8-Geranylumbelliferone isolated from Paramignya trimera triggers RIPK1/RIPK3-dependent programmed cell death upon TNFR1 ligation.

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.

3-O-acetylrubianol C (3AR-C) induces RIPK1-dependent programmed cell death by selective inhibition of IKKß.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKß. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKß phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKß inhibitor to overcome TNF resistance in cancer therpay.

Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis.

Plant-derived lignans have numerous biological effects including anti-tumor and anti-inflammatory activities. Screening of purified constituents of Rubia philippinensis from human glioblastoma cells resistant to TNF-related apoptosis-inducing ligand (TRAIL) has suggested that the lignan pinoresinol was a highly active TRAIL sensitizer. Here we show that treatment with nontoxic doses of pinoresinol in combination with TRAIL induced rapid apoptosis and caspase activation in many types of glioblastoma cells, but not in normal astrocytes. Analyses of apoptotic signaling events revealed that pinoresinol enhanced the formation of TRAIL-mediated death-inducing signaling complex (DISC) and complete processing of procaspase-8 within the DISC in glioblastoma cells, in which caspase-8 was inactivated. Mechanistically, pinoresinol downregulated the expression of cellular FLICE-inhibitory protein (cFLIPL) and survivin through proteasome-mediated degradation, without affecting death receptors or downstream intracellular apoptosis-related proteins. Furthermore, the sensitization of TRAIL-mediated apoptosis by pinoresinol strictly depended on the expression level of cFLIPL, which was regulated through de novo protein synthesis, rather than by NF-κB or p53 signaling. Taken together, our results indicate that pinoresinol facilitates DISC-mediated caspase-8 activation by targeting cFLIPL in an early event in apoptotic signaling, which provides a potential therapeutic module for TRAIL-based chemotherapy.

Post-translational modification of the death receptor complex as a potential therapeutic target in cancer.

Programmed cell death is critical to the physiological function of multi-cellular organisms, controlling development, immunity, inflammation, and cancer progression. Death receptor (DR)-mediated regulation of a protease functions as a second messenger to initiate a death signal cascade to induce apoptosis or necroptosis. Recently, it has become clear that post-translational modifications (PTMs) of signaling components in the DR complex are highly complex, temporally controlled, and tightly regulated, and play an important role in cell death signaling. This review focuses on the molecular mechanisms and pathophysiological consequences of PTMs on the formation of the DR signaling complex, especially with respect to tumor necrosis factor receptor 1 (TNFR1). Furthermore, characterization of the role of PTMs in spatially different TNFR1 complexes (complexes I and II), especially with respect to the role of ubiquitination and phosphorylation of receptor interacting protein 1 (RIP1) in programmed cell death in cancer cells, will be reviewed. By integrating recently gained insight of the functional importance of PTMs in complex I or II, this review discusses how the concerted action of PTMs results in life or death upon DR ligation. Finally, the emerging concept of a sequential cell death checkpoint by the PTMs of RIP1, which may reveal novel therapeutic opportunities for the treatment of some cancers, will be discussed.

C-27-carboxylated oleanane triterpenoids up-regulate TRAIL DISC assembly via p38 MAPK and CHOP-mediated DR5 expression in human glioblastoma cells.

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3ß-hydroxyolean-12-en-27-oic acid (C27OA-1), 3ß,6ß,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3ß-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.

Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma.

Although gene therapy was regarded as a promising approach for glioma treatment, its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems. Mesenchymal stem cells(MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy. Therefore, in this study, we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus. We firstly compared the infectivity of type 3, type 5, and type 35 fiber-modified adenoviruses in MSCs. We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo. Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus. MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups. In conclusion, MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma, making it a potential therapeutic strategy for treating malignant glioma.