bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of T cell subsets to the pathophysiology ofPneumocystis-related immunorestitution disease
Authors: Bhagwat Samir P. , Gigliotti Francis , Xu Haodong , Wright Terry W.
Published: August 2006

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4+and CD8+T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4+cells were more abundant than CD8+cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8+cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4+T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8+T cell response was observed. In contrast, mice depleted of CD8+T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-γ-producing CD4+cells, and a prolonged CD4+T cell response than mice with both CD4+and CD8+cells. These data suggest that CD4+T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8+T cells contributed to neither lung injury nor organism clearance when CD4+cells were present, but instead served to modulate CD4 function. In the absence of CD4+cells, CD8+T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4+and CD8+T cells affects the ultimate outcome of PcP-related IRD.
Hypoxia-induced mitogenic factor has proangiogenic and proinflammatory effects in the lung via VEGF and VEGF receptor-2
Authors: Yamaji-Kegan Kazuyo , Su Qingning , Angelini Daniel J. , Champion Hunter C. , Johns Roger A.
Published: August 2006

From a mouse model of hypoxia-induced pulmonary hypertension, we previously found a highly upregulated protein in the lung that we named hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 (FIZZ1), and resistin-like molecule α (RELMα). However, the mechanisms of HIMF in the pulmonary vascular remodeling remain unknown. We now demonstrate that HIMF promoted cell proliferation, migration, and the production of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) in pulmonary endothelial cells as well as the production of reactive oxygen species in murine monocyte/macrophage cells. HIMF-induced CD31-positive cell infiltrate in in vivo Matrigel plugs was significantly suppressed by VEGF receptor-2 (VEGFR2) blockade. In ex vivo studies, HIMF stimulated the production of VEGF, MCP-1, and stromal cell-derived factor-1 (SDF-1) in the lung resident cells, and VEGFR2 neutralization significantly suppressed HIMF-induced MCP-1 and SDF-1 production. Furthermore, intravenous injection of HIMF showed marked increase of CD68-positive inflammatory cells in the lungs, and these events were attenuated by VEGFR2 neutralization. Intravenous injection of HIMF also downregulated the expression of VEGFR2 in the lung. These results suggest that HIMF plays critical roles in pulmonary inflammation as well as angiogenesis.
p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity
Authors: Siddiqui Shahid S. , Siddiqui Zeba K. , Uddin Shahab , Minshall Richard D. , Malik Asrar B.
Published: August 2006

We show in rat lung microvessel endothelial cells (RLMVEC) that endocytosis is a critical determinant of activation of mitogen-activated protein kinase (MAPK) and thereby regulates endothelial monolayer integrity. In RLMVEC grown in serum-free medium, we observed that albumin supplementation induced the phosphorylation of p38 MAPK within 30 min, which persisted for up to 2 h. Engagement of the endocytic machinery regulated the activation of p38 MAPK that contributed to endothelial cell proliferation and reduction of apoptosis. We also observed an interaction between the caveolar protein caveolin-1 and p38 MAPK with reciprocal coimmunoprecipitation assays and colocalization using double-label immunofluorescence staining. Knockdown of caveolin-1 expression with small interfering RNA significantly reduced endocytosis and activation of p38 MAPK and interfered with the ability of endothelial cells to form a confluent monolayer. Thus caveolae-mediated endocytosis and concomitant activation of p38 MAPK may help to maintain endothelial monolayer integrity by signaling proliferation and survival of endothelial cells.
Extravascular lung water measurements and hemodynamic monitoring in the critically ill: bedside alternatives to the pulmonary artery catheter
Authors: Isakow Warren , Schuster Daniel P.
Published: August 2006

The recently completed Fluid and Catheter Treatment Trial conducted by the National Institutes of Health ARDSNetwork casts doubt on the value of routine pulmonary artery catheterization for hemodynamic management of the critically ill. Several alternatives are available, and, in this review, we evaluate the theoretical, validation, and empirical databases for two of these: transpulmonary thermodilution measurements (yielding estimates of cardiac output, intrathoracic blood volume, and extravascular lung water) that do not require a pulmonary artery catheter, and hemodynamic measurements (including estimates of cardiac output and ejection time, a variable sensitive to intravascular volume) obtained by esophageal Doppler analysis of blood flow through the descending aorta. We conclude that both deserve serious consideration as a means of acquiring useful hemodynamic data for managing shock and fluid resuscitation in the critically ill, especially in those with acute lung injury and pulmonary edema, but that additional study, including carefully performed, prospective clinical trials demonstrating outcome benefit, is needed.
Regulation of ENaC and CFTR expression with K+channel modulators and effect on fluid absorption across alveolar epithelial cells
Authors: Leroy Claudie , Privé Anik , Bourret Jean-Charles , Berthiaume Yves , Ferraro Pasquale , Brochiero Emmanuelle
Published: August 2006

