Cone photoreceptor dysfunction in retinitis pigmentosa revealed by optoretinography.

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerative diseases, whose most debilitating phase is cone photoreceptor death. Perimetric and electroretinographic methods are the gold standards for diagnosing and monitoring RP and assessing cone function. However, these methods lack the spatial resolution and sensitivity to assess disease progression at the level of individual photoreceptor cells, where the disease originates and whose degradation causes vision loss. High-resolution retinal imaging methods permit visualization of human cone cells in vivo but have only recently achieved sufficient sensitivity to observe their function as manifested in the cone optoretinogram. By imaging with phase-sensitive adaptive optics optical coherence tomography, we identify a biomarker in the cone optoretinogram that characterizes individual cone dysfunction by stimulating cone cells with flashes of light and measuring nanometer-scale changes in their outer segments. We find that cone optoretinographic responses decrease with increasing RP severity and that even in areas where cone density appears normal, cones can respond differently than those in controls. Unexpectedly, in the most severely diseased patches examined, we find isolated cones that respond normally. Short-wavelength-sensitive cones are found to be more vulnerable to RP than medium- and long-wavelength-sensitive cones. We find that decreases in cone response and cone outer-segment length arise earlier in RP than changes in cone density but that decreases in response and length are not necessarily correlated within single cones.

Revealing How Color Vision Phenotype and Genotype Manifest in Individual Cone Cells.

Purpose: Psychophysical and genetic testing provide substantial information about color vision phenotype and genotype. However, neither reveals how color vision phenotypes and genotypes manifest themselves in individual cones, where color vision and its anomalies are thought to originate. Here, we use adaptive-optics phase-sensitive optical coherence tomography (AO-PSOCT) to investigate these relationships. Methods: We used AO-PSOCT to measure cone function-optical response to light stimulation-in each of 16 human subjects with different phenotypes and genotypes of color vision (five color-normal, three deuteranopic, two protanopic, and six deuteranomalous trichromatic subjects). We classified three spectral types of cones (S, M, and L), and we measured cone structure-namely cone density, cone mosaic arrangement, and spatial arrangement of cone types. Results: For the different phenotypes, our cone function results show that (1) color normals possess S, M, and L cones; (2) deuteranopes are missing M cones but are normal otherwise; (3) protanopes are missing L cones but are normal otherwise; and (4) deuteranomalous trichromats are missing M cones but contain evidence of at least two subtypes of L cones. Cone function was consistent with the subjects' genotype in which only the first two M and L genes in the gene array are expressed and was correlated with the estimated spectral separation between photopigments, including in the deuteranomalous trichromats. The L/M cone ratio was highly variable in the color normals. No association was found between cone density and the genotypes and phenotypes investigated, and the cone mosaic arrangement was altered in the dichromats. Conclusions: AO-PSOCT is a novel method for assessing color vision phenotype and genotype in single cone cells.

Multi-reference global registration of individual A-lines in adaptive optics optical coherence tomography retinal images.

SIGNIFICANCE: Adaptive optics optical coherence tomography (AO-OCT) technology enables non-invasive, high-resolution three-dimensional (3D) imaging of the retina and promises earlier detection of ocular disease. However, AO-OCT data are corrupted by eye-movement artifacts that must be removed in post-processing, a process rendered time-consuming by the immense quantity of data. AIM: To efficiently remove eye-movement artifacts at the level of individual A-lines, including those present in any individual reference volume. APPROACH: We developed a registration method that cascades (1) a 3D B-scan registration algorithm with (2) a global A-line registration algorithm for correcting torsional eye movements and image scaling and generating global motion-free coordinates. The first algorithm corrects 3D translational eye movements to a single reference volume, accelerated using parallel computing. The second algorithm combines outputs of multiple runs of the first algorithm using different reference volumes followed by an affine transformation, permitting registration of all images to a global coordinate system at the level of individual A-lines. RESULTS: The 3D B-scan algorithm estimates and corrects 3D translational motions with high registration accuracy and robustness, even for volumes containing microsaccades. Averaging registered volumes improves our image quality metrics up to 22 dB. Implementation in CUDA™ on a graphics processing unit registers a 512 × 512 × 512 volume in only 10.6 s, 150 times faster than MATLAB™ on a central processing unit. The global A-line algorithm minimizes image distortion, improves regularity of the cone photoreceptor mosaic, and supports enhanced visualization of low-contrast retinal cellular features. Averaging registered volumes improves our image quality up to 9.4 dB. It also permits extending the imaging field of view (∼2.1 × ) and depth of focus (∼5.6 × ) beyond what is attainable with single-reference registration. CONCLUSIONS: We can efficiently correct eye motion in all 3D at the level of individual A-lines using a global coordinate system.

