Anti-glycating and anti-cytotoxic effect of silibinin on albumin at early glycation: A physiochemical study.

During persistent hyperglycaemia, albumin, one of the major blood proteins, can undergo fast glycation. It can be expected that timely inhibition of protein glycation might be add quality years to diabetic patients' life. Therefore, this study was designed to analyse the role of silibinin to reduced or delay amadori adduct formation at early glycation and its beneficial effect to improve the glycated albumin structure and conformation. We also analysed cytotoxic effect of amadori-albumin in the presence of silibinin on murine macrophage cell line RAW cells by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Formation of early glycated product (furosine) in all samples was confirmed by LCMS. Albumin incubated with glucose only showed presence of furosine like structure. Albumin treated with silibinin in the presence of glucose did not show such furosine like peak. This LCMS result showed the silibinin play a protective role in the formation of early glycated product. HMF contents were also reduced in the presence of silibinin, when albumin was incubated with increasing concentrations of silibinin (100 and 200 µM) in the presence of glucose. ANS binding fluorescence decrease by increasing silibinin concentrations with amadori-albumin. SDS-PAGE was also showed that no significant difference in the band mobility of albumin treated with silibinin as compared to native albumin. The secondary conformational alteration in amadori-albumin due to silibinin were confirmed by FTIR. This spectrum showed slight shift in amide I and Amide II band in albumin co-incubated with glucose and silibinin as compared to albumin incubated with glucose only. We further discussed about cytotoxic effect of amadori albumin and its prevention by silibinin. MTT assay results demonstrated that amadori-albumin showed cytotoxic effect on RAW cells but silibinin showed protective role and increased the cell viability. Moreover, the results showed that silibinin has anti-glycating potential and playing a role to prevent the formation of Amadori-albumin in-vitro. Silibinin possesses strong anti-glycating capacity and can improve albumin structure and function at early stage. It might be useful in delaying the progression of diabetes mellitus and its secondary complications at early stage.

Attenuation of hyperglycemia and amadori products by aminoguanidine in alloxan-diabetic rabbits occurs via enhancement in antioxidant defenses and control of stress.

The micro- and macro-complications in diabetes mellitus (DM) mainly arise from the damage induced by Amadori and advanced glycation end products, as well as the released free radicals. The primary goal of DM treatment is to reduce the risk of micro- and macro-complications. In this study, we looked at the efficacy of aminoguanidine (AG) to prevent the production of early glycation products in alloxan-diabetic rabbits. Type1 DM was induced in rabbits by a single intravenous injection of alloxan (90 mg/kg body weight). Another group of rabbits was pre-treated with AG (100 mg/kg body weight) prior to alloxan injection; this was followed by weekly treatment with 100 mg/kg of AG for eight weeks. Glucose, insulin, and early glycation products (HbA1C and fructosamine) were measured in control, diabetic and AG treated diabetic rabbits. The effects of hyperglycemia on superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), reduced glutathione (rGSH), nitric oxide, lipid peroxides, and protein carbonyl were investigated. Alloxan-diabetic rabbits had lower levels of SOD, CAT, Gpx, and rGSH than control rabbits. Nitric oxide levels were considerably greater. AG administration restored the activities of SOD, CAT, Gpx enzymes up to 70-80% and ameliorated the nitric oxide production. HbA1c and fructosamine levels were considerably lower in AG-treated diabetic rabbits. The observed control of hyperglycemia and amadori adducts in alloxan-diabetic rabbits by AG may be attributed to decrease of stress and restoration of antioxidant defenses.

A study on correlation between oxidative stress parameters and inflammatory markers in type 2 diabetic patients with kidney dysfunction in north Indian population.

BACKGROUND: Research reports support the statement that oxidative stress and inflammation are well-known risk factors for chronic kidney disease (CKD) in patients with diabetes. This study was designed to ascertain the associated role of oxidative stress parameters and inflammatory markers in diabetes and related CKD among the north Indian population. METHODS: The study was divided into three groups as healthy subjects (group 1), patients with diabetes without complication (group 2), and with CKD (group 3). Serum levels of malondialdehyde (MDA) and nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) content were estimated in all individuals. Inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)-α were determined by enzyme-linked immuno-sorbent assay. RESULTS: MDA, protein carbonyl, and NO were significantly elevated in patients with type 2 diabetes as compared with healthy subjects (P ≤ 0.05). Total thiols content were found to be significantly decreased in patients with diabetes with CKD. The activity of antioxidant enzymes SOD, CAT, and GR showed a significant suppression in patients with type 2 diabetes with or without CKD as compared with healthy subjects. Nevertheless, the levels of proinflammatory cytokines IL-6 and TNF-α were significantly upregulated ( P ≤ 0.05) as compared with healthy subjects. CONCLUSION: Determination of antioxidant defense parameters and inflammatory markers contributes to understand the relationship between oxidative stress and inflammation on the development and prevention of chronic kidney disease in Indian patients with diabetes.

