Cheeloo College of Medicine, Shandong University, Jinan, China.
Institute of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, China.
Biochem Biophys Res Commun. 2024 Sep 3;723:150200. doi: 10.1016/j.bbrc.2024.150200. Epub 2024 Jun 1.
OBJECTIVES: This study aimed to explore the involvement of phosphoenolpyruvate carboxykinase 2 (PCK2) in gefitinib-resistant non-small cell lung cancer (NSCLC) cells and assess its feasibility as a therapeutic target against gefitinib resistance. METHODS: Gefitinib-resistant cell lines, PC9GR and HCC827GR, were generated through progressive exposure of parental cells to escalating concentrations of gefitinib. Transcriptomic analysis encompassed the treatment of PC9 and PC9GR cells with gefitinib or vehicle, followed by RNA extraction, sequencing, and subsequent bioinformatic analysis. Cell viability was determined via CCK-8 assay, while clonogenic assays assessed colony formation. Apoptosis was detected utilizing the Annexin V-FITC/7AAD kit. Iron ion concentrations were quantified using FerroOrange. mRNA analysis was conducted through quantitative RT-PCR. Western blotting was employed for protein analysis. H&E and immunohistochemical staining were performed on tumor tissue sections. RESULTS: The results revealed that depletion or inhibition of PCK2 significantly enhanced gefitinib's efficacy in inducing cell growth arrest, apoptosis, and ferroptosis in resistant NSCLC. Moreover, PCK2 knockdown led to the downregulation of key ferroptosis-related proteins, GPX4 and SLC7A11, while upregulating ASCL4. Conversely, overexpression of PCK2 in gefitinib-sensitive cells rendered resistance to gefitinib. In vivo experiments using a gefitinib-resistant xenograft model demonstrated that PCK2 silencing not only reduced tumor growth but also considerably increased the anti-tumor effect of gefitinib. CONCLUSIONS: In conclusion, our study presents compelling evidence indicating that PCK2 plays a pivotal role in gefitinib resistance in NSCLC. The modulation of ferroptosis-related proteins and the involvement of Akt activation further elucidate the mechanisms underlying this resistance. Consequently, PCK2 emerges as a promising therapeutic target for overcoming gefitinib resistance in NSCLC, offering a new avenue for the development of more effective treatment strategies.
目的：本研究旨在探讨磷酸烯醇式丙酮酸羧激酶 2（PCK2）在吉非替尼耐药非小细胞肺癌（NSCLC）细胞中的作用，并评估其作为克服吉非替尼耐药的治疗靶点的可行性。
方法：通过逐步增加吉非替尼浓度对亲本细胞进行处理，生成吉非替尼耐药细胞系 PC9GR 和 HCC827GR。对 PC9 和 PC9GR 细胞进行吉非替尼或载体处理后，进行转录组分析，包括 RNA 提取、测序和随后的生物信息学分析。通过 CCK-8 测定法测定细胞活力，通过集落形成实验评估克隆形成。利用 Annexin V-FITC/7AAD 试剂盒检测细胞凋亡。使用 FerroOrange 定量铁离子浓度。通过定量 RT-PCR 进行 mRNA 分析。采用 Western blot 进行蛋白分析。对肿瘤组织切片进行 H&E 和免疫组织化学染色。
结果：结果表明，PCK2 的耗竭或抑制显著增强了吉非替尼在诱导耐药 NSCLC 细胞生长抑制、凋亡和铁死亡方面的疗效。此外，PCK2 敲低导致关键铁死亡相关蛋白 GPX4 和 SLC7A11 的下调，同时上调 ASCL4。相反，在吉非替尼敏感细胞中过表达 PCK2 会导致对吉非替尼的耐药性。使用吉非替尼耐药异种移植模型的体内实验表明，PCK2 沉默不仅减少了肿瘤生长，而且显著增加了吉非替尼的抗肿瘤效果。
结论：总之，本研究提供了强有力的证据表明，PCK2 在 NSCLC 中的吉非替尼耐药中发挥关键作用。调节铁死亡相关蛋白和 Akt 激活的参与进一步阐明了这种耐药性的机制。因此，PCK2 作为克服 NSCLC 中吉非替尼耐药的有前途的治疗靶点，为开发更有效的治疗策略提供了新的途径。
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