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027–L1037, 2004), we identified an ATP-sensitive K+(KATP) channel in alveolar epithelial cells, formed by inwardly rectifying K+channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of KATP, voltage-dependent K+channel KvLQT1, and calcium-activated K+(KCa) channel modulators modified Na+and Cl−currents in alveolar monolayers. In addition, it was shown previously that a KATPopener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K+channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of KATP, KvLQT1, and KCachannels identified by PCR. Glibenclamide and clofilium (KATPand KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na+current, and forskolin-activated Cl−currents, whereas pinacidil, a KATPactivator, increased them. Interestingly, K+inhibitors or membrane depolarization (induced by valinomycin in high-K+medium) decreased α-, β-, and γ-ENaC and CFTR mRNA. α-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na+transport through ENaC, we tested the impact of K+channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K+channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.
Exercise can be pyrogenic in humans
Authors: Bradford Carl D. , Cotter James D. , Thorburn Megan S. , Walker Robert J. , Gerrard David F.
Published: August 2006

Exercise increases mean body temperature (T̄body) and cytokine concentrations in plasma. Cytokines facilitate PG production via cyclooxygenase (COX) enzymes, and PGE2 can mediate fever. Therefore, we used a COX-2 inhibitor to test the hypothesis that PG-mediated pyrogenicity may contribute to the raised T̄body in exercising humans. In a double-blind, cross-over design, 10 males [age: 23 yr (SD 5), V̇o2 max: 53 ml·kg−1·min−1 (SD 5)] consumed rofecoxib (50 mg/day; NSAID) or placebo (PLAC) for 6 days, 2 wk apart. Exercising thermoregulation was measured on day 6 during 45-min running (∼75% V̇o2 max) followed by 45-min cycling and 60-min seated recovery (28°C, 50% relative humidity). Plasma cytokine (TNF-α, IL-10) concentrations were measured at rest and 30-min recovery. T̄body was similar at rest in PLAC (35.59°C) and NSAID (35.53°C) and increased similarly during running, but became 0.33°C (SD 0.26) lower in NSAID during cycling (37.39°C vs. 37.07°C; P = 0.03), and remained lower throughout recovery. Sweating was initiated at T̄body of ∼35.6°C in both conditions but ceased at higher T̄body in PLAC than NSAID during recovery [36.66°C (SD 0.36) vs. 36.39°C (SD 0.27); P = 0.03]. Cardiac frequency averaged 6·min−1 higher in PLAC ( P < 0.01), whereas exercising metabolic rate was similar (505 vs. 507 W·m−2; P = 0.56). A modest increase in both cytokines across exercise was similar between conditions. COX-2 specific NSAID lowered exercising heat and cardiovascular strain and the sweating (offset) threshold, independently of heat production, indicating that PGE-mediated inflammatory processes may contribute to exercising heat strain during endurance exercise in humans.
Regulatory role of c-Met in insulin-like growth factor-I receptor–mediated migration and invasion of human pancreatic carcinoma cells
Authors: Bauer Todd W. , Somcio Ray J. , Fan Fan , Liu Wenbiao , Johnson Marjorie , Lesslie Donald P. , Evans Douglas B. , Gallick Gary E. , Ellis Lee M.
Published: August 2006

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma. [Mol Cancer Ther 2006;5(7):1676–82]
The effects of the oral, pan-VEGF-R kinase inhibitor CEP-7055 and chemotherapy in orthotopic models of glioblastoma and colon carcinoma in mice
Authors: Jones-Bolin Susan , Zhao Hugh , Hunter Kathryn , Klein-Szanto Andres , Ruggeri Bruce
Published: August 2006

