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 fi brils resulting in diabetes, it is prudent to target insulin as model protein to study its aggregation and folding.
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
©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: 966- 508570982, E-mail: [email protected]
DOI: 10.1007/s11655-015-2153-y ABSTRACT
ABSTRACT ObjectiveObjective: To investigate the aggregation and fibrillation of insulin at low pH and moderate : 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) 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 (
cell line and inhibition of the cytotoxicity by black seeds (Nigella sativaNigella sativa) extract. ) extract. MethodsMethods: Bovine pancreatic : Bovine pancreatic insulin was incubated at pH 2.0, 45 ℃ under stirring condition at 400 r/min for 24 h. Amyloids like structures 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), in the aggregated insulin were characterized using various techniques such as thioflavin T assay (ThT), 1-anilinonaphthalene-8-sulfonic acid (ANS) fl uorescence, circular dichroism (CD) and dynamic light scattering 1-anilinonaphthalene-8-sulfonic acid (ANS) fl uorescence, circular dichroism (CD) and dynamic light scattering (DLS). Moreover, cytotoxicity of aggregated insulin was monitored on SH-SY5Y cell line in the presence (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 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.
bromide (MTT), lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assay kit. ResultsResults: Our : Our fi nding demonstrated that insulin under the mentioned conditions formed amyloid-like structure. ANS binding fi nding 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 to aggregated insulin showed increase in fluorescence, suggesting structural change and increase in hydrophobicity in insulin occurring during the fi bril formation. DLS measurement revealed progressive increase hydrophobicity in insulin occurring during the fi bril formation. DLS measurement revealed progressive increase in hydrodynamic radius of aggregated insulin. Cytotoxicity assays illustrated aggregated insulin induced 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 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 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.
amyloid formation and protected the cells against amyloid toxicity. ConclusionConclusion: Insulin molded into amyloid : Insulin molded into amyloid like structure at low pH and under stirring conditions. Characterization of insulin aggregates illustrated 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 conformational change in insulin and it experiences α-helix to β-sheet transition during the course of fi brillation. Black seeds extract inhibited amyloid progression of insulin via ROS scavenging and restrained fi brillation. Black seeds extract inhibited amyloid progression of insulin via ROS scavenging and restrained the cytotoxicity caused by insulin fi brils suggesting black seeds containing polyphenols may serve as a lead the cytotoxicity caused by insulin fi brils suggesting black seeds containing polyphenols may serve as a lead structure to a novel anti-amyloidogenic drugs.
structure to a novel anti-amyloidogenic drugs.
KEYWORDS
KEYWORDS neurological disease, diabetes, insulin, black seeds, cell lines, amyloid, SH-SY5Y cell lines neurological disease, diabetes, insulin, black seeds, cell lines, amyloid, SH-SY5Y cell lines
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 disulfi de 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 nsulin forms amyloid-like fibrils(13) that 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 fi brils.(19,20) 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.
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
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. Thiofl avin T (ThT) fluorescence, 1-analinonaphthalene-8-sulfonic acid (ANS) fl uorescence, circular dichroism, dynamic light scattering (DLS) and cytotoxicity assays were utilized to characterize insulin fi bril 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 coeffi cient 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
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
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,
shaken a few times before measuring the fl uorescence emission on spectrofluorimeter (JASCO FP-750) at room temperature. A background fl uorescence 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 fl uorescence intensity at 462 nm and the values of maximal wavelength of the emission spectra were plotted against time.
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).
Preparation of Black Seeds Extract
N. sativa seeds were purchased from the local 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 identifi ed, 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 ℃.
Amyloid Inhibition Using Black Seeds
Insulin (4 mg/mL) in 20% acetic acid was
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 fi brillation analysis using ThT fl uorescence assay.
Cell Culture and Cytotoxicity Assay
Human neuroblastoma SH-SY5Y cells were cultured in humidified 5% (v/v) CO2/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 5×104 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.
Lactate Dehydrogenase Determination for Cell Membrane Damage
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.
