Mitsuhashi Yohei, Furusawa Yukihiro, Aradate Tadashi, Zhao Qing-Li, Moniruzzaman Rohan, Kanamori Masahiko, Noguchi Kyo, Kondo Takashi
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama, Japan.
Department of Liberal Arts and Sciences, Toyama Prefectural University, Kurokawa, Toyama, Japan.
PLoS One. 2017 Aug 23;12(8):e0183712. doi: 10.1371/journal.pone.0183712. eCollection 2017.
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.
3 - O - 反式 - 对香豆酰齐墩果酸（3OTPCA）是从植物酸枣（ZJ）中分离出的一种三萜类化合物，已知对癌细胞具有细胞毒性；然而，3OTPCA诱导细胞死亡的分子机制仍不清楚。在此，我们提供了新的证据表明3OTPCA可诱导人白血病细胞发生凋亡性细胞死亡。我们发现，3OPTCA在处理U937细胞24小时内可诱导DNA片段化，在包括Molt - 4和Jurkat细胞在内的其他白血病细胞系中也观察到了这一现象。然后，我们研究了3OTPCA处理的U937细胞中参与凋亡的其他参数，包括磷脂酰丝氨酸外翻和半胱天冬酶 - 3的切割。3OTPCA导致显著的DNA片段化、膜联蛋白 - V结合以及半胱天冬酶 - 3的切割，表明3OTPCA通过诱导凋亡发挥细胞毒性作用。RNA测序分析显示，与未折叠蛋白反应（UPR）相关的转录本，如剪接的XBP - 1和CHOP的表达，在3OTPCA处理后上调。3OTPCA诱导的UPR激活可能是由于内质网（ER）应激，因为3OTPCA和内质网Ca2 + 转运ATP酶抑制剂毒胡萝卜素均增加了细胞内钙水平。3OTPCA下调了CHOP的靶标Bcl - 2的表达，并导致线粒体膜电位丧失，表明3OTPCA可能通过激活UPR触发了内源性（线粒体）凋亡途径。此外，我们发现3OTPCA诱导超氧阴离子生成，并在p38丝裂原活化蛋白激酶磷酸化后导致半胱天冬酶 - 8切割，而不影响Fas表达。它还诱导了随后的Bid切割，这可能增强内源性途径触发的凋亡。这些发现首次揭示了3OTPCA通过产生活性氧和激活UPR诱导凋亡性细胞死亡。
