Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant.

BACKGROUND: Oncogenic mutations in BRAF genes are found in approximately 5-10% of colorectal cancers. The majority of BRAF mutations are located within exons 11-15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. METHODS: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. RESULTS: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. CONCLUSIONS: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Cancer Cell Interaction with Adipose Tissue: Correlation with the Finding of Spiculation at Mammography.

PURPOSE: To investigate the relationship between spiculated masses at mammography and marginal adipose tissue invasion at histologic examination. MATERIALS AND METHODS: The institutional review board approved this retrospective study, and the requirement to obtain informed consent was waived. A total of 478 patients with invasive breast cancer who underwent surgery between 1999 and 2009 were included in this study. Clinical-pathologic findings from patients with spiculated masses on mammograms were compared with those from patients without spiculated masses by using logistic regression models, Cox proportional hazards regression models, and the Kaplan-Meier method. RESULTS: There were 136 spiculated tumors and 342 nonspiculated tumors. All 136 spiculated tumors (100%) were positive for adipose tissue invasion, whereas only 264 of the 342 nonspiculated tumors (77%) were positive for adipose tissue invasion (P < .001). Multivariate analysis revealed that adipose tissue invasion (P < .001), histologic grade (P < .001), dense breast (P = .002), and body mass index (P = .02) were independent factors associated with spiculation. With regard to survival, although many patients with spiculated tumors had a hormone-sensitive (estrogen receptor positive: P = .004; progesterone receptor positive: P = .001) or low-grade (P < .001) tumor, in contrast to the patients without spiculated masses, the prognosis in the two groups was similar (disease-free survival: P = .09; overall survival: P = .23). CONCLUSION: Cancer cell interaction with adipose tissue is crucial for the finding of spiculation at mammography.

Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice.

Trans-3-(3'4'-dimethoxyphenyl)-4-[(E)-3",4"-dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3'4'-dimethoxyphenyl)-4-[(E)-3",4"-dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10-30 µM) or Comp.2 (30 µM) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03-3 µM) or Comp.2 (0.3-3 µM) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 µM) and Comp.2 (3-30 µM) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50mg/kg p.o.), Comp.2 (50mg/kg p.o.), or fluoxetine (10mg/kg i.p.), an antidepressant, were administrated once a day on days 15-28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2'-deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia.

Magnolol and honokiol prevent learning and memory impairment and cholinergic deficit in SAMP8 mice.

The therapeutic use of neurotrophic factors to treat neurodegenerative disorders, including Alzheimer's disease, is considered feasible. Magnolol and honokiol, constituents of the Magnolia plant, are small organic compounds with neurotrophic activity. We investigated whether magnolol and honokiol can prevent age-related learning and memory impairment and cholinergic deficits in senescence-accelerated mice (SAM). Magnolol (1, 10 mg/kg) or honokiol (0.1, 1 mg/kg) were orally administered to SAMP8 mice once a day for 14 days in 2-month-old mice. Learning and memory performance were evaluated by passive avoidance tests and location and object novelty recognition tests. SAMP8 mice showed significant impairment of learning and memory at 4 and 6 months of age. This age-related learning and memory impairment was prevented by pretreatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Cholinergic neuron densities in the medial septum and vertical limb of the diagonal band of the forebrain were evaluated by an immunohistochemical analysis of choline acetyltransferase (ChAT). SAMP8 mice showed a significant cholinergic deficit at 6 months of age. These age-related cholinergic deficits were prevented by treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Moreover, SAMP8 mice showed decreased activity of Akt, a member of the prosurvival pathway, in the forebrain at 2 months of age. A 14-day treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg) enhanced phosphorylation of Akt in the forebrain at 2 months of age. These results suggest that magnolol and honokiol prevent age-related learning and memory impairment by preserving cholinergic neurons in the forebrain. These compounds may have potential therapeutic applications to various neurodegenerative disorders.

Nodular fasciitis with degeneration and regression.

Nodular fasciitis is a benign reactive proliferation that is frequently misdiagnosed as a sarcoma. This article describes a case of nodular fasciitis of 6-month duration located in the cheek, which degenerated and spontaneously regressed after biopsy. The nodule was fixed to the zygoma but was free from the overlying skin. The mass was 3.0 cm in diameter and demonstrated high signal intensity on T2-weighted magnetic resonance imaging. A small part of the lesion was biopsied. Pathological and immunohistochemical examinations identified the nodule as nodular fasciitis with myxoid histology. One month after the biopsy, the mass showed decreased signal intensity on T2-weighted images and measured 2.2 cm in size. The signal on T2-weighted images showed time-dependent decreases, and the mass continued to reduce in size throughout the follow-up period. The lesion presented as hypointense to the surrounding muscles on T2-weighted images and was 0.4 cm in size at 2 years of follow-up. This case demonstrates that nodular fasciitis with myxoid histology can change to that with fibrous appearance gradually with time, thus bringing about spontaneous regression. Degeneration may be involved in the spontaneous regression of nodular fasciitis with myxoid appearance. The mechanism of regression, unclarified at present, should be further studied.

De-repression and comparison of oil-water separation activity of the dibenzothiophene desulfurizing bacterium, Mycobacterium sp. G3.

Expression of the desulfurization genes (dsz) in Mycobacterium sp. G3 is repressed by sulfate, which is the product of biodesulfurization. An expression clone, pSMTABC, was constructed by placing the dsz genes downstream of the hsp60 promoter and the constructed plasmid was electroporated into G3. The recombinant strain G3-1 desulfurized dibenzothiophene in the presence of 0.5 mM: sulfate while the Dsz phenotype was completely repressed in the wild-type strain. However, there was no significant increase in the amount of desulfurization enzymes in G3-1. In addition, G3 had superior separation of diesel oil-water separation activity compared to E. coli, which is superior to desulfurizing rhodococci.

Analyses of microbial desulfurization reaction of alkylated dibenzothiophenes dissolved in oil phase.

The kinetics of the oil/water two-phase reaction system was analyzed, and the reaction was carried out with the desulfurization of alkylated dibenzothiophenes (Cx-DBTs) using the desulfurizing microorganism Mycobacterium sp. G3. In the water-phase reaction system, the desulfurization activities were constant with respect to species of Cx-DBTs as substrates. However, the desulfurization activities in the oil/water two-phase reaction system against DBT, 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT, and 4,6-dibutyl DBT were 49.0, 45.9, 11.5, 1.35, and 0.00 micromol g DCW(-1) h(-1), respectively. The kinetic parameters for the degradation of DBT, 4,6-dimethyl DBT, and 4,6-diethyl DBT were also obtained (V(max) values 90.0, 68.7, and 22.7 micromol g DCW(-1) h(-1) and K(m) values 0.21, 0.70, and 3.03 mM, respectively). The reason for the decrease in activity against Cx-DBTs of high molecular weight was a decrease in the V(max) value and an increase in the K(m) value, the latter being a particularly serious problem. Furthermore, the hydrophobicity of the substrate was evaluated as the capacity factor measured by high-performance liquid chromatography (HPLC). The correlation between substrate hydrophobicity and desulfurization activity indicated that the desulfurization reaction in the oil/water two-phase reaction system is greatly influenced by the hydrophobicity of the substrates. In addition, the influence of the solvent on desulfurization activity was examined, and it was found that not only the hydrophobicity of substrates, but also that of solvents, affected the desulfurization reaction.