Chin J Integr Med


ORIGINAL ARTICLE 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 ) Mohd Shahnawaz Khan1, Shams Tabrez2, Nayyar Rabbani1, Mohammad Oves3, Aaliya Shah4 Mohammad A. Alsenaidy5, and Abdulrahman M. Al-Senaidy1 Objective: To investigate the aggregation and fibrillation of insulin at low pH and moderate ABSTRACT Objective: temperature; and to further test the aggregated insulin for its cytotoxicity on human neuroblastoma (SH-SY5Y) Methods:: Bovine pancreatic cell line and inhibition of the cytotoxicity by black seeds (Nigella sativa ) extract. Methods 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 Results:: Our bromide (MTT), lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assay kit. Results 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 Conclusion:: Insulin molded into amyloid amyloid formation and protected the cells against amyloid toxicity. Conclusion 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. KEYWORDS neurological disease, diabetes, insulin, black seeds, cell lines, amyloid, SH-SY5Y cell lines

Protein aggregation is a prevalent phenomenon encountered in the medical field, and the resulting protein aggregates can cause serious deleterious effects in vivo . It has severe biochemical, physiological and clinical implication. (1,2) More than 20 dreadful diseases have been found to owe their etiology to amyloid formation by protein aggregation. (1) These diseases include hemodialysis amyloidosis, type 2 diabetes, Parkinson's disease, Huntington's disease, and Alzheimer's disease.(3,4) In recent years, several non-pathogenic proteins and peptides have been shown to form amyloid fibrils in vitro including acyclophosphatase, (5) cold-shock protein, (6) hen lysozyme,(7) B1 domain of protein G,(8) SH3 domain,(9) cytochrome C, (10) myoglobin (11) and pancreatic cystatin.(12)

Insulin has tendency to aggregate and form fibrils resulting in diabetes, it is prudent to target insulin as model protein to study its aggregation and folding. ©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2015 Supperted by the King Saud University, Saudi Arabia (No. RGP-215) 1. Department of Biochemistry, Protein Research Chair College of Science, King Saud University, Riyadh (11451), Saudi Arabia; 2. King Fahd Medical Research Center, King Abdulaziz University, Jeddah (21589), Saudi Arabia; 3. Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah (21589), Saudi Arabia; 4. Department of Clinical Biochemistry, Sheri-Kashmir Institute of Medical Sciences, Soura (190011), Srinagar, India; 5. Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh (11451), Saudi Arabia Correspondence to: Dr. Mohd Shahnawaz Khan, Tel: 966508570982, E-mail: [email protected] DOI: 10.1007/s11655-015-2153-y

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Bovine insulin is a 51-amino-acid protein hormone involved in regulating glucose metabolism, which is used to treat diabetes. Its monomeric form consists of a 21-residue A chain containing one disulfide bond and a 30-residue B chain, which are linked together by a pair of inter-chain disulfide bridges.(13) Structurally, insulin primarily adopts a helical conformation at pH 2.0 (–44% α-helix, –9% β-sheet, –30% random coil, and –19% turn)(14) and exists as a mixture of oligomeric states, including hexamers, dimers, and monomers, in a solution the composition of which is strongly depending on the environmental conditions.(13,14) I n s u l i n f o r m s a m y l o i d - l i k e f i b r i l s (13) t h a t pose a variety of problems in its biomedical and biotechnological applications. Amyloid deposits of insulin have been observed both in patients with type 2 diabetes and in normal aging, as well as after subcutaneous insulin infusion and after repeated injection. Recent literature indicates increasing incidence of insulin amyloid in clinical situations.(15,16) Injected insulin seems to form fibrils irrespective of the site of injection. For example, amyloid insulin was observed in thighs,(15) shoulders,(16) arms,(17) and abdominal walls(17,18) of patients at or around the site of repeated injections. The therapeutic strategies for the treatment of amyloidogenic disorders have been proposed in two pathways, which are the inhibition of amyloid formation and disruption of the formed amyloid assemblies. Natural polyphenolic compounds, which are found extensively in food and herbal remedies, have been demonstrated to disrupt amyloid structures and to attenuate the cytotoxicity of amyloid fibrils.(19,20)

also contain nigellidine, nigellimine, nigellcine, and saponine and water soluble triterpene.(21) The black seeds extract has been used to suppress cough,(21) disintegrate renal calculi,(22) retard the carcinogenic process, (23) treat abdominal pain, diarrhea and flatulence,(24) has anti-inflammatory(25) and antioxidant effects.(26) In the present study, the anti-amyloidogenic effect of black seeds was investigated in vitro using bovine insulin as a model protein. Thioflavin T (ThT) fluorescence, 1-analinonaphthalene-8-sulfonic acid (ANS) fluorescence, circular dichroism, dynamic light scattering (DLS) and cytotoxicity assays were utilized to characterize insulin fibril and to determine the effect of black seeds extract on amyloidogenesis of bovine insulin.

