Thiourea compound AW00178 sensitizes human H1299 lung carcinoma cells to TRAIL-mediated apoptosis.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a wide variety of cancer cells. Recently, cancer cell resistance to TRAIL-mediated apoptosis has become a challenging issue in the development of TRAIL-based anti-cancer therapies. In this study, we found that 1-(5-chloro-2-methyl-phenyl)-3-[4-(5-trifluoromethyl-pyrazol-1-yl)-phenyl]-thiourea (AW00178) was able to sensitize TRAIL-resistant human lung cancer H1299 cells to TRAIL-mediated apoptosis. Treatment with AW00178, either alone or in combination with TRAIL, induced the expression of CHOP, a protein related to TRAIL sensitivity, and reduced the expression of survivin, an anti-apoptotic protein involved in TRAIL resistance. Additionally, AW00178, alone or in combination with TRAIL, induced the activation of c-Jun and inactivation of Akt. A pharmacologic inhibition study revealed that c-Jun activation and Akt inactivation were strongly related to CHOP induction and survivin down-regulation, respectively. In summary, these results suggested that AW00178 mediated sensitization to TRAIL-mediated apoptosis in H1299 cells by increasing sensitivity and decreasing resistance to TRAIL via the induction of c-Jun-dependent CHOP expression and the reduction of Akt-dependent survivin expression, respectively.

AW00179 potentiates TRAIL-mediated death of human lung cancer H1299 cells through ROS-JNK-c-Jun-mediated up-regulation of DR5 and down-regulation of anti-apoptotic molecules.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is not effective at treating TRAIL-resistant tumors. This resistance is challenging for TRAIL-based anti-cancer therapies. In this study, we found that 1-(4-trifluoromethoxy-phenyl)-3-[4-(5-trifluoromethyl-2,5-dihydro-pyrazol-1-yl)-phenyl]-urea (AW00179) sensitized human lung cancer H1299 cells to TRAIL-mediated apoptosis. Even in the absence of TRAIL, AW00179 strongly induced DR5 expression and decreased the expression of anti-apoptotic proteins, suggesting that the sensitizing effect of AW00179 on TRAIL-mediated apoptosis is due to increased levels of DR5 protein and decreased anti-apoptotic molecules. AW00179 also induced the activation of c-Jun and ERK; however, a pharmacologic inhibition study revealed that JNK-c-Jun signaling is involved in the induction of DR5 expression. In addition, reactive oxygen species (ROS) appear to be involved in AW00179 activity. In conclusion, AW00179 has the potential to sensitize H1299 cells to TRAIL-mediated apoptosis through two distinct mechanisms: ROS-JNK-c-Jun-mediated up-regulation of DR5, and down-regulation of anti-apoptotic molecules.

Calmodulin inhibition contributes to sensitize TRAIL-induced apoptosis in human lung cancer H1299 cells.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells. However, TRAIL alone is not effective in treating TRAIL-resistant tumors. We evaluated the effect of 180 enzyme inhibitors on TRAIL-induced apoptosis in human lung cancer H1299 cells, and found fluphenazine-N-2-chloroethane (a calmodulin (CaM) antagonist) sensitized TRAIL-induced apoptosis. Interestingly, in the presence of TRAIL, it increased caspase-8 binding to the Fas-associated death domain (FADD), but decreased binding of FADD-like interleukin-1beta-converting enzyme inhibitory proteins (FLIPs). Additionally, its combination with TRAIL inhibited Akt phosphorylation. These results were consistently observed in cells treated with CaM siRNA. We suggested the blockade of CaM could sensitize lung cancer cells to TRAIL-induced apoptosis in at least 2 ways: (i) it can activate death-inducing signaling complex mediated apoptosis by inhibiting TRAIL-induced binding of FLIP and TRAIL-enhanced binding of caspase-8 to FADD; (ii) it can inhibit Akt phosphorylation, consequently leading to decreased expression of anti-apoptotic molecules such as FLIP and members of the inhibitor of apoptosis protein family. This study suggests the combination of CaM antagonists with TRAIL may have the therapeutic potential to overcome the resistance of lung cancers to apoptosis.

Putative therapeutic agents for the learning and memory deficits of people with Down syndrome.

Mental retardation is the most common and debilitating condition for individuals with Down syndrome (DS). The hyper-activation of DYRK1A by overexpression causes significant learning and memory deficits in DS-model mice. Thus far, no mechanism-based drug has been developed to address this. After a combination of in silico and in vitro screenings, two DYRK1A inhibitors were isolated that are active in a cell-based assay. Further optimization could lead to a novel drug discovery that could address DS learning and memory deficits.

Regulation of Dyrk1A kinase activity by 14-3-3.

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.

Inhibition of rho-associated kinase reduces MLC20 phosphorylation and contractility of intact myometrium and attenuates agonist-induced Ca2+ sensitization of force of permeabilized rat myometrium.

The role of rhoA/rho-associated kinase (ROK) signaling pathways in agonist-induced contraction of the rat myometrium was investigated. We measured the [Ca(2+)](i)-force relationship, phosphorylation of myosin regulatory light chains (MLC(20)) in intact tissue and the Ca(2+)-sensitization of force in permeabilized myometrial cells of rat. In measurements of the relationship between [Ca(2+)](i) and tension in intact tissue, Y-27632, a ROK inhibitor, significantly attenuated the carbachol-induced contraction without changing [Ca (2+)](i). Phosphorylation of MLC(20) was increased by carbachol and this increased phosphorylation was blocked by treatment of tissue with Y-27632. In tension measurements of single hyperpermeable cells, carbachol evoked sustained contraction at constant pCa 6.7 and these agonist-induced contractions were decreased by treatment with Y-27632. These results suggest that activation of a ROK-mediated signaling pathway(s) plays an important role in agonist-induced alterations in MLC(20) phosphorylation and force of rat myometrium.