C N Zhi1, L L Lai1, C S Dou1, X H Wang2, P Zhao1, J L Fu1, B Y Yao1. 1. School of Public Health, Peking University, Beijing 100191, China. 2. School of Basic Medical Sciences, Peking University, Beijing 100191, China.
Abstract
Objective: To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells. Methods: SK-N-SH cells were treated with MnCl(2) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl(2) at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results: Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl(2) treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl(2) treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl(2) treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl(2), while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn't observed significant difference of the activity of CTSD between different MnCl(2) treatment groups. Conclusion: MnCl(2) could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.
Objective: To investigate the role of lysosomes in manganese-induced toxicity in humanneuroblastomaSK-N-SH cells. Methods:SK-N-SH cells were treated with MnCl(2) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl(2) at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results: Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl(2) treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl(2) treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl(2) treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl(2), while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn't observed significant difference of the activity of CTSD between different MnCl(2) treatment groups. Conclusion:MnCl(2) could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.
Authors: Karin Tuschl; Richard J White; Chintan Trivedi; Leonardo E Valdivia; Stephanie Niklaus; Isaac H Bianco; Chris Dadswell; Ramón González-Méndez; Ian M Sealy; Stephan C F Neuhauss; Corinne Houart; Jason Rihel; Stephen W Wilson; Elisabeth M Busch-Nentwich Journal: Dis Model Mech Date: 2022-06-15 Impact factor: 5.732