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Aging and Disease    2015, Vol. 6 Issue (6) : 456-465     DOI: 10.14336/AD.2015.0327
Original Article |
Genistein, the Isoflavone in Soybean, Causes Amyloid Beta Peptide Accumulation in Human Neuroblastoma Cell Line: Implications in Alzheimer's Disease
Gargi Chatterjee,Debashree Roy,Vineet Kumar Khemka,Mrittika Chattopadhyay,Sasanka Chakrabarti*
Department of Biochemistry, Institute of Post Graduate Medical Education and Research, 244 AJC Bose Road, Kolkata-700020, India.
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Abstract  

The isoflavone, genistein, present in soybean is being actively investigated for its potential beneficial effect against Alzheimer’s disease. Our data, however, show that in SHSY5Y cells genistein causes increased expression (mRNA and protein) of amyloid precursor protein (APP), increased mRNA expression and activity of β-secretase and diminished level of insulin degrading enzyme (IDE) which also degrades amyloid beta peptide. These effects of genistein lead to enhanced accumulation of amyloid beta peptide (Aβ42) in SHSY5Y cells. The results do not support the view that genistein could be a putative drug against AD and instead strengthen the epidemiological study which implies that genistein content of soybean food product (Tofu) leads to cognitive impairment.

Keywords Alzheimer’s disease      genistein      amyloid precursor protein      β-secretase      insulin degrading enzyme      amyloid β42.     
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Just Accepted Date: 03 April 2015   Issue Date: 18 November 2015
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Gargi Chatterjee
Debashree Roy
Vineet Kumar Khemka
Mrittika Chattopadhyay
Sasanka Chakrabarti
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Gargi Chatterjee,Debashree Roy,Vineet Kumar Khemka, et al. Genistein, the Isoflavone in Soybean, Causes Amyloid Beta Peptide Accumulation in Human Neuroblastoma Cell Line: Implications in Alzheimer's Disease[J]. A&D, 2015, 6(6): 456-465.
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http://www.aginganddisease.org/EN/10.14336/AD.2015.0327     OR     http://www.aginganddisease.org/EN/Y2015/V6/I6/456
Figure 1.  Changes in APP expression by genistein in SHSY5Y cells. SHSY5Y cells were incubated without (control) or with varying concentrations of genistein (10 - 30 µM) for 48 h followed by the measurement of APP mRNA expressions or intracellular level of APP as described in the MATERIALS AND METHODS. (A) RT-qPCR analysis of APP mRNA expression. (B) Representative immunoblot of APP in control or 20 µM genistein-treated cells. The two lanes were aligned from different parts of the same blot. C. Densitometric analysis of APP band intensity. The expression level was presented as fold increase over control. The results are the means ± SEM of 6 observations. Statistically significant, Ψp< 0.05 vs. control.
Figure 2.  β-Secretase expression and activity in SHSY5Y cells after exposure to genistein. The SHSY5Y cells were incubated without (control) or with varying concentrations of genistein (10 - 30 µM) for 48 h. The cell lysate was analyzed for β-secretase mRNA expression or activity as described in the text. (A) RT-qPCR analysis of β-secretase mRNA expression. The results, expressed as fold increase over control, are the means ± SEM of 4 observations. Statistically significant, Ψp< 0.05 vs. control. (B) β-Secretase enzyme activity. The results are the means ± SEM of 6 observations. Statistically significant, ϕp< 0.01 vs. control.
Figure 3.  Genistein effect on IDE and neprilysin level in SHSY5Y cells. SHSY5Y cells were grown in the absence (control) or in the presence of genistein (20 µM) for 48 h. The cell lysate was analysed for IDE content by immunoblotting using γ-actin as the loading control or neprilysin enzyme activity as described in the ‘MATERIALS AND METHODS’. (A) Representative blot. (B) Densitometric analysis of IDE immunoblots with the band intensity expressed as the fold changes with respect to the control. C. Neprilysin enzyme activity. The results are the means ± SEM of 6 observations. Statistically significant, *p< 0.001 vs. control; ϕp< 0.01 vs. control.
Figure 4.  Effect of genistein on Aβ42 content of SHSY5Y cells. SHSY5Y cells were incubated without (control) or with 20 µM of genistein for 48 h. (A) The cell lysate was analyzed for Aβ oligomers by immunoblotting as mentioned in the text. (B) Amido black stained membrane from a parallel blot. (C) Oligomerized Aβ42 analyzed by 5 - 15% SDS-PAGE followed by immunoblotting.
Figure 5.  Immunoassay of Aβ42. Amyloid β42 in the cell lysate as well as that released in the culture medium was measured as described in ‘MATERIALS AND METHODS’ from control and genistein (20 µM) treated SHSY5Y cells. A. Released Aβ42 (extracellular) in culture supernatant. B. Intracellular Aβ42 in cell lysate. The results are the mean ± SEM of four experimental observations. Statistically significant, ϕp< 0.01 vs. control; Ψp< 0.05 vs. control.
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