Suite of methods for assessing inner retinal temporal dynamics across spatial and temporal scales in the living human eye.

Significance: There are no label-free imaging descriptors related to physiological activity of inner retinal cells in the living human eye. A major reason is that inner retinal neurons are highly transparent and reflect little light, making them extremely difficult to visualize and quantify. Aim: To measure physiologically-induced optical changes of inner retinal cells despite their challenging optical properties. Approach: We developed an imaging method based on adaptive optics and optical coherence tomography (AO-OCT) and a suite of postprocessing algorithms, most notably a new temporal correlation method. Results: We captured the temporal dynamics of entire inner retinal layers, of specific tissue types, and of individual cells across three different timescales from fast (seconds) to extremely slow (one year). Time correlation analysis revealed significant differences in time constant (up to 0.4 s) between the principal layers of the inner retina with the ganglion cell layer (GCL) being the most dynamic. At the cellular level, significant differences were found between individual GCL somas. The mean time constant of the GCL somas ( 0.69 ± 0.17 s ) was ∼ 30 % smaller than that of nerve fiber bundles and inner plexiform layer synapses and processes. Across longer durations, temporal speckle contrast and time-lapse imaging revealed motion of macrophage-like cells (over minutes) and GCL neuron loss and remodeling (over one year). Conclusions: Physiological activity of inner retinal cells is now measurable in the living human eye.

Cone photoreceptor classification in the living human eye from photostimulation-induced phase dynamics.

Human color vision is achieved by mixing neural signals from cone photoreceptors sensitive to different wavelengths of light. The spatial arrangement and proportion of these spectral types in the retina set fundamental limits on color perception, and abnormal or missing types are responsible for color vision loss. Imaging provides the most direct and quantitative means to study these photoreceptor properties at the cellular scale in the living human retina, but remains challenging. Current methods rely on retinal densitometry to distinguish cone types, a prohibitively slow process. Here, we show that photostimulation-induced optical phase changes occur in cone cells and carry substantial information about spectral type, enabling cones to be differentiated with unprecedented accuracy and efficiency. Moreover, these phase dynamics arise from physiological activity occurring on dramatically different timescales (from milliseconds to seconds) inside the cone outer segment, thus exposing the phototransduction cascade and subsequent downstream effects. We captured these dynamics in cones of subjects with normal color vision and a deuteranope, and at different macular locations by: (i) marrying adaptive optics to phase-sensitive optical coherence tomography to avoid optical blurring of the eye, (ii) acquiring images at high speed that samples phase dynamics at up to 3 KHz, and (iii) localizing phase changes to the cone outer segment, where photoactivation occurs. Our method should have broad appeal for color vision applications in which the underlying neural processing of photoreceptors is sought and for investigations of retinal diseases that affect cone function.

Global gene expression of histologically normal primary skin cells from BCNS subjects reveals "single-hit" effects that are influenced by rapamycin.

