bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential expression of NKCC2, TonEBP, aldose reductase, and urea transporter-A in developing mouse kidney
Authors: Lee Hyun-Wook , Kim Wan-Young , Song Hyun-Kuk , Yang Chul-Woo , Han Ki-Hwan , Kwon H. Moo , Kim Jin
Published: August 2006

This study was conducted to test the hypothesis that, during renal development, the Na-K-2Cl cotransporter type 2 (NKCC2) activates the tonicity-responsive enhancer binding protein (TonEBP) transcription factor by creating medullary hypertonicity. TonEBP, in turn, drives the expression of aldose reductase (AR) and urea transporter-A (UT-A). Kidneys from 13- to19-day-old fetuses ( F13– F19), 1- to 21-day-old pups ( P1– P21), and adult mice were examined by immunohistochemistry. NKCC2 was first detected on F14 in differentiating macula densa and thick ascending limb (TAL). TonEBP was first detected on F15 in the medullary collecting duct (MCD) and surrounding endothelial cells. AR was detected in the MCD cells of the renal medulla from F15. UT-A first appeared in the descending thin limb (DTL) on F16 and in the MCD on F18. After birth, NKCC2-positive TALs disappeared gradually from the tip of the renal papilla, becoming completely undetectable in the inner medulla on P21. TonEBP shifted from the cytoplasm to the nucleus in both vascular endothelial cells and MCD cells on P1, and its abundance increased gradually afterward. Immunoreactivity for AR and UT-A in the renal medulla increased markedly after birth. Treatment of neonatal animals with furosemide dramatically reduced expression of TonEBP, AR, and UT-A1. Furosemide also prevented the disappearance of NKCC2-expressing TALs in the papilla. The sequential expression of NKCC2, TonEBP, and its targets AR and UT-A and the reduced expression TonEBP and its targets in response to furosemide treatment support the hypothesis that local hypertonicity produced by the activity of NKCC2 activates TonEBP during development.
Sensitization of pelvic nerve afferents and mast cell infiltration in the urinary bladder following chronic colonic irritation is mediated by neuropeptides
Authors: Ustinova Elena E. , Gutkin Dmitriy W. , Pezzone Michael A.
Published: August 2006

Irritable bowel syndrome and interstitial cystitis frequently overlap. We have shown that acute colitis sensitizes urinary bladder afferents to both mechanical and chemical stimuli and that chronic colitis similarly produces neurogenic cystitis. We hypothesize that chronic irritation of the colon releases neuropeptides from bladder afferents, leading to receptor sensitization and neurogenic inflammation. Female Sprague-Dawley rats received intrarectal trinitrobenzenesulfonic acid (TNBS) or vehicle 3 days following either systemic capsaicin (CP) pretreatment or vehicle. Ten days later, action potentials of single-unit pelvic C-fiber afferents with receptive fields in the bladder were recorded under urethane anesthesia during graded bladder distensions (UBD) or intravesical capsaicin (vCP) administration. In controls, UBD increased bladder afferent firing in proportion to intravesical pressure. At intravesical pressures of 30 mmHg and above, the percent increase in afferent firing was significantly accentuated following TNBS compared with controls (1,222 ± 176 vs. 624 ± 54%, P < 0.01). The response to vCP was also enhanced (4,126 ± 775 vs. 1,979 ± 438%, P < 0.01). Systemic depletion of neuropeptides from sensory nerves abolished these effects. Histological examination of the bladders revealed an increase in mast cell density in TNBS-treated animals compared with controls (18.02 ± 1.25 vs. 3.11 ± 0.27 mast cells/×100 field, P < 0.01). This effect was significantly ameliorated with CP (10.25 ± 0.95, P < 0.5 vs. TNBS-treated animals). In summary, chronic colonic irritation in the rat sensitizes urinary bladder afferents to noxious stimuli and causes mast cell infiltration in the bladder. Depletion of neuropeptides from sensory afferents diminishes these effects, suggesting they play an important role.
Interaction of epithelial ion channels with the actin-based cytoskeleton
Authors: Mazzochi C. , Benos D. J. , Smith P. R.
Published: August 2006

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells not only maintains the polarized expression of ion channels within specific membrane domains, it also functions in the intracellular trafficking and regulation of channel activity. Initial evidence supporting an interaction between epithelial ion channels and the actin-based cytoskeleton came from patch-clamp studies examining the effects of cytochalasins on channel activity. Cytochalasins were shown to either activate or inactivate epithelial ion channels. An interaction between the actin-based cytoskeleton and epithelial ion channels was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that the interactions between epithelial ion channels and actin can either be direct or indirect, the latter being mediated through scaffolding or actin-binding proteins that serve as links between the channels and the actin-based cytoskeleton. This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.
Functional rescue of vasopressin V2 receptor mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes insipidus
Authors: Robben J. H. , Sze M. , Knoers N. V. A. M. , Deen P. M. T.
Published: August 2006