Study of IL4-590C/T and IL6-174G/C Gene Polymorphisms in Type 2 Diabetic Patients With Chronic Kidney Disease in North Indian Population.

To explore the associations between potential functional promoter polymorphisms in pro-inflammatory and anti-inflammatory (IL-4(-590C/T) and IL-6(-174G/C) cytokine genes, and kidney dysfunction in North Indian type 2 diabetic subjects with chronic kidney disease. A total of 150 subjects aged 25-75 year were included in this study. The glomerular filtration rate (GFR) and serum creatinine were estimated. PCR was performed to analyse genotype distribution in IL-4 (-590T/C) and IL-6 (-174G/C) among healthy, type 2 diabetic patients with or without CKD. The genotype distributions were determined by Hardy-Weinberg equilibrium. CKD patients showed lower GFR (59.36 ± 1.33 ml/min/1.73 m2 ) and higher serum creatinine (1.93 ± 0.99% mg) level in comparison to diabetic patients without CKD and healthy subjects. Genotypic distribution of the different genotypes among the study groups in IL-4 gene was genotype CC = 30, TC = 12, and TT = 8 in CKD patients. In type 2 diabetic patients without CKD, genotype distribution was CC = 38, TC = 10, and TT = 2. In healthy subjects, distribution of genotype was CC = 35, TC = 14, and TT = 1. The distribution of different genotype among the study groups for IL-6 gene was GG = 27, GC = 20, and CC = 3 in healthy subjects; GG = 28, GC = 19, and CC = 3 in diabetic patients without CKD and GG = 38, GC = 11, and CC = 1 in diabetic patients with CKD. There was no significant difference in the distribution of genotype frequencies between healthy subjects and diabetic patients without CKD but a significant difference was found in diabetic patients with CKD. The functional promoter polymorphisms IL4-590C/T and IL6-174G/C, which affect the IL-4 and IL-6 levels in north Indian subjects, were associated with kidney dysfunction and CKD. J. Cell. Biochem. 118: 1803-1809, 2017. © 2016 Wiley Periodicals, Inc.

Hypermethylation Status of E-Cadherin Gene in Gastric Cancer Patients in a High Incidence Area.

Gastric cancer (GC) is the fourth most prevalant cancer and the second leading cause of cancer-related mortality worldwide. As in other cancers gastric carcinogenesis is multifactorial involving environmental, genetic and epigenetic components. Epigenetic silencing due to hypermethylation of tumour suppressor genes is one of the key events in gastric carcinogenesis. This study was aimed to analyse the hypermethylation status of the E-Cadherin (CDH1) gene promoter in GCs in the ethnic Kashmiri population. In this study a total of 80 GC patients were recruited. Hypermethylation in tumour tissue was detected by methylation specific PCR (MS-PCR). Hypermethylation of CDH1 promoter was observed in 52 (65%) of gastric carcinoma cases which was significantly much higher than adjacent normal tissue [p≤0.0001]. Further the frequency of CDH1 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [55.7% vs 82.1%; <0.05]. Moreover females and cases with lymph node invasion had higher frequencies of CDH1 hypermethylation [P≤0.05]. Thus the current data indicate a vital role of epigenetic alteration of CDH1 in the causation and development of gastric cancer, particularly of diffuse type, in our population.

Structural changes in histone H2A by methylglyoxal generate highly immunogenic amorphous aggregates with implications in auto-immune response in cancer.

