Differential Inhibitory Effects Research Articles

Evaluation of Inhibitory Effect of Organic and Inorganic Salts Against Ilyonectria liriodendri, The Causal Agent of Root Rot Disease of Kiwifruit

AbstractThis study evaluated efficacy of 42 organic and inorganic salts as possible alternatives to synthetic fungicides for the control of Ilyonectria root rot of kiwifruit. Preliminary in vitro tests showed that ammonium bicarbonate, ammonium carbonate, potassium benzoate, potassium sorbate, sodium benzoate and sodium metabisulphite at 2% completely inhibited mycelial growth of the fungus. No significant differences were observed among these salts and disodium EDTA (P ≤ 0.05). However, the ED50, minimum inhibition concentration (MIC), and minimum fungicidal concentration (MFC) values indicated that sodium metabisulphite was more toxic to Ilyonectria liriodendri than these other six salts. Soil bioassays showed that sodium metabisulphite, sodium benzoate and potassium sorbate at 0.25% completely inhibited mycelial growth of the fungus, whereas potassium benzoate reduced the mycelial growth of fungus by 90.30%; however, the differences in inhibitory effects were statistically insignificant (P ≤ 0.05). Moreover, there was no significant difference between 0.1% sodium metabisulphite and 0.5% ammonium carbonate, 0.75% ammonium bicarbonate and 1.5–2.0% disodium EDTA (P ≤ 0.05). Unlike disodium EDTA, complete inhibitory was observed with ammonium carbonate and ammonium bicarbonate at higher concentrations. However, in root bioassays, applications of 2% ammonium bicarbonate, 1.5% ammonium carbonate and 2% disodium EDTA were phytotoxic to kiwifruit seedlings, but 0.25% four other salts were neither phytotoxic to kiwifruit seedlings nor did it adversely affect root length, root fresh weight and root dry weight of seedling. This study also showed I. liriodendri to be capable of growth in both acidic and basic environments. However, while the fungus showed uninhibited growth at pH values of 5–11, growth decreased significantly at both higher and lower pH values (P ≤ 0.05) and was completely inhibited at pH 12.

Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations.

Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV–vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the structural diversity of the isozymes. These findings may represent additional mechanisms for the differential inhibitory effects arising from the coincubated AuNPs on the metabolic activities of the hepatic CYP isozymes.

The amino acid at residue 155 in nonstructural protein 4 of porcine reproductive and respiratory syndrome virus contributes to its inhibitory effect for interferon-β transcription in vitro

Type I interferons (IFNs), predominantly IFN-α and β, play important roles in both innate and adaptive immune responses against viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be able to down-regulate the IFN response during in vivo and in vitro infection. In this study, we first analyzed inhibitory effect of each NSP of low pathogenic PRRSV HB-1/3.9 on IFN-β transcription in MARC-145cells, and the results showed that the IFN-β promoter activation could be suppressed by NSP1α, NSP2, NSP1β, NSP3, NSP4, NSP5 and NSP11. We next confirmed that the inhibitory effect of NSP4 was mainly mediated through suppressing NF-κB activation, whereas not hindering NF-κB phosphorylation and nuclear translocation, and nuclear-localized NSP4 was responsible for inhibiting IFN-β activation. We further found that the NSP4 of different pathogenic PRRSV strains exhibited differential inhibitory effect on IFN-β, NF-κB, and IRF3 transcription, and the NSP4 of highly pathogenic (HP)-PRRSV could display more strong inhibitory effect. Finally, we determined that the amino acid at residue 155 in NSP4 contributed to its inhibitory effect for IFN-β transcription in vitro by altering its subcellular distribution. Our findings suggest that the nucleus-localized NSP4 of PRRSV participates in the modulation of the host type I IFNs system, and also provide novel insight for understanding the pathogenesis of the Chinese HP-PRRSV.

Selective tumor cell killing by triptolide in p53 wild-type and p53 mutant ovarian carcinomas.

