Abstract
Background
The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho‐physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4‐hydroxynonenal (HNE). 4‐HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes.
Methods
The present study examines to probe the presence of auto‐antibodies against 4‐hydroxynonenal damaged DNA (HNE–DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4‐HNE. The DNA was incubated with 4‐HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto‐antibodies were assessed by direct binding and competitive inhibition ELISA.
Results
DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4‐HNE modified calf thymus DNA as compared to its native conformer. The 4‐HNE modified DNA presents unique epitopes which may be one of the factors for the auto‐antibody induction in cancer patients.
Conclusion
The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients.
Keywords: 4‐hydroxynonenal (HNE), auto‐antibody, cancer, DNA, glycation
1. Introduction
Earlier studies have drawn in reactive oxygen and nitrogen species (ROS & RNS) in carcinogenesis.1, 2 Reactive oxygen species initiate carcinogenesis by virtue of their competence to react with DNA and cause mutation and structural changes in the molecule. Beside direct actions, ROS elicit lipid peroxidation, leading to the production of many aldehydes including 4‐hydroxynoneal (4‐HNE). Unlike reactive free radicals, aldehydes are rather long lived and can therefore diffuse from the site of their origin (ie membranes) and reach and attack targets intracellularly or extracellularly which are distant from the initial free radical event. Lipid peroxidation on its own is an amplifier for the initial free radicals and the reactive aldehydes generated in this process may well act as “second toxic messengers” of the complex chain reactions which are initiated if polyunsaturated fatty acids of the membrane bilayer are converted to lipid hydroperoxides.3 These products further react and modify both proteins and DNA resulting in toxicity or even mutagenesis and therefore have been associated with aging, cardiovascular diseases, neurological disorders and cancer.4, 5 However, their effects are not only toxic, but rather homeostatic as they participate in signal transduction pathways.6, 7 Among the many different aldehydes which can be formed during lipid peroxidation, the best studied are malondialdehyde (MDA) and 4‐HNE. 4‐HNE is a 9‐carbon α,β unsaturated aldehyde formed when n‐6 polyunsaturated fatty acids such as arachidonic and linoleic acid are attacked by peroxidative free radicals during lipid peroxidation (LPO). 4‐HNE is a highly chemically reactive molecule and is considered as one of the major generators of oxidative stress and is often used as a bioactive marker of oxidative stress and LPO.8, 9, 10 4‐HNE reacts with all four DNA bases but with different efficiency. HNE induces DNA damage predominantly at deoxyguanosine (dG) resulting in the formation of HNE‐dG. Two types of mutagenic lesions arising by fatty acid oxidation have been described—bulky adducts to DNA bases, eg, HNE‐DNA adducts and relatively small etheno‐adducts to G, A, and C.11, 12 Previous studies have shown that the endogenous DNA damage induced by lipid peroxidation may play an important role in carcinogenesis.13, 14 HNE‐DNA adducts have been shown to induce site‐specific mutations and may contribute greatly to the G:C to T:A mutations at codon 249 of the p53 gene and thus play an important role in carcinogenesis.11, 15 HNE‐DNA adducts have been proposed as oxidative stress markers in carcinogenesis.10, 16
In our study calf thymus DNA was modified by HNE. We report that damage to the DNA caused by HNE was sufficient to behave this nucleic acid as a foreign substance. This study investigates the presence/prevalence of antibodies against 4‐HNE‐modified calf thymus DNA in the serum samples of patient having cancer of different tissue origins. The antibody analysis was undertaken by direct binding and competition ELISA. Serum antibodies from healthy individuals were used as control. The changes in DNA on HNE modification were confirmed through spectroscopic and fluorometric sensitivity assays.
2. Materials and Methods
2.1. Materials
Calf thymus DNA, ethidium bromide, nuclease S1, Tween‐20, agarose, anti‐human IgG‐alkaline phosphatase conjugate, and para‐nitrophenyl phosphate were purchased from Sigma Chemical Company, St. Louis, MO, USA. 4‐Hydroxynonenal was purchased from Cayman Chemical Company, Ann Arbor, MI, USA. Flat bottom ELISA modules were purchased from NUNC, Roskilde, Denmark. All other chemicals used in this study were of the highest analytical grade available.
