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ORIGINAL ARTICLE
Year : 2012  |  Volume : 3  |  Issue : 1  |  Page : 34-37  

Negative effect of noscapine on human serum albumin glycation


Cell and molecular group, School of biology, College of science, University of Tehran, Tehran, Iran

Date of Web Publication11-Aug-2012

Correspondence Address:
Alireza Ahmadzadeh
Cell and molecular group, School of biology, College of science, University of Tehran, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.99654

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   Abstract 

Introduction: Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of sugar to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leads to the formation of Amadori compounds. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. It has recently become clear that glycation also affects diabetes-related complications and Alzheimer's disease. The main aim of this investigation is to provide direct evidence for the effects of Noscapine on HSA glycation. Materials and Methods: In this study human serum albumin (HSA) (10 mg / ml) was incubated in phosphate buffered saline (PBS) (50 mM) with Glucose (40 mM) and different concentrations of Noscapine (25, 100, 250, 500 μM), for 42 days, at 37°C, as also HSA was incubated alone (control or H), with Glucose (40mM) (glycated or H + G), respectively, under the same conditions. After incubation the samples were prepared for circular dichroism (CD) and Fluorescencetechniques. Statistical Analysis Used: The results were expressed as mean ± SEM and chi square test was significant at P < 0.05. Results: In glycated sample CD and Fluorescence showed more change relative to the control sample, but in the presence of different Noscapine concentrations and Glucose there were no significant changes relative to the glycated sample. Conclusions: Thus, this study suggests that Noscapine is not responsible for the antiglycation effect.

Keywords: Advanced glycation end products, antiglycation, Alzeimer′s, human serum albumin, Noscapine


How to cite this article:
Ahmadzadeh A. Negative effect of noscapine on human serum albumin glycation. J Pharm Negative Results 2012;3:34-7

How to cite this URL:
Ahmadzadeh A. Negative effect of noscapine on human serum albumin glycation. J Pharm Negative Results [serial online] 2012 [cited 2019 Nov 12];3:34-7. Available from: http://www.pnrjournal.com/text.asp?2012/3/1/34/99654


   Introduction Top


In various biological settings, reducing sugars can react nonenzymatically with the protein amino groups, forming Schiff bases and Amadori products, to produce advanced glycation end-products (AGEs). During AGE formation, Amadori rearrangements to more reactive intermediates induce protein cross-linking, which not only affects protein function and half-life, but also engages signaling cascades transduced by the AGE receptor (RAGE). [1] Compelling evidence from the epidemiological studies indicates that hyperglycemia is an independent risk factor for cardiovascular disease and that this risk is reduced in patients with diabetes, in whom strict glycemic control is maintained [2] AGEs can also originate from exogenous sources such as tobacco smoke and diet. [3] Extracellular matrix (ECM) proteins are susceptible to AGE modification because of their slow turnover rate. For example, AGE cross-linking on type I collagen and elastin leads to increased stiffness of the blood vessels. [4] The composition of ECM is also modified by AGE, with increased expression of ECM proteins, including fibronectin, types III, IV, and VI collagen, and laminin, possibly mediated through the upregulation of key profibrotic cytokines such as TGF- – [5] and the connective tissue growth factor. [6] In vitro studies have identified that AGEs can modulate renal tubular growth and migration via an ezrin-dependent pathway. [7] Carboxymethyl Lysine (CML) and other AGEs have been localized to retinal blood vessels in patients with type 2 diabetes and found to correlate with the degree of retinopathy. [8] Administration of AGEs and the subsequent Receptor for Advanced Glycation End-product (RAGE) activation replicates the effects of hyperglycemia, whereas, RAGE antibody administration suppresses both NF-κB activation and expression of IL-6 transcripts in sciatic nerve studies. [9] There is mounting evidence to suggest that the antiatherogenic properties of high-density lipoprotein (HDL) are decreased in patients with diabetes. [10],[11] Intracellular AGE formation also reduces the expression of endothelial nitric oxide synthase (eNOS) through increased eNOS mRNA degradation. In addition, studies have shown that AGEs quench NO, and thereby, directly lead to the inactivation of NO. Reduced eNOS activity and inactivation of NO by the AGEs may play an important role in the defective vasodilatory responses that occur in diabetes. [12] Nonenzymatic glycation of serum albumin in vivo occurs at multiple sites. Glucose gets attached to lys-199, lys-281, lys-439, and lys-525, as well as to some other lysine residues. The principal glycosylated site is lys-525 [13] .Noscapine (also called narcotine, nectodon, nospen, or anarcotine) is a benzylisoquinoline alkaloid derived from the opium poppy Papaver somniferum. This alkaloid lacks the sedative activity and has been used as a cough suppressant for decades, with no evidence of toxicity or side effects. [14] We found that noscapine potentiates apoptosis induced by cytokines and chemotherapeutic agents in tumor cells. Noscapine alone suppressed proliferation of human leukemia and myeloma cells and downregulated the constitutive expression of cell survival proteins. Noscapine also abrogated the inducible expression of proteins involved in survival, proliferation, invasion, and angiogenesis, all of which are regulated by NF-κB. [15] We identified noscapine as a small molecule inhibitor of the hypoxia-inducible factor-1 pathway in hypoxic human glioma cells and human umbilical vein endothelial cells. [16]


