Department of Respiratory Medicine, Kanazawa University, Ishikawa, Japan
Department of Respiratory Medicine, Kanazawa University, Ishikawa, Japan.
Cancer Genomics Proteomics. 2021 Jul-Aug;18(4):579-590. doi: 10.21873/cgp.20282.
BACKGROUND/AIM: Cisplatin combined with pemetrexed disodium heptahydrate (pemetrexed) is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer. However, its growth-inhibitory effects on KRAS-dependent lung cancer as monotherapy and combination therapy are not well understood. The aim of this study was to compare the effects of cisplatin and pemetrexed on A549 cells as mono- and combination therapies and elucidate the underlying mechanisms.
For in vitro studies, A549 cells were exposed to cisplatin with/without pemetrexed for 72 h. The results were then evaluated by cell viability, apoptosis, reactive oxygen species, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and western blotting assays.
Our results revealed that cisplatin monotherapy was most cytotoxic to A549 cells, while cisplatin plus pemetrexed combination had an intermediate effect, and pemetrexed monotherapy induced a minimal cytotoxic effect on A549 cells. This effect was evidenced by cell viability results, inhibition of KRAS proto-oncogene, GTPase (KRAS)/Raf proto-oncogene, serine/threonine kinase/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways and apoptosis induction triggered by reactive oxygen species-mediated DNA damage. The immunoblotting result of conversion of microtubule-associated protein 1 light chain 3 alpha (LC3)-I to -II indicated that the greatest inducer of autophagy was combined treatment with cisplatin plus pemetrexed, while pemetrexed monotherapy had the lowest effect on autophagy induction, with cisplatin monotherapy having an intermediate effect. We found that the AKT serine/threonine kinase 1/mechanistic target of rapamycin kinase (mTOR) and AMP-activated protein kinase/mTOR signaling pathways were associated with autophagy activation. Interestingly, combination therapy with cisplatin plus pemetrexed was the primary eliminator of cellular senescence; cisplatin monotherapy had an intermediate effect, while pemetrexed monotherapy increased cellular senescence of A549 cells, as assessed by the expression of β-galactosidase protein.
Cisplatin monotherapy may be more effective than pemetrexed monotherapy or cisplatin plus pemetrexed combination therapy against KRAS-dependent lung cancer.
背景/目的：顺铂联合培美曲塞二钠（培美曲塞）被认为是治疗晚期非鳞状非小细胞肺癌患者的标准治疗方法。然而，其作为单药和联合治疗对 KRAS 依赖性肺癌的生长抑制作用尚不清楚。本研究旨在比较顺铂和培美曲塞作为单药和联合治疗对 A549 细胞的作用，并阐明其潜在机制。
对于体外研究，A549 细胞用顺铂加/不加培美曲塞处理 72 小时。然后通过细胞活力、细胞凋亡、活性氧、末端脱氧核苷酸转移酶 dUTP 缺口末端标记和 Western blot 分析评估结果。
我们的结果表明，顺铂单药治疗对 A549 细胞的细胞毒性最大，而顺铂加培美曲塞联合治疗的效果居中，培美曲塞单药治疗对 A549 细胞的细胞毒性最小。这一作用通过细胞活力结果、KRAS 原癌基因，GTP 酶（KRAS）/Raf 原癌基因，丝氨酸/苏氨酸激酶/丝裂原活化蛋白激酶激酶/细胞外信号调节激酶途径的抑制以及活性氧介导的 DNA 损伤诱导的细胞凋亡得到证实。微管相关蛋白 1 轻链 3 alpha（LC3）-I 向 -II 的免疫印迹结果表明，自噬最强诱导剂是顺铂加培美曲塞联合治疗，而培美曲塞单药治疗对自噬诱导的作用最低，顺铂单药治疗的作用居中。我们发现 AKT 丝氨酸/苏氨酸激酶 1/雷帕霉素靶蛋白激酶（mTOR）和 AMP 激活的蛋白激酶/mTOR 信号通路与自噬的激活有关。有趣的是，顺铂加培美曲塞联合治疗是细胞衰老的主要消除剂；顺铂单药治疗的效果居中，而培美曲塞单药治疗增加了 A549 细胞的细胞衰老，β-半乳糖苷酶蛋白的表达表明了这一点。
顺铂单药治疗可能比培美曲塞单药治疗或顺铂加培美曲塞联合治疗对 KRAS 依赖性肺癌更有效。
