Diacylglycerol kinase α‐selective inhibitors induce apoptosis and reduce viability of melanoma and several other cancer cell lines

Abstract

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)

Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

“…A,B). An inhibitor of type I DGK isozymes (DGKα, β, and γ), Compound A (IC 50 for DGKβ: 0.02 μ m ) , nullified the colocalization of EGFP‐DGKβ and DsRed‐α‐Syn‐N (Fig. C,D), indicating that the colocalization occurred in a DGK activity (PA)‐dependent manner and that α‐Syn‐N does not recognize the DGKβ protein itself.…”

“…We recently demonstrated that DGKδ produced 14:0/16:0‐, 14:0/16:1‐, 16:0/16:0‐, 16:0/16:1‐, 16:0/18:0‐, and 16:0/18:1‐PA in glucose‐stimulated C2C12 cells . Moreover, the production of 16:0/16:0‐ and 16:0/18:0‐PA species was attenuated by DGKα‐specific inhibitors . DGKζ exclusively generates very restricted PA species, 16:0/16:0‐PA, during the initial/early stage of neuroblastoma cell differentiation .…”

1‐Stearoyl‐2‐docosahexaenoyl‐phosphatidic acid interacts with and activates Praja‐1, the E3 ubiquitin ligase acting on the serotonin transporter in the brain

“…Phosphatidic acid species in the extracted lipids (10 μL) were analyzed by LC‐MS and LC‐MS/MS as previously described [16‐19]. Diacylglycerol species in the extracted lipids (10 μL) were analyzed by LC‐MS and LC‐MS/MS as previously described [20].…”

Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography–Tandem Mass Spectrometry

“…COS‐7 cell lysates (20 μg) were separated by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE). Western blotting was performed as described previously (Liu et al, ; Yamaki et al, ) using anti‐GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) antibodies.…”