Reactive Oxygen Species Measurement
The levels of intracellular reactive oxygen species
(ROS) were determined by the change in fl uorescence 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.
Statistical Analysis
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 signifi cant.
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 fi gure 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 fl uorescence.
This supposedly corresponded to the nucleation phase. The second phase of incubation (4–24 h) showed rapid increase in fluorescence (elongation phase) and the fi nal plateau region (24–32 h) refl ected the maturation phase of insulin fibrillation. Insulin under acidic condition perturbed a sensitive balance of net charge and induced its fi brillation.
Figure 1. Fibrillogenesis of Insulin by ThT Fluorescence Measurment
Time (h) 30
25 20 15 10 5
0 0 1 2 4 21 27 30 32
ThT Fluorescence Intensity
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)
Wavelength (nm) 40
30 20 10 0 –10 –20 –30 –40 –50
200 210 220 230 240 250
Circular dichroism (millidegree)
CD Analysis
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.
Figure 3. Structural Changes Accompanying the Aggregation/Fibrillation of Insulin by
ANS Fluorescence Analysis
Wavelength (nm)
400 420 440 460 480 500 520 540 560 580 600 0 1 4 10 20 25
Time (h) Insulin 0 h
Insulin 1 h Insulin 4 h Insulin 20 h Insulin 25 h
140 120 100 80 60 40 20 0
505 500 495 490 485 480
ANS fl uorescence intensity Wavelength (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 fi brillated stages. This indicated protein structural changes due to acidic condition exposing hydrophobic regions in the process of insulin fi brillation. 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 fi brillation.
DLS of Aggregated Insulin
DLS measurement results demonstrated an increase in the size of insulin aggregation during fi brillation. 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 fi brillation.
Amyloid Inhibition Using Black Seeds Extract Simultaneous incubation of various concentrations of black seeds extract with insulin, resulted in dose- dependent decrease in the ThT fl uorescence intensity indicating black seeds protection of insulin against fi brillation (Figure 5).
Figure 4. DLS Measurement of Fibrillated Insulin
(a) Control
Intensity (%)
0.1 1 10 100
217 (d.nm)
1000 10000 15
10
5
0
Size (d.nm) (c) 5 h
Intensity (%)
0.1 1 10 100
560 (d.nm)
1000 10000 20
15
10
5
0
Size (d.nm)
(d) 27 h
Intensity (%)
0.1 1 10 100
290 (d.nm)
1000 10000 10
8 6 4 2 0
Size (d.nm) (b) 1 h
Intensity (%)
0.1 1 10 100
242 (d.nm)
1000 10000 25
20 15 10 5 0
Size (d.nm)
Figure 5. Amyloid Inhibition Study Using Fluorescence Measurement
Insulin (0 h) Insulin (24 h) Insulin+25 μg black seeds Insulin+50 μg black seeds Insulin+200 μg black seeds
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ThT fl uorescence intensity 0
Wavelength (nm)
460 470 480 490 500 510 520 530 540 550
Whereas, at 1 mmol/L concentration the cells viability was reduced to 10% as compared with control.
Cytotoxicity Inhibition by Black Seeds Extract P r e - t r e a t m e n t o f t h e c e l l s w i t h v a r i o u s concentrations of black seeds extract followed by incubation with 50 μmol/L insulin fibrils showed dose- dependent 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 fi bril-treated cells.
LDH Determination
Release of LDH in the cells treated with insulin fi brils suggested that insulin fi rbrils 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
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 Cytotoxicity of Fibrillated Insulin
As shown in Figure 6A, addition of fi brillated 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 fi bril concentration.