The role of aberrant protein modifications in cancer and its diagnosis have emerged as a promising research field. Nonenzymatic glyco-oxidation of proteins under oxidative stress has been associated with carcinogenesis through advanced glycation end products (AGE)-receptors for advanced glycation end products (RAGE) axis. Modified proteins that are immunogenic and stimulate cellular and humoral immune responses are being studied to develop early detection markers of cancer. This study has probed the structural alternations; leading to the formation of adducts and aggregates, in histone H2A upon in vitro modification by methylglyoxal (MG). The immunogenicity of modified histone H2A and its binding with cancer autoantibodies was also assessed. MG induced lysine side chain modifications, blocking of free amino groups and the formation of condensed cross structures in histone H2A; and its effect was inhibited by carbonyl scavengers. It led to the adduct formation and generation of N-epsilon-(carboxyethyl)lysine (CEL) and its decomposition forms as revealed by Matrix-assisted laser desorption ionization-mass spectrometry, high-performance liquid chromatography and LC-MS. MG-H2A showed amorphous aggregate formation under electron microscopy and altered binding with DNA in circular dichroism studies. The modified histone elicited high titer immunogen-specific antibodies in rabbits when compared with the native, thus pointing toward the generation of neo-epitopes in MG-H2A. The autoantibodies derived from cancer patients exhibited enhanced binding with MG-H2A as compared with the native histone in enzyme-linked immunosorbent assay and gel retardation assay. This reflects sharing of epitopes on MG-H2A and histones in cancer patients. The neo-epitopes on H2A may be responsible for induction and elevated levels of antibodies in cancer patients. Thus, MG-H2A may be considered as potential antigenic candidate for auto-immune response in cancer.

Dicarbonyl Induced Structural Perturbations Make Histone H1 Highly Immunogenic and Generate an Auto-Immune Response in Cancer.

Increased oxidative stress under hyperglycemic conditions, through the interaction of AGEs with RAGE receptors and via activation of interleukin mediated transcription signalling, has been reported in cancer. Proteins modifications are being explored for their roles in the development and progression of cancer and autoantibody response against them is gaining interest as a probe for early detection of the disease. This study has analysed the changes in histone H1 upon modification by methylglyoxal (MG) and its implications in auto-immunopathogenesis of cancer. Modified histone showed modifications in the aromatic residues, changed tyrosine microenvironment, intermolecular cross linking and generation of AGEs. It showed masking of hydrophobic patches and a hypsochromic shift in the in ANS specific fluorescence. MG aggressively oxidized histone H1 leading to the accumulation of reactive carbonyls. Far UV CD measurements showed di-carbonyl induced enhancement of the alpha structure and the induction of beta sheet conformation; and thermal denaturation (Tm) studies confirmed the thermal stability of the modified histone. FTIR analysis showed amide I band shift, generation of a carboxyethyl group and N-Cα vibrations in the modified histone. LCMS analysis confirmed the formation of Nε-(carboxyethyl)lysine and electron microscopic studies revealed the amorphous aggregate formation. The modified histone showed altered cooperative binding with DNA. Modified H1 induced high titre antibodies in rabbits and the IgG isolated form sera of rabbits immunized with modified H1 exhibited specific binding with its immunogen in Western Blot analysis. IgG isolated from the sera of patients with lung cancer, prostate cancer, breast cancer and cancer of head and neck region showed better recognition for neo-epitopes on the modified histone, reflecting the presence of circulating autoantibodies in cancer. Since reports suggest a link between AGE-RAGE axis and carcinogenesis, glycoxidation of histone H1 and its immunogenicity paves ways for understanding role of glycoxidatively damaged nuclear proteins in cancer.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

PURPOSE: Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. METHODS: In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. RESULTS: Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). CONCLUSIONS: Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

Human DNA damage by the synergistic action of 4-aminobiphenyl and nitric oxide: an immunochemical study.

4-Aminobiphenyl (4-ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4-ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4-ABP + SNP-mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4-ABP-SNP-modified human DNA-induced high-titer immunogen-specific antibodies, which showed Cross-reaction with modified/unmodified DNA bases and ss-DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4-ABP-SNP-modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti-modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule.

Genotoxic effect of N-hydroxy-4-acetylaminobiphenyl on human DNA: implications in bladder cancer.

BACKGROUND: The interaction of environmental chemicals and their metabolites with biological macromolecules can result in cytotoxic and genotoxic effects. 4-Aminobiphenyl (4-ABP) and several other related arylamines have been shown to be causally involved in the induction of human urinary bladder cancers. The genotoxic and the carcinogenic effects of 4-ABP are exhibited only when it is metabolically converted to a reactive electrophile, the aryl nitrenium ions, which subsequently binds to DNA and induce lesions. Although several studies have reported the formation of 4-ABP-DNA adducts, no extensive work has been done to investigate the immunogenicity of 4-ABP-modified DNA and its possible involvement in the generation of antibodies in bladder cancer patients. METHODOLOGY/PRINCIPAL FINDINGS: Human DNA was modified by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), a reactive metabolite of 4-ABP. Structural perturbations in the N-OH-AABP modified DNA were assessed by ultraviolet, fluorescence, and circular dichroic spectroscopy as well as by agarose gel electrophoresis. Genotoxicity of N-OH-AABP modified DNA was ascertained by comet assay. High performance liquid chromatography (HPLC) analysis of native and modified DNA samples confirmed the formation of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4ABP) in the N-OH-AABP damaged DNA. The experimentally induced antibodies against N-OH-AABP-modified DNA exhibited much better recognition of the DNA isolated from bladder cancer patients as compared to the DNA obtained from healthy individuals in competitive binding ELISA. CONCLUSIONS/SIGNIFICANCE: This work shows epitope sharing between the DNA isolated from bladder cancer patients and the N-OH-AABP-modified DNA implicating the role of 4-ABP metabolites in the DNA damage and neo-antigenic epitope generation that could lead to the induction of antibodies in bladder cancer patients.

