Abstract
Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIPL negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIPL inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIPL) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIPL, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of c-FLIPL prevented TNF-α-induced JNK inactivation, whereas c-FLIPL overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIPL expression, thus inducing TK-10 cell apoptosis.
Highlights
Introduction cFLICEinhibitory protein (FLIP), a master anti-apoptotic mediator that acts via preventing the activation of caspase-8/10 homologue [1], is involved in TRAIL, Fas, TNF-α, and chemotherapeutic drug resistance in various human cancers [2]. Cellular FLICE inhibitory protein (c-FLIP) has 13 splice variants, three of which are expressed as proteins: c-FLIP long form (c-FLIPL) (55 kDa, long form), c-FLIPS (26 kDa, short form), and c-FLIPR (24-kDa form) [1]
While 20 μg/ml cordycepin caused no drastic change in the Annexin V-stained viable fraction of TK-10 cells (95% to 84%), 40 μg/ml cordycepin shifted the cells to the late apoptotic stage (3% to 14%)
Several recent reports suggested that cordycepin exerts its anticancer and antimetastatic effects by inhibiting the expression of some inflammatory genes [28] and some molecules critically involved in tumor growth and metastasis [29,30], by suppressing NF-κB
Summary
Introduction cFLIP, a master anti-apoptotic mediator that acts via preventing the activation of caspase-8/10 homologue [1], is involved in TRAIL, Fas, TNF-α, and chemotherapeutic drug resistance in various human cancers [2]. c-FLIP has 13 splice variants, three of which are expressed as proteins: c-FLIPL (55 kDa, long form), c-FLIPS (26 kDa, short form), and c-FLIPR (24-kDa form) [1]. C-FLIP has 13 splice variants, three of which are expressed as proteins: c-FLIPL (55 kDa, long form), c-FLIPS (26 kDa, short form), and c-FLIPR (24-kDa form) [1]. These proteins affect other cellular functions, including increasing cell growth rate and decreasing cell differentiation [2,3]. The pro-apoptotic JNK is the downstream component of major mitogenactivated protein kinase (MAPK) cascades, including the extracellular signal-regulated kinase 1 and 2 (ERK 1 and 2) and p38 cascades. JNK and p38 MAPK family members function in a cell-type- and context-specific manner to integrate signals that affect proliferation, differentiation, survival, and migration [13,14]. GADD45B, a pivotal survival factor downstream of NF-κB, is involved in the crosstalk between NF-κB and JNK and modulates JNK activation by binding to and inhibiting the JNK kinase, MKK7 [16,17]
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