Effect of trans-2,3-dimethoxycinnamoyl azide on enhancing antitumor activity of romidepsin on human bladder cancer

PURPOSE: Romidepsin (FK228, depsipeptide, FR901228), a unique cyclic depsipeptide with a histone deacetylase inhibitor (HDACI) activity, is a potential cancer therapeutic agent and currently under clinical trials for several types of cancer. For bladder cancer, romidepsin seems to be a potent antitumor agent from our recent study. In this study, we further delineate a new agent that can enhance both HDACI and antitumor activity of romidepsin. EXPERIMENTAL DESIGN: We screened a chemical library to identify candidate(s) that could enhance romidepsin activity. Chemical synthesis and purification were carried out to produce pure compound to examine its biochemical and antitumor effect on bladder cancer cell lines both in vitro and in vivo. RESULTS: Tranilast, N-(acetoacetyl) anthranilic acid, was first identified as a lead compound from screening, and then, one of the analogues, 2,3-dimethoxycinnamoyl azide (DMCA), seems to be more potent than tranilast. Our data indicate that DMCA can potentiate the HDACI activity of romidepsin and other biological activities, such as cell cycle arrest and apoptosis; these effects were accompanied with the expression of various key cell cycle regulators in different bladder cancer cells. Consistently, DMCA can enhance the in vivo antitumor effect of romidepsin without causing any more weight loss than romidepsin alone. CONCLUSION: DMCA is able to enhance the antitumor effect of romidepsin on bladder cancer from in vitro and in vivo.     

Deletions of CDKN2C in multiple myeloma: biological and clinical implications

PURPOSE: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. EXPERIMENTAL DESIGN: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. RESULTS: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. CONCLUSIONS: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.     

Factors influencing delay in the diagnosis of colorectal cancer: a study protocol

Background  

Colorectal cancer (CRC) is the second most frequent tumor in developed countries. Since survival from CRC depends mostly on disease stage at the time of diagnosis, individuals with symptoms or signs suspicious of CRC should be examined without delay. Many factors, however, intervene between symptom onset and diagnosis. This study was designed to: 1) Describe the diagnostic process of CRC from the onset of first symptoms to diagnosis and treatment. 2) Establish the time interval from initial symptoms to diagnosis and treatment, globally and considering patient's and doctors' delay, with the latter due to family physician and/or hospital services. 3) Identify the factors related to defined types of delay. 4) Assess the concordance between information included in primary health care and hospital clinical records regarding onset of first symptoms.     

4E-binding protein 1, a cell signaling hallmark in breast cancer that correlates with pathologic grade and prognosis.

PURPOSE: Cell signaling pathways include a complex myriad of interconnected factors from the membrane to the nucleus, such as erbB family receptors and the phosphoinositide-3-kinase/Akt/mTOR and Ras-Raf-ERK cascades, which drive proliferative signals, promote survival, and regulate protein synthesis. EXPERIMENTAL DESIGN: To find pivotal factors in these pathways, which provide prognostic information in malignancies, we studied 103 human breast tumors with an immunohistochemical profile, including total and phosphorylated (p) proteins: human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor, extracellular signal-regulated kinase 1/2, Akt, 4E-binding protein 1 (4EBP1), eukaryotic initiation factor 4E, phosphorylated ribosomal protein S6 kinase 1, phosphorylated ribosomal protein S6, and Ki67. Western blot and reverse lysate protein arrays were also done in a subset of tumors. RESULTS: Significantly, activation of the phosphoinositide-3-kinase/Akt/mTOR cascade was detected in a high proportion of tumors (41.9%). Tumors with HER2 overexpression showed higher p-Akt as compared with negative tumors (P < 0.001). Levels of p-Akt correlated with the downstream molecules, p-4EBP1 (P = 0.001) and p-p70S6K (P = 0.05). Although 81.5% of tumors expressed p-4EBP1, in 16.3% of these tumors, concomitant activation of the upstream factors was not detected. Interestingly, p-4EBP1 was mainly expressed in poorly differentiated tumors (P < 0.001) and correlated with tumor size (P < 0.001), presence of lymph node metastasis (P = 0.002), and locoregional recurrences (P = 0.002). Coexpression of p-4EBP1 and p-eIF4G correlated with a high tumor proliferation rate (P = 0.012). CONCLUSION: In this study, p-4EBP1 was the main factor in signaling pathways that associate with prognosis and grade of malignancy in breast tumors. Moreover, p-4EBP1 was detected in both HER2-positive and HER2-negative tumors. This factor seems to be a channeling point at which different upstream oncogenic alterations converge and transmit their proliferative signal, modulating protein translation.     

Diode laser-induced mitosis in the rabbit retinal pigment epithelium.

