Olajossy Bartlomiej, Wronski Norbert, Madej Ewelina, Komperda Joanna, Szczygieł Małgorzata, Wolnicka-Glubisz Agnieszka
Department of Biophysics and Cancer Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa Street 7, 30-387 Krakow, Poland.
Doctoral School of Exact and Natural Sciences, Jagiellonian University, 30-387 Krakow, Poland.
Biomolecules. 2024 Dec 10;14(12):1573. doi: 10.3390/biom14121573.
Melanoma cells remain resistant to chemotherapy with cisplatin (CisPt) and doxorubicin (DOX). The abnormal expression of Receptor-Interacting Protein Kinase 4 (RIPK4) in certain melanomas contributes to tumour growth through the NFκB and Wnt/β-catenin signalling pathways, which are known to regulate chemoresistance and recurrence. Despite this, the role of RIPK4 in response to chemotherapeutics in melanoma has not been reported. In this study, we examined how the downregulation and overexpression of RIPK4 affect the sensitivity of BRAF-mutated melanoma cells (A375 and WM266.4) to CisPt and DOX along with determining the underlying mechanism. Using two RIPK4 silencing methods (siRNA and CRISPR/Cas9) and overexpression (dCas9-VPR), we assessed CisPt and DOX-induced apoptosis using caspase 3/7 activity, annexin V/7AAD staining, and FASC analysis. In addition, qRT-PCR and Western blotting were used to detect apoptosis-related genes and proteins such as cleaved PARP, p53, and cyclin D1. We demonstrated that the overexpression of RIPK4 inhibits, while its downregulation enhances, CisPt- or DOX-induced apoptosis in melanoma cells. The effects of downregulation are similar to those observed with pre-incubation with cyclosporin A, an ABCG2 inhibitor. Additionally, our findings provide preliminary evidence of crosstalk between RIPK4, BIRC3, and ABCG2. The results of these studies suggest the involvement of RIPK4 in the observed resistance to CisPt or DOX.
黑色素瘤细胞对顺铂（CisPt）和阿霉素（DOX）化疗仍具有抗性。受体相互作用蛋白激酶4（RIPK4）在某些黑色素瘤中的异常表达通过NFκB和Wnt/β-连环蛋白信号通路促进肿瘤生长，已知这些信号通路可调节化疗耐药性和复发。尽管如此，RIPK4在黑色素瘤对化疗药物反应中的作用尚未见报道。在本研究中，我们研究了RIPK4的下调和过表达如何影响BRAF突变的黑色素瘤细胞（A375和WM266.4）对CisPt和DOX的敏感性，并确定其潜在机制。我们使用两种RIPK4沉默方法（siRNA和CRISPR/Cas9）和过表达方法（dCas9-VPR），通过caspase 3/7活性、膜联蛋白V/7AAD染色和FASC分析评估CisPt和DOX诱导的细胞凋亡。此外，使用qRT-PCR和蛋白质免疫印迹法检测凋亡相关基因和蛋白，如裂解的PARP、p53和细胞周期蛋白D1。我们证明，RIPK4的过表达抑制，而其下调增强CisPt或DOX诱导的黑色素瘤细胞凋亡。下调的效果与用ABCG2抑制剂环孢菌素A预孵育观察到的效果相似。此外，我们的研究结果提供了RIPK4、BIRC3和ABCG2之间相互作用的初步证据。这些研究结果表明RIPK4参与了观察到的对CisPt或DOX的抗性。
