METHODS: Alamar Blue proliferation and colony formation assay (CFA) were used to determine the radioresponse of a cohort of 38 murine PDAC cell lines. A gene set enrichment analysis was performed to reveal differentially expressed pathways. CFA, cell cycle distribution, γH2AX FACS analysis, and Caspase 3/7 SYTOX assay were used to examine the effect of a combination treatment using KIRA8 as an IRE1α-inhibitor and Ceapin-A7 as an inhibitor against ATF6.
RESULTS: The unfolded protein response (UPR) was identified as a pathway highly expressed in radioresistant cell lines. Using the IRE1α-inhibitor KIRA8 or the ATF6-inhibitor Ceapin-A7 in combination with radiation, a radiosensitizing effect was observed in radioresistant cell lines, but no substantial alteration of the radioresponse in radiosensitive cell lines. Mechanistically, increased apoptosis by KIRA8 in combination with radiation and a cell cycle arrest in the G1 phase after ATF6 inhibition and radiation have been observed in radioresistant cell lines.
CONCLUSIONS: So, our data show evidence that the UPR is involved in radioresistance of PDAC. Increased apoptosis and a G1 cell cycle arrest seem to be responsible for the radiosensitizing effect of UPR inhibition. These findings are supportive for developing novel combination treatment concepts in PDAC to overcome radioresistance.
方法:AlamarBlue增殖和集落形成试验(CFA)用于测定一组38只鼠PDAC细胞系的放射反应。进行基因集富集分析以揭示差异表达的途径。CFA,细胞周期分布,γH2AXFACS分析,和半胱天冬酶3/7SYTOX测定用于检查使用KIRA8作为IRE1α抑制剂和Ceapin-A7作为ATF6抑制剂的组合治疗的效果。
结果:未折叠蛋白反应(UPR)被鉴定为在耐放射细胞系中高度表达的途径。使用IRE1α抑制剂KIRA8或ATF6抑制剂Ceapin-A7与辐射联合使用,在抗放射性细胞系中观察到放射增敏作用,但放射敏感细胞系的放射反应没有实质性改变。机械上,在抗辐射细胞系中观察到ATF6抑制和辐射后,KIRA8增加的细胞凋亡和G1期的细胞周期停滞。
结论:所以,我们的数据显示有证据表明UPR与PDAC的耐辐射性有关.凋亡增加和G1细胞周期停滞似乎是UPR抑制的放射增敏作用的原因。这些发现支持在PDAC中开发新的组合治疗概念以克服辐射抗性。