   Materials and Methods Top


Chemicals

Human serum albumin (≥ 96%, free fatty acid) and sodium bicinchoninic acid (BCA) (were purchased from Sigma). The membrane filters (0.2 μm pore size, 25 mm in diameter) and dialysis tubing (cut-off 10,000 MW) were obtained from Whatman (UK). 2, 4, 6-trinitrobenzene sulfonic acid (TNBSA) was acquired from Fluka. Noscapine was obtained from the pharmacy. D(+) glucose, phosphate-buffered saline (PBS), sodium azide , Ethylenediaminetetraacetic acid (EDTA), and other materials were purchased from Merck (Germany). All solutions were prepared with deionized water.

In vitro glycation of human serum albumin

Human serum albumin of 10mg / ml was incubated in PBS 50 mM (pH7.4), containing NaN3 and EDTA 1 mM, using Glucose 40 mM and different concentrations of Noscapine 25, 100, 250, 500 μM, for 42 days, at 37°C. Also HSA was incubated alone (control or H), with glucose (glycated sample or H + G) and the same Noscapine concentration, without any additive, respectively, under the same condition. Following this the 42-day samples were dialyzed extensively against PBS at 4°C and stored at - 20°C. Protein concentration was determined using the bicinchoninic acid (BCA) assay according to the supplier's protocol. [17],[18]

Circular dichroism experiments

The Far-UV (ultraviolet) CD was used to measure changes in the secondary structure of HSA (0.2 mg / ml), using the Aviv Circular dichromis spectropolarimeter model 215 with a path length of 0.1 cm, at a range of 190 - 260 nm, using ammonium d-10-camphorsulfonic acid for calibration. The results were expressed as the molar ellipticity[θ], which was defined as [θ] = θ millidegrees / {10 × number of amino acid (lc)}, where c is the molar concentration of the sample, and l is the length of the light path (cm). The amount of secondary structure associated with the samples was calculated by the CDNN software. [19] The curve in each sample was obtained from the mean of three independent experiments.

Detection of advanced glycation end product-specific fluorescence

The AGE-fluorescence of all samples (1 mg / ml) was obtained on a Cary Eclipse fluorescence spectrophotometer at Ex / Em (380 / 390-540 nm). Each point represented the mean of three independent experiments. [20] The curve in each sample was obtained from the mean of three independent experiments.

Statistical analysis

The results were expressed as mean ± SEM. The significance of the antiglycation effect was determined by a chi square test and the results were regarded as significant at P < 0.05.


   Results Top


The Far-UV CD spectrum of HSA is characterized by the presence of two strong negative bands at 208 and 222 nm, which represent the helical characteristics of HSA. As seen in [Figure 1], there is a loss of helical structure, as shown by a decrease in the negative ellipticity at 208 and 222 nm following the 42-day incubation of HSA with Glucose (H + G or glycated) compared to the control (HSA without any additive or H). However the CD data for HSA incubated for 42 days, with the mixture of Glucose + different concentrations of Noscapine do not show a significant change relative to the glycated (H + G) sample.
Figure 1: Circular dichroism spectra of all samples that were incubated for 42 days in PBS 50 mM , pH7.4, at 37¢ªC, H, G, and N indicate HSA, Glucose, and Noscapine, respectively.

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The advanced glycation end product fluorescence spectra of the glycated (H + G) sample, at Ex / Em (380 / 390 - 500 nm), in [Figure 2], increased relative to the control (H) . Also [Figure 2] and [Figure 3] do not show any significant difference in the AGE fluorescence spectra for HSA incubated with the mixture of glucose and Noscapine 25, 100, 250, 500 μM compared to the glycated sample.
Figure 2: AGE fluorescence spectra of all samples that were incubated for 42 days in PBS 50 mM, pH 7.4, at 37¢ªC, Ex / Em(380 / 390 - 550nm). H,G, and N are the same as in Figure 1

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Figure 3: Max AGE fluorescence in all samples at 420 nm. H, G, and N are the same as in Figure 1

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   Discussion Top


The main aim of this investigation was to provide direct evidence for the antiglycation effects of Noscapine on HSA glycation. This information was obtained by AGE-fluorescence and the study of the secondary structure of HSA after incubation with glucose, in the absence and presence of different concentrations of Noscapine. Noscapine (also called narcotine, nectodon, nospen or anarcotine) is a benzylisoquinoline alkaloid derived from the opium poppy Papaver somniferum. [14] The formation of AGEs is the result of the reaction of reducing sugars with proteins. [21] The Far-UV CD spectrum [Figure 1] reveals the glycation of HSA following the 42-day incubation, which induces a loss of helical structure in HSA, as compared to the control. [22] However, the presence of Noscapine in the mixture of HSA and Glucose does not show a more significant change relative to the glycated sample (H + G). To confirm this, as shown in [Figure 2] and [Figure 3], AGE-specific fluorescence has been seen to increase in the glycated sample (H+ G) following the 42-day incubation relative to the control (H). However, in the presence of Noscapine + Glucose, no significant change relative to the glycated sample (H + G) has been seen. It appears that Noscapine does not have a significant effect on AGE formation .Collectively, the results in this article confirm that the presence of Noscapine does not have an anti-glycation effect on the pathway of AGE formation.