Intracellular retention of a functional vasopressin V2 receptor (V2R) is a major cause of congenital nephrogenic diabetes insipidus (NDI) and rescue of V2R mutants by nonpeptide antagonists may restore their basolateral membrane (BM) localization and function. However, the criteria for efficient functional rescue of G protein-coupled receptor (GPCR) mutants at clinically feasible antagonist concentrations are unknown. We found that the four nonpeptide antagonists SR49059, OPC31260 , OPC41061 , and SR121463B induced maturation and rescued the BM expression of eight of nine different V2R mutants, stably expressed in physiologically relevant polarized cells. The extent of maturation and rescued BM expression correlated with the antagonists' concentration and affinity for the V2R. Displacement of the antagonists by AVP and subsequent cAMP generation inversely correlated with the antagonists' affinities for the V2R but is partially influenced by antagonist-specific aspects. Despite limited increases in maturation and cell-surface expression of V2R mutants, the low-affinity SR49059 optimally induced functional rescue at high concentrations, due to its easy displacement by vasopressin. At clinically feasible antagonist concentrations, however, only the high-affinity antagonists OPC31260 and OPC41061 induced functional rescue, as at these concentrations the extent of BM expression became limited. In conclusion, functional rescue of mutant V2Rs at clinically feasible concentrations is most effective with high-affinity antagonists. As OPC31260 and OPC41061 are clinically safe, they are promising candidates to relieve NDI. Moreover, as numerous other diseases are caused by endoplasmic reticulum-retained GPCRs for which cell-permeable antagonists become available, our finding that high-affinity antagonists are superior is anticipated to be important for pharmacotherapy development of these diseases.
Peritubular capillary dysfunction and renal tubular epithelial cell stress following lipopolysaccharide administration in mice
Authors: Wu Liping , Tiwari Manish M. , Messer Kurt J. , Holthoff Joseph H. , Gokden Neriman , Brock Robert W. , Mayeux Philip R.
Published: August 2006

The mortality rate for septic patients with acute renal failure is extremely high. Since sepsis is often caused by lipopolysaccharide (LPS), a model of LPS challenge was used to study the development of kidney injury. Intravital video microscopy was utilized to investigate renal peritubular capillary blood flow in anesthetized male C57BL/6 mice at 0, 2, 6, 10, 18, 24, 36, and 48 h after LPS administration (10 mg/kg ip). As early as 2 h, capillary perfusion was dramatically compromised. Vessels with continuous flow were decreased from 89 ± 4% in saline controls to 57 ± 5% in LPS-treated mice ( P < 0.01), and vessels with intermittent flow were increased from 6 ± 2% to 31 ± 5% ( P < 0.01). At 2 h, mRNA for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were elevated 50- and 27-fold, respectively, suggesting that vascular inflammation is an early event that may contribute to capillary dysfunction. By 10 h, vessels with no flow increased from 5 ± 2% in saline controls to 19 ± 3% in LPS-treated mice ( P < 0.05). By 48 h, capillary function was returning toward control levels. The decline in functional capillaries preceded the development of renal failure and was paralleled by induction of inducible nitric oxide synthase in the kidney. Using NAD(P)H autofluorescence as an indicator of cellular redox stress, we found that tubular cell stress was highly correlated with the percentage of dysfunctional capillaries ( r 2 = 0.8951, P < 0.0001). These data show that peritubular capillary dysfunction is an early event that contributes to tubular stress and renal injury.
Integration of Target and Effector Information in the Human Brain During Reach Planning
Authors: Beurze S. M. , de Lange F. P. , Toni I. , Medendorp W. P.
Published: August 2006

To plan a reaching movement, the brain must integrate information about the location of the target with information about the limb selected for the reach. Here, we applied rapid event-related 3-T fMRI to investigate this process in human subjects ( n = 16) preparing a reach following two successive visual instruction cues. One cue instructed which arm to use; the other cue instructed the location of the reach target. We hypothesized that regions involved in the integration of target and effector information should not only respond to each of the two instruction cues, but should respond more strongly to the second cue due to the added integrative processing to establish the reach plan. We found bilateral regions in the posterior parietal cortex, the premotor cortex, the medial frontal cortex, and the insular cortex to be involved in target–arm integration, as well as the left dorsolateral prefrontal cortex and an area in the right lateral occipital sulcus to respond in this manner. We further determined the functional properties of these regions in terms of spatial and effector specificity. This showed that the posterior parietal cortex and the dorsal premotor cortex specify both the spatial location of a target and the effector selected for the response. We therefore conclude that these regions are selectively engaged in the neural computations for reach planning, consistent with the results from physiological studies in nonhuman primates.
Spike-Timing-Dependent Plasticity of Inhibitory Synapses in the Entorhinal Cortex
Authors: Haas Julie S. , Nowotny Thomas , Abarbanel H.D.I.
Published: August 2006

