Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, China; Orthopedic Laboratory of Chongqing Medical University, Yuzhong, Chongqing 400016, China; Department of Orthopedics, Chongqing University Three Gorges Hospital, Chongqing Municipality Clinical Research Center for Geriatric diseases, Wanzhou, Chongqing 404000, China.
Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, China; Orthopedic Laboratory of Chongqing Medical University, Yuzhong, Chongqing 400016, China.
Photodiagnosis Photodyn Ther. 2022 Sep;39:102964. doi: 10.1016/j.pdpdt.2022.102964. Epub 2022 Jun 12.
This study was designed to explore the effects of Yes-associated protein (YAP) knockdown on human osteosarcoma (HOS) cell sensitivity to Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT), and to assess how YAP silencing in combination with treatment with the ferroptosis inducer Erastin improves HOS cell sensitivity to MPPa-PDT in an effort to better clarify the molecular mechanisms underlying these phenotypes.
At 12 h post-MPPa-PDT, Hoechst staining and flow cytometry were conducted to evaluate the apoptotic death of HOS cells. The expression of YAP in these cells at 12 h post-MPPa-PDT treatment was assessed via Western blotting and immunofluorescent staining. BODIPY-C11 was used to evaluate lipid peroxidation. Following shYAP lentiviral transduction, Western blotting was conducted to assess the expression of proteins associated with proliferation, apoptosis, and ferroptosis. EdU assays and clonogenic assays were performed to analyze cellular proliferation. Erastin-treated HOS cells were used to establish a ferroptosis model. Western blotting was used to measure ferroptosis-associated protein levels following shYAP and erastin treatment, while changes in proliferation and MDA levels in each group were examined using an MDA kit.
At 12 h post-MPPa-PDT, HOS cells exhibited apoptotic characteristics including nuclear fragmentation and pyknosis, with concomitant increases in apoptosis-associated proteins as detected via Western blotting and apoptotic induction as measured via flow cytometry. Phosphorylated YAP levels fell and non-phosphorylated YAP levels rose following such treatment. Transfection with shYAP was successful as a means of generating stable HOS cell lines, and Western blotting analyses of these cells revealed reductions in proteins associated with cellular proliferation together with the upregulation of apoptosis-related proteins. MDA assays indicated that erastin combined with YAP knockdown enhanced the sensitivity of HOS cells to MPPa-PDT treatment.
These data indicate that ferroptosis and YAP knockdown can enhance osteosarcoma cell sensitivity to MPPa-PDT therapy.
本研究旨在探讨 Yes 相关蛋白（YAP）敲低对人骨肉瘤（HOS）细胞对 Pyropheophorbide-α 甲酯介导的光动力疗法（MPPa-PDT）敏感性的影响，并评估 YAP 沉默联合铁死亡诱导剂 Erastin 治疗如何提高 HOS 细胞对 MPPa-PDT 的敏感性，以更好地阐明这些表型的分子机制。
MPPa-PDT 后 12 小时，通过 Hoechst 染色和流式细胞术评估 HOS 细胞的凋亡死亡。通过 Western blot 和免疫荧光染色评估 MPPa-PDT 后 12 小时细胞中 YAP 的表达。使用 BODIPY-C11 评估脂质过氧化。转导 shYAP 慢病毒后，通过 Western blot 评估与增殖、凋亡和铁死亡相关的蛋白表达。EdU 检测和集落形成检测分析细胞增殖。用 Erastin 处理 HOS 细胞建立铁死亡模型。Western blot 检测 shYAP 和 erastin 处理后铁死亡相关蛋白水平的变化，用 MDA 试剂盒检测各组细胞增殖和 MDA 水平的变化。
MPPa-PDT 后 12 小时，HOS 细胞表现出凋亡特征，包括核碎裂和固缩，同时 Western blot 检测到凋亡相关蛋白增加，流式细胞术检测到凋亡诱导增加。这种处理后磷酸化 YAP 水平下降，非磷酸化 YAP 水平上升。shYAP 转染成功，可用于生成稳定的 HOS 细胞系，这些细胞的 Western blot 分析显示细胞增殖相关蛋白减少，凋亡相关蛋白上调。MDA 检测表明，Erastin 联合 YAP 敲低增强了 HOS 细胞对 MPPa-PDT 治疗的敏感性。
这些数据表明铁死亡和 YAP 敲低可增强骨肉瘤细胞对 MPPa-PDT 治疗的敏感性。