Studies of dominantly heritable cancers enabled insights about tumor progression. BCNS is a dominantly inherited disorder that is characterized by developmental abnormalities and postnatal neoplasms, principally BCCs. We performed an exploratory gene expression profiling of primary cell cultures derived from clinically unaffected skin biopsies of BCNS gene-carriers (PTCH1 +/-) and normal individuals. PCA and HC of untreated keratinocytes or fibroblasts failed to clearly distinguish BCNS samples from controls. These results are presumably due to the common suppression of canonical HH signaling in vitro. We then used a relaxed threshold (p-value <0.05, no FDR cut-off; FC 1.3) that identified a total of 585 and 857 genes differentially expressed in BCNS keratinocytes and fibroblasts samples, respectively. A GSEA identified pancreatic ß cell hallmark and mTOR signaling genes in BCNS keratinocytes, whereas analyses of BCNS fibroblasts identified gene signatures regulating pluripotency of stem cells, including WNT pathway. Significantly, rapamycin treatment (FDR<0.05), affected a total of 1411 and 4959 genes in BCNS keratinocytes and BCNS fibroblasts, respectively. In contrast, rapamycin treatment affected a total of 3214 and 4797 genes in normal keratinocytes and normal fibroblasts, respectively. The differential response of BCNS cells to rapamycin involved 599 and 1463 unique probe sets in keratinocytes and fibroblasts, respectively. An IPA of these genes in the presence of rapamycin pointed to hepatic fibrosis/stellate cell activation, and HIPPO signaling in BCNS keratinocytes, whereas mitochondrial dysfunction and AGRN expression were uniquely enriched in BCNS fibroblasts. The gene expression changes seen here are likely involved in the etiology of BCCs and they may represent biomarkers/targets for early intervention.

The National Cancer Institute ALMANAC: A Comprehensive Screening Resource for the Detection of Anticancer Drug Pairs with Enhanced Therapeutic Activity.

To date, over 100 small-molecule oncology drugs have been approved by the FDA. Because of the inherent heterogeneity of tumors, these small molecules are often administered in combination to prevent emergence of resistant cell subpopulations. Therefore, new combination strategies to overcome drug resistance in patients with advanced cancer are needed. In this study, we performed a systematic evaluation of the therapeutic activity of over 5,000 pairs of FDA-approved cancer drugs against a panel of 60 well-characterized human tumor cell lines (NCI-60) to uncover combinations with greater than additive growth-inhibitory activity. Screening results were compiled into a database, termed the NCI-ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations), publicly available at https://dtp.cancer.gov/ncialmanac Subsequent in vivo experiments in mouse xenograft models of human cancer confirmed combinations with greater than single-agent efficacy. Concomitant detection of mechanistic biomarkers for these combinations in vivo supported the initiation of two phase I clinical trials at the NCI to evaluate clofarabine with bortezomib and nilotinib with paclitaxel in patients with advanced cancer. Consequently, the hypothesis-generating NCI-ALMANAC web-based resource has demonstrated value in identifying promising combinations of approved drugs with potent anticancer activity for further mechanistic study and translation to clinical trials. Cancer Res; 77(13); 3564-76. ©2017 AACR.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

The Effects of Cell Phone and Text Message Conversations on Simulated Street Crossing.

OBJECTIVE: A fully immersive, high-fidelity street-crossing simulator was used to examine the effects of texting on pedestrian street-crossing performance. BACKGROUND: Research suggests that street-crossing performance is impaired when pedestrians engage in cell phone conversations. Less is known about the impact of texting on street-crossing performance. METHOD: Thirty-two young adults completed three distraction conditions in a simulated street-crossing task: no distraction, phone conversation, and texting. A hands-free headset and a mounted tablet were used to conduct the phone and texting conversations, respectively. Participants moved through the virtual environment via a manual treadmill, allowing them to select crossing gaps and change their gait. RESULTS: During the phone conversation and texting conditions, participants had fewer successful crossings and took longer to initiate crossing. Furthermore, in the texting condition, smaller percentage of time with head orientation toward the tablet, fewer number of head orientations toward the tablet, and greater percentage of total characters typed before initiating crossing predicted greater crossing success. CONCLUSION: Our results suggest that (a) texting is as unsafe as phone conversations for street-crossing performance and (b) when subjects completed most of the texting task before initiating crossing, they were more likely to make it safely across the street. APPLICATION: Sending and receiving text messages negatively impact a range of real-world behaviors. These results may inform personal and policy decisions.