Actions of inhibitory interneurons organize and modulate many neuronal processes, yet the mechanisms and consequences of plasticity of inhibitory synapses remain poorly understood. We report on spike-timing-dependent plasticity of inhibitory synapses in the entorhinal cortex. After pairing presynaptic stimulations at time tpre with evoked postsynaptic spikes at time tpost under pharmacological blockade of excitation we found, via whole cell recordings, an asymmetrical timing rule for plasticity of the remaining inhibitory responses. Strength of response varied as a function of the time interval Δ t = tpost − tpre: for Δ t > 0 inhibitory responses potentiated, peaking at a delay of 10 ms. For Δ t < 0, the synaptic coupling depressed, again with a maximal effect near 10 ms of delay. We also show that changes in synaptic strength depend on changes in intracellular calcium concentrations and demonstrate that the calcium enters the postsynaptic cell through voltage-gated channels. Using network models, we demonstrate how this novel form of plasticity can sculpt network behavior efficiently and with remarkable flexibility.
Visual Spatial Summation in Macaque Geniculocortical Afferents
Authors: Sceniak Michael P. , Chatterjee Soumya , Callaway Edward M.
Published: August 2006

The spatial summation properties of visual signals were analyzed for geniculocortical afferents in the primary visual cortex (V1) of anesthetized paralyzed macaque monkeys. Afferent input responses were recorded extracellularly during cortical inactivation through superfusion of the cortex with muscimol, allowing investigation of lateral geniculate nucleus of the thalamus (LGN) cell properties in the absence of cortical feedback. Responses from afferent inputs were classified as magno-, parvo-, or koniocellular based on anatomical organization within the cortex, established through histological reconstructions, and visual response wavelength sensitivity. More than 80% of afferents showed strong surround suppression [suppression index (SI) >0.5] and 14% showed negligible surround suppression (SI < 0.2). Afferent responses with weak and strong surround suppression were found throughout cortical input layers 4C and 4A. High-contrast estimates of the spatial extent of the classical surround were similar to the nonclassical surround. The classical and nonclassical surrounds were, on average, 1.5-fold larger than the excitatory center. Unlike neurons within V1, the spatial extent of excitatory summation for geniculocortical afferents was contrast invariant. Nonclassical surround suppression showed slight contrast dependency with estimates larger (20%) at lower contrasts and stronger at higher contrasts (13%). Surround suppression is inherent in cortical input responses and likely derives from lateral inhibition in either the LGN or retina. Although surround suppression within afferent responses increases slightly with contrast, the spatial spread of excitation remains fixed with contrast. This argues for distinct mechanisms of action for contrast-dependent modulation in cortical and subcortical responses.
Comparative analysis of the paired immunoglobulin-like receptor (PILR) locus in six mammalian genomes: duplication, conversion, and the birth of new genes
Authors: Wilson Michael D. , Cheung Joseph , Martindale Duane W. , Scherer Stephen W. , Koop Ben F.
Published: August 2006

Manyaspects of the immune system are controlled by homologous cell surface receptors that mediate inhibitory and activating pathways. The paired immunoglobulin-like receptor ( PILR) locus at 7q22 encodes both PILRA, an inhibitory receptor, and PILRB, its activating counterpart. Mouse Pilrb1 is a novel immune system regulator, and its ligand Cd99 participates in the recruitment of T-cells to inflamed tissue. We characterized the PILR locus in six mammalian genomes and investigated the structure and mRNA expression of human PILRB. Synteny at the PILR locus is conserved in the human, chimpanzee, dog, mouse and rat genomes. The absence of the PILR locus in opossum and chicken genomes suggests it arose after the divergence of placental and nonplacental mammals. In humans, a Williams-Beuren syndrome-related segmental duplication has created a complex chimeric transcript representing the predominantly expressed form of PILRB. Unlike PILRA, PILRB transcripts were detected in a wide variety of tissues including cells of the lymphoid lineage. In the mouse genome, a second activating gene, Pilrb2, and six pseudogenes were found. Extensive gene duplications in the rat genome have resulted in at least 27 Pilrb genes and or pseudogenes. Abundant gene duplication events involving novel CD99-related genes were also detected in the rat genome. In addition to duplication, we show that gene conversion has played a persistent role in the evolution of the PILR genes. Overall, we demonstrate that the PILR locus is dynamically evolving via multiple evolutionary mechanisms in several mammalian genomes.
Evolution of thermoTRP ion channel homologs in vertebrates
Authors: Saito Shigeru , Shingai Ryuzo
Published: August 2006

In mammalian thermosensation, nine temperature-sensitive ion channels that are activated by distinct temperature thresholds have been identified as thermosensors. These ion channels belong to the transient receptor potential (TRP) superfamily and are referred to as “thermoTRPs” (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, and TRPA1). To elucidate the evolutionary processes of thermoTRPs, we conducted comprehensive searches for mammalian thermoTRP gene homologs in the draft genome sequences of chicken ( Gallus gallus), western clawed frog ( Xenopus tropicalis), zebrafish ( Danio rerio), and pufferfish ( Fugu rubripes). Newly identified homologs were compared with known thermoTRPs, and phylogenetic analyses were conducted. Our comparative analyses revealed that most of the mammalian thermo-TRP members already existed in the common ancestor of fishes and tetrapods. Tetrapods shared almost the same repertoire, except that the western clawed frog expanded TRPV4s (six copies) and TRPM8s (two copies), which were diversified considerably. Comparisons of nonsynonymous and synonymous substitution rates among TRPV4s suggested that one copy of the TRPV4 channel in the western clawed frog retained its original function, while the other copies diversified and obtained slightly different properties. In fish lineages, several members of thermo-TRPs have duplicated in the whole genome duplication occurred in the ancestral ray-finned fish; however, some of the copies have subsequently been lost. Furthermore, fishes do not possess the three members of thermoTRPs existed in mammals, e.g., thermoTRPs activated by noxious heat, warm, and cool temperatures. Our results suggest that thermosensation mechanisms have changed through vertebrate evolution with respect to thermosensor repertoires.
Bortezomib inhibits docetaxel-induced apoptosis via a p21-dependent mechanism in human prostate cancer cells
Authors: Canfield Steven E. , Zhu Keyi , Williams Simon A. , McConkey David J.
Published: August 2006

