"Ion Chiricuta" Oncology Institute Cluj-Napoca, Romania.
"Ion Chiricuta" Oncology Institute Cluj-Napoca, Romania.
Hear Res. 2020 Mar 15;388:107893. doi: 10.1016/j.heares.2020.107893. Epub 2020 Jan 17.
Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line.
We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test.
CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79 μM, compared to 611.7 μM for L-OHP and 882.9 μM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 μM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations.
The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.
基于铂的抗肿瘤药物在治疗多种癌症方面已经占据了重要地位。但它们对健康组织也有严重的毒性作用。耳毒性是导致听力损伤的严重副作用，是影响患者生活质量的一个重要问题。目前使用的铂类化疗药物对耳蜗细胞有不同的毒性。本研究旨在回答一些关于顺铂（CDDP）、卡铂（CBDCA）和奥沙利铂（L-OHP）在 HEI-OC1 耳蜗细胞系中的摄取和细胞药代动力学差异的问题。
我们通过免疫荧光显微镜和流式细胞术研究了铜转运体 CTR1、ATP7A 和 ATP7B 的表达，这些转运体可能参与了铂化合物的摄取、细胞内转运和外排。通过原子吸收光谱法测定细胞内铂浓度来评估化合物的细胞摄取。还评估了铂化合物处理 HEI-OC1 细胞的效果：用 Cell Titer Blue 活力试验评估细胞毒性，用 2'，7' -二氯荧光素二乙酸酯评估活性氧的形成，用彗星试验评估遗传毒性，用 cleaved PARP ELISA 试验评估细胞凋亡。
HEI-OC1 细胞表达 CTR1、ATP7A 和 ATP7B。铂化合物的处理导致其表达的调节，表现为铂的摄取差异。与奥沙利铂和卡铂相比，顺铂处理导致细胞内铂浓度最高。用 CuSO4 处理可显著降低所有化合物的铂摄取，尤其是顺铂和卡铂。与 L-OHP（611.7 μM）和 CBDCA（882.9 μM）相比，CDDP 对 HEI-OC1 细胞的细胞毒性最强，IC50 为 65.79 μM。CDDP 后 ROS 的产生最为剧烈，其次是 L-OHP 和 CBDCA。在彗星试验中，在 100 μM 浓度下，L-OHP 和 CBDCA 诱导 DNA 加合物，而 CDDP 诱导加合物和 DNA 链断裂。在 24 小时时，CBDCA 和 L-OHP 导致 cleaved PARP 显著增加（p < 0.001），表明在使用的浓度下这些化合物诱导了重要的凋亡过程。
本研究结果表明，局部铜转运体的调节可能代表一种对抗铂类药物耳毒性的新策略。