Are gamers better crossers? An examination of action video game experience and dual task effects in a simulated street crossing task.

OBJECTIVE: A high-fidelity street crossing simulator was used to test the hypothesis that experienced action video game players are less vulnerable than non-gamers to dual task costs in complex tasks. BACKGROUND: Previous research has shown that action video game players outperform nonplayers on many single task measures of perception and attention. It is unclear, however, whether action video game players outperform nonplayers in complex, divided attention tasks. METHOD: Experienced action video game players and nongamers completed a street crossing task in a high-fidelity simulator. Participants walked on a manual treadmill to cross the street. During some crossings, a cognitively demanding working memory task was added. RESULTS: Dividing attention resulted in more collisions and increased decision making time. Of importance, these dual task costs were equivalent for the action video game players and the nongamers. CONCLUSION: These results suggest that action video game players are equally susceptible to the costs of dividing attention in a complex task. APPLICATION: Perceptual and attentional benefits associated with action video game experience may not translate to performance benefits in complex, real-world tasks.

The effect of landmarks in human path integration.

The effect of landmarks in human path integration was investigated using virtual hallway-mazes. Participants traveled along random 5-segment paths either with or without distinctive landmarks at some segment intersections. They were required to directly return to the origin or to one of the landmark locations from the end of the path. Results showed that knowledge of the return targets prior to the outbound trip significantly reduced RTs to both the origin and the landmarks. Moreover, RTs in the return-to-origin trials were longer with landmarks present than without landmarks. This effect was eliminated when the return target was given prior to the trip. These results suggest that processing of the origin and the landmarks interfere with each other. However this interference is not obligatory and can be eliminated or reduced by prior knowledge about the target. The influences of landmarks on path integration and spatial updating were discussed.

Synthesis and biological activity of acetyl-protected hydroxybenzyl diethyl phosphates (EHBP) as potential chemotherapeutic agents.

Several acetyl-protected hydroxybenzyl diethyl phosphates (EHBPs) that are capable of forming quinone methide intermediates were synthesized and their cell growth inhibitory properties were evaluated in four different human cancer cell lines. Compounds 1, 1a, and 1b, corresponding to (4-acetyloxybenzyl diethylphosphate), (3-methyl-4-acetyloxybenzyl diethylphosphate), and (3-chloro-4-acetyloxybenzyl diethylphosphate), were significantly more potent than compounds 2 and 3, (2-acetyloxybenzyl diethylphosphate) and (3-acetyloxybenzyl diethylphosphate), respectively. Using HT-29 human colon cancer cells, compounds 1 and 3 increased apoptosis, inhibited proliferation, and caused a G(2)/M block in the cell cycle. Our data suggest that these compounds merit further investigation as potential anti-cancer agents.

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.

APC +/- alters colonic fibroblast proteome in FAP.

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.

Walking and talking: dual-task effects on street crossing behavior in older adults.

The ability to perform multiple tasks simultaneously has become increasingly important as technologies such as cell phones and portable music players have become more common. In the current study, we examined dual-task costs in older and younger adults using a simulated street crossing task constructed in an immersive virtual environment with an integrated treadmill so that participants could walk as they would in the real world. Participants were asked to cross simulated streets of varying difficulty while either undistracted, listening to music, or conversing on a cell phone. Older adults were more vulnerable to dual-task impairments than younger adults when the crossing task was difficult; dual-task costs were largely absent in the younger adult group. Performance costs in older adults were primarily reflected in timeout rates. When conversing on a cell phone, older adults were less likely to complete their crossing compared with when listening to music or undistracted. Analysis of time spent next to the street prior to each crossing, where participants were presumably analyzing traffic patterns and making decisions regarding when to cross, revealed that older adults took longer than younger adults to initiate their crossing, and that this difference was exacerbated during cell phone conversation, suggesting impairments in cognitive planning processes. Our data suggest that multitasking costs may be particularly dangerous for older adults even during everyday activities such as crossing the street.

Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics, and effect on the insulin-like growth factor axis.

Resveratrol, a naturally occurring polyphenol, has cancer chemopreventive properties in preclinical models. It has been shown to downregulate the levels of insulin-like growth factor-1 (IGF-I) in rodents. The purpose of the study was to assess its safety, pharmacokinetics, and effects on circulating levels of IGF-I and IGF-binding protein-3 (IGFBP-3) after repeated dosing. Forty healthy volunteers ingested resveratrol at 0.5, 1.0, 2.5, or 5.0 g daily for 29 days. Levels of resveratrol and its metabolites were measured by high performance liquid chromatography-UV in plasma obtained before and up to 24 hours after a dose between days 21 and 28. IGF-I and IGFBP-3 were measured by ELISA in plasma taken predosing and on day 29. Resveratrol was safe, but the 2.5 and 5 g doses caused mild to moderate gastrointestinal symptoms. Resveratrol-3-O-sulfate, resveratrol-4'-O-glucuronide, and resveratrol-3-O-glucuronide were major plasma metabolites. Maximal plasma levels and areas under the concentration versus time curve for the metabolites dramatically exceeded those for resveratrol, in the case of areas under the concentration versus time curve, by up to 20.3-fold. Compared with predosing values, the ingestion of resveratrol caused a decrease in circulating IGF-I and IGFBP-3 (P<0.04 for both), respectively, in all volunteers. The decrease was most marked at the 2.5 g dose level. The results suggest that repeated administration of high doses of resveratrol generates micromolar concentrations of parent and much higher levels of glucuronide and sulfate conjugates in the plasma. The observed decrease in circulating IGF-I and IGFBP-3 might contribute to cancer chemopreventive activity.

The effect of active selection in human path integration.

Path integration refers to the ability to integrate self-motion information to estimate one's current position and orientation relative to the origin. To investigate the effect of active selection in path integration, we used a virtual homing task in which participants traveled along hallways and attempted to directly return to the origin. Two groups of participants differed in the voluntary selection of the path structure, but received the same perceptual and motor information. Information about distance traveled was purely visual via optic flow, whereas turnings were specified both visually and through body senses. The active group made free (Experiment 1) or forced (Experiment 2) selections to determine the structure of the outbound path, whereas the passive group followed these outbound paths. We found no facilitation effects of the active selection on homing performance, suggesting that humans' limited path integration abilities cannot be attributed to the nature of the task.

Clinical pharmacology of resveratrol and its metabolites in colorectal cancer patients.

Resveratrol is a phytochemical with chemopreventive activity in preclinical rodent models of colorectal carcinogenesis. Antiproliferation is one of the many chemopreventive modes of action it has been shown to engage in. Concentrations of resveratrol, which can be achieved in human tissues after p.o. administration, have not yet been defined. The purpose of this study was to measure concentrations of resveratrol and its metabolites in the colorectal tissue of humans who ingested resveratrol. Twenty patients with histologically confirmed colorectal cancer consumed eight daily doses of resveratrol at 0.5 or 1.0 g before surgical resection. Resveratrol was found to be well tolerated. Normal and malignant biopsy tissue samples were obtained before dosing. Parent compound plus its metabolites resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, resveratrol-3-O-sulfate, resveratrol-4'-O-sulfate, resveratrol sulfate glucuronide, and resveratrol disulfate were identified by high-performance liquid chromatography (HPLC) with UV or mass spectrometric detection in colorectal resection tissue. Quantitation was achieved by HPLC/UV. Cell proliferation, as reflected by Ki-67 staining, was compared in preintervention and postintervention tissue samples. Resveratrol and resveratrol-3-O-glucuronide were recovered from tissues at maximal mean concentrations of 674 and 86.0 nmol/g, respectively. Levels of resveratrol and its metabolites were consistently higher in tissues originating in the right side of the colon compared with the left. Consumption of resveratrol reduced tumor cell proliferation by 5% (P = 0.05). The results suggest that daily p.o. doses of resveratrol at 0.5 or 1.0 g produce levels in the human gastrointestinal tract of an order of magnitude sufficient to elicit anticarcinogenic effects. Resveratrol merits further clinical evaluation as a potential colorectal cancer chemopreventive agent.

