目的:巨细胞增殖是一种非选择性的内吞形式,可以促进细胞外物质的摄取,如营养素和大分子,进入细胞。在KRAS驱动的癌症中,包括胰腺导管腺癌,已知巨生胞吞作用和随后的溶酶体利用被增强以克服代谢应激。在这项研究中,我们研究了酪蛋白激酶2(CK2)抑制在KRAS突变胆管癌(CCA)细胞系中的巨噬细胞吞噬和随后的代谢过程中的作用。
方法:使用HuCCT1KRAS突变体CCA细胞系通过流式细胞术进行指示巨细胞胞吞作用的牛血清白蛋白(BSA)摄取。为了验证macropinosome,标记Rab7和LAMP2,并通过免疫细胞化学和蛋白质印迹进行分析。CX-4945(Silmitasertib),CK2抑制剂,用于研究CK2在巨噬细胞增多和随后的溶酶体代谢中的作用。
结果:TFK-1,一种KRAS野生型CCA细胞系,仅显示凋亡形态变化。然而,HuCCT1细胞系显示巨噬细胞增多症。尽管CX-4945诱导了伴随着细胞内液泡积累和细胞死亡的形态学变化,巨噬细胞增多水平没有变化.这些细胞内液泡被鉴定为晚期巨角质蛋白体,代表与溶酶体融合前的Rab7囊泡。此外,CX-4945在抑制Akt-mTOR信号通路后抑制LAMP2表达,这中断了成熟的宏染色体和溶酶体代谢利用。
结论:在KRAS突变体CCA细胞系HuCCT1中,巨噬细胞胞吞作用被用作能量来源。CX-4945对CK2的抑制通过溶酶体依赖性代谢的改变导致HuCCT1细胞的细胞死亡。
OBJECTIVE: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines.
METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism.
RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization.
CONCLUSIONS: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.