Bortezomib (PS-341, Velcade) is a peptide boronate inhibitor of the 20S proteasome that is currently being combined with taxanes in several clinical trials in patients with prostate cancer. Here, we report that bortezomib inhibited docetaxel-induced M-phase arrest and apoptosis in androgen-dependent LNCaP-Pro5 cells. Direct analysis of kinase activity in immune complex kinase assays revealed that docetaxel activated cyclin-dependent kinase (CDK) 1 (CDC2) and that bortezomib blocked this activation. The effects of bortezomib were associated with accumulation of p21 and mimicked by chemical CDK inhibitors or by transfecting cells with a small interfering RNA construct specific for CDK1. Transient transfection with p21 also inhibited docetaxel-induced apoptosis; conversely, p21 silencing reversed the antagonistic effects of bortezomib on docetaxel-induced apoptosis. Together, our data show that bortezomib interferes with docetaxel-induced apoptosis via a p21-dependent mechanism that is associated with CDK1 inhibition. These observations may have important implications for the ongoing bortezomib-docetaxel combination trials as well as trials using bortezomib and other cell cycle–sensitive agents. [Mol Cancer Ther 2006;5(8):2043–50]
Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2
Authors: Xu Changjiang , Yuan Xiaoling , Pan Zui , Shen Guoxiang , Kim Jung-Hwan , Yu Siwang , Khor Tin Oo , Li Wenge , Ma Jianjie , Kong Ah-Ng Tony
Published: August 2006

The up-regulation of phase II detoxifying and stress-responsive genes is believed to play an important role in cancer prevention, and many natural compounds have been shown to be potent inducers of these genes. Previous studies showed that the antioxidant responsive element (ARE), found in these genes, can be bound by the transcription factor Nrf2, and is responsive to the activation by chemopreventive compounds and by oxidative stress. In the present study, we investigated the roles of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-kinase (JNK) in the regulation of phenethyl isothiocyanate (PEITC)–induced and Nrf2-dependent ARE activity and ARE-driven heme oxygenase-1 (HO-1) gene expression in PC-3 cells. ARE activity and HO-1 expression were strongly increased after treatment with PEITC. PEITC also increased the phosphorylation of ERK1/2 and JNK1/2 and caused release of Nrf2 from sequestration by Keap1, and its subsequent translocation into the nucleus. Importantly, Nrf2 was also translocated into the nucleus after transfection with ERK or JNK and that these activated ERK and JNK colocalized with Nrf2 in the nucleus. Activation of ERK and JNK signaling also resulted in the elevation of ARE activity and HO-1 expression. Importantly, PEITC-induced ARE activity was attenuated by inhibition of ERK and JNK signaling. In vitro kinase assays showed that both ERK2 and JNK1 could directly phosphorylate glutathione S-transferase–Nrf2 protein. Taken together, these results strongly suggest a model in which PEITC treatment of PC-3 cells activates ERK and JNK, which, in turn, phosphorylate Nrf2 and induce its translocation to the nucleus. Nuclear Nrf2 activates ARE elements and induces expression of stress-responsive genes, including HO-1. [Mol Cancer Ther 2006;5(8):1918–26]
Therapeutic advantage of combining calcium channel blockers and TRAIL in prostate cancer
Authors: Kaddour-Djebbar Ismail , Lakshmikanthan Vijayabaskar , Shirley Robert B. , Ma Yulin , Lewis Ronald W. , Kumar M. Vijay
Published: August 2006