Triptolide is a traditional Chinese medicinal herb-derived antineoplastic agent. However, its antitumor activity against gynecologic carcinomas has not yet been well described. It is the purpose of this article to investigate the effect and mechanism of triptolide in human ovarian cancer using both A2780 (p53 wild) and OVCAR-3 (p53 mutated) cells. Our results showed that triptolide exerted a potent inhibitory effect on the growth and proliferation of both cell lines in a dose- and time-dependent manner and that the effect was independent of the expression of p53. In contrast, triptolide had only a marginal cytotoxicity in noncancerous ovary cells, lung fibroblast cells, and macrophage cells, indicating differential inhibitory effects of the drug on cell growth between ovarian cancer cells and normal tissue cells. Exposure of the ovarian cancer cells to triptolide induced apoptosis, as evaluated by annexin V/propidium iodide-labeled flow cytometry. Triptolide-induced apoptosis was accompanied by cytochrome c release and caspase-3 activation and was associated with downregulation of Bcl-2 and upregulation of Bax. Cell cycle analysis demonstrated that treatment with triptolide induced cell cycle S phase arrest in A2780 cells and G2/M phase arrest in OVCAR-3 cells. Further detection by Western blotting revealed that the cell cycle arrest by triptolide in both cell lines occurred in concert with increased expression of p21(CIP1/WAF1). This study shows that triptolide selectively kills ovarian cancer cells with different p53 status predominantly through regulating the coordinate and dynamic cellular processes of proliferation and apoptosis, thereby making it a promising chemotherapeutic agent against a broad spectrum of ovarian carcinomas.

SULF1/SULF2 splice variants differentially regulate pancreatic tumour growth progression

This study highlights the highly dynamic nature of SULF1/SULF2 splice variants in different human pancreatic cancers that regulate the activities of multiple cell signalling pathways in development and disease. Most pancreatic tumours expressed variable levels of both SULF1 and SULF2 variants including some expression during inflammation and pancreatitis. Many ductal and centro-acinar cell-derived pancreatic tumours are known to evolve into lethal pancreatic ductal adenocarcinomas but the present study also detected different stages of such tumour progression in the same tissue biopsies of not only acinar cell origin but also islet cell-derived cancers. The examination of caerulein-induced pancreatic injury and tumorigenesis in a Kras-driven mouse model confirmed the activation and gradual increase of SULF1/SULF2 variants during pancreatitis and tumorigenesis but with reduced levels in Stat3 conditional knockout mice with reduced inflammation. The significance of differential spatial and temporal patterns of specific SULF1/SULF2 splice variant expression during cancer growth became further apparent from their differential stimulatory or inhibitory effects on growth factor activities, tumour growth and angiogenesis not only during in vitro but also in vivo growth thus providing possible novel therapeutic targets.

Prediction of the likelihood of drug interactions with kinase inhibitors based on in vitro and computational studies

Kinase inhibitors (KIs) represent an important group of anticancer drugs, and many of them are substrates and inhibitors of human cytochrome P450s (CYPs), raising the potential of harmful drug interactions. This study investigated the effect of a library of KIs (n = 91) including 11 FDA-approved KIs on human CYP1A2, 2D6, 2C9, and 3A4 using high-throughput screening kits and the binding modes with CYPs using the Discovery Studio program 3.1. The KIs exhibited differential inhibitory effect on CYP1A2, 2D6, 2C9, and 3A4, while some of them showed activating effect on CYP2C9 and 3A4. For example, SP 600125 was a potent inhibitor for CYP1A2, but enhanced the activity of CYP2C9 fourfolds. Among the 80 KIs that are not used clinically, about 13% showed significant inhibition to CYPs. Nilotinib, sunitinib, and imatinib were found to be potent CYP1A2 inhibitor. Our docking studies have demonstrated the importance of multiple amino acid residues in the active sites of CYP1A2, 2C9, 2D6, and 3A4 in binding with various KIs. Finally, the in vitro data were used to predict potential KI-drug interactions. These findings indicate that many KIs can serve as CYP inhibitors, and further studies are needed to examine the clinical impact.

