Preliminary study of photodynamic diagnosis using 5-aminolevulinic acid in gastric and colorectal tumors

AIM: To investigate the utility of photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) to detect gastric/colorectal tumors.METHODS: This prospective single-center study investigated inter-subject variability in patients with early-stage gastric/colorectal tumor indicated for endoscopic resection. Subjects were patients with gastric or colorectal tumors who had undergone endoscopic resection between November 2012 and November 2013. Selection criteria included age 20-80 years, either sex, and provision of informed consent. Patients were orally administered 20 mg/kg of 5-ALA enteric-coated capsules (SBI ALApromo Co., Tokyo, Japan). Administration of 5-ALA was followed by endoscopic resection of gastric or colorectal tumors, and the resected specimens were examined using a video autofluorescence processor and a fluorescence endoscope (SAFE-3000 and EB-1970AK, respectively; Pentax, Tokyo, Japan). The primary endpoint was the presence of fluorescence in tumors. Endoscopic, macroscopic, and histopathologic findings of tumors were assessed. We also evaluated adverse events of the present procedure as a secondary endpoint and examined each patient for the presence of known adverse effects of 5-ALA, namely, hematocytopenia, liver dysfunction, hypotension, nausea, and photosensitivity.RESULTS: We enrolled 10 patients (7 men, 3 women) (n = 13 lesions: 10 gastric/3 colorectal tumors). Fluorescence was detected in 7/13 (53.8%) lesions. No significant differences in sex (male: 55.6% vs female: 50.5%, P = 1.00), age (67.1 ± 1.9 years vs 65.0 ± 2.0 years, P = 0.45), tumor color (reddish: 60.0% vs discolored: 33.3%, P = 0.56), tumor diameter (15.0 ± 2.1 mm vs 14.2 ± 2.3 mm, P = 0.80), macroscopic type (protruded: 70.0% vs depressed 0%, P = 0.07), histologic type (differentiated type: 58.3% vs 0%, P = 0.46), invasion depth (mucosal layer: 55.6% vs submucosal layer: 33.3%, P = 1.00), lymphatic invasion (present: 33.3% vs absent: 50.0%, P = 1.00), venous invasion (present: 0% vs absent: 54.5%, P = 1.00) or procedure time of endoscopic resection (36.3 ± 8.3 min vs 36.7 ± 9.0 min, P = 0.98) were observed between the patients with and without fluorescence. Fluorescence detection rate tended to be high for elevated lesions. Liver dysfunction developed in 4/10 (40.0%) patients. The extent of the liver dysfunction was a slight increase in transaminases and total bilirubin levels, which spontaneously improved in the patients. None of the patients developed photosensitivity.CONCLUSION: Results of this preliminary study suggest the utility of PDD using 5-ALA for screening of gastric and colorectal cancers.     

Prolonged circulation half-life of interferon γ activity by gene delivery of interferon γ-serum albumin fusion protein in mice

Gene delivery of mouse interferon (IFN) γ has been shown to inhibit metastatic tumor growth and onset of atopic dermatitis in mouse models. In this study, we tried to increase the circulation half-life of IFNγ after its gene delivery by designing a novel fusion protein of IFNγ with mouse serum albumin (MSA). Western blot analysis confirmed that IFNγ-MSA was expressed as a fusion protein, but hardly formed dimer as IFNγ did. The biological activity of IFNγ-MSA, which was examined using a plasmid expressing luciferase under the control of gamma-activated sequence elements, was about 200-fold lower than the activity of IFNγ. Intravenous injection of the proteins into mice confirmed that the circulation half-life of IFNγ was significantly prolonged by the modification. A hydrodynamic injection of a plasmid expressing IFNγ-MSA resulted in a sustained concentration in mouse serum; it resulted in about sixfold greater area under the concentration-time curve and about threefold longer mean residence time of IFNγ activity than those of IFNγ. Gene delivery of IFNγ-MSA inhibited tumor metastasis to a similar level to that of IFNγ despite the reduced activity of IFNγ-MSA. These results indicate that gene delivery of IFNγ-MSA is a promising approach to prolong the circulation half-life of IFNγ activity.     