Disruption of intracellular calcium initiates multiple cell-damaging processes, such as apoptosis. In normal cells, the levels of Ca2+ are low in the mitochondria, whereas in apoptotic cells, Ca2+ increases. Mitochondria uptake Ca2+ via an inner membrane channel called the uniporter and extrude it into the cytoplasm through a Na+/Ca2+ exchanger. Overload of Ca2+ in the mitochondria in CGP-treated cells leads to its damage, thus affecting cellular function and survival. The goal of these experiments was to determine the importance of mitochondrial calcium ([Ca2+]m) in apoptosis of prostate cancer cells. Furthermore, we have examined the advantages of increasing the [Ca2+]m and treating the cells with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a potent apoptotic agent. Our results show that, under these treatment conditions, inhibiting the Na+/Ca2+ exchanger using benzothiazepin CGP-37157 (CGP) did not induce apoptosis. However, combination of CGP and TRAIL increased the apoptotic response ∼25-fold compared with control. Increase in apoptosis followed enhanced levels of [Ca2+]m and was accompanied by pronounced mitochondrial changes characteristic of mitochondria-mediated apoptosis. Experiments with calcium ionophores showed that mere increase in cytosolic and/or mitochondrial Ca2+ was not sufficient to induce apoptosis. These results have therapeutic implications as inhibitors of Na+/Ca2+ exchanger are being used for treating some neurologic and cardiologic ailments, and TRAIL induces apoptosis preferentially in cancer cells. Furthermore, this system provides an excellent model to investigate the role of [Ca2+]m in apoptosis. [Mol Cancer Ther 2006;5(8):1958–66]
Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of γ-H2AX foci
Authors: Munshi Anupama , Tanaka Toshimitsu , Hobbs Marvette L. , Tucker Susan L. , Richon Victoria M. , Meyn Raymond E.
Published: August 2006

Vorinostat (suberoylanilide hydroxamic acid) is the prototype of a family of hybrid polar compounds that can induce growth arrest in transformed cells and shows promise for the treatment of cancer. Vorinostat specifically binds to and inhibits the activity of histone deacetylases resulting in acetylation of nucleosomal histones and an activation of gene transcription. Because histone deacetylases modulate chromatin structure and gene expression, both of which can influence radioresponse, this study was designed to examine the capacity of Vorinostat to influence radiation response in human tumor cells and investigate the mechanism underlying these interactions. Vorinostat induced hyperacetylation of histone H4 in a dose-dependent manner. We tested its ability to radiosensitize three human tumor cell lines (A375, MeWo, and A549) using clonogenic cell survival assays. Clonogenic cell survival assay showed that Vorinostat significantly radiosensitized all three tumor cell lines, substantially reducing the surviving fraction at 2 Gy. We examined potential mechanisms that may contribute to the enhanced radiation response induced by Vorinostat. Vorinostat and radiation alone did not induce apoptosis in the melanoma cell line. However, enhanced apoptosis was observed when cells were exposed to both Vorinostat and radiation, suggesting that Vorinostat renders tumor cells more susceptible to radiation-induced apoptosis. Results from DNA damage repair analysis in cultured A375 cells showed that Vorinostat had a strong inhibitory effect on the nonhomologous end joining pathway after radiation. A detailed examination of the involvement of the DNA repair pathway following Vorinostat treatment showed that Vorinostat reduced the expression of the repair-related genes Ku70, Ku80, and Rad50 in A375 cells as detected by Western blot analysis. We also examined γ-H2AX phosphorylation as a predictive marker of radiotherapy response to Vorinostat and observed that the combination of Vorinostat and radiation caused a prolongation of expression of DNA repair proteins such as γ-H2AX. Overall, we conclude that Vorinostat enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation. [Mol Cancer Ther 2006;5(8):1967–74]
Sequential oral 9-nitrocamptothecin and etoposide: a pharmacodynamic- and pharmacokinetic-based phase I trial
Authors: Simon George R. , Lush Richard M. , Gump Jana , Tetteh Leticia , Williams Charles , Cantor Alan , Antonia Scott , Garrett Christopher , Rocha-Lima Caio , Fishman Mayer , Sullivan Daniel M. , Munster Pamela N.
Published: August 2006

Purpose: Resistance to topoisomerase (topo) I inhibitors has been related to down-regulation of nuclear target enzyme, whereas sensitization to topo II inhibitors may result from induction of topo II by topo I inhibitors. Here, we evaluated a sequence-specific administration of a topo I inhibitor followed by a topo II inhibitor. Experimental Design: Twenty-five patients with advanced or metastatic malignancies were treated with increasing doses (0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 mg/m2) of 9-nitrocamptothecin (9-NC) on days 1 to 3, followed by etoposide (100 or 150 mg/d) on days 4 and 5. At the maximally tolerated dose, 20 additional patients were enrolled. The median age was 60 years (range, 40–84 years). Endpoints included pharmacokinetic analyses of 9-NC and etoposide, and treatment-induced modulations of topo I and II expression in peripheral blood mononuclear cells. Results: Neutropenia, thrombocytopenia, nausea, vomiting, diarrhea, and fatigue were dose-limiting toxicities and occurred in six patients. Despite a median number of four prior regimens (range 1–12), 2 (4%) patients had an objective response and 13 (29%) patients had stable disease. In contrast to the expected modulation in topo I and IIα levels, we observed a decrease in topo IIα levels, whereas topo I levels were not significantly altered by 9-NC treatment. Conclusions: Sequence-specific administration of 9-NC and etoposide is tolerable and active. However, peripheral blood mononuclear cells may not be a predictive biological surrogate for drug-induced modulation of topo levels in tumor tissues and should be further explored in larger studies. [Mol Cancer Ther 2006;5(8):2130–7]
Inhibition of vascular endothelial growth factor (VEGF)-165 and semaphorin 3A–mediated cellular invasion and tumor growth by the VEGF signaling inhibitor ZD4190 in human colon cancer cells and xenografts
Authors: Nguyen Quang-Dé , Rodrigues Sylvie , Rodrigue Christelle M. , Rivat Christine , Grijelmo Clara , Bruyneel Erik , Emami Shahin , Attoub Samir , Gespach Christian
Published: August 2006

