Yamamoto Vicky, Wang Bintao, Lee Amy S
Department of Biochemistry and Molecular Medicine, University of Southern California, Keck School of Medicine, Los Angeles, CA, United States.
USC Norris Comprehensive Cancer Center, Los Angeles, CA, United States.
Front Oncol. 2023 Jan 11;12:1044699. doi: 10.3389/fonc.2022.1044699. eCollection 2022.
Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related death worldwide. Surgical resection, radiation and chemotherapy are the mainstay of HNSCC treatment but are often unsatisfactory. Cisplatin is a commonly used chemotherapy in HNSCC; however, cisplatin resistance is a major cause of relapse and death. The 78-kD glucose-regulated protein (GRP78) is the master regulator of the unfolded protein response (UPR) and is implicated in therapeutic resistance in cancer. The role of GRP78 in cisplatin resistance in HNSCC remains unclear. YUM70 is a newly discovered hydroxyquinoline analogue and found to be an inhibitor of GRP78. The effect of YUM70 in HNSCC cell lines is unknown.
Knockdown of GRP78 by siRNAs was performed to investigate the effect of GRP78 reduction in endoplasmic reticulum (ER)-stress induced and general apoptosis. Western blots examining apoptotic markers were performed on three HPV-negative HNSCC cell lines. WST-1 assay was performed to determine cell viability. In reverse, we utilized AA147, an ER proteostasis regulator to upregulate GRP78, and apoptotic markers and cell viability were determined. To test the ability of YUM70 to reverse cisplatin resistance, cisplatin-resistant HNSCC cell lines were generated by prolonged, repeated exposure to increasing concentrations of cisplatin. Colony formation assay using the cisplatin-resistant HNSCC cell line was performed to assess the reproductive cell survival. Furthermore, to test the ability of YUM70 to reverse cisplatin resistance in a physiologically relevant system, we subjected the 3D spheroids of the cisplatin-resistant HNSCC cell line to cisplatin treatment with or without YUM70 and monitored the onset of apoptosis.
Reduction of GRP78 level induced HNSCC cell death while GRP78 upregulation conferred higher resistance to cisplatin. Combined cisplatin and YUM70 treatment increased apoptotic markers in the cisplatin-resistant HNSCC cell line, associating with reduced cell viability and clonogenicity. The combination treatment also increased apoptotic markers in the 3D spheroid model.
The GRP78 inhibitor YUM70 reduced HNSCC cell viability and re-sensitized cisplatin-resistant HNSCC cell line in both 2D and 3D spheroid models, suggesting the potential use of YUM70 in the treatment of HNSCC, including cisplatin-resistant HNSCC.
头颈部鳞状细胞癌（HNSCC）是全球癌症相关死亡的主要原因之一。手术切除、放疗和化疗是HNSCC治疗的主要手段，但往往不尽人意。顺铂是HNSCC常用的化疗药物；然而，顺铂耐药是复发和死亡的主要原因。78-kD葡萄糖调节蛋白（GRP78）是未折叠蛋白反应（UPR）的主要调节因子，与癌症的治疗耐药有关。GRP78在HNSCC顺铂耐药中的作用尚不清楚。YUM70是一种新发现的羟基喹啉类似物，被发现是GRP78的抑制剂。YUM70对HNSCC细胞系的作用尚不清楚。
通过小干扰RNA（siRNAs）敲低GRP78，以研究GRP78水平降低对内质网（ER）应激诱导的凋亡和一般凋亡的影响。对三种人乳头瘤病毒（HPV）阴性的HNSCC细胞系进行检测凋亡标志物的蛋白质免疫印迹法。进行WST-1检测以确定细胞活力。相反，我们利用内质网蛋白稳态调节剂AA147上调GRP78，并测定凋亡标志物和细胞活力。为了测试YUM70逆转顺铂耐药的能力，通过长时间、反复暴露于浓度递增的顺铂来建立顺铂耐药的HNSCC细胞系。使用顺铂耐药的HNSCC细胞系进行集落形成试验，以评估生殖细胞存活情况。此外，为了测试YUM70在生理相关系统中逆转顺铂耐药的能力，我们将顺铂耐药的HNSCC细胞系的三维球体用或不用YUM70进行顺铂处理，并监测凋亡的发生。
GRP78水平降低诱导HNSCC细胞死亡，而GRP78上调赋予对顺铂更高的耐药性。顺铂与YUM70联合治疗增加了顺铂耐药的HNSCC细胞系中的凋亡标志物，与细胞活力和克隆形成能力降低相关。联合治疗在三维球体模型中也增加了凋亡标志物。
GRP78抑制剂YUM70在二维和三维球体模型中均降低了HNSCC细胞活力，并使顺铂耐药的HNSCC细胞系重新敏感，提示YUM70在治疗HNSCC（包括顺铂耐药的HNSCC）中的潜在应用。
