Physico-chemical stress induced amyloid formation in insulin: Amyloid characterization, cytotoxicity analysis against human neuroblastoma cell lines and its prevention using black seeds (Nigella sativa)

Abstract

Objective: To investigate the aggregation and fibrillation of insulin at low pH and moderate temperature; and to further test the aggregated insulin for its cytotoxicity on human neuroblastoma (SH-SY5Y) cell line and inhibition of the cytotoxicity by black seeds (Nigella sativa) extract. Methods: Bovine pancreatic insulin was incubated at pH 2.0, 45 ℃ under stirring condition at 400 r/min for 24 h. Amyloids like structures in the aggregated insulin were characterized using various techniques such as thioflavin T assay (ThT), 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, circular dichroism (CD) and dynamic light scattering (DLS). Moreover, cytotoxicity of aggregated insulin was monitored on SH-SY5Y cell line in the presence and absence of black seeds extract using standard 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assay kit. Results: Our finding demonstrated that insulin under the mentioned conditions formed amyloid-like structure. ANS binding to aggregated insulin showed increase in fluorescence, suggesting structural change and increase in hydrophobicity in insulin occurring during the fibril formation. DLS measurement revealed progressive increase in hydrodynamic radius of aggregated insulin. Cytotoxicity assays illustrated aggregated insulin induced apoptosis in SH-SY5Y cell through ROS formation. Moreover, LDH measurement showed aggregated insulin triggered membrane damage in SH-SY5Y cell lines. Black seeds extract was found to inhibit amyloid formation and protected the cells against amyloid toxicity. Conclusion: Insulin molded into amyloid like structure at low pH and under stirring conditions. Characterization of insulin aggregates illustrated conformational change in insulin and it experiences α-helix to β-sheet transition during the course of fibrillation. Black seeds extract inhibited amyloid progression of insulin via ROS scavenging and restrained the cytotoxicity caused by insulin fibrils suggesting black seeds containing polyphenols may serve as a lead structure to a novel anti-amyloidogenic drugs.