NO-releasing NSAIDs suppress NF-κB signaling in vitro and in vivo through S-nitrosylation.

NO-NSAIDs are promising anticancer drugs, comprising an NSAID, an NO-releasing moiety, and a spacer linking them. Although the effect of NO-NSAIDs on a wide variety of signaling and other cellular mechanisms has been deciphered, a key question remains unanswered, that being the role of NO to the overall biological effect of these agents. It has been shown that NO can directly modify sulfhydryl residues of proteins through S-nitrosylation and induce apoptosis. We studied 3 NO-NSAIDs having a different NSAID, spacer, and NO-releasing moiety. In vitro: aspirin, NO-ASA, naproxen, and NO-naproxen inhibited HT-29 human colon cancer cell growth, the IC(50)s being >5000, 192±6, 2800±210 and 95±5µM at 24h, respectively. NO-Aspirin and NO-naproxen reduced NF-κB protein levels, and activated caspase-3 enzyme in a dose- and time-dependent manner. Based on the biotin switch assay, NO-ASA and NO-naproxen S-nitrosylated NF-κB p65 in a time-dependent manner. Pretreatment of the cells with carboxy-PTIO, abrogated the S-nitrosylation of NF-κB p65. In vivo: rats treated with NO-ASA, NONO-ASA, and NO-naproxen showed S-nitrosylation of NF-κB p65 in the stomach tissue, increases in plasma TNF-α, and reductions in mucosal PGE(2) levels. These data provide a mechanistic role for NO and a rational for the chemopreventive effects of NO-NSAIDs.

Resveratrol modulates drug- and carcinogen-metabolizing enzymes in a healthy volunteer study.

Resveratrol has been shown to exhibit cancer-preventive activities in preclinical studies. We conducted a clinical study to determine the effect of pharmacologic doses of resveratrol on drug- and carcinogen-metabolizing enzymes. Forty-two healthy volunteers underwent baseline assessment of cytochrome P450 (CYP) and phase II detoxification enzymes. CYP1A2, CYP2D6, CYP2C9, and CYP3A4 enzyme activities were measured by the metabolism of caffeine, dextromethorphan, losartan, and buspirone, respectively. Blood lymphocyte glutathione S-transferase (GST) activity and GST-pi level and serum total and direct bilirubin, a surrogate for UDP-glucuronosyl transferase (UGT) 1A1 activity, were measured to assess phase II enzymes. After the baseline evaluation, study participants took 1 g of resveratrol once daily for 4 weeks. Enzyme assessment was repeated upon intervention completion. Resveratrol intervention was found to inhibit the phenotypic indices of CYP3A4, CYP2D6, and CYP2C9 and to induce the phenotypic index of 1A2. Overall, GST and UGT1A1 activities were minimally affected by the intervention, although an induction of GST-pi level and UGT1A1 activity was observed in individuals with low baseline enzyme level/activity. We conclude that resveratrol can modulate enzyme systems involved in carcinogen activation and detoxification, which may be one mechanism by which resveratrol inhibits carcinogenesis. However, pharmacologic doses of resveratrol could potentially lead to increased adverse drug reactions or altered drug efficacy due to inhibition or induction of certain CYPs. Further clinical development of resveratrol for cancer prevention should consider evaluation of lower doses of resveratrol to minimize adverse metabolic drug interactions.