Abstract
Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.
Highlights
Sorafenib (Nexavar, BAY43-9006) was initially identified as a Raf1 kinase inhibitor by high-throughput screening, subsequent studies showed that it has additional activity against the serine/threonine kinases B-Raf, B-Raf(V600E); p38 mitogen-activated protein kinase; the receptor tyrosine kinases c-kit, Flt3, RET; and the proangiogenic receptor kinases vascular endothelial growth factor receptors 1, 2, 3; and platelet-derived growth factor receptor
Either simultaneous exposure to sorafenib/tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) (24 h) or preexposure to TRAIL followed by sorafenib (T24 h!S24 h) resulted in modest activity (e.g., f45–50% lethality; data not shown), 24 h exposure to sorafenib followed by addition of TRAIL concentrations z50 ng/mL (i.e., 50, 75, 100 ng/mL) markedly increased cell death, ranging from 60% for 5 Amol/L sorafenib to >80% to 90% in the case of 7.5 Amol/L sorafenib (Fig. 1A)
The rationale for the present studies stemmed from recent observations showing that, first, exposure of human leukemia cells to sorafenib induced apoptosis through a mechanism involving Mcl-1 down-regulation via inhibition of translation [5]; and second, evidence that Mcl-1 plays an important role in regulating the sensitivity of cells to TRAIL-induced apoptosis [11,12,13]
Summary
Sorafenib (Nexavar, BAY43-9006) was initially identified as a Raf kinase inhibitor by high-throughput screening, subsequent studies showed that it has additional activity against the serine/threonine kinases B-Raf, B-Raf(V600E); p38 mitogen-activated protein kinase; the receptor tyrosine kinases c-kit, Flt, RET; and the proangiogenic receptor kinases vascular endothelial growth factor receptors 1, 2, 3; and platelet-derived growth factor receptor (reviewed in ref. 1). Sorafenib was recently approved by the Food and Drug Administration for treatment of advanced renal cell carcinoma [2]. Through these actions, sorafenib blocks neoplastic cell signaling pathways, those involved in growth and vascularization [3, 4] and avoidance of cell death [3, 5,6,7]. The ability of sorafenib to down-regulate Mcl-1 and inactivate eIF4E has been confirmed in vivo in a human hepatocellular carcinoma tumor xenograft model [3]
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