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)
Changes in the conformation of insulin were monitored by a variety of probes during incubation of monomeric insulin under acidic conditions leading to fibrillation. The apparent pH of a 20% acetic acid solution is 1.8, such low pHs are used in the commercial production of recombinant human insulin, thus supporting our experimental design and conditions of insulin fibrillation. At low acidic pH, charge on insulin gets perturbed and thereby destabilize its secondary structure leading to aggregation. Our finding is well supported by the facts that proteins involved in neurological diseases like α-synuclein, Aβ, and several other natively unfolded proteins transform into β-sheet rich fi brils rapidly after gaining a critical amount of ordered secondary structure,(31) whereas globular proteins undergoes faster fi brillation upon destabilization of their secondary structure. In some cases, perturbing a sensitive balance of net charge of the protein (e.g.
acylphosphatase(32) and α-synuclein(31)) induces fi brillation.
CD, DLS and ANS binding studies illustrated conformational change in insulin and formation of intermediates (oligomer) occured during fibrillation.
There is ample evidence favoring the existence of a partially folded intermediate on the fi brillation pathway, thus supporting the proposed mechanism that partially folded conformations are critical for amyloidosis.(31)
We also examined the aggregated state of insulin induced by 20% acetic acid for its biological toxicity.
Although amyloid fi brils are associated with pathological symptoms in a diverse range of diseases,(33) their specific role in inducing certain disease remains unclear. In the present study, the aggregated state of insulin was observed to exert toxicity and reduce cell viability of SH-SY5Y. The interaction of various types of aggregates/fibrils with cell membranes might be leading to the production of ROS which then disrupt the membrane and other biomolecules along with paving way for aggregates into the interior of the cell.(34) All these processes then lead to the destruction and death of the cell. Protein amyloids are reported to be toxic to Figure 7. Effect of Different Concentrations of
Black Seeds on IA-Induced Cell Membrane Damage by LDH Release
Notes: P<0.05, compare with the control (IA) group
Figure 8. Effect of Different Concentrations of Black Seeds on IA-Induced ROS Generation in
SH-SY5Y Cells by DCFH-DA Notes: P<0.05, compare with the control (IA) group
IA (100 μmol/L) IA+BS (25 μg) IA+BS (50 μg) IA+BS (200 μg) 70
60 50 40 30 20 10 0
LDH release (%)
IA (100 μmol/L) IA+BS (25 μg) IA+BS (50 μg) IA+BS (200 μg) 90
80 70 60 50 40 30 20 10 DCF Fluoresence (ROS Level) 0
Figure 6. Cytotoxicity of Amyloid and Its Attenuation with Black Seeds
Notes: (A) Viability of SH-SY5Y cells in the presence of fi brillated 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<0.05, compare with the control group
Insulin fi brills (μmol/L)
0 1 5 10 25 50 100 200 500 1000 120
100 80 60 40 20 0
Cell viability (% of control)
A
Black seeds (μg/mL) ControlBSC IA 10 25 50 100 200 120
100 80 60 40 20 0
Cell viability (% of control)
B
various types of cells including neurons. Their amount and degree of fi brillation contribute to cell toxicity.(35-37)
Since black seeds contain quinones and thymoquinones(21) as their main constituent, seeds extract was tested for its possible anti-aggregating application. Our results showed that black seeds extract inhibited amyloid formation. Further, black seeds extract- incubated cells showed increased cell viability and decrease in LDH activity. This protective effect of black seeds extract against insulin fi brils-mediated cytotoxicity may be attributed to the anti-oxidative property of different constituents of black seeds. Numerous reports have proposed that the anti-oxidant property of polyphenols is involved in anti-amyloid activity.(38,39) Moreover, previous investigations also confirmed that decrease in protein fi bril formation by polyphenols.(40,41) Recent literature demonstrating quinones like EGCG, benzoquinone (BQ) and napthoquinoes (NQ) redirect the amyloid formation and reduces cytotoxicity.(42) Quinones have been also reported to inhibit the classical amyloid formation pathways of insulin, both in the oligomerization and fibrillation stages.(42) ROS scavenging mechanism and presence of quinones in black seeds extract might be the significant aspects for its anti-aggregating and anti-apoptotic function.