English

Antibiotic resistance in pathogenic bacteria is emerging as an issue of serious concern in bio-medical research as well as food and health organizations. Metal complexes are currently being employed in medical devices for their inhibition to bacterial adherence and antibacterial activities. The primary aim of this study was to evaluate the antibacterial activities of pure metals, including selenium, germanium and lithium on planktonic cultures and biofilms of three bacterial species: S. aureus SH1000, P aeruginosa PA01 and E. coli. O157:H7. The antagonistic effects of selenium, germanium and lithium on these three bacterial species were examined using zone of inhibition assay. The minimum inhibitory concentrations and minimum bactericidal concentrations of antibiotics (rifampicin, mupirocin and ciprofloxacin) and metals (selenium, germanium and lithium) were measured. The minimum biofilm eradication concentrations (MBEC) of metals were determined against biofilms composed of S. aureus SH1000 and P. aeruginosa PA01. Metal susceptibility tests suggested that biofilms displayed increased resistance over their planktonic state. Differential inhibitory effects were observed for different strains of planktonic and biofilm bacteria in response to different metals and their varying concentrations. Amongst the three metals tested, selenium proved to be the most active against all three species, whereas lithium demonstrated the least inhibitory effects. Scanning electron microscope (SEM) image analysis revealed several detrimental structural changes in bacterial cells exposed to metals compared to those grown in the metal-free culture. In conclusion, the results demonstrate the antibacterial efficacy of pure metals against planktonic and biofilm bacteria paving the way for further similar investigations in search of alternative antibacterial agents.    Key words: Antibiotic resistance, antibacterial, metal, biofilm, planktonic.

Efferent Inhibitory Effect Observed in Otoacoustic Emissions and Auditory Brainstem Response in the Neonatal Population

Objective: To characterize the inhibitory effect (IE) in the otoacoustic emission (OAE) and auditory brainstem response (ABR) in newborns at high and low risk for hearing loss. Design: Cross-sectional study. Patients and Methods: Seventy-nine newborns at low risk for hearing loss and 46 at high risk underwent transient evoked OAE (TEOAE), distortion product OAE (DPOAE) and ABR testing with or without the presence of contralateral white noise presented at a level of 60 dB SPL. Results: For both low- and high-risk newborns, there were no significant differences in IE between the left and right ears. There was a statistically significant difference in the right-ear IE between the low- and high-risk group for DPOAE and ABR testing. There was also greater agreement of the efferent system evaluation outcomes between TEOAE and ABR. Conclusions: ABR testing detected IE in a greater number of newborns in the low-risk, as compared to the high-risk group.

Glutathione-dependent cell cycle G1 arrest and apoptosis induction in human lung cancer A549 cells caused by methylseleninic acid: comparison with sodium selenite.

The aim of the present study was to clarify the mechanism underlying the inhibition of cell proliferation in human lung cancer A549 cells by selenium (Se) compounds. Methylseleninic acid (CH3SeO2H, abbreviated as MSA), a synthetic Se compound, is a direct precursor of active methylselenol (CH3SeH) and is considered to be one of beneficial agents for cancer prevention and therapy. Sodium selenite (Na2SeO3), an inorganic Se form, is utilized in clinical Se supplementation. MSA markedly inhibited the growth of A549 cells at a concentration of 2.5×10(-6) mol/L for 1 d. On Day 1, Na2SeO3 also inhibited A549 cell growth at the concentration of 7.5×10(-6) mol/L. These compounds induced cell cycle arrest at the G1 phase and apoptosis under the inhibitory condition. Reduced glutathione (GSH) is critical to MSA or Na2SeO3 metabolism. The depletion of intracellular GSH suppressed Na2SeO3-induced G1 arrest, but promoted Na2SeO3-induced apoptosis. Therefore, Na2SeO3 appears to have directly induced apoptosis. In contrast, the MSA-induced G1 arrest was ameliorated by a marked decrease in GSH content. Additionally, the depletion of GSH slightly suppressed MSA-induced apoptosis. The difference in inhibitory effects between MSA and Na2SeO3 may be due to this variation in GSH-related metabolism. After exposure of A549 cells to MSA, the GSH content was significantly decreased. These results indicate that because MSA-induced G1 arrest and apoptosis induction are enhanced by GSH, the maintenance of GSH is essential for the effective anticancer action of MSA in A549 cells.

