Elucidation of structure-activity relationship of humulanolides and identification of humulanolide analog as a novel HSP90 inhibitor.

Humulanolides are natural products isolated from Asteriscus, and the isolation and total synthesis of many types of humulanolides have been reported. In this study, we evaluated anti-proliferative activity of twelve humulanolides against various human cancer cell lines and found that humulanolide analog E, which was newly designed and synthesized, exhibited the highest anti-proliferative activity. Structure-activity relationship analysis revealed that α,ß-unsaturated carbonyl moieties in humulanolides play an important role for anti-proliferative activity. To identify molecular targets of humulanolide analog E, we investigated various cell-based and in vitro assays. Treatment with humulanolide analog E against human fibrosarcoma HT1080 cells increased the expression level of HSP70 protein and decreased the levels of AKT and CDK4, which are HSP90 client proteins. Moreover, humulanolide analog E inhibited refolding of denatured luciferase protein via suppression of HSP90 activity in vitro. These results suggest that humulanolide analog E possesses the anti-proliferative activity against human cancer cells by inhibiting HSP90 functions.

The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4.

BACKGROUND: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation. METHODS: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope. RESULTS: We found that MFAP4 is C-mannosylated at Trp235 in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion. CONCLUSIONS: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.

Cell-dependent regulation of vasculogenic mimicry by carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1).

Vasculogenic mimicry (VM) promotes tumor migration, metastasis, and invasion in various types of cancer, but the relationship between VM and these phenotypes remains undefined. In this study, we examined carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) as a novel target of VM. We found that ectopic expression of CEACAM1 in HT1080 human fibrosarcoma cells suppressed the formation of a VM-like network. Further, cell migration and proliferation were abated by the introduction of CEACAM1 into HT1080 cells. Conversely, knockout (KO) of the CEACAM1 gene in SK-MEL-28 melanoma cells, which normally express high levels of CEACAM1, inhibited formation of a VM-like network, which was covered on reintroduction of CEACAM1. These results suggest that CEACAM1 differentially regulates formation of the VM-like network between cancer cell types and implicate CEACAM1 as a novel therapeutic target in malignant cancer.

[Feasibility Study of a One-Day Educational Program to Train Advance Care Planning Facilitators(ACPFs)in Regional Areas].

Promoting advance care planning in regional areas is important. Education For Implementing End-of-Life Discussion(EFIELD) is a two-day educational program for Advance Care Planning Facilitators(ACPFs)developed by the National Center for Geriatrics and Gerontology. Unfortunately, some trainers experience difficulties implementing the content of the program, and some trainees feel the program is too long for implementation in many regional areas. The purpose of the research is to clarify the feasibility of ACPFs education using a one-day program in regional areas. The methods involved documenting the process of a one-day program from implementation to evaluation from May of 2015 to March of 2016 and then evaluating the effectiveness of the program 3 months after the implementation using meeting minutes from 7 local hospitals. The results indicated a need for 5 steps from program implementation to evaluation as well as 5 categories for final evaluation. The most important finding is that E-FIELD challenged trainers to shorten and simplify their expressions in order to teach the content more efficiently. The second finding is that Group for Promoting Advance Care Planning & End Of Life Discussion in Chita(GACPEL) activities encouraged ACPimplementation within each hospital. The limitations of this research are related to small regional areas. In conclusion, a one-day regional ACPFs educational program is feasible.