CEP-7055, a fully synthetic, orally active N,N-dimethylglycine ester of CEP-5214, a C3-(isopropylmethoxy)–fused pyrrolocarbazole with potent pan–vascular endothelial growth factor receptor (VEGFR) kinase inhibitory activity, has recently completed phase I clinical trials in cancer patients. These studies evaluated the antitumor efficacy of CEP-7055 using orthotopic models of glioblastoma and colon carcinoma in combination with temozolomide, and irinotecan and oxaliplatin, respectively, for their effects on primary and metastatic tumor burden and median survival. Chronic administration of CEP-7055 (23.8 mg/kg/dose) and temozolomide resulted in improvement of median survival of nude mice bearing orthotopic human glioblastoma xenografts compared with temozolomide alone (261 versus 192 days, respectively; P ≤ 0.02). Reductions in neurologic dysfunction, brain edema, hemorrhage, and intratumoral microvessel density (CD34 staining) were observed in glioma-bearing mice receiving CEP-7055 alone, temozolomide alone, and the combination of CEP-7055 and temozolomide relative to vehicle and to temozolomide monotherapy. The administration of CEP-7055 in combination with irinotecan (20 mg/kg/dose i.p. × 5 days), and to a lesser degree with oxaliplatin (10 mg/kg/dose i.v.), showed reductions on primary colon carcinoma and hepatic metastatic burden in the CT-26 tumor model relative to that achieved by irinotecan and oxaliplatin monotherapy. These data show the significant efficacy and tolerability of optimal efficacious doses of CEP-7055 when given in combination with temozolomide and irinotecan relative to monotherapy with these cytotoxic agents in preclinical orthotopic glioma and colon carcinoma models and lend support for the use of these treatment regimens in a clinical setting in patients with glioblastoma and colon carcinoma. [Mol Cancer Ther 2006;5(7):1744–53]
SU14813: a novel multiple receptor tyrosine kinase inhibitor with potent antiangiogenic and antitumor activity
Authors: Patyna Shem , Laird A. Douglas , Mendel Dirk B. , O'Farrell Anne-Marie , Liang Chris , Guan Huiping , Vojkovsky Tomas , Vasile Stefan , Wang Xueyan , Chen Jeffrey , Grazzini Maren , Yang Cheng Y. , Haznedar Joshua Ö. , Sukbuntherng Juthamas , Zhong Wei-Zhu , Cherrington Julie M. , Hu-Lowe Dana
Published: August 2006

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-β, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies. [Mol Cancer Ther 2006;5(7):1774–82]
AGRO100 inhibits activation of nuclear factor-κB (NF-κB) by forming a complex with NF-κB essential modulator (NEMO) and nucleolin
Authors: Girvan Allicia C. , Teng Yun , Casson Lavona K. , Thomas Shelia D. , Jüliger Simone , Ball Mark W. , Klein Jon B. , Pierce William M. , Barve Shirish S. , Bates Paula J.
Published: August 2006

AGRO100, also known as AS1411, is an experimental anticancer drug that recently entered human clinical trials. It is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO), which are non-antisense, guanosine-rich phosphodiester oligodeoxynucleotides that form stable G-quadruplex structures. The biological activity of GROs results from their binding to specific cellular proteins as aptamers. One important target protein of GROs has been previously identified as nucleolin, a multifunctional protein expressed at high levels by cancer cells. Here, we report that AGRO100 also associates with nuclear factor-κB (NF-κB) essential modulator (NEMO), which is a regulatory subunit of the inhibitor of κB (IκB) kinase (IKK) complex, and also called IKKγ. In the classic NF-κB pathway, the IKK complex is required for phosphorylation of IκBα and subsequent activation of the transcription factor NF-κB. We found that treatment of cancer cells with AGRO100 inhibits IKK activity and reduces phosphorylation of IκBα in response to tumor necrosis factor-α stimulation. Using a reporter gene assay, we showed that AGRO100 blocks both tumor necrosis factor-α-induced and constitutive NF-κB activity in human cancer cell lines derived from cervical, prostate, breast, and lung carcinomas. In addition, we showed that, in AGRO100-treated cancer cells, NEMO is coprecipitated by nucleolin, indicating that both proteins are present in the same complex. Our studies suggest that abrogation of NF-κB activity may contribute to the anticancer effects of AGRO100 and that nucleolin may play a previously unknown role in regulating the NF-κB pathway. [Mol Cancer Ther 2006;5(7):1790–9]
Cyclohexylpiperazine derivative PB28, a σ2 agonist and σ1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer
Authors: Azzariti Amalia , Colabufo Nicola A. , Berardi Francesco , Porcelli Letizia , Niso Mauro , Simone Grazia M. , Perrone Roberto , Paradiso Angelo
Published: August 2006

σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ2 agonist/σ1 antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ2 receptor expression, by high and low σ1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ2 receptor affinity and low σ1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC50 in nanomolar range), a consistent time exposure-independent increase of G0-G1-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ2 agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]
Enzastaurin (LY317615), a protein kinase Cβ inhibitor, inhibits the AKT pathway and induces apoptosis in multiple myeloma cell lines
Authors: Rizvi Mujahid A. , Ghias Kulsoom , Davies Katharine M. , Ma Chunguang , Weinberg Frank , Munshi Hidayatullah G. , Krett Nancy L. , Rosen Steven T.
Published: August 2006

Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the protein kinase Cβ isozyme. The objective of this study was to assess the efficacy of enzastaurin in inducing apoptosis in multiple myeloma (MM) cell lines and to investigate possible mechanisms of apoptosis. Cell proliferation assays were done on a variety of MM cell lines with unique characteristics (dexamethasone sensitive, dexamethasone resistant, chemotherapy sensitive, and melphalan resistant). The dexamethasone-sensitive MM.1S cell line was used to further assess the effect of enzastaurin in the presence of dexamethasone, insulin-like growth factor-I (IGF-I), interleukin-6, and the pan-specific caspase inhibitor ZVAD-fmk. Enzastaurin increased cell death in all cell lines at clinically significant low micromolar concentrations (1–3 μmol/L) after 72 hours of treatment. Dexamethasone and enzastaurin were shown to have an additive effect on MM.1S cell death. Although IGF-I blocked the effect of 1 μmol/L enzastaurin, IGF-I did not abrogate cell death induced with 3 μmol/L enzastaurin. Moreover, enzastaurin-induced cell death was not affected by interleukin-6 or ZVAD-fmk. GSK3β phosphorylation, a reliable pharmacodynamic marker for enzastaurin activity, and AKT phosphorylation were both decreased with enzastaurin treatment. These data indicate that enzastaurin induces apoptosis in MM cell lines in a caspase-independent manner and that enzastaurin exerts its antimyeloma effect by inhibiting signaling through the AKT pathway. [Mol Cancer Ther 2006;5(7):1783–9]
Identification of a lead small-molecule inhibitor of the Aurora kinases using a structure-assisted, fragment-based approach
Authors: Warner Steven L. , Bashyam Sridevi , Vankayalapati Hariprasad , Bearss David J. , Han Haiyong , Von Hoff Daniel D. , Hurley Laurence H.
Published: August 2006

Aurora A and Aurora B are potential targets for anticancer drug development due to their roles in tumorigenesis and disease progression. To identify small-molecule inhibitors of the Aurora kinases, we undertook a structure-based design approach that used three-dimensional structural models of the Aurora A kinase and molecular docking simulations of chemical entities. Based on these computational methods, a new generation of inhibitors derived from quinazoline and pyrimidine-based tricyclic scaffolds were synthesized and evaluated for Aurora A kinase inhibitory activity, which led to the identification of 4-(6,7-dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioic acid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide. The lead compound showed selectivity for the Aurora kinases when it was evaluated against a panel of diverse kinases. Additionally, the compound was evaluated in cell-based assays, showing a dose-dependent decrease in phospho-histone H3 levels and an arrest of the cell cycle in the G2-M fraction. Although biological effects were observed only at relatively high concentrations, this chemical series provides an excellent starting point for drug optimization and further development. [Mol Cancer Ther 2006;5(7):1764–72]
Assessment of gefitinib- and CI-1040-mediated changes in epidermal growth factor receptor signaling in HuCCT-1 human cholangiocarcinoma by serial fine needle aspiration
Authors: Hidalgo Manuel , Amador Maria Luz , Jimeno Antonio , Mezzadra Heather , Patel Pina , Chan Audrey , Nielsen Matthew E. , Maitra Anirban , Altiok Soner
Published: August 2006

