Obakan-Yerlikaya Pinar, Arisan Elif Damla, Coker-Gurkan Ajda, Adacan Kaan, Ozbey Utku, Somuncu Berna, Baran Didem, Palavan-Unsal Narcin
Department of Molecular Biology and Genetics, Istanbul Kultur University, Ataköy Campus, Istanbul, Turkey.
Mol Carcinog. 2017 Jun;56(6):1603-1619. doi: 10.1002/mc.22616. Epub 2017 Feb 16.
Epibrassinolide (EBR), a member of brassinostreoids plant hormones with cell proliferation promoting role in plants, is a natural polyhydroxysteroid with structural similarity to steroid hormones of vertebrates. EBR has antiproliferative and apoptosis-inducing effect in various cancer cells. Although EBR has been shown to affect survival and mitochondria-mediated apoptosis pathways in a p53-independent manner, the exact molecular targets of EBR are still under investigation. Our recent SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) data showed that the most significantly altered protein after EBR treatment was calreticulin (CALR). CALR, a chaperone localized in endoplasmic reticulum (ER) lumen, plays role in protein folding and buffering Ca ions. The alteration of CALR may cause ER stress and unfolded protein response correspondingly the induction of apoptosis. Unfolded proteins are conducted to 26S proteasomal degradation following ubiquitination. Our study revealed that EBR treatment caused ER stress and UPR by altering CALR expression causing caspase-dependent apoptosis in HCT 116, HT29, DLD-1, and SW480 colon cancer cells. Furthermore, 48 h EBR treatment did not caused UPR in Fetal Human Colon cells (FHC) and Mouse Embryonic Fibroblast cells (MEF). In addition our findings showed that HCT 116 colon cancer cells lacking Bax and Puma expression still undergo UPR and related apoptosis. CALR silencing and rapamycin co-treatment prevented EBR-induced UPR and apoptosis, whereas 26S proteasome inhibition further increased the effect of EBR in colon cancer cells. All these findings showed that EBR is an ER stress and apoptotic inducer in colon cancer cells without affecting non-malignant cells.
表油菜素内酯（EBR）是油菜素甾体类植物激素的一种，在植物中具有促进细胞增殖的作用，它是一种天然的多羟基甾体，其结构与脊椎动物的甾体激素相似。EBR在多种癌细胞中具有抗增殖和诱导凋亡的作用。尽管已表明EBR以不依赖p53的方式影响细胞存活和线粒体介导的凋亡途径，但其确切的分子靶点仍在研究中。我们最近的细胞培养中氨基酸稳定同位素标记（SILAC）数据显示，EBR处理后变化最显著的蛋白质是钙网蛋白（CALR）。CALR是一种定位于内质网（ER）腔的伴侣蛋白，在蛋白质折叠和缓冲钙离子方面发挥作用。CALR的改变可能相应地导致内质网应激和未折叠蛋白反应，进而诱导凋亡。未折叠的蛋白质在泛素化后被导向26S蛋白酶体降解。我们的研究表明，EBR处理通过改变CALR表达在HCT 116、HT29、DLD-1和SW480结肠癌细胞中引起内质网应激和未折叠蛋白反应，导致半胱天冬酶依赖性凋亡。此外，48小时的EBR处理在人胎儿结肠细胞（FHC）和小鼠胚胎成纤维细胞（MEF）中未引起未折叠蛋白反应。此外，我们的研究结果表明，缺乏Bax和Puma表达的HCT 116结肠癌细胞仍会发生未折叠蛋白反应和相关凋亡。CALR沉默与雷帕霉素联合处理可预防EBR诱导的未折叠蛋白反应和凋亡，而26S蛋白酶体抑制则进一步增强了EBR对结肠癌细胞的作用。所有这些研究结果表明，EBR是结肠癌细胞内质网应激和凋亡的诱导剂，而不影响非恶性细胞。
