Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa, 751024, India.
Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA.
Cell Oncol (Dordr). 2017 Dec;40(6):593-607. doi: 10.1007/s13402-017-0347-3. Epub 2017 Sep 21.
Previously, we reported that quinacrine (QC) may cause apoptosis in breast and colon cancer cells by activating the death receptor 5 (DR5), resulting in autophagic cell death through p21 modulation. Here, we systematically evaluated the combined role of p21 and DR5 and their crosstalk in QC-mediated autophagy and apoptosis in breast cancer cells using in vitro and in vivo models.
Multiple breast cancer-derived cell lines (MCF-7, ZR-75-1, T47D, MDA-MB-231 and MCF-10A-Tr) and a mouse xenograft model were used. Also, multiple assays, including Western blotting, immunoprecipitation, staining for autophagy and apoptosis, gene silencing, hematoxylin and eosin staining, immunohistochemistry, cell viability assessment, fluorescence imaging and cell sorting were used.
We found that QC activates p21 and DR5 in combination with the apoptosis inducer TRAIL in the breast cancer-derived cells tested. Combined TRAIL and QC treatment increased autophagy and apoptosis by increasing the interaction between, and co-localization of, p21 and DR5 in the death-inducing signaling complex (DISC). We found that this combination also inhibited the mTOR/PI3K/AKT signaling cascade and modulated reactive oxygen species (ROS) and nitric oxide (NO) production. Reductions in autophagy and apoptosis in DR5-knockout cells and a lack of change in p21-DR5-silenced cells were noted after TRAIL + QC treatment. This result explains dependence of the death (autophagy and apoptosis) cascade on these two key regulatory proteins. In addition, we found in an in vivo mouse xenograft model that increased expression and enhanced co-localization of p21 and DR5 after TRAIL + QC treatment supported a joint regulatory role of these proteins in the co-prevalence of autophagy and apoptosis.
Our data suggest that a combined treatment of TRAIL and QC causes cell death in breast cancer-derived cells via autophagy and apoptosis by increasing the interaction of p21 and DR5, as indicated by both in vitro and in vivo studies.
此前，我们报道了吖啶酮（QC）通过激活死亡受体 5（DR5）可能导致乳腺癌和结肠癌细胞发生凋亡，从而通过 p21 调节导致自噬细胞死亡。在这里，我们使用体外和体内模型系统地评估了 p21 和 DR5 及其在 QC 介导的乳腺癌细胞自噬和凋亡中的相互作用的联合作用。
使用了多种乳腺癌衍生细胞系（MCF-7、ZR-75-1、T47D、MDA-MB-231 和 MCF-10A-Tr）和小鼠异种移植模型。还使用了多种检测方法，包括 Western 印迹、免疫沉淀、自噬和凋亡染色、基因沉默、苏木精和伊红染色、免疫组织化学、细胞活力评估、荧光成像和细胞分选。
我们发现 QC 与凋亡诱导剂 TRAIL 联合激活了所测试的乳腺癌衍生细胞中的 p21 和 DR5。联合 TRAIL 和 QC 处理通过增加 p21 和 DR5 在诱导死亡信号复合物（DISC）中的相互作用和共定位来增加自噬和凋亡。我们发现，这种组合还抑制了 mTOR/PI3K/AKT 信号级联，并调节了活性氧（ROS）和一氧化氮（NO）的产生。在 TRAIL + QC 处理后，DR5 敲除细胞中的自噬和凋亡减少，而 p21-DR5 沉默细胞中的变化则没有。这一结果解释了死亡（自噬和凋亡）级联对这两种关键调节蛋白的依赖性。此外，我们在体内小鼠异种移植模型中发现，在 TRAIL + QC 处理后，p21 和 DR5 的表达增加和共定位增强支持了这两种蛋白在自噬和凋亡共同发生中的联合调节作用。
我们的数据表明，TRAIL 和 QC 的联合治疗通过增加 p21 和 DR5 的相互作用，导致乳腺癌衍生细胞发生细胞死亡，通过体外和体内研究均表明，这是通过自噬和凋亡发生的。