One specific limitation to the clinical development of targeted cancer therapeutics is the lack of well-validated pharmacodynamic markers. Such tools might conceivably provide a framework within which to better evaluate the selection of specific molecules as therapeutic targets. Nevertheless, the practical application of this hypothesis in clinical development remains elusive. In this study, we present a minimally invasive pharmacodynamic assay for monitoring therapy-mediated changes in the activity of target signaling pathways by using fine needle aspiration (FNA) samples and quantitative ELISA methods. To this end, we used the HuCCT-1 cholangiocarcinoma cell line treated with gefitinib (ZD1839, Iressa), a selective blocker of the epidermal growth factor receptor (EGFR), and CI-1040, a selective inhibitor of the mitogen extracellular regulated kinase [mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase 1/2]. HuCCT-1 cells were resistant to gefitinib and CI-1040 alone but susceptible to the combination of these drugs in vitro and in vivo. This effect was associated with a greater inhibition of ERK1/2 activation, a downstream mediator in the EGFR–mitogen-activated protein/ERK kinase pathway. Using this model, we sought to assess whether FNA-obtained tumor biopsies could be used to measure signaling pathway activation. Cellular extracts prepared from FNA samples yielded adequately cellular, high-quality samples to assess therapy-mediated changes in EGFR and ERK1/2 phosphorylation by Western blotting and quantitative ELISA assays. Treatment with gefitinib alone effectively inhibited EGFR activation but failed to block ERK1/2 phosphorylation and tumor growth. Blocking was achieved by the addition of CI-1040 to the treatment regimen. These results show that the combination of serial FNA sampling with highly sensitive quantitative ELISA assays permits assessment of therapy-mediated changes in signaling pathways, which correlate well with antitumor effects. This assay is simple to implement and broadly applicable to diverse tumor types in clinical studies with cancer patients and may be useful in the development of targeted anticancer agents. [Mol Cancer Ther 2006;5(7):1895–903]
Mannosylated chitosan nanoparticle–based cytokine gene therapy suppressed cancer growth in BALB/c mice bearing CT-26 carcinoma cells
Authors: Kim Tae Hee , Jin Hua , Kim Hyun Woo , Cho Myung-Haing , Cho Chong Su
Published: August 2006

Cancer immunotherapy relies on the ability of the immune system to destroy tumor cells selectively and to elicit a long-lasting memory of such activity. Interleukin-12 (IL-12) is an immunomodulatory cytokine produced primarily by antigen-presenting cells, which play an important role in promoting Th1-type immune response and cell-mediated immunity. To augment the antitumor immune action by in vivo IL-12 gene delivery, mannosylated chitosan (MC) was prepared to induce mannose receptor–mediated endocytosis of IL-12 gene directly into dendritic cells which reside within the tumor. Upon characterization, MC was proven to be suitable for IL-12 gene delivery due to good physicochemical properties and low cytotoxicity. In addition, MC exhibited much enhanced IL-12 gene transfer efficiency to dendritic cells rather than chitosan itself in terms of the induction of murine IL-12 p70 and murine IFN-γ. In animal studies, intratumoral injection of MC/plasmid encoding murine IL-12 complex into BALB/c mice bearing CT-26 carcinoma cells clearly suppressed tumor growth and angiogenesis, and significantly induced cell cycle arrest and apoptosis. Therefore, this study provides a new MC-mediated cytokine gene delivery system for cancer immunotherapy. [Mol Cancer Ther 2006;5(7):1723–32]
Microsomal prostaglandin E synthase-1 regulates human glioma cell growth via prostaglandin E2–dependent activation of type II protein kinase A
Authors: Payner Troy , Leaver H. Anne , Knapp Brian , Whittle Ian R. , Trifan Ovidiu C. , Miller Steven , Rizzo Maria Teresa
Published: August 2006

Dysregulation of enzymes involved in prostaglandin biosynthesis plays a critical role in influencing the biological behavior and clinical outcome of several tumors. In human gliomas, overexpression of cyclooxygenase-2 has been linked to increased aggressiveness and poor prognosis. In contrast, the role of prostaglandin E synthase in influencing the biological behavior of human gliomas has not been established. We report that constitutive expression of the microsomal prostaglandin E synthase-1 (mPGES-1) is associated with increased prostaglandin E2 (PGE2) production and stimulation of growth in the human astroglioma cell line U87-MG compared with human primary astrocytes. Consistently, pharmacologic and genetic inhibition of mPGES-1 activity and expression blocked the release of PGE2 from U87-MG cells and decreased their proliferation. Conversely, exogenous PGE2 partially overcame the antiproliferative effects of mPGES-1 inhibition and stimulated U87-MG cell proliferation in the absence of mPGES-1 inhibitors. The EP2/EP4 subtype PGE2 receptors, which are linked to stimulation of adenylate cyclase, were expressed in U87-MG cells to a greater extent than in human astrocytes. PGE2 increased cyclic AMP levels and stimulated protein kinase A (PKA) activity in U87-MG cells. Treatment with a selective type II PKA inhibitor decreased PGE2-induced U87-MG cell proliferation, whereas a selective type I PKA inhibitor had no effect. Taken together, these results are consistent with the hypothesis that mPGES-1 plays a critical role in promoting astroglioma cell growth via PGE2-dependent activation of type II PKA. [Mol Cancer Ther 2006;5(7):1817–26]
Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells
Authors: Rapozzi Valentina , Cogoi Susanna , Xodo Luigi E.
Published: August 2006

Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA)–modified oligonucleotides to silence BCR/ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79°C and 75°C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts (∼20% of control) and protein p210BCR/ABL (∼30% of control). Moreover, the antisense oligonucleotides suppressed cell growth up to 40% of control and induced apoptosis, as indicated by the increase of caspase-3/7 activity in the treated cells. Finally, the b2a2-specific antisense gapmer used in combination with STI571 (imatinib mesylate), a tyrosine kinase inhibitor of p210BCR/ABL, produced an enhanced antiproliferative effect in KYO-1 cells, which compared with K562 cells are refractory to STI571. The data of this study support the application of BCR/ABL antisense LNA-DNA gapmers, used either alone or in combination with STI571, as potential antileukemic agents. [Mol Cancer Ther 2006;5(7):1683–92]
The determinants of sensitivity and acquired resistance to gemcitabine differ in non–small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity
Authors: Oguri Tetsuya , Achiwa Hiroyuki , Sato Shigeki , Bessho Yuji , Takano Yuko , Miyazaki Mikinori , Muramatsu Hideki , Maeda Hiroyoshi , Niimi Takashi , Ueda Ryuzo
Published: August 2006

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non–small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC. [Mol Cancer Ther 2006;5(7):1800–6]
Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells
Authors: Cerone Maria Antonietta , Londoño-Vallejo J. Arturo , Autexier Chantal
Published: August 2006

Breast cancer is the most common malignancy among women. Current therapies for breast tumors are based on the use of chemotherapeutic drugs that are quite toxic for the patients and often result in resistance. Telomerase is up-regulated in 95% of breast carcinomas but not in adjacent normal tissues. Therefore, it represents a very promising target for anticancer therapies. Unfortunately, the antiproliferative effects of telomerase inhibition require extensive telomere shortening before they are fully present. Combining telomerase inhibition with common chemotherapeutic drugs can be used to reduce this lag phase and induce tumor cell death more effectively. Few studies have analyzed the effects of telomerase inhibition in combination with anticancer drugs in breast cancer cells. In this study, we inhibited telomerase activity in two breast cancer cell lines using a dominant-negative human telomerase reverse transcriptase and analyzed cell viability after treatment with different anticancer compounds. We found that dominant-negative human telomerase reverse transcriptase efficiently inhibits telomerase activity and causes telomere shortening over time. Moreover, cells in which telomerase was suppressed were more sensitive to anticancer agents independently of their mechanism of action and this sensitization was dependent on the presence of shorter telomeres. Altogether, our data show that blocking telomere length maintenance in combination with anticancer drugs can be used as an effective way to induce death of breast cancer cells. [Mol Cancer Ther 2006;5(7):1669–75]
CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo
Authors: Cao Z. Alexander , Bass Kathryn E. , Balasubramanian Sriram , Liu Liang , Schultz Brian , Verner Erik , Dai YuQin , Molina Rafael A. , Davis Jack R. , Misialek Shawn , Sendzik Martin , Orr Christine J. , Leung Ling , Callan Ondine , Young Peter , Dalrymple Stacie A. , Buggy Joseph J.
Published: August 2006

CRA-026440 is a novel, broad-spectrum, hydroxamic acid–based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of α-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity. [Mol Cancer Ther 2006;5(7):1693–701]
Effect of an epidermal growth factor receptor tyrosine kinase inhibitor on actin remodeling in anin vitrobladder cancer carcinogenesis model
Authors: Jin Yusheng , Iwata Kenneth K. , Belldegrun Arie , Figlin Robert , Pantuck Allan , Zhang Zuo-Feng , Lieberman Ronald , Rao Jianyu
Published: August 2006