   Acknowledgment Top


The financial support from the Research Council of the University of Tehran is gratefully acknowledged.

 
   References Top

1.Ulrich P, Cerami A.Protein glycation, diabetes, and aging. Recent Prog Horm Res 2001;56:1-21.  Back to cited text no. 1
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2.Nathan DM, Cleary PA, Backlund JY, Genuth SM, Lachin JM, Orchard TJ,et al. Intensive diabetes treatment and cardiovascular disease in patients with type1 diabetes. N Engl J Med 2005;353:2643-53.  Back to cited text no. 2
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3.Vlassara H, Cai W, Crandall J, Goldberg T, Oberstein R, Dardaine V,et al. Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy. Proc Natl Acad Sci USA 2002;99:15596-15601.  Back to cited text no. 3
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5.Forbes JM, Cooper ME, Oldfield MD, Thomas MC. Role of advanced glycation end products in diabetic nephropathy. J Am Soc Nephrol 2003;14 (8 Suppl3): S254-8.  Back to cited text no. 5
    
6.Twigg SM, Cao Z, MCLennan SV, Burns WC, Brammar G, Forbes JM, et al. Renal connective tissue growth factor induction in experimental diabetes is prevented by aminoguanidine. Endocrinology2002;143:4907-4915.  Back to cited text no. 6
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10.Nobécourt E, Jacqueminet S, Hansel B, Chantepie S, Grimaldi A, Chapman MJ, et al. Defective antioxidative activity of small dense HDL3 particles in type 2 diabetes: Relationship to elevated oxidative stress and hyperglycemia. Diabetologia 2005;48:529-538.  Back to cited text no. 10
    
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12.Soro-Paavonen A, Zhang WZ, Venardos K, Coughlan MT, Harris E, Tong DC, et al. Advanced glycation end-products induce vascular dysfunction via resistance to nitric oxide and suppression of endothelial nitric oxide synthase. J Hypertens 2010;28:780-788.  Back to cited text no. 12
    
13.Iberg N, Fluckiger R.Nonenzymatic Glycosylation of Albumin in vivo.JBiol Chem 1996;261:13542-13545.  Back to cited text no. 13
    
14.Mahmoudian M, Mehrpour M, Benaissa F, Siadatpour Z. A preliminary report on the application of noscapine in the treatment of stroke. Eur J Clin Pharmacol2003;59:579-581.  Back to cited text no. 14
    
15.Sung B, Ahn KS, Aggarwal B .Noscapine, a benzylisoquinoline alkaloid, sensitizes leukemic cells to chemotherapeutic agents and cytokines by modulating the NF-êB signaling pathway.J Cancer Res 2010 ; 70:3259-3268.  Back to cited text no. 15
    
16.Newcomb EW, Lukyanov Y, Smirnova I, Schnee T, Zagzag D.Noscapine induces apoptosis in human glioma cells by an apoptosis-inducing factor-dependent pathway.J Anti Cancer Drug 2008;19:553-56317.  Back to cited text no. 16
    
17.Schmitt A, Gasic Milenkovic J, Schmitt J.Characterization of advanced glycation end products:Mass changes in Correlation to side chain modification. J Anal Biochem 2005;338:201-215.  Back to cited text no. 17
    
18.Valencia JV, Weldon SC, Quinn D, Kiers GH, DeGroot J, TeKoppele JM, et al. Advanced glycation end product ligands for the receptor for advanced glycation endproducts: Biochemical characterization and formation kinetics. Anal Biochem 2004;324:68-78.  Back to cited text no. 18
    
19.Sattarahmady N, Khodagholi F, Moosavi-Movahedi AA, Heli H, Hakimelahi GH.Alginate as an anti glycating agent for HSA.JBiol Macromol 2007;41:180-184.  Back to cited text no. 19
    
20.Yan SF, Ramasamy R,Schmidt AM. Mechanisms of disease: Advanced glycation end products and their receptor in inflammation and diabetes complications. Nat Clin Pract Endocrinol Metab 2008;4:285-293.  Back to cited text no. 20
    
21.Kumar PA, Kumar MS, Reddy GB. Effect of glycation on á-crystallin structure and chaperone-like function. JBiochem 2007;408:251-258.  Back to cited text no. 21
    
22.Khazaei MR, Bakhti M. Nicotine reduces the cytotoxic effect of glycated proteins on microglial cells.J Neurochem Res 2010;35: 548-558.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]


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