We recently showed by DNA microarray analysis that vascular endothelial growth factor (VEGF) receptor (VEGFR) is expressed in HCT8/S11 human colon cancer cells, suggesting that several angiogenic factors may target colon cancer cells themselves. In this study, transcripts encoding the VEGF-165 and semaphorin 3A (Sema3A) receptors and coreceptors Flt-1, KDR/Flk-1, plexin A1, and neuropilins NP-1 and NP-2 were identified by reverse transcription-PCR in the human colon cancer cell lines HCT8/S11, HT29, HCT116, and PCmsrc. Collagen invasion induced by VEGF-165 and Sema3A in HCT8/S11 cells (EC50, 0.4–1 nmol/L) required p42/44 mitogen-activated protein kinase and signaling through RhoA/Rho-kinase–dependent and –independent pathways, respectively. As expected, the VEGFR signaling inhibitor ZD4190 selectively abrogated the proinvasive activity of VEGF in collagen gels (IC50, 10 nmol/L) and chick heart fragments. We identify a novel function for VEGF-165 and Sema3A as proinvasive factors for human colorectal cancer cells. Interestingly, oral administration of the single drug ZD4190 to athymic mice (50 mg/kg/d, once daily) inhibited by 70% the growth of HCT8/S11 tumor cell xenografts. Combinations between the src tyrosine kinase inhibitor M475271 and ZD4190 or cisplatin resulted in additive therapeutic activity against LNM35 human lung tumor xenografts. Our data have significant implications for new therapeutic approaches and individualized treatment targeting VEGFR and src signaling pathways in combination with established clinical drugs at primary tumors and distant metastases in colon and lung cancer patients. [Mol Cancer Ther 2006;5(8):2070–7]
Differential effect of silibinin on E2F transcription factors and associated biological events in chronically UVB-exposed skin versus tumors in SKH-1 hairless mice
Authors: Gu Mallikarjuna , Singh Rana P. , Dhanalakshmi Sivanandhan , Mohan Sarumathi , Agarwal Rajesh
Published: August 2006

UVB radiation–induced DNA damage in skin activates cellular pathways involved in DNA repair, cell cycle regulation, and apoptosis, important events that prevent conversion of damaged skin cells into cancer. We reported recently the efficacy of silibinin against photocarcinogenesis along with altered molecular events in tumors (Cancer Research, 64:6349–56, 2004). The molecular and biological events modulated by silibinin in chronically UVB-irradiated skin leading to cancer prevention, however, are not known. Herein, we describe effect of silibinin on skin 15 and 25 weeks after UVB exposure and compared them with molecular alterations in skin tumors. UVB decreased E2F1 but increased E2F2 and E2F3 protein levels in skin, and these were reversed by silibinin treatment. Silibinin-induced E2F1 was accompanied by an inhibition of apoptosis and decreases in p53 and cyclin-dependent kinase inhibitors. Silibinin-caused decrease in E2F2 and E2F3 was accompanied by reduced levels of cyclin-dependent kinases, cyclins, CDC25C, and mitogen-activated protein kinases and Akt signaling and inhibition of cell proliferation. In tumorigenesis protocols, topical or dietary silibinin significantly inhibited tumor appearance and growth. As opposed to UVB-exposed skin, UVB-induced tumors showed elevated levels of E2F1, but these were reduced in silibinin-treated tumors without any effect on E2F2 and E2F3. Contrary to the inhibition of apoptosis and p53 expression in UVB-exposed skin cells, silibinin increased these variables in tumors. These differential effects of silibinin on E2F1 versus E2F2 and E2F3 and their associated molecular alterations and biological effects in chronic UVB-exposed skin suggest their role in silibinin interference with photocarcinogenesis. [Mol Cancer Ther 2006;5(8):2121–9]
Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/β-catenin signaling
Authors: Roy Hemant K. , Kunte Dhananjay P. , Koetsier Jennifer L. , Hart John , Kim Young L. , Liu Yang , Bissonnette Marc , Goldberg Michael , Backman Vadim , Wali Ramesh K.
Published: August 2006

