Triptolide Sensitizes Leukemic Cells to TRAIL Induced Apoptosis Via Decrease of XIAP and p53-Mediated Increase of DR5.
Authors: Carter Bing Z. , Mak Duncan H. , Schober Wendy D. , Dietrich Martin , Pinilla Clemencia , Reed John C. , Andreeff Michael 
Published: October 2019

Abstract
Triptolide, a recently identified anticancer agent from a Chinese herb, has been shown to synergistically enhance TRAIL-induced cell death in various solid tumor cell lines. We have found that triptolide potently induces apoptosis in leukemic cell lines and blasts from AML patients at least in part by decreasing XIAP levels. XIAP is known to be a resistance factor in TRAIL-induced cell death. XIAP is highly expressed in AML blasts and primary AML cells generally are insensitive to TRAIL. We therefore hypothesize that triptolide will sensitize AML cells to TRAIL-induced apoptosis. We treated OCI-AML3, U937, and Jurkat cells with sub-optimal concentrations of triptolide, TRAIL, and their combination. At concentrations showing no or minimal effects by each agent alone, their combinations significantly promoted cell death with combination indices (CI) <1.0 for all three leukemia cell lines. To ensure that XIAP contributes to TRAIL resistance, we treated U937 cells overexpressing XIAP (U937XIAP) and the control cells (U937neo) with TRAIL. U937XIAP cells were 6-fold more resistant to TRAIL (IC50=455.8 ng/ml) than U937neo cells (IC50=74.3 ng/ml). We then treated these cells with TRAIL and 1396–11, a small molecule XIAP antagonist that binds to BIR2 of XIAP and promotes caspase-dependent apoptosis. At 3μM, 1396–11 had no significant effect on survival of either U937neo or U939XIAP cells. When combined with TRAIL, 1396–11 augmented TRAIL-induced cell death of both U937neo (IC50=45.0 ng/ml with 1396-11 vs. IC50=74.3 ng/ml without) and U939XIAP cells (IC50=318.9 ng/ml with 1396–11 vs. IC50=455.8 ng/ml without). Furthermore, we observed that triptolide decreased MDM2 and increased p53 protein levels in p53 wild type OCI-AML3 but not in p53 null U937 cells. TRAIL receptor DR5 has been shown to be regulated by p53. To elucidate the role of p53 in TRAIL-induced cell death sensitized by triptolide, we treated OCI-AML3 and U937 cells with triptolide and examined DR5 expression. We found that triptolide induced DR5 protein levels in OCI-AML3, but much less so in U937 cells. Treatment of OCI-AML3 cells with Nutlin3a, a MDM2 inhibitor that binds to MDM2 and stabilizes p53, increased DR5 protein levels and sensitized to TRAIL-induced cell death. Knockdown of p53 with retrovirus expressing p53siRNA in OCI-AML3 cells abolished cellular responses to nutlin3a and significantly decreased the sensitization to TRAIL by nutlin3a. Finally, we treated OCI-AML3 cells with 1396–11, nutlin3a, and TRAIL and our results showed that the triple combination (CI=0.045 at 24 hours, average of ED50, ED75, and ED90) was more effective in inducing cell death than either 1396–11 and TRAIL (CI=0.066) or nutlin3a and TRAIL (CI=0.190) combinations supporting our notion that triptolide sensitizes to TRAIL-induced cell death by modulating both XIAP expression and p53 signaling. Collectively, our studies suggest that inhibition of XIAP and induction of DR5 mediated by p53 activation both independently sensitize leukemic cells to TRAIL-induced apoptosis. Triptolide not only inhibits XIAP, which is overexpressed in AML, but also activates p53 signaling, which is intact in the majority of AMLs. Thus, combinations of triptolide and TRAIL may provide a novel strategy for treating AML patients by overcoming critical mechanisms of apoptosis resistance.