Binding of circulating autoantibodies in breast cancer to native and peroxynitrite-modified RNA.

Peroxynitrite (ONOO(-)) is a powerful oxidant and nitrosative agent and has in vivo existence. The half life of ONOO(-) at physiological pH is less than 1 s. It can react with nucleic acids, proteins, lipoproteins, saccharides, cardiolipin, etc., and can modify their native structures. Action of ONOO(-), synthesized in the authors' laboratory by a rapid quenched flow process, on structural changes of commercially available RNA was studied by ultraviolet (UV), fluorescence, and agarose gel electrophoresis. Compared to native RNA, the ONOO(-)-modified RNA showed hyperchromicity at 260 nm. Furthermore, the ethidium bromide (EtBr) assisted emission intensities of ONOO(-)-modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex. Agarose gel electrophoresis of ONOO(-)-modified RNA showed a gradual decrease in band intensities compared to native RNA, an observation clearly due to the poor intercalation of EtBr with ONOO(-)-modified RNA. Native and ONOO(-)-modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer. Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO(-)-modified RNA specific autoantibodies in breast cancer patients. Moreover, the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO(-)-modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs. The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO(-)-modified RNA. It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules. Higher concentrations of nitrogen reactive species can be detrimental to RNA. However, the emergence of native as well as 0.8 mmol/L ONOO(-)-modified RNA as a novel antigen/substrate for autoantibodies in breast cancer patients indicates that, in future, these molecules might find a place on the panel of antigens for early diagnosis of breast cancer.

Acquired immunogenicity of human DNA damaged by N-hydroxy-N-acetyl-4-aminobiphenyl.

4-Aminobiphenyl, a known carcinogen, has many environmental sources like cigarette smoke, industrial waste, and so forth. It can be metabolized to form a potent mutagen, N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP) that undergoes further processing to form electrophilic nitrenium ions which interact with DNA-forming covalent adducts, thereby exerting genotoxic effects. While the mutagenicity of N-OH-AABP has been amply reported, no extensive studies have been performed to assess the immunogenicity of N-OH-AABP-modified DNA. In this study, human placental DNA was modified with N-OH-AABP, and the structural perturbations in the DNA molecule were evaluated by ultraviolet spectroscopy and nuclease S1 digestion. Native and N-OH-AABP-modified DNA were used as antigens for immunizing female rabbits. The modified DNA was found to be highly immunogenic, eliciting high titer immunogen-specific antibodies, while the native form was almost nonimmunogenic. The induced antibodies exhibited wide range of heterogeneity in recognizing various nucleic acid conformers and DNA bases. We also detected deposits of immune complex in glomerular basement membrane in rabbits immunized with N-OH-AABP-DNA. Possible role of N-OH-AABP-DNA in the induction of antibodies in cancer patients and the related consequences have been discussed.

Catechol-estrogen modified DNA: a better antigen for cancer autoantibody.

Estrogens are known mutagenic and carcinogenic risk factors. Non-enzymatic oxidation of catechol-estrogens in the presence of copper is reported to generate reactive oxygen species (ROS) that can cause DNA damage. We show that DNA modification in the presence of 4-hydroxyestradiol (4-OHE(2)) and copper (Cu-II) results in single and double strand breaks, base modification, hyperchromicity and change in ellipticity. Modified DNA (4-OHE(2)-Cu(II)-DNA) was highly immunogenic in experimental animals. Induced anti-4-OHE(2)-Cu(II)-DNA antibodies were effectively used as a probe for detecting oxidative lesions in human genomic DNA and for the estimation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the urine of cancer patients. Circulating antibodies from cancer patients showed high binding to 4-OHE(2)-Cu(II)-DNA as compared to native DNA. Our results imply that interaction of catechol-estrogen and copper leads to the production of potent ROS, capable of causing DNA damage, thus playing an important role in carcinogenesis. The modified DNA presents unique epitopes which may be one of the factors for autoantibody induction in cancer.