METHODS Materials Bovine insulin was purchased from Fluka, USA. All the chemicals (ANS, ThT) were analytical grade, and were from Sigma, USA.

Preparation of Protein Samples Solutions of monomeric human insulin, 4 mg/mL, was freshly prepared in 20% acetic acid. The concentration of insulin was determined using an extinction coefficient of 1.0 for 1 mg/mL at 276 nm.(13) 1 mmol/L ThT was prepared by dissolving ThT in double-distilled water and the concentration was determined using a molar extinction coefficient of 24,420 mol-1•cm-1 at 420 nm.

Fibrillation of Insulin Black seed plant (Nigella sativa ) is an annual herb of the Ranunculaceae family, which grows in countries bordering the Mediterranean Sea, Pakistan and India. This widely distributed plant is native to Arab countries and other parts of the Mediterranean region.

Solutions of insulin (500 μL, 4 mg/mL) in 20% acetic acid was incubated at 45 ℃ in a glass vial on a stirrer with a small magnetic bead spinning at the bottom of the vial at 400 r/min. Aliquots from this solution were taken at desired time intervals.

Kinetics of Insulin Fibrillation Chief constituents/ingredients of N . sativa are thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (THY); p-cymene, 4-terpineol, and t-anethol. N . sativa seeds contain other ingredients as well, such as carbohydrates, fats, vitamins, mineral elements, proteins, and essential amino acids.(21) Black seed

Monomeric insulin at low pH 1.8 and under stirring conditions leads to insulin fibrillation as indicated by ThT fluorescence kinetics. ThT is a fluorescent dye and is frequently used as a specific probe for fibril formation in vitro . (27) A sample of 5 μL aliquots were added to solution containing 20 μmol/L ThT in 20 mmol/L Tris-HCl buffer, pH 7.4,

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shaken a few times before measuring the fluorescence emission on spectrofluorimeter (JASCO FP-750) at room temperature. A background fluorescence spectrum obtained by running a blank buffer was subtracted from each sample fluorescence spectrum. The excitation wavelength was at 444 nm, and the emission was recorded at 482 nm. Fluorescence intensity at 482 nm was plotted against time.

Circular Dichroism Aliquot (40 μL, 0.2 mg/mL) were placed in a cuvette with 0.1 mm path length, and CD spectra were recorded on an applied Photophysics spectrophotometer (Chirascan, UK) at 25 ℃. Spectrum of the buffer was subtracted from the sample spectra for background correction.

Hyrophobicity Measurement (ANS Fluorescence) Aliquots (5 μL) from the incubated mixture were added to solution containing 5 μmol/L ANS in 20 mmol/L Tris-HCl buffer, pH 7.4 in a total volume of 1 mL, and the fluorescence was measured with a spectrofluorometer at room temperature. The excitation wavelength was 350 nm, and the emission was measured at 460 nm. The values of fluorescence intensity at 462 nm and the values of maximal wavelength of the emission spectra were plotted against time.


pre-treated with 0–200 μg of black seeds extract and incubated at 37 ℃ in a glass vial on a stirrer with a small magnetic bead spinning at the bottom of the vial at 400 r/min. Aliquots from this solution were taken after 24 h incubation for fibrillation analysis using ThT fluorescence assay.

Cell Culture and Cytotoxicity Assay Human neuroblastoma SH-SY5Y cells were cultured in humidified 5% (v/v) CO 2/air at 37 ℃ in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/mL penicillin. Cells were plated at a density of 5×10 4 cells/mL in 96-well plates. Pre-formed insulin amyloid (20 μmol/L) with or without black seeds extract was diluted with fresh medium and added to individual wells. The final concentration of fibrillated insulin in each culture well was 2 μmol/L. The same volume of medium was added to control cultures. The plates were then incubated for an additional 48 h at 37 ℃. Cell viability was determined using a MTT toxicity assay by adding 10 mL of 5 mg/mL MTT to each well. After 3 h of incubation at 37 ℃, the medium was gently removed, and then 100 mL dimethyl sulphoxide was added to each well. Plates were shaken at room temperature for 10 min to dissolve the crystals before the absorbance at 490 nm was measured using a microplate reader.