Polyethylene glycol (PEG) is one of the most potent chemopreventive agents against colorectal cancer; however, the mechanisms remain largely unexplored. In this study, we assessed the ability of PEG to target cyclin D1–β-catenin–mediated hyperproliferation in the azoxymethane-treated rat model and the human colorectal cancer cell line, HT-29. Azoxymethane-treated rats were randomized to AIN-76A diet alone or supplemented with 5% PEG-8000. After 30 weeks, animals were euthanized and biopsies of aberrant crypt foci and uninvolved crypts were subjected to immunohistochemical and immunoblot analyses. PEG markedly suppressed both early and late markers of azoxymethane-induced colon carcinogenesis (fractal dimension by 80%, aberrant crypt foci by 64%, and tumors by 74%). In both azoxymethane-treated rats and HT-29 cells treated with 5% PEG-3350 for 24 hours, PEG decreased proliferation (45% and 52%, respectively) and cyclin D1 (78% and 56%, respectively). Because β-catenin is the major regulator of cyclin D1 in colorectal cancer, we used the T-cell factor (Tcf)–TOPFLASH reporter assay to show that PEG markedly inhibited β-catenin transcriptional activity. PEG did not alter total β-catenin expression but rather its nuclear localization, leading us to assess E-cadherin expression (a major determinant of β-catenin subcellular localization), which was increased by 73% and 71% in the azoxymethane-rat and HT-29 cells, respectively. We therefore investigated the effect of PEG treatment on levels of the negative regulator of E-cadherin, SNAIL, and observed a 50% and 75% decrease, respectively. In conclusion, we show, for the first time, a molecular mechanism through which PEG imparts its antiproliferative and hence profound chemopreventive effect. [Mol Cancer Ther 2006;5(8):2060–9]
Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action
Authors: Husbeck Bryan , Bhattacharyya Rumi S. , Feldman David , Knox Susan J.
Published: August 2006

The development of prostate cancer and its progression to a hormone-refractory state is highly dependent on androgen receptor (AR) expression. Recent studies have shown that the selenium-based compound methylseleninic acid (MSeA) can disrupt AR signaling in prostate cancer cells. We have found that selenite can inhibit AR expression and activity in LAPC-4 and LNCaP prostate cancer cells as well but through a different mechanism. On entering the cell, selenite consumes reduced glutathione (GSH) and generates superoxide radicals. Pretreatment with N-acetylcysteine, a GSH precursor, blocked the down-regulation of AR mRNA and protein expression by selenite and restored AR ligand binding and prostate-specific antigen expression to control levels. MSeA reacts with reduced GSH within the cell; however, N-acetylcysteine did not effect MSeA-induced down-regulation of AR and prostate-specific antigen. The superoxide dismutase mimetic MnTMPyP was also found to prevent the decrease in AR expression caused by selenite but not by MSeA. A Sp1-binding site in the AR promoter is a key regulatory component for its expression. Selenite decreased Sp1 expression and activity, whereas MSeA did not. The inhibition of Sp1 by selenite was reversed in the presence of N-acetylcysteine. In conclusion, we have found that selenite and MSeA disrupt AR signaling by distinct mechanisms. The inhibition of AR expression and activity by selenite occurs via a redox mechanism involving GSH, superoxide, and Sp1. [Mol Cancer Ther 2006;5(8):2078–85]
Differential roles of checkpoint kinase 1, checkpoint kinase 2, and mitogen-activated protein kinase–activated protein kinase 2 in mediating DNA damage–induced cell cycle arrest: implications for cancer therapy
Authors: Xiao Zhan , Xue John , Sowin Thomas J. , Zhang Haiying
Published: August 2006

Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus preserving their genomic integrity. The protein kinases checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and mitogen-activated protein kinase–activated protein kinase 2 (MK2) have all been shown to be involved in cell cycle checkpoint control. Recently, cell cycle checkpoint abrogation has been proposed as one way to sensitize cancer cells to DNA-damaging agents due to the expected induction of mitotic catastrophe. Due to their overlapping substrate spectra and redundant functions, it is still not clear which kinase is mainly responsible for the cell cycle arrests conferred by clinically relevant chemotherapeutics. Thus, the issue remains about which kinase is the most therapeutically relevant target and, more importantly, whether multiple kinases might need to be targeted to achieve the best efficacy in light of recent studies showing superior efficacy for pan-receptor tyrosine kinase inhibitors. To clarify this issue, we investigated the roles of the three kinases in response to different genotoxic stresses through small interfering RNA–mediated specific target knockdowns. Our result showed that only the down-regulation of Chk1, but not of Chk2 or MK2, abrogated camptothecin- or 5-fluorouracil–induced S-phase arrest or doxorubicin-induced G2-phase arrest. This was followed by mitotic catastrophe and apoptosis. Moreover, double inhibition of Chk1 and Chk2 failed to achieve better efficacy than Chk1 inhibition alone; surprisingly, inhibition of MK2, in addition to Chk1 suppression, partially reversed the checkpoint abrogation and negated mitotic catastrophe. We further showed that this is due to the fact that in MK2-deficient cells, Cdc25A protein, which is critically required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other checkpoint kinases in addition to Chk1 would be nonproductive. [Mol Cancer Ther 2006;5(8):1935–43]
Modulation of the function of the multidrug resistance–linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin
Authors: Chearwae Wanida , Shukla Suneet , Limtrakul Pornngarm , Ambudkar Suresh V.
Published: August 2006

