Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid ( 1-6 ). To date, 10 mammalian DGK isozymes ( ␣ , ␤ , ␥ , ␦ , , , , , , and ) have been identifi ed. These DGK isozymes are divided into fi ve groups (type I-V) according to their structural features ( 1-6 ). Type I DGK isozymes (DGKs ␣ , ␤ , and ␥ ) commonly contain tandem repeats of two EFhand motif domains and are classifi ed as members of the EF-hand family of Ca 2+ binding proteins. In addition to the Ca 2+ binding EF-hand motifs, all type I DGK isozymes contain an N-terminal recoverin homology domain, two cysteine-rich C1 domains, and the C-terminal catalytic region ( 1-6 ).DGK ␣ ( 7, 8 ) is highly expressed in hepatocellular carcinoma and melanoma cells ( 9, 10 ). DGK ␣ expression is involved in hepatocellular carcinoma progression and is a Abstract Diacylglycerol kinase (DGK) consists of 10 isozymes. The ␣ -isozyme enhances the proliferation of cancer cells. However, DGK ␣ facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGK ␣ enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK ␣ activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. Abbreviations: Con A, concanavalin A; CU-3, 5-[(2E)-3-(2-furyl) prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one; DG, diacylglycerol; DGK, diacylglycerol kinase; HTS, high-through put screening; IL-2, interleukin-2; PA, phosphatidic acid; PS, phosphatidylserine ; R59022, 6-(2-{4-[(4-fluorophenyl)phenylmethylene]1-piperidinyl}ethyl)-7-methyl-5 H -thiazolo-(3,2-a )pyrimidin-5-one; R59949, 3-(2-{4-[bis-(4-fl uorophenyl)methylene]1-piperidinyl}ethyl)-2,3-dihydro-2-thioxo-4(1 H )quinazolinone. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also supported by MEXT/JSPS KAKENHI Grant Numbers 22370047 [Grant-in-Aid for Scientifi c Research (B)], 23116505 (Grant-in-Aid for Scientifi c Research on Innovative Areas), 25116704 (Grant-in-Aid for Scientifi c Research on Innovative Areas), 26291017 [Grant-in-Aid for Scientifi c Research (B)], and 15K14470 (Grant

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

Background: Diacylglycerol (DG) kinase (DGK) ␦ is activated by acute high glucose stimulation. Results: DGK␦ high glucose-dependently phosphorylates 30:0-, 32:0-, and 34:0-DG and interacts with phosphatidylcholinespecific phospholipase C (PC-PLC). Conclusion: DGK␦ utilizes palmitic acid-containing DG species and metabolically connects with PC-PLC. Significance: The newly identified PC-PLC/DGK␦ pathway could play an important role in insulin signaling and glucose uptake.

Diacylglycerol (DG) kinase (DGK), which phosphorylates DG to generate phosphatidic acid (PA), consists of ten isozymes (α-к). Recently, we identified a novel small molecule inhibitor, CU-3, that selectively inhibits the activity of the α isozyme. In addition, we newly obtained Compound A, which selectively and strongly inhibits type I DGKs (α, β, and γ). In the present study, we demonstrated that both CU-3 and Compound A induced apoptosis (caspase 3/7 activity and DNA fragmentation) and viability reduction of AKI melanoma cells. Liquid chromatography-mass spectrometry revealed that the production of 32:0-and 34:0-PA species was commonly attenuated by CU-3 and Compound A, suggesting that lower levels of these PA molecular species are involved in the apoptosis induction and viability reduction of AKI cells. We determined the effects of the DGKα inhibitors on several other cancer cell lines derived from refractory cancers. In addition to melanoma, the DGKα inhibitors enhanced caspase 3/7 activity and reduced the viability of hepatocellular carcinoma, glioblastoma, and pancreatic cancer cells, but not breast adenocarcinoma cells.Interestingly, Western blot analysis indicated that the DGKα expression levels were positively correlated with the sensitivity to the DGK inhibitors. Because both CU-3 and Compound A induced interleukin-2 production by T cells, it is believed that these two compounds can enhance cancer immunity. Taken together, our results suggest that DGKα inhibitors are promising anticancer drugs. K E Y W O R D S apoptosis, cancer, diacylglycerol kinase (DGK), inhibitor, melanoma, phosphatidic acid (PA)