Dynamic Light Scattering Aggregate analysis of insulin incubated under 20% acetic acid and 400 r/min stirring agitation was measured by DLS. Different aliquots after 0 (control), 1, 5 and 27 h were analyzed. Hydrodynamic particle size of insulin aggregate was determined by measuring the DLS by use of a ZetaSizer-HT (Malvern, UK).

Lactate Dehydrogenase Determination for Cell Membrane Damage

herbalist in Riyadh, Saudi Arabia. The seeds were botanically authenticated by a specialist of plant taxonomy in Botany Department, King Saud University, Riyadh. The extraction procedure of N . sativa seeds was according to Musa, et al.(28) Briefly, the seeds were identified, cleaned, dried, mechanically powdered and extracted with 96% ethanol and evaporated with rotary evaporator to render the extract alcohol free. The extract was kept in a domestic refrigerator at 4 ℃.

Lactate dehydrogenase (LDH) released into the medium is an index of cell membrane damage because of the enzyme's high intracellular localization is used as marker. The plasma membrane damage was evaluated by measuring extracellular LDH activity in the medium. SH-SY5Y cells were pretreated with different concentrations of the black seeds extract (25–200 μg), and then exposed to 100 μmol/L insulin amyloid for 24 h. After the incubation, 50 μL of culture supernatants were collected from each well. The LDH activity was determined with a colorimetric LDH assay kit. Total cellular LDH activity was determined by solubilizing the cell with 0.2% Triton X-100. The release of intracellular LDH to the extracellular medium is expressed as a percentage of total cellular LDH activity.

Amyloid Inhibition Using Black Seeds

Reactive Oxygen Species Measurement

Preparation of Black Seeds Extract N . sativa seeds were purchased from the local

Insulin (4 mg/mL) in 20% acetic acid was

The levels of intracellular reactive oxygen species

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RESULTS Kinetics of Insulin Fibrillation by ThT Assay Changes in ThT emission at 482 nm as a function of time of insulin incubation is shown in Figure 1. The figure indicated that the increase in ThT fluorescence intensity followed a typical sigmoidal pattern. The initial period of incubation showed no substantial change (0–4 h incubation) in fluorescence. This supposedly corresponded to the nucleation phase. The second phase of incubation (4–24 h) showed rapid increase in fluorescence (elongation phase) and the final plateau region (24–32 h) reflected the maturation phase of insulin fibrillation. Insulin under acidic condition perturbed a sensitive balance of net charge and induced its fibrillation. ThT Fluorescence Intensity

30 25 20 15 10 5 0




4 21 Time (h)




Figure 1. Fibrillogenesis of Insulin by ThT Fluorescence Measurment

40 30 20 10 0 –10 –20 –30 –40 –50 200






Wavelength (nm)

Figure 2. Structural Changes Accompanying the Process of Insulin Fibrillation as Detected by Far-UV CD Notes: Insulin samples (0.2 mg/mL) at pH 2.0 were incubated at 0, (- - - -); 3 (. . . .) and 27 (—) h. Changes in insulin secondary structure were monitored in far-UV-CD spectrum (190–250 nm)

Hydrophobicity Measurement (ANS Binding Fluoresence) The kinetic profile of ANS binding to insulin under acidic condition is shown in Figure 3. The data illustrated an increase in the fluorescence intensity from native to fibrillated stages. This indicated protein structural changes due to acidic condition exposing hydrophobic regions in the process of insulin fibrillation. Analysis of ANS-Insulin binding exhibited initially a red shift (between 0–4 h) followed by a blue shift thereafter, suggesting biphasic sequential structural change during insulin fibrillation. 140 120 100 80

505 Insulin 0 h Insulin 1 h Insulin 4 h Insulin 20 h Insulin 25 h

Wavelength (nm)

Results were expressed as mean±standard error of at least three independent experiments (each in triplicate). One way ANOVA was employed to detect differences between the groups of treated and control. P values less than 0.05 were considered statistically significant.

Far-UV CD spectra obtained for insulin at various time of incubation is shown in Figure 2. The CD spectrum measured for the sample at 0 h has double minima at 208 and 222 nm, which is a characteristic of α-helical structure. Spectra obtained from samples incubated up to 3 h did not exhibit any significant difference from the native monomer. The CD spectrum showed a characteristic predominant β-structured trough at 218 nm after 27 h of incubation. Circular dichroism (millidegree)

Statistical Analysis

CD Analysis

ANS fluorescence intensity

(ROS) were determined by the change in fluorescence resulting from the oxidation of the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). (29) Briefly, SH-SY5Y cells were pretreated with different concentrations of the black seeds extract, and then exposed to 100 μmol/L insulin amyloid for 24 h. After the medium was removed, the cells were incubated with 100 μmol/L DCFH-DA for 30 min, and the cells were washed to remove the extracellular DCFH-DA. The cells were then suspended in phosphate buffered saline (PBS). The fluorescence intensity was determined using a CytoFluor multi-well plate reader (Fluoroskan Ascent, Thermo Lab Systems, USA) at the excitation wavelength of 485 nm and emission wavelength of 535 nm.