Alteration of actin remodeling is a marker of malignant-associated field defect and a potential surrogate biomarker for chemoprevention trials. We tested erlotinib, a specific tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), on actin remodeling in a bladder carcinogenic model consisting of untransformed HUC-PC cells and transformed MC-T11 cells, both derived from the same normal human urothelial clone immortalized by SV40. Erlotinib had a selective growth inhibitory and actin remodeling effect on MC-T11 cells over HUC-PC cells, as examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and immunofluorescence labeling with laser scan cytometer analysis, respectively. The IC50 of untransformed HUC-PC cells was significantly higher than that of transformed MC-T11 cells (P < 0.05, t test). The actin remodeling effect was more prominent at lower dosage levels (1/8-1/4 of IC50), which was accompanied by an increased cell adhesion and decreased motility. At higher dosage levels (1/2 of IC50), erlotinib induced a decreased adhesion and anoikis (detachment-associated apoptosis). The transformed MC-T11, but not HUC-PC, showed a weak constitutive EGFR phosphorylation activity, which was inhibited by erlotinib in a dose-response manner. However, on epidermal growth factor stimulation, both cell lines showed a similar dose-response inhibitory effect on phosphorylated EGFR and mitogen-activated protein kinase (MAPK; P44/P42) activities, and MAPK inhibitor PD98059 showed no specific effect on erlotinib-induced actin remodeling, suggesting that pathways other than MAPK (P44/P42) may be responsible for erlotinib-induced actin remodeling. The findings provide evidence to support erlotinib-based bladder cancer chemoprevention and using actin remodeling as a marker for erlotinib-based intervention trials. [Mol Cancer Ther 2006;5(7):1754–63]
Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL
Authors: Cheng Jinrong , Hylander Bonnie L. , Baer Maria R. , Chen Xing , Repasky Elizabeth A.
Published: August 2006

Targeting death receptors with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has the remarkable potential to selectively kill malignant cells whereas normal cells are largely unaffected by this treatment. However, some tumor cells, including leukemia cells, exhibit resistance to this molecule. To investigate the basis for resistance of leukemia cells to the zinc-bound form of Apo2 ligand (Apo2L)/TRAIL, which is currently being evaluated in clinical trial, we isolated several resistant HL60 clones from parental HL60 cells by selection using the recombinant Apo2L/TRAIL. Differing resistance mechanisms were identified and characterized in these Apo2L/TRAIL-resistant clones. In one case, the level of the cell-surface death receptor DR4, but not DR5, was significantly decreased. However, these cells did undergo apoptosis in response to another form of recombinant TRAIL, histidine-tagged TRAIL, suggesting differing contributions of DR4 and DR5 in the response to these two forms of TRAIL. In the case of other clones, expression of procaspase-8 protein was lost and this was associated with a novel Leu22→Phe22 point mutation in CASP-8 gene. These results show that cells within a given tumor can have widely distinct mechanisms underlying resistance to Apo2L/TRAIL. [Mol Cancer Ther 2006;5(7):1844–53]
Sequence-dependent inhibition of human colon cancer cell growth and of prosurvival pathways by oxaliplatin in combination with ZD6474 (Zactima), an inhibitor of VEGFR and EGFR tyrosine kinases
Authors: Troiani Teresa , Lockerbie Owen , Morrow Mark , Ciardiello Fortunato , Eckhardt S. Gail
Published: August 2006

To date, clinical studies combining the new generation of targeted therapies and chemotherapy have had mixed results. Preclinical studies can be used to identify potential antagonism/synergy between certain agents, with the potential to predict the most efficacious combinations for further investigation in the clinical setting. In this study, we investigated the sequence-dependent interactions of ZD6474 with oxaliplatin in two human colon cell lines in vitro. We evaluated the in vitro antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) and RET tyrosine kinase activity, and oxaliplatin using three combination schedules: ZD6474 before oxaliplatin, oxaliplatin before ZD6474, and concurrent exposure. Cell proliferation studies showed that treatment with oxaliplatin followed by ZD6474 was highly synergistic, whereas the reverse sequence was clearly antagonistic as was concurrent exposure. Oxaliplatin induced a G2-M arrest, which was antagonized if the cells were previously or concurrently treated with ZD6474. ZD6474 enhanced oxaliplatin-induced apoptosis but only when added after oxaliplatin. The sequence-dependent antitumor effects appeared, in part, to be based on modulation of compensatory prosurvival pathways. Thus, expression of total and active phosphorylated EGFR, as well as AKT and extracellular signal-regulated kinase, was markedly increased by oxaliplatin. This increase was blocked by subsequent treatment with ZD6474. Furthermore, the synergistic sequence resulted in reduced expression of insulin-like growth factor-I receptor and a marked reduction in secretion of vascular endothelial growth factor protein. ZD6474 in combination with oxaliplatin has synergistic antiproliferative properties in human colorectal cancer cell lines in vitro when oxaliplatin is administered before ZD6474. [Mol Cancer Ther 2006;5(7):1883–94]
Inorganic selenium sensitizes prostate cancer cells to TRAIL-induced apoptosis through superoxide/p53/Bax-mediated activation of mitochondrial pathway
Authors: Hu Hongbo , Jiang Cheng , Schuster Todd , Li Guang-Xun , Daniel Peter T. , Lü Junxuan
Published: August 2006

