USP14 inhibition regulates tumorigenesis by inducing apoptosis in gastric cancer.

Deubiquitinases (DUBs) are an essential component of the ubiquitin-proteasome system (UPS). They trim ubiquitin from substrate proteins, thereby preventing them from degradation, and modulate different cellular processes. Ubiquitin-specific protease 14 (USP14) is a DUB that has mainly been studied for its role in tumorigenesis in several cancers. In the present study, we found that the protein levels of USP14 were remarkably higher in gastric cancer tissues than in the adjacent normal tissues. We also demonstrated that the inhibition of USP14 activity using IU1 (an USP14 inhibitor) or the inhibition of USP14 expression using USP14-specific siRNA markedly reduced the viability of gastric cancer cells and suppressed their migratory and invasive abilities. The reduction in gastric cancer cell proliferation due to the inhibition of USP14 activity was a result of the increase in the degree of apoptosis, as evidenced by the increased expression levels of cleaved caspase-3 and cleaved PARP. Furthermore, an experiment using the USP14 inhibitor IU1 revealed that the inhibition of USP14 activity suppressed 5-fluorouracil (5-FU) resistance in GC cells. Collectively, these findings indicate that USP14 plays critical roles in gastric cancer progression and suggest its potential to serve as a novel therapeutic target for gastric cancer treatment. [BMB Reports 2023; 56(8): 451-456].

Sulforaphane inhibits PDGF-induced proliferation of rat aortic vascular smooth muscle cell by up-regulation of p53 leading to G1/S cell cycle arrest.

Vascular diseases such as atherosclerosis and restenosis artery angioplasty are associated with vascular smooth muscle cell (VSMC) proliferation and intimal thickening arterial walls. In the present study, we investigated the inhibitory effects of sulforaphane, an isothiocyanate produced in cruciferous vegetables, on VSMC proliferation and neointimal formation in a rat carotid artery injury model. Sulforaphane at the concentrations of 0.5, 1.0, and 2.0 µM significantly inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation in a concentration-dependent manner, determined by cell count. The IC50 value of sulforaphane-inhibited VSMC proliferation was 0.8 µM. Sulforaphane increased the cyclin-dependent kinase inhibitor p21 and p53 levels, while it decreased CDK2 and cyclin E expression. The effects of sulforaphane on vascular thickening were determined 14 days after the injury to the rat carotid artery. The angiographic mean luminary diameters of the group treated with 2 and 4 µM sulforaphane were 0.25±0.1 and 0.09±0.1 mm², respectively, while the value of the control groups was 0.40±0.1 mm², indicating that sulforaphane may inhibit neointimal formation. The expression of PCNA, maker for cell cycle arrest, was decreased, while that of p53 and p21 was increased, which showed the same pattern as one in in-vitro study. These results suggest that sulforaphane-inhibited VSMC proliferation may occur through the G1/S cell cycle arrest by up-regulation of p53 signaling pathway, and then lead to the decreased neointimal hyperplasia thickening. Thus, sulforaphane may be a promising candidate for the therapy of atherosclerosis and post-angiography restenosis.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 µM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5-2 µM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway.

Anti-platelet activity of diacetylated obovatol through regulating cyclooxygenase and lipoxygenase activities.

Obovatol has been reported biological activities such as muscle relaxative, anti-gastric ulcer, anti-allergic and anti-bacterial activities. The present study was undertaken to investigate the effect of diacetylated obovatol, an obovatol derivative, on rabbit platelet aggregation, and their possible molecular mechanisms. Effects of diacetylated obovatol on platelet activation including aggregation and serotonin secretion were examined. In addition, we investigated the effect of diacetylated obovatol on archidonic acid and metabolites liberation and intracellular calcium mobilization. Diacetylated obovatol concentration-dependently inhibited the washed rabbit platelet aggregation induced by collagen and arachidonic acid, suggesting that diacetylated obovatol may selectively inhibits collagen- and arachidonic acid-mediated signal transduction. In accordance with these results, diacetylated obovatol showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization and serotonin secretion. However, diacetylated obovatol did not inhibit arachidonic acid liberation; on the other hand, diacetylated obovatol inhibited the formation of arachidonic acid metabolites such as thromboxane A(2), prostaglandin D(2) and 12-HETE through interfering with cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. The results demonstrated that diacetylated obovatol has antiplatelet activities through inhibition of COX-1 and LOX activities.

JJK694, a synthesized obovatol derivative, inhibits platelet activation by suppressing cyclooxygenase and lipoxygenase activities.

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

Inhibitory effects of OD 78 [3-(4-bromo-phenoxy)-4,5-dihydroxybenzoic acid-methyl ester] on the proliferation and migration of TNF-α-induced rat aortic smooth muscle cells.

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the formation and progression of intimal thickening in early-phase atherosclerosis and in restenosis after vascular injury. Tumor necrosis factor-α (TNF-α) is released from macrophages in atherosclerotic lesions and from neointimal vascular smooth muscle cells after balloon-injury. Obovatol, a major biphenolic component isolated from the Magnolia obovata leaf, is known to have anti-inflammatory and antitumor activities. The goal of this study was to examine the cardioprotective effects of the obovatol derivative OD 78 on the TNF-α-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs). The antiproliferative effects of OD 78 on RASMCs were examined by cell counting and [(3)H]-thymidine incorporation assays. Treatment of cells with 1-4 µM OD 78 inhibited the proliferation and DNA synthesis of TNF-α-stimulated RASMCs in a concentration-dependent manner, without cytotoxicity. Treatment with OD 78 inhibited TNF-α-mediated p38 phosphorylation, but did not change the activation of extracellular signal-regulated kinase or c-Jun N-terminal kinase. Furthermore, treatment with OD 78 decreased TNF-α-induced levels of cyclin E, cyclin D1, CDK2, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein, but not the CDK4 expression level. Also, OD 78 inhibits the migration of TNF-α-induced RASMC in transwells. OD 78 treatment strongly decreased matrix metalloproteinase-9 (MMP-9) expression in a dose-dependent manner, but the MMP-2 expression was unchanged. These results show that OD 78 may be developed as a potential antiproliferative agent for the treatment of angioplasty restenosis and atherosclerosis.

Antiplatelet activity of J78 (2-Chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization.

We previously reported that J78 (2-chloro-3-[2'-bromo, 4'-fluoro-phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, exhibited a potent antithrombotic effect, which might be due to antiplatelet rather than anticoagulation activity. In the present study, possible anti-platelet mechanism of J78 was investigated. J78 concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/ml), thrombin (0.05 U/ml), arachidonic acid (100 microM), and U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F(2); 1 microM), a thromboxane (TX) A(2) mimic, with IC(50) values of 0.32 +/- 0.01, 0.44 +/- 0.02, 0.50 +/- 0.04, and 0.36 +/- 0.02 microM, respectively. J78 also produced a shift to the right of the concentration-response curve of U46619, indicating an antagonistic effect on the TXA(2) receptor. J78 concentration-dependently inhibited collagen-induced arachidonic acid liberation. In addition, J78 potently suppressed TXA(2) formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner but had no effect on the production of PGD(2), indicating an inhibitory effect on TXA(2) synthase. This was supported by a TXA(2) synthase activity assay that J78 concentration-dependently inhibited TXB(2) formation converted from PGH(2). Furthermore, J78 was also able to inhibit the [Ca(2+)](i) mobilization induced by collagen or thrombin at such a concentration that completely inhibited platelet aggregation. Taken together, these results suggest that the antiplatelet activity of J78 may be mediated by TXA(2) receptor blockade with TXA(2) synthase inhibition and suppression of cytosolic Ca(2+) mobilization.