500 495 490 485 480

0 1 4 10 20 25 Time (h)

60 40 20 0 400 420 440 460 480 500 520 540 560 580 600 Wavelength (nm)

Figure 3. Structural Changes Accompanying the Aggregation/Fibrillation of Insulin by ANS Fluorescence Analysis

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217 (d.nm)

(a) Control

242 (d.nm)

(b) 1 h

Intensity (%)

Intensity (%)

20 10


15 10 5

0 0.1



10 100 Size (d.nm)


0 0.1



560 (d.nm)

(c) 5 h

Intensity (%)

Intensity (%)

10 100 Size (d.nm)



290 (d.nm)

(d) 27 h





6 4 2

0 0.1


10 100 Size (d.nm)


Figure 4.


DLS measurement results demonstrated an increase in the size of insulin aggregation during fibrillation. The size of insulin aggregate formed after 27 h-incubation was 590 nm in diameter, in contrast to control insulin (217 nm, Figure 4), which suggested a change in the hydrodynamic radii of insulin during fibrillation.

Amyloid Inhibition Using Black Seeds Extract Simultaneous incubation of various concentrations of black seeds extract with insulin, resulted in dosedependent decrease in the ThT fluorescence intensity indicating black seeds protection of insulin against fibrillation (Figure 5). 4.0

0 0.1


10 100 Size (d.nm)



DLS Measurement of Fibrillated Insulin

DLS of Aggregated Insulin

ThT fluorescence intensity


Insulin (0 h)


Insulin (24 h)


Insulin+25 μg black seeds


Insulin+50 μg black seeds


Insulin+200 μg black seeds


Whereas, at 1 mmol/L concentration the cells viability was reduced to 10% as compared with control.

Cytotoxicity Inhibition by Black Seeds Extract Pre-treatment of the cells with various concentrations of black seeds extract followed by incubation with 50 μmol/L insulin fibrils showed dosedependent protection of cell viability by defibrillating insulin fibrils (Figure 6B). Black seeds extract at concentrations of 10, 25, 50, 100, and 200 μg increased cell viabilities by 10%, 24%, 44%, 60% and 84%, respectively, as compared with insulin fibril-treated cells.

LDH Determination Release of LDH in the cells treated with insulin fibrils suggested that insulin firbrils resulted in membrane damage and decrease in cell viability (Figure 7). However, black seeds extract-incubated cells showed increased cell viability and decrease in LDH activity.


Reactive Oxygen Measurement

0.5 0 460










Wavelength (nm)

Figure 5. Amyloid Inhibition Study Using Fluorescence Measurement

Cytotoxicity of Fibrillated Insulin As shown in Figure 6A, addition of fibrillated insulin to the cell culture resulted in decrease of MTT reduction by SH-SY5Y cells. A marked reduction in cell viability was observed (50%) at 50 μmol/L insulin fibril concentration.

Interestingly, the intracellular ROS accumulation resulting from insulin fibrils treatment was reduced when cells were treated with various concentrations of black seeds extract (Figure 8), suggesting that black seeds may have the ability to scavenge ROS.

DISCUSSION Insulin aggregation studies are vital to understand the factors involved in the insulin pathway

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leading to its aggregation in vivo and possible therapeutic agents for amyloidosis. The number of people suffering from diabetes and its complications is immense. In 2000, 171 million people were estimated to be suffering from diabetes and the number is expected to increase to 300 million in 2030.(30)

Cell viability (% of control)

120 100 80 60 40 20 0




5 10 25 50 100 200 500 1000 Insulin fibrills (μmol/L)

Cell viability (% of control)

120 100 80 60 40 20 0

Control BSC







Black seeds (μg/mL)

Figure 6. Cytotoxicity of Amyloid and Its Attenuation with Black Seeds Notes: (A) Viability of SH-SY5Y cells in the presence of fibrillated insulin. (B) Fibrillated insulin (100 μmol/L) incubated with or without black seeds was added to SH-SY5Y cells and the cell viability was measured using MTT assays. BSC: black seeds control; IA: insulin amyloid. P

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).

To investigate the aggregation and fibrillation of insulin at low pH and moderate temperature; and to further test the aggregated insulin for its cyto...
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