BACKGROUND AND OBJECTIVE: The induction of retinal pigment epithelium (RPE) proliferation without damaging the inner layers of the retina might be helpful in patients with RPE atrophic changes and degeneration. This study aimed to induce mitosis in the RPE of the rabbit after subthreshold photocoagulation with the diode laser. MATERIALS AND METHODS: Twenty-five male Dutch rabbits received retinal photocoagulation using an 810-nm diode laser with different power settings and exposure times. The eyes were processed for light microscopy, electron transmission microscopy, and immunohistochemistry. RESULTS: Neither morphological alterations nor mitotic activity was found after 5-mJ energies. Retinal layers were not affected and RPE hyperplasia appeared in the treated areas associated with mitotic activity when 10 mJ was used. Mitosis induction and retinal damage appeared with 20, 50, and 100 mJ and were associated with ophthalmoscopic damage. CONCLUSIONS: The use of subthreshold 810-nm diode laser treatment may induce mitosis in the RPE without causing damage to the neighboring layers.     

Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland.

The physiological response of the PSP toxin producing dinoflagellate Alexandrium minutum isolated from the Irish coast was assessed after modulating the initial concentrations of nitrate and phosphate in batch cultures. The cell growth in cultures of strain CK.A02 was primarily controlled by nitrate availability. In all experiments, only gonyautoxins 2 and 3 (GTX2 and 3) were synthesized along the different growth phases, with GTX3 dominating ( approximately 80%) at all stages, making the GTX2-3 toxin profile a possible population marker of A. minutum in Cork Harbour. The cellular toxin quotas remained low and relatively stable at around 2pgcell(-1), except when high N:P ratios were initially used for culture inoculations; in these conditions PSP toxins accumulated up to 14pgcell(-1). Due to the composition of the toxin profile, the toxicity of strain CK.A02 was generally relatively low (from 1.1 to 1.7pg STX eqcell(-1)) in comparison with strains from other geographic areas except when phosphate limiting culture conditions were applied (maximum of 12.5pg STX eqcell(-1)). Results showed that sufficient soluble protein quotas were necessary to observe the intra-cellular accumulation of PSP toxins in phosphate limiting conditions, highlighting also the requirement of adequate nitrogen supplies. The possible existence of localized toxicity hot spots in the field, linked to the accumulation of PSP toxins within A. minutum cells as a metabolic response to adverse environmental conditions, could potentially increase risks for shellfish farming operations.     

Changes in intraocular pressure after topical anaesthetic instillation

To determine the influence of topical anaesthetic drops, age and central corneal thickness (CCT) in the determination of intraocular pressure (IOP) by non contact tonometry (NCT). · METHODS: Ninety-three eyes from 47 patients were examined for CCT and IOP by NCT before and after the instillation of topical anaesthetic drops. · RESULTS: Average age was 66.4 (SD 16, range 34 to 88 years-of-age). Thirty one patients were female and 16 were male. Average basal IOP was 16.0mmHg (SD 4.0, range 8.5 to 26.1). IOP one minute after topical anesthesia instillation was 15.0mmHg (SD 3.8, range 7.7 to 26.7), and 14.9mmHg (SD 3.9, range 7.6 to 26.3) five minutes after the instillation. The differences were statistically significant for the 0 to 1 minute lapse(P =0.0007) and for the 0 to 5 minute lapse (P = 0.0003), but not for the 1 to 5 minute lapse (P =0.27) (Student's t test for paired data). Average CCT before topical anaesthetic drops was 565.4 microns. Simple linear regression analysis demonstrated absence of significant variation between age and IOP changes and between CCT and IOP changes. · CONCLUSION: Our study confirms that the instillation of topical anaesthetics causes a reduction in IOP, which is progressive during the first 5 minutes after instillation. This IOP reduction does not seem to be associated with basal CCT or age.     

The role of apoptosis in Listeria monocytogenes neural infection: listeriolysin O interaction with neuroblastoma Neuro-2a cells.

Listeria monocytogenes is the etiological agent of meningitis that affects individuals at high risk such as pregnant women, neonates, the elderly and immunocompromised individuals. Infection by this intracellular pathogen can be lethal if not diagnosed and treated. Mouse neuroblastoma Neuro-2a cells, a neuron-like cell line, were infected with L. monocytogenes. In this study apoptotic changes of neuroblastoma Neuro-2a cells infected with strains of Listeria producing different listeriolysin O levels are investigated by cytotoxicity assay, cellular viability assay, DAPI staining, intranucleosomal DNA fragmentation test, and monoclonal antibodies against ss-DNA. Results show that after internalization, the bacteria induced morphological, functional and genetic changes in the cells characteristic of apoptosis, which was dose-and time-dependent on listeriolysin O. Neuroblastoma Neuro-2a cells represent an interesting model cell line to further the understanding of Listeria pathogenesis within the central nervous system.