Li Peng, Zhao Qing-Li, Jawaid Paras, Rehman Mati Ur, Ahmed Kanwal, Sakurai Hiroaki, Kondo Takashi
a Department of Radiological Sciences , Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama , Toyama , Japan.
c Department of Basic Medical Sciences , College of Medicine, King Saud Bin Abdulaziz University of Health Sciences , Jeddah , Kingdom of Saudi Arabia.
Int J Hyperthermia. 2017 Jun;33(4):411-418. doi: 10.1080/02656736.2017.1278629. Epub 2017 Jan 22.
Transforming growth factor-β-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line.
Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity.
Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis.
Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.
转化生长因子-β激活激酶1（TAK1）在应对多种应激时发挥抗凋亡作用。TAK1抑制剂5Z-7-氧代玉米烯醇（OZ）的凋亡效应已得到研究。然而，OZ与物理应激的联合作用仍不清楚。因此，在本研究中，我们聚焦于使用Molt-4细胞系来确定OZ与热疗（HT）的联合作用。
用OZ预处理Molt-4细胞1小时，随后进行热暴露（44°C，10分钟），并在37°C孵育24小时后收获细胞，通过流式细胞术使用Annexin V-FITC/PI双染法测定细胞凋亡，并通过细胞计数法观察细胞生长。通过测量线粒体膜电位（MMP）、细胞内活性氧生成、凋亡相关蛋白的表达、细胞内钙离子水平和Fas活性来研究联合作用涉及的进一步机制。
OZ与HT联合显著增强MMP丧失和超氧化物生成。此外，OZ预处理促进半胱天冬酶-8的切割、Fas外化、半胱天冬酶3活性和细胞内钙离子水平。OZ预处理降低了HT诱导的Bcl-2表达，并增加了促凋亡蛋白Bax的表达，同时显著抑制JNK和p38的磷酸化。此外，联合处理后CHOP表达增加表明内质网应激可能也参与增强HT诱导的细胞凋亡。
我们的数据首次表明，OZ通过外源性和内源性凋亡途径使Molt-4细胞对HT诱导的细胞凋亡敏感。此外，活性氧和内质网应激也可能在OZ增强HT诱导的细胞凋亡中发挥作用。
