Zuo Hui, Chen Cheng, Ma Ling, Min Qiu-Xia, Shen Yue-Hai
Department of Pharmacology, The First People's Hospital of Yunnan Province Kunming 650032, Yunnan Province, China.
Department of Pharmaceutical Science, The Affiliated Hospital of Kunming University of Science and Technology Kunming 650032, Yunnan Province, China.
Am J Transl Res. 2020 Oct 15;12(10):6478-6489. eCollection 2020.
Drug resistance remains a major cause of relapse and therapeutic failure in non-small cell lung cancer (NSCLC). The purpose of this investigation is to explore the relationship between caspase-8 level and chemo-sensitivity, as well as its underlying mechanism in NSCLC cells.
NSCLC cell line, A549 cells was used to investigate the influence of caspase-8 on the biological behavior . The abundance of caspase-8 in A549 cells was manipulated by transfection lentivirus containing specific caspase-8 short hairpin RNA (sh-caspase-8) and caspase-8 overexpressed plasmid. Cell viability and the percentage of apoptotic cells was quantified using cell counting kit-8 (CCK-8) assay and flow cytometry following Annexin V-FITC/PI staining, respectively. The formation of acidic vesicle organelles (AVOs) was examined by acridine orange staining and visualized under a fluorescence microscope. The mRNA and protein levels of relative genes were determined by qRT-PCR and western blotting.
Our results indicated that cells infected with sh-caspase-8 exhibited high knockdown efficiency. Knockdown of caspase-8 significantly reduced apoptosis of A549 cells. As evidenced by the decreased number of apoptotic cells and the reduction of Bcl-2/bax ratio. Interestingly, caspase-8 knockdown also enhanced autophagy in A549 cells. Additionally, knockdown of caspase-8 reduced the doxorubicin, carboplatin, cisplatin, and etoposide sensitivity towards A549 cells.
In summary, our results revealed that knockdown of caspase-8 could promote cell growth and autophagy, while reduce chemo-sensitivity and apoptotic cell death. These finding suggest caspase-8 might serve as a potential target to improve the chemo-sensitivity for NSCLC patients in clinical setting.
耐药性仍然是非小细胞肺癌（NSCLC）复发和治疗失败的主要原因。本研究的目的是探讨caspase-8水平与化疗敏感性之间的关系及其在NSCLC细胞中的潜在机制。
使用NSCLC细胞系A549细胞来研究caspase-8对生物学行为的影响。通过转染含有特异性caspase-8短发夹RNA（sh-caspase-8）的慢病毒和caspase-8过表达质粒来调控A549细胞中caspase-8的丰度。分别使用细胞计数试剂盒-8（CCK-8）法和Annexin V-FITC/PI染色后的流式细胞术对细胞活力和凋亡细胞百分比进行定量。通过吖啶橙染色检查酸性囊泡细胞器（AVO）的形成，并在荧光显微镜下观察。通过qRT-PCR和蛋白质印迹法测定相关基因的mRNA和蛋白质水平。
我们的结果表明，感染sh-caspase-8的细胞表现出高敲低效率。敲低caspase-8显著降低了A549细胞的凋亡。凋亡细胞数量减少和Bcl-2/bax比值降低证明了这一点。有趣的是，敲低caspase-8还增强了A549细胞中的自噬。此外，敲低caspase-8降低了阿霉素、卡铂、顺铂和依托泊苷对A549细胞的敏感性。
总之，我们的结果表明，敲低caspase-8可促进细胞生长和自噬，同时降低化疗敏感性和凋亡细胞死亡。这些发现表明，caspase-8可能作为一个潜在靶点，在临床环境中提高NSCLC患者的化疗敏感性。