In conclusion, insulin under acidic condition undergoes conformational change leading to fi brillation which is toxic to cell viability. Black seeds extract showed inhibition of insulin fibrillation as well as protected against its cytotoxicity in cultured human neuroblastoma cells (SH-SY5Y). Anti-fibrillogenic potential of black seeds might be due to its anti-oxidant activity showed by thymoquinone or other related components present in black seeds. Thus, future research on specific component of black seeds may serve as a lead structure to novel anti-cancer and/or anti-amyloidogenic drugs.
Confl ict of Interest
The authors confirm that this article content has no confl icts of interest.
Author Contributions
Dr. Mohd Shahnawaz khan has designed and perform the work. All other authors contributed equally in experimental work.
Acknowledgement
The authors extend their appreciation to the deanship of scientifi c research at King Saud University for funding the work
through the research group project No. RGP-215.
REFERENCES
1. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 2006;75:333-336.
2. Fink AL. Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 1998;3:9-23.
3. Dobson CM. Principles of protein folding, misfolding and aggregation. Cell Dev Biol 2004;15:3-16.
4. Uversky VN, Fink AL. Conformational constraints for amyloid fibrillation: the importance of being unfolded.
Biochim Biophys Acta 2004;1698:131-153.
5. Chiti F, Taddei N, Bucciantini M, White P, Rampovi G, Dobson CM. Mutational analysis of the propensity for amyloid formation by a globular protein. EMBO J 2000;19:1441-1449.
6. Wilkins DK, Dobson CM, Gross M. Biophysical studies of the development of amyloid fi brils from a peptide fragment of cold shock protein B. Eur J Biochem 2000;267:2609-2616.
7. Krebs MR, Wilkins DK, Chung EW, Pitkeathy MC, Chamberlin AK, Zurdo J, et al. Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain. J Mol Biol 2000;300:541-549.
8. Ramirez-Alvarado M, Merkel JS, Regan LA. A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro. Proc Natl Acad Sci USA.
2000;97:8974-8984.
9. Zurdo J, Guijarro JI, Jimenez JL, Saibil HR, Dobson CM.
Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain. J Mol Biol 2001;311:325-340.
10. Pertinhez TA, Bouchard M, Tomlinson EJ, Wain R, Ferguson SJ, Dobson CM. Amyloid fibril formation by a helical cytochrome. FEBS Lett 2001;495:184-186.
11. Fandrich M, Flectcher MA, Dobson CM. Amyloid fi brils from muscle myoglobin. Nature 2001;410:165-166.
12. Priyadarshini M, Bano B. Conformational changes during amyloid fi bril formation of pancreatic thiol proteinase inhibitor:
effect of copper and zinc. Mol Biol Rep 2012;39:2945-2955.
13. Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, et al. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 2001;40:6036-6046.
14. Hua QX, Weiss MA. Mechanism of insulin fibrillation:
the structure of insulin under amyloidogenic conditions resembles a protein-folding intermediate. J Biol Chem 2004;279:21449-21460.
15. Swift B. Examination of insulin injection sites: an unexpected fi nding of localized amyloidosis. Diabet Med 2002;19:881-882.
16. Sahoo S, Reeves W, DeMay RM. Amyloid tumor: a clinical and cytomorphologic study. Diagn Cytopathol 2003;28:325-328.
17. Yumlu S, Barany R, Eriksson, M, Rocken C. Localized insulin-derived amyloidosis in patients with diabetes mellitus: a case report. Hum Pathol 2009;40:1655-1660.
18. Shikama Y, Kitazawa, Yagihashi NU, ehara O, Murata Y, Yajima N, et al. Localized amyloidosis at the site of repeated insulin injection in a diabetic patient. Intern Med 2010;49:397-401
19. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, et al. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat Struct Mol Biol 2008;15:558-566.
20. Akaishi T, Morimotoa T, Shibaoa M, Watanabea S, Sakai- Katob K, Utsunomiya-Tateb N, et al. Structural requirements for the fl avonoid fi setin in inhibiting fi bril formation of amyloid β protein. Neurosci Lett 2008;444:280-285.