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells through DR4 and DR5 death receptors, but not in normal prostate cells, which do not express these receptors. Therefore, TRAIL has excellent potential to be a selective prostate cancer therapeutic agent with minimal toxic side effects. However, prostate cancer cells, as many other cancer types, develop resistance to TRAIL, and the underlying molecular mechanisms require further investigation. We hypothesize that selenium may sensitize TRAIL-resistant cells to undergo caspase-mediated apoptosis and increase therapeutic efficacy. Here, we report that TRAIL signaling in LNCaP prostate cancer cells stalled at downstream of caspase-8 and BID cleavage, as indicated by the lack of Bax translocation into mitochondria, and no subsequent activation of the caspase-9 cascade. Selenite induced a rapid generation of superoxide and p53 Ser15 phosphorylation and increased Bax abundance and translocation into the mitochondria. Selenite and TRAIL combined treatment led to synergistic increases of Bax abundance and translocation into mitochondria, loss of mitochondrial membrane potential, cytochrome c release, and cleavage activation of caspase-9 and caspase-3. Inactivating p53 with a dominant-negative mutant abolished apoptosis without affecting superoxide generation, whereas a superoxide dismutase mimetic agent blocked p53 activation, Bax translocation to mitochondria, cytochrome c release, and apoptosis induced by selenite/TRAIL. In support of Bax as a crucial target for cross-talk between selenite and TRAIL pathways, introduction of Bax into p53 mutant DU145 cells enabled selenite to sensitize these cells for TRAIL-induced apoptosis. Taken together, the results indicate that selenite induces a rapid superoxide burst and p53 activation, leading to Bax up-regulation and translocation into mitochondria, which restores the cross-talk with stalled TRAIL signaling for a synergistic caspase-9/3 cascade-mediated apoptosis execution. [Mol Cancer Ther 2006;5(7):1873–82]
Clioquinol and docosahexaenoic acid act synergistically to kill tumor cells
Authors: Ding Wei-Qun , Liu Bolin , Vaught Joshua L. , Palmiter Richard D. , Lind Stuart E.
Published: August 2006

Clioquinol, an 8-hydroxyquinoline derivative (5-chloro-7-iodo-8-hydroxyquinoline) with antimicrobial properties, has recently been found to have cytotoxic activity towards human cancer cell lines at concentrations achieved by oral administration. This study was initiated to determine whether clioquinol could potentiate the antitumor effects of two drugs, doxorubicin and docosahexaenoic acid (DHA), believed to act in part via the generation of reactant oxidant species. At low micromolar concentrations, clioquinol had little effect upon cell viability and did not potentiate doxorubicin's cytotoxicity. Clioquinol significantly enhanced DHA's cytotoxic effects, an interaction that was shown to be synergistic by isobolographic analysis. Clioquinol exhibited a synergistic interaction with DHA in reducing nuclear factor-κB activity and inducing apoptosis, and the combination reduced the level of several molecules that promote cell survival, including Akt, p65, and Bcl-2. Interestingly, clioquinol neither induced lipid peroxidation itself nor increased peroxidation brought about by the addition of DHA. However, when cells were pretreated with antioxidant vitamin E, the synergism of clioquinol and DHA was blocked, indicating the essential role of lipid peroxidation for their action. These findings reveal a novel antitumor drug combination that synergistically targets major cell survival signaling pathways. [Mol Cancer Ther 2006;5(7):1864–72]