Differential effects of amino acid surface decoration on the anticancer efficacy of selenium nanoparticles

The use of selenium for anticancer therapy has been heavily explored during the last decade. Amino acids (AAs) play central roles both as building blocks of proteins and intermediates in metabolism. In the present study, AAs-modified selenium nanoparticles (SeNPs@AAs) have been successfully synthesized in a simple redox system. Typical neutral (valine), acidic (aspartic acid) and basic (lysine) amino acids were used to decorate SeNPs, and the stable and homodisperse nanoparticles were characterized by zeta potential and transmission electron microscope. The result of X-ray photoelectron spectra (XPS) showed that the interaction of -NH3(+) groups of the amino acids with negative-charged SeNPs could be a driving force for dispersion of the nanoparticles. The screening of in vitro anticancer activities demonstrated that SeNPs@AAs exhibited differential growth inhibitory effects on various human cancer cell lines. Among them, SeNPs decorated by Lys displayed higher anticancer efficacy than those of valine and aspartic acid. The studies on the in vitro cellular uptake mechanisms revealed that SeNPs@AAs were internalized by cancer cells through endocytosis. Flow cytometric analysis and the determination of caspase activity indicated that treatment of the MCF-7 breast adenocarcinoma cells with SeNPs@AAs led to a dose-dependent increase in apoptosis. Moreover, it was found that SeNPs@AAs-induced ROS overproduction could be the upstream signal of caspase activation and mitochondrial dysfunction in cancer cells. Taken together, our results suggest that these amino acid biocompatible nanoparticles might have potential application as chemopreventive and chemotherapeutic agents for human cancers.

Differential Inhibitory Effects of Cyanovirin-N, Griffithsin, and Scytovirin on Entry Mediated by Envelopes of Gammaretroviruses and Deltaretroviruses

The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect.

Differential adhesion-inhibitory patterns of antibodies raised against two major variants of the NTS-DBL2X region of VAR2CSA

BackgroundVAR2CSA is a large polymorphic Plasmodium falciparum protein expressed on infected erythrocytes (IE) that allows their binding in the placenta, thus precipitating placental malaria (PM). The N-terminal part of VAR2CSA that contains the binding site to placental chondroitin sulfate A (CSA) is currently recognized as the most attractive region for vaccine development. An ultimate challenge is to define epitopes in this region that induce a broad cross-reactive adhesion inhibitory antibody response. MethodsBased on phylogenetic data that identified a dimorphic sequence motif in the VAR2CSA DBL2X, we raised antibodies against the NTS-DBL2X constructs containing one sequence or the other (3D7 and FCR3) and tested their functional properties on P. falciparum isolates from pregnant women and on laboratory-adapted strains. ResultsThe CSA binding inhibitory capacity of the antibodies induced varied from one parasite isolate to another (range, 10%–100%), but the combined analysis of individual activity highlighted a broader functionality that increased the total number of isolates inhibited. Interestingly, the differential inhibitory effect of the antibodies observed on field isolates resulted in significant inhibition of all field isolates tested, suggesting that optimal inhibitory spectrum on field isolates from pregnant women might be achieved with antibodies targeting limited variants of the N-terminal VAR2CSA. ConclusionsOur findings indicate that the NTS-DBL2X region of VAR2CSA can elicit strain-transcending anti-adhesion antibodies and suggest that the combination of the two major variants used here could represent the basis for an effective bivalent VAR2CSA-based vaccine.