Curcumin (curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the major forms of curcuminoids found in the turmeric powder, which exhibit anticancer, antioxidant, and anti-inflammatory activities. In this study, we evaluated the ability of purified curcuminoids to modulate the function of either the wild-type 482R or the mutant 482T ABCG2 transporter stably expressed in HEK293 cells and drug-selected MCF-7 FLV1000 and MCF-7 AdVp3000 cells. Curcuminoids inhibited the transport of mitoxantrone and pheophorbide a from ABCG2-expressing cells. However, both cytotoxicity and [3H]curcumin I accumulation assays showed that curcuminoids are not transported by ABCG2. Nontoxic concentration of curcumin I, II, and III sensitized the ABCG2-expressing cells to mitoxantrone, topotecan, SN-38, and doxorubicin. This reversal was not due to reduced expression because ABCG2 protein levels were unaltered by treatment with 10 μmol/L curcuminoids for 72 hours. Curcumin I, II, and III stimulated (2.4- to 3.3-fold) ABCG2-mediated ATP hydrolysis and the IC50s were in the range of 7.5 to 18 nmol/L, suggesting a high affinity of curcuminoids for ABCG2. Curcuminoids also inhibited the photolabeling of ABCG2 with [125I]iodoarylazidoprazosin and [3H]azidopine as well as the transport of these two substrates in ABCG2-expressing cells. Curcuminoids did not inhibit the binding of [α-32P]8-azidoATP to ABCG2, suggesting that they do not interact with the ATP-binding site of the transporter. Collectively, these data show that, among curcuminoids, curcumin I is the most potent modulator of ABCG2 and thus should be considered as a treatment to increase the efficacy of conventional chemotherapeutic drugs. [Mol Cancer Ther 2006;5(8):1995–2006]
Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies
Authors: Qian Xiaozhong , LaRochelle William J. , Ara Gulshan , Wu Frank , Petersen Kamille Dumong , Thougaard Annemette , Sehested Maxwell , Lichenstein Henri S. , Jeffers Michael
Published: August 2006

Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC50 potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of α-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer. [Mol Cancer Ther 2006;5(8):2086–95]
Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer
Authors: Yuan Qing-an , Simmons Heidi H. , Robinson Matthew K. , Russeva Maria , Marasco Wayne A. , Adams Gregory P.
Published: August 2006

The Müllerian inhibiting substance type II receptor (MISIIR) is involved in Müllerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Müllerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the production of recombinant human MISIIR extracellular domain-human immunoglobulin Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) molecules from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based molecules. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease. [Mol Cancer Ther 2006;5(8):2096–105]
Enhanced killing of pancreatic cancer cells by expression of fusogenic membrane glycoproteins in combination with chemotherapy
Authors: Hoffmann Dennis , Wildner Oliver
Published: August 2006

Pancreatic cancer has a poor prognosis with an annual mortality rate close to the annual incidence rate. We evaluated whether the expression of measles virus fusogenic membrane glycoproteins (FMG) H and F will enhance chemotherapy. Using Chou-Talalay analysis, we showed in vitro in pancreatic cancer cells that the expression of FMG often synergistically enhances clinically relevant chemotherapy. Furthermore, cell fusion in combination with chemotherapy resulted in strongly enhanced Annexin V binding, an early marker for apoptosis, when compared with single treatment. We showed in an i.p. and s.c. pancreatic xenograft model that the administration of a replication-defective adenoviral vector Ad.H/F encoding tumor-restricted FMG in combination with gemcitabine significantly enhanced treatment outcome when compared with treatment with each compound individually. To improve tumor transduction efficiency, the Ad.H/F vector was also transcomplemented with an oncolytic replication-restricted adenovirus (Ad.COX•MK), resulting in significantly improved treatment efficacy. We assessed treatment efficacy by survival analysis or measuring growth, respectively. In the i.p. model, on day 120, three of eight animals treated with this novel triple therapy consisting of Ad.H/F, gemcitabine, and Ad.COX•MK were alive and tumor free. Treatment with Ad.H/F and Ad.COX•MK resulted in one long-term survivor. In all other treatment groups, there were no long-term survivors. The significantly improved therapeutic outcome of animals receiving the triple therapy was attributed to multiple factors, including most likely improved FMG expression throughout the tumor and enhanced sensitivity of the tumor cells to gemcitabine by adenoviral gene products but also FMG expression. Qualitatively similar results were obtained in a s.c. pancreatic xenograft model. [Mol Cancer Ther 2006;5(8):2013–22]
Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery
Authors: Sinha Rajni , Kim Gloria J. , Nie Shuming , Shin Dong M.
Published: August 2006

Nanotechnology refers to the interactions of cellular and molecular components and engineered materials—typically, clusters of atoms, molecules, and molecular fragments into incredibly small particles—between 1 and 100 nm. Nanometer-sized particles have novel optical, electronic, and structural properties that are not available either in individual molecules or bulk solids. The concept of nanoscale devices has led to the development of biodegradable self-assembled nanoparticles, which are being engineered for the targeted delivery of anticancer drugs and imaging contrast agents. Nanoconstructs such as these should serve as customizable, targeted drug delivery vehicles capable of ferrying large doses of chemotherapeutic agents or therapeutic genes into malignant cells while sparing healthy cells. Such “smart” multifunctional nanodevices hold out the possibility of radically changing the practice of oncology, allowing easy detection and then followed by effective targeted therapeutics at the earliest stages of the disease. In this article, we briefly discuss the use of bioconjugated nanoparticles for the delivery and targeting of anticancer drugs. [Mol Cancer Ther 2006;5(8):1909–17]