21. Rahmani AH, Mohammad A, Alzohairy, Khan MA, Salah MA. Therapeutic implications of black seed and its constituent thymoquinone in the prevention of cancer through inactivation and activation of molecular pathways.
Evid Based Complement Alternat Med 2014:724658.
22. Hashem FM, El-Kiey MA. Nigella sativa seeds of Egypt. J Pharm Sci United Arab Repub 1982;3:121-133.
23. Worthen D, Ghosheh O, Crooks P. The in vitro anti-tumor activity of some crude and purified components of black seed, Nigella sativa L. Anticancer Res 1998;18:1527-1532.
24. Enomoto S, Asano R, Iwahori Y, Narui T, Okada Y, Singab AN, et al. Hematological studies on black cumin oil from the seeds of Nigella sativa L. Biol Pharm Bull 2001;24:307-310.
25. Chakravarty N. Inhibition of histamine release from mast cells by nigellone. Ann Allergy 1993;70:237-242.
26. Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM.
Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem Funct 2002;20:143-151.
27. LeVine H. Thiofl avine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 1993;2:404-410.
28. Musa D, Dilsiz N, Gumushan H, Ulakoglu G, Bitiren M.
Antitumor effects of ethanol extract of Nigella sativa seeds.
Biolagia Bratisla 2005; 59:735-40.
29. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofl uorescein assay using microplate reader. Free Radic Biol Med 1999;7:612-616.
30. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for 2000 and projections for 2030. Diabetes Care 2004;27:1047-1053.
31. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in α-synuclein fibril formation. J Biol Chem 2001;276:10737-10744.
32. Chiti F, Webster P, Taddei N, Clark A, Stefani M, Ramponi G, et al. Designing conditions for in vitro formation of amyloid protofi laments and fi brils. Proc Natl Acad Sci USA 1999;96:3590-3594.
33. Pepys MB. Pathogenesis, diagnosis and treatment of systemic amyloidosis. Philos Trans R Soc Lond B Biol Sci 2001;356:203-210.
34. Sciacca MF, Kotler SA, Brender JR, Chen J, Lee DK, Ramamoorthy A. Two-step mechanism of membrane disruption by Aβ through membrane fragmentation and pore formation. Biophys J 2012;103:702-710.
35. Caughey B, Lansbury PT. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Ann Rev Neurosci 2003;26:267-298.
36. Scrocchi LA, Ha KBN, Wang F, Chen Y, Wu L, Tremblay P, et al. Identifi cation of multiple domains that participate in the fibrillogenesis and cytotoxicity of human islet amyloid polypeptide (MAPP). Diabetologia 2003;46:A49-A49.
37. Zako T, Sakono M, Hashimoto N, Ihara M, Maeda M. Bovine insulin filaments induced by reducing disulfide bonds show a different morphology, secondary structure, and cell toxicity from intact insulin amyloid fi brils. Biophys J 2009;96:3331-3340.
38. Shoval H, Weiner L, Gazit E, Levy M, Pinchuk I, Lichtenberg D. Polyphenol induced dissociation of various amyloid fi brils results in a methionine independent formation of ROS. Biochim Biophys Acta 2008;1784:1570-1577.
39. Rattanajarasroj S, Unchern S. Comparable attenuation of A beta (25–35)-induced neurotoxicity by quercitrin and 17beta-estradiol in cultured rat hippocampal neurons.
Neurochem Res 2010;35:1196-1205.
40. Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, et al. EGCG remodels mature alpha- synuclein and amyloid-beta fibrils and reduces cellular toxicity. Proc Natl Acad Sci USA 2010;107:7710-7715.
41. Lemkul JA, Bevan DR. Destabilizing Alzheimer's Aβ42 protofi brils with morin: mechanistic insights from molecular dynamics simulations. Biochemistry 2010;49:3935-3946.
42. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, et al. EGCG redirects amyloidogenic polypeptides into unstructured, off pathway oligomers. Nat Struct Mol Biol 2008;15:558-566.
(Received July 30, 2014) Edited by YUAN Lin
