Qin Haihong, Li Zheng, Wu Jinfeng, Liu Xiao, Wang Ruilong, Xu Jinhua, Zhu Xiaohua
Department of Dermatology, Fudan University Huashan Hospital, Shanghai, China.
Clin Exp Pharmacol Physiol. 2025 Mar;52(3):e70022. doi: 10.1111/1440-1681.70022.
BRAF inhibitors (BRAFi) represent a cornerstone in melanoma therapy due to their high efficacy. However, the emergence of resistance causes a significant challenge to their clinical utility. This study aims to investigate the potential of diclofenac as a sensitizer for BRAFi therapy in melanoma and to elucidate its underlying mechanism. BRAFi-acquired resistant melanoma cell lines SK-MEL-5R and A375R were established and treated with diclofenac in combination with BRAFi PLX4032. Cell viability was assessed using the MTT assay, cell proliferation was determined by crystal violet staining, cell apoptosis was evaluated by flow cytometry, and intracellular ROS levels were measured using the DCFH-DA probe-labeled and flow cytometry. Mitochondrial membrane potential was assessed by JC-1 staining and flow cytometry, and protein expression levels were detected by western blotting. Our results demonstrated that diclofenac significantly augmented the cytotoxicity of PLX4032 and enhanced its ability to induce apoptosis in SK-MEL-5R and A375R cells. Diclofenac treatment led to the release of intracellular reactive oxygen species (ROS), consequently reducing transmembrane potential, promoting mitochondrial apoptosis, and activating the ROS downstream p38/p53 signaling pathway. Pretreatment with N-acetylcysteine significantly reversed the sensitizing effect of diclofenac on PLX4032 in SK-MEL-5R cells. These findings suggested that diclofenac sensitized BRAFi-resistant melanoma cells to BRAFi by increasing ROS release and activating p38/p53 signaling pathway. Diclofenac might serve as a promising adjunct therapy to overcome BRAFi resistance in melanoma treatment.
BRAF抑制剂（BRAFi）因其高效性而成为黑色素瘤治疗的基石。然而，耐药性的出现对其临床应用构成了重大挑战。本研究旨在探讨双氯芬酸作为黑色素瘤BRAFi治疗增敏剂的潜力，并阐明其潜在机制。建立了BRAFi获得性耐药黑色素瘤细胞系SK-MEL-5R和A375R，并用双氯芬酸与BRAFi PLX4032联合处理。使用MTT法评估细胞活力，通过结晶紫染色测定细胞增殖，通过流式细胞术评估细胞凋亡，使用DCFH-DA探针标记和流式细胞术测量细胞内ROS水平。通过JC-1染色和流式细胞术评估线粒体膜电位，通过蛋白质印迹法检测蛋白质表达水平。我们的结果表明，双氯芬酸显著增强了PLX4032的细胞毒性，并增强了其诱导SK-MEL-5R和A375R细胞凋亡的能力。双氯芬酸处理导致细胞内活性氧（ROS）释放，从而降低跨膜电位，促进线粒体凋亡，并激活ROS下游的p38/p53信号通路。用N-乙酰半胱氨酸预处理可显著逆转双氯芬酸对SK-MEL-5R细胞中PLX4032的增敏作用。这些发现表明，双氯芬酸通过增加ROS释放和激活p38/p53信号通路，使BRAFi耐药的黑色素瘤细胞对BRAFi敏感。双氯芬酸可能作为一种有前景的辅助治疗方法，用于克服黑色素瘤治疗中的BRAFi耐药性。
