3-O-trans-p-coumaroyl-alphitolic acid, a triterpenoid from Zizyphus jujuba, leads to apoptotic cell death in human leukemia cells through reactive oxygen species production and activation of the unfolded protein response.

Abstract

3-O-trans-p-coumaroyl-alphitolic acid (3OTPCA), a triterpenoid isolated from the plant Zizyphus jujuba (ZJ), is known to be cytotoxic to cancer cells; however, the molecular mechanism underlying 3OTPCA-induced cell death remains unknown. Here, we provide novel evidence that 3OTPCA induces apoptotic cell death in human leukemia cells. We found that 3OPTCA induces DNA fragmentation within 24 h after treatment in U937 cells, which was also observed in other leukemia cell lines, including Molt-4 and Jurkat cells. We then investigated other parameters involved in apoptosis, including phosphatidylserine externalization and caspase-3 cleavage in U937 cells treated with 3OTPCA. 3OTPCA caused significant DNA fragmentation, annexin-V binding, and caspase-3 cleavage, indicating that 3OTPCA exerts cytotoxicity through apoptosis induction. RNA-seq analysis revealed that the expression of transcripts associated with the unfolded protein response (UPR), such as spliced XBP-1 and CHOP, were up-regulated by 3OTPCA treatment. 3OTPCA-induced UPR activation may be due to endoplasmic reticulum (ER) stress because both 3OTPCA and thapsigargin, an endoplasmic Ca2+ transport ATPase inhibitor, increased intracellular calcium levels. 3OTPCA down-regulated the expression of Bcl-2, a target of CHOP, and led to the loss of the mitochondrial membrane, indicating that the intrinsic (mitochondrial) apoptotic pathway was triggered by 3OTPCA, likely through UPR activation. Furthermore, we found that 3OTPCA induced superoxide anion generation and, following p38 MAPK phosphorylation, caspase-8 cleavage without affecting Fas expression. It also induced subsequent Bid cleavage, which may enhance the apoptosis triggered by the intrinsic pathway. These findings reveal for the first time that 3OTPCA induces apoptotic cell death through the generation of reactive oxygen species and activation of UPR.