Differential Profiles and Inhibitory Effect on Rotavirus Vaccines of Nonantibody Components in Breast Milk From Mothers in Developing and Developed Countries

Live oral rotavirus vaccines have been less immunogenic and efficacious for children of developing countries than for those in middle income and industrialized countries, and the basis for these differences is not fully understood. Recently, we demonstrated that breastmilk from mothers in India had significantly higher IgA and neutralizing activity against rotavirus that could reduce the effective titer of rotavirus vaccines reaching the gut when compared with that from mothers in the United States. We extended our study to understand the specific contribution of those nonantibody components in breastmilk to the neutralizing activity against rotavirus vaccine we observed. Breastmilk samples were collected from mothers of breast-feeding infants aged between 4 and 29 weeks (ie, vaccine eligible age) in India (N = 40), South Africa (N = 50) and the United States (N = 51). We examined breastmilk for lactoferrin, lactadherin, rotavirus-specific IgA and neutralizing activity against 3 rotavirus vaccine strains (Rotarix, RotaTeq G1 and 116E) using enzyme immunoassays, a plaque reduction assay or a microneutralization assay. We observed higher levels of lactoferrin, lactadherin, IgA and neutralizing activity in breastmilk specimens from Indian and South African women than those from American women. We demonstrated positive associations between levels of lactoferrin or IgA and neutralizing activity in Indian and South African specimens, but not in American specimens. We demonstrated that the inhibitory effect of lactoferrin was dose- or species-dependent, as evidenced by greater reduction in titer of Rotarix and 116E by human lactoferrin. Lactadherin also exhibited inhibitory activity to rotavirus vaccines but appeared to be less effective. The lower immunogenicity and efficacy of rotavirus vaccines in developing countries could be explained, in part, by synergistic inhibitory effect of high levels of antibody and nonantibody components in breastmilk consumed by infants at the time of immunization. Therefore, there is a need for alternative rotavirus vaccine strategies in breast-feeding populations.

Differential Growth Inhibitory Effects of Highly Oxygenated Guaianolides Isolated from the Middle Eastern Indigenous Plant Achillea falcata in HCT-116 Colorectal Cancer Cells

Medicinal plants play a crucial role in traditional medicine and in the maintenance of human health worldwide. Sesquiterpene lactones represent an interesting group of plant-derived compounds that are currently being tested as lead drugs in cancer clinical trials. Achillea falcata is a medicinal plant indigenous to the Middle Eastern region and belongs to the Asteraceae family, which is known to be rich in sesquiterpene lactones. We subjected Achillea falcata extracts to bioassay-guided fractionation against the growth of HCT-116 colorectal cancer cells and identified four secotanapartholides, namely 3-β-methoxy- isosecotanapartholide (1), isosecotanapartholide (2), tanaphallin (3), and 8-hydroxy-3-methoxyisosecotanapartholide (4). Three highly oxygenated guaianolides were isolated for the first time from Achillea falcata, namely rupin A (5), chrysartemin B (6), and 1β, 2β-epoxy- 3β,4α,10α-trihydroxyguaian- 6α,12-olide (7). These sesquiterpene lactones showed no or minor cytotoxicity while exhibiting promising anticancer effects against HCT-116 cells. Further structure-activity relationship studies related the bioactivity of the tested compounds to their skeleton, their lipophilicity, and to the type of functional groups neighboring the main alkylating center of the molecule.

Structural requirements within protoporphyrin IX in the inhibition of heat shock protein 90

Porphyrins are used for photodynamic therapy for their light-absorbing properties, and some of them were approved for the treatment of certain types of cancers. Porphyrins prevent activation of hypoxia inducible factor-1α (HIF-1α) by inhibiting heat shock protein 90 (HSP90). This study investigated the structural requirements within protoporphyrin IX (PPIX) for the inhibition of HSP90 activity. In HCT116, HT29 and DLD-1 cells, PPIX treatment directly hindered the binding between HSP90 and HIF-1α; PPIX treatment inhibited the chaperone activity of HSP90, accelerating protein degradation of HIF-1α. In addition, PPIX treatment suppressed cancer cell migration, endothelial cell tube formation, and aortic ring sprouting, being consistent with its anti-tumor and anti-angiogenic activities. In silico analysis, molecular docking model indicated that a tetrapyrrole macrocycle and two propionate chains within PPIX are necessary for the binding to the adenosine triphosphate (ATP)-binding pocket of HSP90. The predicted structural requirement was verified by the differential inhibitory effects of PPIX analogs, or the precursor of PPIX, on HIF-1α; compounds lacking either the tetrapyrrole macrocycle or the propionate chains were inactive. Our results show that a tetrapyrrole macrocycle and two attached propionate chains in PPIX coordinately interact with the ATP-binding pocket of HSP90, offering structural information on the inhibitory effect of porphyrins on angiogenesis.

