Koustas Evangelos, Sarantis Panagiotis, Trifylli Eleni-Myrto, Dikoglou-Tzanetatou Eleftheria, Ioakeimidou Evangelia, Anastasiou Ioanna A, Karamouzis Michalis V, Theocharis Stamatios
Oncology Department, General Hospital Evangelismos, Ipsilantou 45-47, 106 76 Athens, Greece.
First Department of Pathology, Medical School, National and Kapodistrian University of Athens, 115 27 Athens, Greece.
Genes (Basel). 2025 Jul 28;16(8):892. doi: 10.3390/genes16080892.
Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. Although chemotherapy is an effective treatment for colorectal cancer (CRC), its effectiveness is frequently hindered by the emergence of resistant cancer cells. Studies have demonstrated a linkage between drug resistance and the pregnane X receptor (PXR), which influences the metabolism and the transport of chemotherapeutic agents. Likewise, autophagy is also a well-established mechanism that contributes to chemotherapy resistance, and it is closely tied to tumor progression. This pre-clinical study aims to investigate the role of mtKRAS-dependent autophagy with PXR expression after treatment with Irinotecan in colorectal cancer. CRC lines were treated with specific inhibitors, such as 3-methyladeninee, hydroxychloroquine PI-103, and irinotecan hydrochloride, and subjected to various assays, including MTT for cell viability, Western blot for protein expression, siRNA-mediated PXR knock-out, and confocal microscopy for autophagic vacuole visualization. Protein quantification, gene knockdown, and subcellular localization studies were performed under standardized conditions to investigate treatment effects on autophagy and apoptosis pathways. Our experiments showed that PXR knockdown does not alter autophagy levels following Irinotecan treatment, but it promotes apoptotic cell death despite elevated autophagy. Moreover, late-stage autophagy inhibition reduces PXR expression, whereas induction through PI3K/AKT/mTOR inhibition leads to increased expression of PXR. Our experiments uncover a mechanism by which autophagy facilitates the nuclear translocation of the PXR, thereby promoting resistance to Irinotecan across multiple cell lines.
结直肠癌（CRC）是全球最常被诊断出的恶性肿瘤之一。尽管化疗是治疗结直肠癌（CRC）的有效方法，但其有效性常常因耐药癌细胞的出现而受到阻碍。研究表明耐药性与孕烷X受体（PXR）之间存在联系，PXR会影响化疗药物的代谢和转运。同样，自噬也是一种公认的导致化疗耐药的机制，并且它与肿瘤进展密切相关。这项临床前研究旨在探究伊立替康治疗后，线粒体KRAS依赖性自噬与PXR表达在结直肠癌中的作用。用特定抑制剂（如3-甲基腺嘌呤、羟氯喹啉PI-103和盐酸伊立替康）处理结直肠癌细胞系，并进行各种检测，包括用于细胞活力检测的MTT法、用于蛋白质表达检测的蛋白质印迹法、小干扰RNA介导的PXR基因敲除以及用于自噬泡可视化的共聚焦显微镜检测。在标准化条件下进行蛋白质定量、基因敲低和亚细胞定位研究，以探究对自噬和凋亡途径的治疗效果。我们的实验表明，PXR基因敲低在伊立替康治疗后不会改变自噬水平，但尽管自噬水平升高，它却能促进凋亡性细胞死亡。此外，晚期自噬抑制会降低PXR表达，而通过抑制PI3K/AKT/mTOR诱导自噬则会导致PXR表达增加。我们的实验揭示了一种机制，即自噬促进PXR的核转位，从而增强多种细胞系对伊立替康的耐药性。
