YAP knockdown in combination with ferroptosis induction increases the sensitivity of HOS human osteosarcoma cells to pyropheophorbide-α methyl ester-mediated photodynamic therapy

Fangbiao Zhan; Ye Zhang; Qiang Zuo; Chaozheng Xie; Huanhuan Li; Ling Tian; Chunrong Wu; Zhiyu Chen; Chaohua Yang; Yang Wang; Qiaochu Li; Tao He; Haoyang Yu; Jian Chen; Jiangxia Xiang; Yunsheng Ou

doi:10.1016/j.pdpdt.2022.102964

YAP knockdown in combination with ferroptosis induction increases the sensitivity of HOS human osteosarcoma cells to pyropheophorbide-α methyl ester-mediated photodynamic therapy

Photodiagnosis Photodyn Ther. 2022 Sep:39:102964. doi: 10.1016/j.pdpdt.2022.102964. Epub 2022 Jun 12.

Authors

Fangbiao Zhan¹, Ye Zhang², Qiang Zuo³, Chaozheng Xie⁴, Huanhuan Li⁵, Ling Tian⁶, Chunrong Wu⁷, Zhiyu Chen², Chaohua Yang², Yang Wang², Qiaochu Li², Tao He², Haoyang Yu², Jian Chen⁸, Jiangxia Xiang⁹, Yunsheng Ou¹⁰

Affiliations

¹ 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.
² 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.
³ 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; West China-Guang'an Hospital, Sichuan University, Guang'an, Sichuan 638000, China.
⁴ Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
⁵ Department of Emergency Medicine, Chongqing University Three Gorges Hospital, Wanzhou, Chongqing 404000, China.
⁶ Department of Clinical Laboratory, Chongqing University Three Gorges Hospital, Wanzhou, Chongqing 404000, China.
⁷ Department of Oncology, Jiangjin Central Hospital of Chongqing, Chongqing 402260, China.
⁸ Department of Orthopedics, Chongqing University Three Gorges Hospital, Chongqing Municipality Clinical Research Center for Geriatric diseases, Wanzhou, Chongqing 404000, China.
⁹ 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; Traumatology department, Chongqing university central hospital. 1#, Jiankong road, Yuzhong district, Chongqing 400014, China.
¹⁰ 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. Electronic address: ouyunsheng2001@163.com.

PMID: 35705143
DOI: 10.1016/j.pdpdt.2022.102964

Abstract

Background and aims: 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.

Methods: 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^581/591-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.

Results: 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.

Conclusions: These data indicate that ferroptosis and YAP knockdown can enhance osteosarcoma cell sensitivity to MPPa-PDT therapy.

Keywords: Apoptosis; Ferroptosis; MPPa-PDT; Osteosarcoma; Photodynamic therapy; YAP.

MeSH terms

Apoptosis
Bone Neoplasms* / drug therapy
Cell Line, Tumor
Esters
Ferroptosis*
Humans
Osteosarcoma* / drug therapy
Photochemotherapy* / methods
Photosensitizing Agents / pharmacology
Photosensitizing Agents / therapeutic use
Porphyrins* / pharmacology

Substances

Esters
Photosensitizing Agents
Porphyrins