Abstract 2574: Curcumin either stimulates or inhibits expression of short isoform of stem-cell-marker DCLK1 (DCLK1-S) in non-transformed vs transformed cells, resulting in stabilization/inhibition of growth, respectively.

Abstract Inhibitory effects of curcumin are well documented on colon cancer cells, with minimal side effects on normal cells. Mechanisms mediating opposite effects of curcumin on normal vs cancer cells, remain unknown. We examined possible differential effects of curcumin on stem-cell-marker, DCLK1 in non-transformed (HEK-C, NCM460) vs transformed/cancer (HEKmGAS, HCT-116) cells. Curcumin (10-25μM) significantly inhibited growth of HEKmGAS/HCT-116 cells in a dose-dependent manner, with no significant effects on HEK-C cells. Surprisingly, transcript levels of DCLK1-S were increased by 3-10 fold in non-transformed cells, but decreased by 50-80% in transformed cells, which correlated with activation/inhibition of transiently transfected intron V promoter-reporter constructs in response to curcumin. Several cis elements, including NFκB binding sites, identified in intron V promoter, may play a role in transcribing DCLK1-S. Significant down-regulation of DCLK1-S by curcumin correlated with significant inhibition of NFκBp65 binding to intron V promoter in a ChIP assay. L-isoform of DCLK1 (DCLK1-L) was mainly expressed by non-transformed/normal cells, due to possible epigenetic silencing of DCLK1-L 5’promoter in cancer cells. The 5’ promoter was positive for several cis elements including TCF/β-catenin binding sites. Since Wnt/β-catenin pathway is up-regulated in many cancer cells, transiently transfected 5’ promoter-reporter constructs were activated by several fold in HEKmGAS/HCT-116 cells compared to that in non-transformed cells; curcumin significantly inhibited TCF expression and β-catenin binding to the 5’ promoter in a ChIP assay. However, since 5’ promoter is silenced in many cancer cells, significant effects of curcumin were not measured on DCLK1-L transcript in cancer cells. Curcumin did not inhibit DCLK1-L transcript in non-transformed cells, suggesting that pathways other than Wnt/β-catenin likely up-regulate DCLK1-L expression in normal cells, which may not be inhibited by curcumin. Role of other transcriptional factors in differential expression of S and L forms in non-transformed vs transformed cells remains unknown, which likely contributes to stimulatory vs inhibitory effects of curcumin on non-transformed vs transformed cells. It is speculated that significant differences in inhibitory effects of curcumin on transformed vs non-transformed cells may be due to significant differences in pathways inducing transcriptional activation of the promoters transcribing DCLK1-S/L isoforms. These differences may allow us to develop promoter-specific inhibitors for targeting cancer stem cells while sparing normal stem cells. This work was supported by NIH Grant to PS (R01CA09795909). Citation Format: Shubhashish Sarkar, Malaney O’ Connell1, Stephanie M. Moya, Pomila Singh. Curcumin either stimulates or inhibits expression of short isoform of stem-cell-marker DCLK1 (DCLK1-S) in non-transformed vs transformed cells, resulting in stabilization/inhibition of growth, respectively. [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 2574. doi:10.1158/1538-7445.AM2013-2574

Abstract 83: Differential inhibitory effects of galeterone, abiraterone, orteronel and ketoconazole on CYP17A1 and steroidogenesis.