HERC2 Interacts with Claspin and regulates DNA origin firing and replication fork progression

DNA replication, recombination, and repair are highly interconnected processes the disruption of which must be coordinated in cancer. HERC2, a large HECT protein required for homologous recombination repair, is an E3 ubiquitin ligase that targets breast cancer suppressor BRCA1 for degradation. Here, we show that HERC2 is a component of the DNA replication fork complex that plays a critical role in DNA elongation and origin firing. In the presence of BRCA1, endogenous HERC2 interacts with Claspin, a protein essential for G(2)-M checkpoint activation and replication fork stability. Claspin depletion slowed S-phase progression and additional HERC2 depletion reduced the effect of Claspin depletion. In addition, HERC2 interacts with replication fork complex proteins. Depletion of HERC2 alleviated the slow replication fork progression in Claspin-deficient cells, suppressed enhanced origin firing, and led to a decrease in MCM2 phosphorylation. In a HERC2-dependent manner, treatment of cells with replication inhibitor aphidicolin enhanced MCM2 phosphorylation. Taken together, our results suggest that HERC2 regulates DNA replication progression and origin firing by facilitating MCM2 phosphorylation. These findings establish HERC2 as a critical function in DNA repair, checkpoint activation, and DNA replication.     

SHP-2/PTPN11 mediates gliomagenesis driven by PDGFRA and INK4A/ARF aberrations in mice and humans

Recent collaborative efforts have subclassified malignant glioblastomas into 4 clinical relevant subtypes based on their signature genetic lesions. Platelet-derived growth factor receptor α (PDGFRA) overexpression is concomitant with a loss of cyclin-dependent kinase inhibitor 2A (CDKN2A) locus (encoding P16INK4A and P14ARF) in a large number of tumors within one subtype of glioblastomas. Here we report that activation of PDGFRα conferred tumorigenicity to Ink4a/Arf-deficient mouse astrocytes and human glioma cells in the brain. Restoration of p16INK4a but not p19ARF suppressed PDGFRα-promoted glioma formation. Mechanistically, abrogation of signaling modules in PDGFRα that lost capacity to bind to SHP-2 or PI3K significantly diminished PDGFRα-promoted tumorigenesis. Furthermore, inhibition of SHP-2 by shRNAs or pharmacological inhibitors disrupted the interaction of PI3K with PDGFRα, suppressed downstream AKT/mTOR activation, and impaired tumorigenesis of Ink4a/Arf-null cells, whereas expression of an activated PI3K mutant rescued the effect of SHP-2 inhibition on tumorigenicity. PDGFRα and PDGF-A are coexpressed in clinical glioblastoma specimens, and such co-expression is linked with activation of SHP-2/AKT/mTOR signaling. Together, our data suggest that in glioblastomas with Ink4a/Arf deficiency, overexpressed PDGFRα promotes tumorigenesis through the PI3K/AKT/mTOR-mediated pathway regulated by SHP-2 activity. These findings functionally validate the genomic analysis of glioblastomas and identify SHP-2 as a potential target for treatment of glioblastomas.     

Asian sand dust enhances murine lung inflammation caused by Klebsiella pneumoniae

Inhaling concomitants from Asian sand dust (ASD) may result in exacerbation of pneumonia by the pathogen. The exacerbating effect of ASD on pneumonia induced by Klebsiella pneumoniae (KP) was investigated in ICR mice. The organic substances adsorbed onto ASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360 °C for 30 min. ICR mice were instilled intratracheally with ASD at doses of 0.05 mg or 0.2 mg/mouse four times at 2-week intervals (total dose of 0.2 mg or 0.8 mg/mouse) and were administrated with ASD in the presence or absence of KP at the last intratracheal instillation. Pathologically, ASD caused exacerbation of pneumonia by KP as shown by increased inflammatory cells within the bronchiolar and the alveolar compartments. ASD enhanced the neutrophil number dose dependently as well as the expression of cytokines (IL-1β, IL-6, IL-12, IFN-γ, TNF-α) and chemokines (KC, MCP-1, MIP-1α) related to KP in BALF. In an in vitro study using RAW264.7 cells, combined treatment of ASD and KP increased gene expression of IL-1β, IL-6, IFN-β, KC, MCP-1, and MIP-1α. The same treatment tended to increase the protein level of IL-1β, TNF-α and MCP-1 in a culture medium compared to each treatment alone. The combined treatment tended to increase the gene expression of Toll-like receptor 2 (TLR2), and NALP3, ASC and caspase-1 compared with KP alone. These results suggest that the exacerbation of pneumonia by ASD + KP was due to the enhanced production of pro-inflammatory mediators via activation of TLR2 and NALP3 inflammasome pathways in alveolar macrophages.     

