNA-based enzyme greatly reduced the mortality of HSV-1-infected mice by decreasing viral loads in tissues. This study provides what we believe is the first evidence that Egr-1 increases the mortality of HSV-1 encephalitis by enhancing viral replication. Moreover, blocking this cellular machinery exploited by the virus could prevent host mortality.

Design of an amide N-glycoside derivative of β-glucogallin: a stable, potent, and specific inhibitor of aldose reductase.

β-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.

Role for NAD(P)H:quinone Oxidoreductase 1 and Manganese-Dependent Superoxide Dismutase in 17-(Allylamino)-17-demethoxygeldanamycin-Induced Heat Shock Protein 90 Inhibition in Pancreatic Cancer Cells

Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH2 were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH2 could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH2), reduction of 17AAG to 17AAGH2 by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH2. In addition, oxidation of 17AAGH2 could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH2 was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH2 with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH2, the active Hsp90 inhibitor derived from 17AAG.

Indolequinone inhibitors of NRH:quinone oxidoreductase 2. Characterization of the mechanism of inhibition in both cell-free and cellular systems.

We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems.

Characterization of Emodin as a Therapeutic Agent for Diabetic Cataract

Aldose reductase (AR) in the lens plays an important role in the pathogenesis of diabetic cataract (DC) by contributing to osmotic and oxidative stress associated with accelerated glucose metabolism through the polyol pathway. Therefore, inhibition of AR in the lens may hold the key to prevent DC formation. Emodin, a bioactive compound isolated from plants, has been implicated as a therapy for diabetes. However, its inhibitory activity against AR remains unclear. Our results showed that emodin has good selectively inhibitory activity against AR (IC50 = 2.69 ± 0.90 μM) but not other aldo-keto reductases and is stable at 37 °C for at least 7 days. Enzyme kinetic studies demonstrated an uncompetitive inhibition against AR with a corresponding inhibition constant of 2.113 ± 0.095 μM. In in vivo studies, oral administration of emodin reduced the incidence and severity of morphological markers of cataract in lenses of AR transgenic mice. Computational modeling of the AR-NADP(+)-emodin ternary complex indicated that the 3-hydroxy group of emodin plays an essential role by interacting with Ser302 through hydrogen bonding in the specificity pocket of AR. All the findings above provide encouraging evidence for emodin as a potential therapeutic agent to prevent cataract in diabetic patients.

Influence of aldose reductase on epithelial-to-mesenchymal transition signaling in lens epithelial cells

Cataract is the most frequent cause of blindness worldwide and is treated by surgical removal of the opaque lens to restore the light path to the retina. While cataract surgery is a safe procedure, some patients develop a complication of the surgery involving opacification and wrinkling of the posterior lens capsule. This process, called posterior capsule opacification (PCO), requires a second clinical treatment that can in turn lead to additional complications. Prevention of PCO is a current unmet need in the vision care enterprise. The pathogenesis of PCO involves the transition of lens epithelial cells to a mesenchymal phenotype, designated epithelial-to-mesenchymal transition (EMT). Our previous studies showed that transgenic mice designed for overexpression of human aldose reductase developed lens defects reminiscent of PCO. In the current study, we evaluated the impact of aldose reductase (AR) on expression of expression of EMT markers in the lens. Primary lens epithelial cells from AR-transgenic mice showed downregulated expression of Foxe3 and Pax6 and increased expression of α-SMA, fibronectin and snail, a pattern of gene expression typical of cells undergoing EMT. A role for AR in these changes was further confirmed when we observed that they could be normalized by treatment of cells with Sorbinil, an AR inhibitor. Smad-dependent and Smad-independent pathways are known to contribute to EMT. Interestingly, AR overexpression induced ERK but not Smad-2 activation. These results suggest that elevation of AR may lead to activation of ERK signaling and thus play a role in TGF-β/Smad independent induction of EMT in lens epithelial cells.

Abstract 570: Role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the cytotoxicity of the Hsp90 inhibitor 17AAG in human pancreatic cancer cells

Previous work has demonstrated that NQO1 can reduce the benzoquinone ansamycin Hsp90 inhibitor 17-allyamino-demethoxy geldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a more water soluble molecule and that binds with greater affinity to Hsp90 when compared to the parent quinone 17AAG. Experiments were conducted with the human pancreatic cancer cell line Panc1 which is homozygous for the NQO1*2 polymorphism and contains only trace levels of NQO1 protein and no NQO1 catalytic activity. An isogenic cell line expressing high levels of NQO1 (Panc1-C5) was generated by stable transfection of wild type NQO1 in Panc1 cells. 17AAG was more cytotoxic to Panc1-C5 cells with high levels of NQO1 when compared to NQO1-null parental Panc1 cells. Pretreatment of Panc1-C5 cells with the NQO1 mechanism-based inhibitor ES936 reduced the cytotoxicity of 17AAG to the level observed in Panc1 cells. No difference in the cytotoxicity of the non-quinone Hsp90 inhibitor radicicol was observed between the two cell lines Biomarkers of Hsp90 inhibition were measured in both Panc1 and Panc1-C5 cells following treatment with 17AAG. Higher protein levels of Hsp70 and lower amounts of cRaf, phosphorylated ERK1/2 and phosphylated MEK were observed in Panc1-C5 cells compared to Panc1 cells. In addition, cell cycle analysis revealed a greater number of Panc1-C5 cells arrested in G2M following 17AAG treatment compared to Panc1 cells. Cytotoxicity studies were also performed in additional human pancreatic cancer cell lines (BxPC3 and MiaPaCa2) that contain high levels of catalytically active NQO1*1 protein and in agreement with the studies described above the cytotoxicity of 17AAG could be reduced significantly following pretreatment with the NQO1 inhibitor ES936. The intracellular concentrations of 17AAG and 17AAH2 were measured in human pancreatic cancer cell lines and it was observed that greater amounts of both 17AAG and 17AAGH2 could be detected in cell lines with catalytically active NQO1 when compared to cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that in addition to generating a more potent Hsp90 inhibitor (17AAGH2) the reduction of 17AAG to 17AAGH2 by NQO1 leads to significantly greater intracellular concentrations of 17AAG and 17AAGH2. These data confirm the role of NQO1 in sensitivity to 17AAG and suggest that genotyping for the NQO1*2 polymorphism should be considered in patients undergoing 17AAG treatment for pancreatic cancer or other solid tumors that are known to contain high levels of NQO1 activity (Supported by CA51210). 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 570.