Abstract Prostate cancer growth is dependent on androgens. Because the CYP17A1 enzyme plays a central role in converting progestogens to androgens, inhibitors of CYP17A1 have been developed to treat castration-resistant prostate cancer (CRPC). CYP17A1 has both hydroxylase and lyase catalytic functions. However, hydroxylase inhibition causes build-up of progestogens and mineralocorticoids while also reducing cortisol. This syndrome, referred to secondary mineralocorticoid excess, causes edema, hypokalemia and hypertension and is treated clinically with concomitant steroids. We analyzed four inhibitors for their effects on CYP17A1 hydroxylase and lyase activities, and more globally on the steroidogenic pathway. Human CYP17A1 expressed in yeast microsomes was incubated with pregnenolone or 17α-hydroxypregnenolone and products quantitated using LC/MS/MS. Galeterone, abiraterone, orteronel or ketoconazole were added, and hydroxylase or lyase IC50 values calculated. In a separate experiment, H295R human adrenocortical carcinoma cells were incubated with a range of drug concentrations between 0.316 nM and 10 μM for 24 hours and media was analyzed for steroid production by LC/MS/MS. We observed that all drugs inhibited CYP17A1. However, potency and selectivity for hydroxylase and lyase varied significantly. Galeterone was the most potent CYP17A1 lyase inhibitor that also exhibited selectivity (3x more selective for lyase than hydroxylase). In contrast, abiraterone most selectively inhibited CYP17A1 hydroxylase (by 1.7-fold), while orteronel and ketoconazole were less potent lyase inhibitors than galeterone. In the cell-based assay, all drugs at 1 μM inhibited testosterone synthesis by ≥94%. However, while galeterone produced only moderate changes in cortisol (decreased 14%) and other intermediate precursors, evidence of hydroxylase inhibition for the other inhibitors was supported by significant elevation of progesterone (increased 293-fold by abiraterone), mineralocorticoids (orteronel increased DOC 74-fold), or reductions in cortisol (abiraterone, orteronel, ketoconazole reduced by 91%, 70% and 94%, respectively). Steroid changes across a range of drug concentrations from 0.316 nM to 10 μM showed evidence of different potencies for CYP17 hydroxylase and lyase, as well as potential off-target effects on other enzymes in the steroidogenic pathway. In summary, we found that galeterone is a selective and potent CYP17A1 lyase inhibitor that also antagonizes and degrades the AR, and exhibits minimal evidence of deleterious steroid changes associated with secondary mineralocorticoid excess. These experimental data recapitulate the Phase 1 clinical experience where no mineralocorticoid excess was observed, or prednisone required in CRPC patients receiving galeterone therapy. ARMOR2, a phase 2 clinical study in CRPC, is being initiated in 2012. Citation Format: Douglas Jacoby, Martin Williams. Differential inhibitory effects of galeterone, abiraterone, orteronel and ketoconazole on CYP17A1 and steroidogenesis. [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 83. doi:10.1158/1538-7445.AM2013-83

Differential lasting inhibitory effects of oxytocin and food-deprivation on mediobasal hypothalamic polydipsia

This study analyzed the effects of systemic oxytocin (OT) administration and 48-h food deprivation on the polydipsia, hyperphagia, and polyuria produced by electrolytic lesions of the mediobasal hypothalamus (MBH). In a first experiment, food deprivation transiently decreased the polydipsic response, whereas food deprivation plus OT administration reduced the water intake and urine excretion of polydipsic animals but not their subsequent food intake. These results were replicated in a second experiment (20 days), which also showed that OT potentiates sodium excretion, reducing the estimated plasma sodium levels in food-deprived MBH-lesioned animals. Administration of OT on day 21 to food-deprived (from day 20 to 22) animals (second period of the experiment 2) blocked the differences in water intake and urine excretion volumes between MBH and control animals on days 21 and 22. Subsequently, this 48-h food deprivation induced an additional and lasting (days 23-40) reduction in the intake of water and food of MBH animals. According to these findings, OT administration and/or food deprivation may potentially exert enduring reducing effects on the polydipsia, polyuria, and hyperphagia of MBH syndrome.

Differential Inhibitory Effects of 2-Azafluorenones on PI-PLC Activation but not on PC-PLC- or PC-PLD-Activation Induced by Histamine, PAF, PMA or A23187 in C6 Glioma Cells

In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.