2.2. Collection of sera samples
Blood and serum samples of cancer patients (n=79) were collected from IIMS&R Integral University, Lucknow. Age‐ and sex‐matched healthy individuals (n=20) served as negative control. Informed consent was obtained from all the patients and normal healthy individuals. The work had clearance from the Institutional Ethical Committee. All the serum samples were heated at 56°C for 30 minutes to inactivate complement proteins and stored at −20°C with 0.2% sodium azide.
2.3. Purification and modification of DNA
Commercially available calf thymus DNA was purified free of proteins, RNA, and single‐stranded regions. Purity of DNA was ascertained by A260/A280 ratio which was found to be in the range 1.8‐2.0. Purified calf thymus DNA (10 mg/mL, final concentration) was incubated in 10 mmol/L sodium phosphate buffer, pH 7.4 containing 150 mmol/L NaCl for 24 hours at 37°C containing 1 mmol/L HNE, and was followed by extensive dialysis against phosphate buffer to remove unbound constituents. The modified form of DNA was characterized through UV absorption, fluorometric, and circular dichroism.17, 18
2.4. Spectral studies of native and modified DNA
The ultraviolet absorption profile of native and modified calf thymus DNA was recorded in the wavelength range 200‐400 nm on Eppendorf spectrophotometer.
Fluorescence emission spectral analysis of native and modified DNA samples (5 μg/mL) was undertaken in the wavelength range 300‐700 nm using quartz cuvette on Agilent Spectrofluorophotometer at an excitation wavelength of 325 nm. Ethidium bromide (2.5 μg/mL) was used as an external chromophore for the process.
2.5. Enzyme linked immunosorbent assay (ELISA)
Direct binding ELISA was carried out on flat bottom polystyrene modules (maxisorp). Microtiter wells were coated with 100 μL of native or modified DNA (2.5 μg/mL in TBS, pH 7.4) and incubated for 2 hours at 37°C and overnight at 4°C. Each sample was coated in duplicate and half of the plate devoid of antigen served as control. The remaining steps were performed as described earlier.19
2.6. Competitive ELISA
Competition ELISA was performed to evaluate the specific binding of circulating cancer auto‐antibodies to native and modified DNA.14 Briefly, 100 μL of HNE‐DNA (2.5 μg/mL) was coated onto microtiter plates, incubated for 2 hours at room temperature and overnight at 4°C. The plates were washed with TBS‐T and blocked with 150 μL of 1.5% bovine serum albumin (BSA). Immune complexes were prepared by mixing 100 μL of 1:100 dilutions of cancer sera with increasing amount (0‐20 μg/mL) of native DNA and HNE‐DNA, respectively, and incubated at 37°C for 2 hours and overnight at 4°C. One hundred microliters of immune complex was added to each well, followed by anti‐human IgG‐alkaline phosphatase conjugate. The remaining steps were same as in direct binding ELISA.
3. Results
3.1. UV‐Vis characterization of modified DNA
The UV absorption spectra showed increasing hyperchromicity upon increasing the concentration of HNE. 100 and 500 μmol/L HNE‐modified calf thymus DNA showed 10.1% and 34.6% hyperchromicities, however, when the concentration of HNE was increased to 1 mmol/L, the DNA showed further increase in hyperchromicity (44.1%) at 260 nm as compared to native calf thymus DNA (Figure 1). Further increase in the concentration of HNE leads to no further change in hyperchromicity of the DNA macromolecule. The increase in absorbance at 260 nm in case of modified DNA is due to damage to bases which results in the exposure of chromophoric groups.
Figure 1.
Ultraviolet absorption spectra of native calf thymus DNA (─), calf thymus DNA modified with 100 μmol/L HNE (─▲─), 500 mol/L HNE (─■─), 1 mmol/L HNE (─●─)
3.2. Fluorescence studies of native and modified DNA
Fluorescence spectra of native and modified DNA were taken using ethidium bromide (Figure 2). The decrease in the fluorescence intensity was seen in case of modified DNA as compared to its native form. Destruction of the structure of DNA due to modification by HNE may attribute to the decrease in fluorescence intensity.
