Mehdi Rabiee Valashedi1, Amaneh Mohammadi Roushandeh2, Kazuo Tomita3, Yoshikazu Kuwahara4, Zahra Pourmohammadi-Bejarpasi2, Pouya Safarzadeh Kozani1, Tomoaki Sato3, Mehryar Habibi Roudkenar5. 1. Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran. 2. Burn and Regenerative Medicine Research Center, Velayat Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran. 3. Department of Applied Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. 4. Division of Radiation Biology and Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan. 5. Burn and Regenerative Medicine Research Center, Velayat Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran; Cardiovascular Diseases Research Center, Department of Cardiology, Heshmat Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran. Electronic address: roudkenar@gums.ac.ir.
Abstract
AIMS: Lipocalin 2 (Lcn2) is an antioxidant-related protein upregulated in various cellular stress conditions, especially cancer. In this study, we abrogated Lcn2 expression in MDA-MB-231 breast cancer cells using the CRISPR/Cas9 technology and evaluated its effect on cellular proliferation, migration, and ferroptotic cell death. MAIN METHODS: Validated human Lcn2 CRISPR/Cas9 knockout (KO) and homology-directed repair (HDR) plasmids were co-transfected into MDA-MB-231 breast cancer cells. Lcn2 gene knockout was confirmed at the transcriptional and protein levels using reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cytotoxicity assay was performed in the presence or absence of erastin, cisplatin (CDDP), and ferrostatin-1 using the CCK-8 method. Ferroptosis level was measured using the malondialdehyde assay lipid peroxidation kit. The migration capacity of the cells was also evaluated using the scratch assay. KEY FINDINGS: Targeting Lcn2 using CRISPR/Cas9 reduced cellular proliferation and migration capability, and elevated the vulnerability of MDA-MB-231 cells to cisplatin. Furthermore, Lcn2 expression loss effectively promoted erastin-mediated ferroptosis in MDA-MB-231 cells. SIGNIFICANCE: Inhibition of Lcn2 is a potentially useful strategy for sensitizing MDA-MB-231 tumor cells to ferroptotic cell death.
AIMS: Lipocalin 2 (Lcn2) is an antioxidant-related protein upregulated in various cellular stress conditions, especially cancer. In this study, we abrogated Lcn2 expression in MDA-MB-231 breast cancer cells using the CRISPR/Cas9 technology and evaluated its effect on cellular proliferation, migration, and ferroptotic cell death. MAIN METHODS: Validated human Lcn2 CRISPR/Cas9 knockout (KO) and homology-directed repair (HDR) plasmids were co-transfected into MDA-MB-231 breast cancer cells. Lcn2 gene knockout was confirmed at the transcriptional and protein levels using reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cytotoxicity assay was performed in the presence or absence of erastin, cisplatin (CDDP), and ferrostatin-1 using the CCK-8 method. Ferroptosis level was measured using the malondialdehyde assay lipid peroxidation kit. The migration capacity of the cells was also evaluated using the scratch assay. KEY FINDINGS: Targeting Lcn2 using CRISPR/Cas9 reduced cellular proliferation and migration capability, and elevated the vulnerability of MDA-MB-231 cells to cisplatin. Furthermore, Lcn2 expression loss effectively promoted erastin-mediated ferroptosis in MDA-MB-231 cells. SIGNIFICANCE: Inhibition of Lcn2 is a potentially useful strategy for sensitizing MDA-MB-231 tumor cells to ferroptotic cell death.