Establishment and validation of a rabbit model for in vivo pharmacodynamic screening of tachykinin NK(2) antagonists

We attempted to establish and validate an in vivo pharmacodynamic (PD) rabbit model to screen tachykinin NK(2) receptor (NK(2)-R) antagonists using pharmacological and pharmacokinetic (PK)/PD analyses. Under urethane anesthesia, changes in intracolonic pressure associated with intravenous (i.v.) administration of a selective NK(2)-R agonist, βAla(8)-neurokinin A(4-10) (βA-NKA), was monitored as a PD marker. The analgesic effects of NK(2)-R antagonists were evaluated by monitoring visceromotor response (VMR) to colorectal distension in a rabbit model of visceral hypersensitivity induced by intracolonic treatment of acetic acid. Intravenous administration of βA-NKA induced transient colonic contractions dose-dependently, which were inhibited by the selective NK(2)-R antagonists in dose- and/or plasma concentration-dependent manners. The correlation between PD inhibition and plasma concentration normalized with the corresponding in vitro binding affinity was relatively high (r(2) = 0.61). Furthermore, the minimum effective doses on the VMR and ID(50) values calculated in the PD model were highly correlated (r(2) = 0.74). In conclusion, we newly established and validated a rabbit model of agonist-induced colonic contractions as a screening tool for NK(2)-R antagonists. In a drug discovery process, this PD model could enhance the therapeutic candidate selection for irritable bowel syndrome, pharmacologically connecting in vitro affinity for NK(2)-R with in vivo therapeutic efficacy.     

Serotonin potentiates high-glucose-induced endothelial injury: the role of serotonin and 5-HT(2A) receptors in promoting thrombosis in diabetes

To clarify the involvement of 5-hydroxytryptamine (5-HT) in promotion of thrombogenesis in diabetes, we examined the inhibitory effect of sarpogrelate, a 5-HT(2A) receptor antagonist, on thrombus formation in diabetic rats. In streptozotocin-induced diabetic rats, polyethylene tube-induced thrombus formation was enhanced compared with that in normal rats. The thrombogenesis was inhibited by sarpogrelate; cilostazol, a PDE3 inhibitor; and aspirin, a COX inhibitor, by 75.8%, 42.3%, and 34.3%, respectively. The inhibition by sarpogrelate was more pronounced in diabetic rats than normal ones. High glucose and 5-HT increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and combination of both high glucose and 5-HT further potentiated the effect. Sarpogrelate but not aspirin inhibited the increase in VCAM-1 expression induced by high glucose and 5-HT. These findings suggest that 5-HT mediates the enhanced thrombogenesis in diabetes and suggests that a 5-HT(2A) receptor antagonist may have novel therapeutic potential for the treatment of diabetic complications.     

Effect of renal impairment on the pharmacokinetics of memantine

The effect of renal impairment on the pharmacokinetics of a single oral dose of memantine (10 mg) was determined in Japanese subjects. Subjects were assigned to four groups based on baseline creatinine clearance (CL(CR)): normal renal function (> 80 mL/min, n = 6), and mild (50 to ≤ 80 mL/min, n = 6), moderate (30 to < 50 mL/min, n = 6), and severe renal impairment (5 to < 30 mL/min, n = 7). Mean memantine maximum plasma concentration (C(max)) was similar in the groups (12.66, 17.25, 15.75, and 15.83 ng/mL, respectively), as was mean time to C(max) (6.2, 5.2, 4.3, and 5.4 h, respectively). However, exposure to memantine determined from mean area under the plasma concentration-time curve was 1.62-, 1.97-, and 2.33-times higher in subjects with mild, moderate, and severe renal impairment, respectively, as compared to controls with normal renal function. Mean memantine plasma elimination half-life increased according to increasing renal impairment (61.15, 83.00, 100.13, and 124.31 h, respectively), while mean cumulative urinary recovery of unchanged memantine in 72 h after dosing decreased according to increasing renal impairment (33.68%, 33.47%, 23.60%, and 16.17%, respectively). These results are the same as those in the previous study on caucasian individuals, when compared per body weight. It is suggested that the dose of memantine should be halved in patients with renal impairment.