Uncorrected Proof Int J Cancer Manag. 2022 August; 15(8):e117962. Published online 2022 September 12. doi: 10.5812/ijcm-117962. Research Article The Expression Pattern of Non-apoptotic Cell Death Pathway in Osteosarcoma: Necroptosis and Autophagy as Backup Mechanisms for Therapeutics Strategy Marzieh Neykhonji 1 , Shima Nazem 1 , Hamid Ghaedi 2 , Alireza Mirzaei 3 , Masoumeh Tavakoli-Yaraki 4, * and Zahra Shahsavari 5, ** 1 Department of Laboratory Medicine, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Bone and Joint Reconstruction Research Center, Shafa Orthopedic Hospital, Iran University of Medical Sciences, Tehran, Iran 4 Department of Biochemistry, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 5 Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran * Corresponding author: Department of Biochemistry, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. Email: masoumeh.tavakoli@gmail.com ** Corresponding author: Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Email: shahsavari.z@gmail.com Received 2021 July 16; Revised 2022 August 10; Accepted 2022 August 30. Abstract Background: Among the primary bone tumors, osteosarcoma accounts for a malignant tumor with a high rate of progression and poor prognosis. Despite the achievement of combined therapy regimens in improving patients’ overall survival, patients with osteosarcoma confront the chemoresistance obstacle. Objectives: This study aimed at determining the expression pattern of autophagy and necroptosis pathways mediators in osteosar- coma tumors. Methods: The expression level of autophagy main mediators such as autophagy-associated protein 5 (ATG5), Beclin 1 (BECN1), and microtubule-associated protein 1A/1B-light chain 3 (LC3), necroptosis biomarkers such as receptor-interacting protein kinases (RIPK1 and RIPK3), and mixed lineage kinase domain-like (MLKL) were evaluated in 80 bone tissues including 60 bone tumors (40 malig- nant tumors and 20 benign tumors) and 20 margin tissues, using real-time PCR. The correlations of gene expression levels with the patient’s clinical and pathological features were considered. Results: Based on our data, ATG5, BECN1 and LC3 expression were down-regulated in osteosarcoma tumors compared to margin tissues. Also, malignant osteosarcoma tumors showed a significant decrease in the expression level of RIPK1 and MLKL as necroptosis regulators, which revealed a correlation with tumor malignancy. In addition, the higher expression levels of BECN1, LC3, RIPK1, and MLKL were observed in tumor tissues of patients under the chemotherapy regimen, indicating the relevance of autophagy and necroptosis pathways with the patient’s response to therapy. Conclusions: Reduction in the expression level of autophagy and necroptosis mediators in high-grade osteosarcoma tumors indi- cates the possible impact of these pathways on the rate of proliferation and growth of osteosarcoma tumor cells and can emphasize the importance of cell death alternative pathways for treatment when apoptosis machinery is mutated and cause chemoresistance. Keywords: Autophagy, Necroptosis, Osteosarcoma, Chemotherapy Resistance, Therapeutic Strategy 1. Background Primary bone tumors are considered a leading cause of death and impose a burden of morbidity on patients, worldwide. Among the different types of primary bone tumors, osteosarcoma is a common type with the abil- ity to induce a high degree of malignancy, metastasis at early stages, poor prognosis, and high rate of progres- sion in individuals with different age groups (1). Cur- rent therapeutic and detection approaches failed to im- prove the survival rate of osteosarcoma and its consider- able death rate remains (2). Notably, the combined therapy approaches such as surgery in combination with multiple chemotherapy regimens reached a remarkable improve- ment in the patient’s survival rate; however; chemoresis- tance is observed in some patients and worth to be con- sidering (3). Determining how cell death pathways can overcome the problem of resistance to chemotherapy in these patients can make treatment more effective. It has Copyright © 2022, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.