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Exposure of cells to ionizing radiation induces activation of multiple signaling pathways that play critical roles in determining cell fate. However, the molecular basis for cell death or survival signaling in response to radiation is unclear at present. Here, we show opposing roles of the c-jun NH 2 -terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways in the mitochondrial cell death in response to ionizing radiation in human cervical cancer cells. Ionizing radiation triggered Bax and Bak activation, Bcl-2 down-regulation, and subsequent mitochondrial cell death. Inhibition of JNK completely suppressed radiation-induced Bax and Bak activation and Bcl-2 down-regulation. Dominant-negative forms of stress-activated protein kinase/extracellular signal-regulated kinase kinase 1 (SEK-1)/mitogen-activated protein kinase kinase-4 (MKK-4) inhibited JNK activation. Radiation also induced phosphoinositide 3-kinase (PI3K) activation. Interestingly, inhibition of PI3K effectively attenuated radiation-induced mitochondrial cell death and increased clonogenic survival. Inhibition of PI3K also suppressed SEK-1/MKK-4 and JNK activation, Bax and Bak activation, and Bcl-2 down-regulation. In contrast, inhibition of p38 MAPK led to enhanced Bax and Bak activation and mitochondrial cell death. RacN17, a dominant-negative form of Rac1, inhibited p38 MAPK activation and increased Bax and Bak activation. Exposure of cells to radiation also induced selective activation of c-Src among Src family kinases. Inhibition of c-Src by pretreatment with Src family kinase inhibitor PP2 or small interfering RNA targeting of c-Src attenuated radiation-induced p38 MAPK and Rac1 activation and enhanced Bax and Bak activation and cell death. Our results support the notion that the PI3K-SEK-1/MKK-4-JNK pathway is required for the mitochondrial cell death in response to radiation, whereas the c-Src-Rac1-p38 MAPK pathway plays a cytoprotective role against mitochondrial cell death.

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Abstract. Sensitization of cancer cells to TRAIL could improve the effectiveness of TRAIL as an anticancer agent. We explored whether TRAIL in combination with phytosphingosine could sensitize cancer cells to TRAIL. The combined treatment enhanced synergistic apoptotic cell death of Jurkat T cells, compared to TRAIL or phytosphingosine alone. Enhanced apoptosis in response to the combination treatment was associated with caspase-8 activation-mediated Bax and Bak activation and mitochondrial dysfunction. The combination treatment also resulted in synergistic up-regulation of TRAIL receptor R1 (DR4) and R2 (DR5). siRNA targeting of DR5 significantly attenuated the combination treatmentinduced caspase-8 activation, mitochondrial dysfunction, and apoptotic cell death. Upon stimulation of cells with the combination treatment, NF-κB was activated. Moreover, siRNA targeting of NF-κB significantly attenuated the combination treatment-induced DR4 and DR5 expression and receptor-mediated caspase-8 activation. These results indicate that phytosphingosine sensitizes cancer cells to TRAIL through the synergistic up-regulation of DR4 and DR5 in an NF-κB-dependent fashion resulting in caspase-8 activation and subsequent mitochondrial dysfunction. These findings support the potential application of combination treatment with TRAIL and phytosphingosine in the treatment of cancers that are less sensitive to TRAIL. Introduction Cytotoxic molecules have been considered as potential new therapeutics for drug-resistant cancer cells. Tumor necrosis factor (TNF)1 -related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, which includes TNF, FasL, CD27L, OX40, CD30L and CD40L. All members in this superfamily are type II membrane proteins, and many of them play an important role in regulating important biological processes such as apoptosis, cytokine production, and other cellular activities (1,2). Whereas other anti-cancer molecules such as TNF-· and Fas3L cause life-threatening toxicities, TRAIL induces apoptosis in various tumor cells but not in normal cells (3-5). Due to its apparent highly selective tumoricidal activity, TRAIL may be useful as a new therapeutic agent against cancer (6-8).TRAIL can interact with at least 4 cell membrane-anchored receptors, including the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which contain an intracytoplasmic death domain and are therefore capable of triggering the apoptotic death signal, and the decoy receptors TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2), which do not contain an intact cytoplasmic death domain and are therefore incapable of transmitting a death signal. Triggering of proapoptotic receptors by TRAIL induces recruitment of a DISC consisting of FADD and procaspase-8, resulting in cleavage and activation of caspase-8 followed by proteolytic initiation of effector caspases, such as caspase-3 (9).Although TRAIL is a potent inducer of apoptosis in certain tumor cell types, some cancer cells are not sensitive to TRAILmediated apoptosis (10-12). We previously showed tha...