Zhang Li, Cheng Xian, Xu Shichen, Bao Jiandong, Yu Huixin
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, China.
Medicine (Baltimore). 2018 Jun;97(24):e11095. doi: 10.1097/MD.0000000000011095.
Thyroid cancer is the most common endocrine tumor. Our previous studies have demonstrated that curcumin can induce apoptosis in human papillary thyroid carcinoma BCPAP cells. However, the underlined mechanism has not been clearly elucidated. Endoplasmic reticulum (ER) is a major organelle for synthesis, maturation, and folding proteins as well as a large store for Ca. Overcoming chronically activated ER stress by triggering pro-apoptotic pathways of the unfolded protein response (UPR) is a novel strategy for cancer therapeutics. Our study aimed to uncover the ER stress pathway involved in the apoptosis caused by curcumin.
BCPAP cells were treated with different doses of curcumin (12.5-50 μM). Annexin V/PI double staining was used to determine cell apoptosis. Rhod-2/AM calcium fluorescence probe assay was performed to measure the calcium level of endoplasmic reticulum. Western blot was used to examine the expression of ER stress marker C/EBP homologous protein 10 (CHOP) and glucose-regulated protein 78 (GRP78). X-box binding protein1 (XBP-1) spliced form was examined by reverse transcriptase-polymerase chain reaction (RT-PCR).
Curcumin significantly inhibited anchorage-independent cell growth and induced apoptosis in BCPAP cells. Curcumin induced ER stress and UPR responses in a dose- and time-dependent manner, and the chemical chaperone 4-phenylbutyrate (4-PBA) partially reversed the antigrowth activity of curcumin. Moreover, curcumin significantly increased inositol-requiring enzyme 1α (IRE1α) phosphorylation and XBP-1 mRNA splicing to induce a subsets of ER chaperones. Increased cleavage of activating transcription factor 6 (ATF6), which enhances expression of its downstream target CHOP was also observed. Furthermore, curcumin induced intracellular Ca influx through inhibition of the sarco-endoplasmic reticulum ATPase 2A (SERCA2) pump. The increased cytosolic Ca then bound to calmodulin to activate calcium/calmodulin-dependent protein kinase II (CaMKII) signaling, leading to mitochondrial apoptosis pathway activation. Ca chelator BAPTA partially reversed curcumin-induced ER stress and growth suppression, confirming the possible involvement of calcium homeostasis disruption in this response.
Curcumin inhibits thyroid cancer cell growth, at least partially, through ER stress-associated apoptosis. Our observations provoked that ER stress activation may be a promising therapeutic target for thyroid cancer treatment.(Figure is included in full-text article.).
甲状腺癌是最常见的内分泌肿瘤。我们之前的研究表明姜黄素可诱导人甲状腺乳头状癌BCPAP细胞凋亡。然而，其潜在机制尚未完全阐明。内质网（ER）是蛋白质合成、成熟和折叠的主要细胞器，也是钙的重要储存库。通过触发未折叠蛋白反应（UPR）的促凋亡途径来克服长期激活的内质网应激是癌症治疗的一种新策略。我们的研究旨在揭示姜黄素诱导凋亡所涉及的内质网应激途径。
用不同剂量的姜黄素（12.5 - 50 μM）处理BCPAP细胞。采用膜联蛋白V/碘化丙啶（Annexin V/PI）双染法检测细胞凋亡。用罗丹明-2/AM钙荧光探针法检测内质网钙水平。采用蛋白质免疫印迹法检测内质网应激标志物C/EBP同源蛋白10（CHOP）和葡萄糖调节蛋白78（GRP78）的表达。通过逆转录聚合酶链反应（RT-PCR）检测X-box结合蛋白1（XBP-1）的剪接形式。
姜黄素显著抑制BCPAP细胞的非锚定依赖性生长并诱导其凋亡。姜黄素以剂量和时间依赖性方式诱导内质网应激和UPR反应，化学伴侣4-苯基丁酸（4-PBA）部分逆转了姜黄素的抗生长活性。此外，姜黄素显著增加肌醇需求酶1α（IRE1α）磷酸化和XBP-1 mRNA剪接，从而诱导内质网伴侣蛋白亚群。还观察到激活转录因子6（ATF6）的切割增加，这增强了其下游靶标CHOP的表达。此外，姜黄素通过抑制肌浆内质网ATP酶2A（SERCA2）泵诱导细胞内钙内流。增加的胞质钙随后与钙调蛋白结合以激活钙/钙调蛋白依赖性蛋白激酶II（CaMKII）信号，导致线粒体凋亡途径激活。钙螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）部分逆转了姜黄素诱导的内质网应激和生长抑制，证实钙稳态破坏可能参与了这一反应。
姜黄素至少部分通过内质网应激相关的凋亡抑制甲状腺癌细胞生长。我们的观察结果提示内质网应激激活可能是甲状腺癌治疗的一个有前景的治疗靶点。（图见全文）