Figure 2.
Fluorescence emission spectra of native calf thymus DNA (─) and modified calf thymus DNA with 1 mmol/L HNE (‐ ‐ ‐)
3.3. Recognition of modified DNA by cancer auto‐antibodies
In this study, we have analyzed randomly selected 28 cases of head and neck cancer, 22 cases of breast cancer, 16 cases of cervical cancer, and 13 cases of lung carcinoma. Sera from normal healthy individuals (n=20) served as control. All sera were diluted to 1:100 in PBS and subjected to direct binding ELISA on solid phase separately coated with equal amounts of native and HNE‐modified DNA. Of 79 cancer sera, 57 samples showed enhanced binding with modified DNA. In head and neck cancer patients, 18 of 28 serum samples (64.2%) showed enhanced binding with the modified DNA as compared to the native form. Direct binding ELISA for 16 samples of 22 samples (72.2%) of breast cancer patients revealed appreciably better binding with the modified DNA. Similarly in the cervical cancer patients 11 of 16 sera (68.8%) showed higher binding to the modified DNA. While in the lung cancer patients nine of 13 serum samples (69.2%) showed enhanced binding to the modified DNA. The auto‐antibodies showed appreciably higher binding to modified DNA as compared to the native analog (Figure 3).
Figure 3.
Binding of various cancer sera to native (■) and modified DNA (■). Normal human sera (NHS) served as negative control. The histogram shows mean absorbance values for binding of NHS and sera from patients with different types of cancer (1) normal human sera, (2) Head and Neck Cancer, (3) Breast Cancer, (4) Cervical Cancer, (5) Lung Cancer. Microtiter plates were coated with HNE‐DNA (2.5 μg/mL)
3.4. Specificity of circulating antibodies in cancer patients
Competition ELISA was carried out to analyze the specific binding of circulating antibodies in cancer patients to native and HNE‐modified DNA. Cancer sera showing enhanced binding with the modified DNA were assessed for their specific binding characteristics in competitive binding assay. Appreciably higher inhibition in the binding of these auto‐antibodies was observed with modified DNA as compared to the native form (Figure 4). The competition ELISA data indicate that the HNE‐modified DNA is a better inhibitor of naturally occurring antibodies in the majority of cancer patients in comparison to native DNA (Table 1). However, the inhibition caused by native DNA in the binding of cancer auto‐antibodies was quite low. Inhibition (mean±SD) in serum antibodies from normal healthy individuals was 11.3±2.9% and 15.4±2.2% when native and modified DNA were, respectively, used as inhibitors.
Figure 4.
Inhibition of serum antibodies from healthy controls and cancer patients by modified DNA (■). The microtiter ELISA plates were coated with HNE‐DNA (2.5 μg/mL). Curves represent mean values for serum antibodies from different individuals with head and neck cancer (Ο), breast cancer (■), lung cancer (filled Δ), and normal subjects (Δ)
Table 1.
Inhibition of binding of auto‐antibodies in cancer sera to HNE‐DNA by native and HNE‐DNA
| Type of cancer | Maximum percent inhibition at 20 μg/mL | |
|---|---|---|
| Native DNA | Modified DNA | |
| Normal human sera (20)a | 22.6±2.6% | 27.6±4.5% |
| Head and neck (18) | 30.8±2.3% | 51.2±4.1% |
| Breast (16) | 33.3±2.5% | 54.2±3.9% |
| Cervical (11) | 30.3±3.1% | 50.0±4.9% |
| Lung (9) | 32.1±2.5% | 50.7±4.9% |
Values in parenthesis () indicate the number of sera samples.
4. Discussion
The highly reactive electrophile 4‐hydroxynonenal (HNE) is a product of lipid peroxidation, and plays an important role in cancer, diabetes, aging, and neurodegenerative diseases. DNA and protein adducts of HNE have been detected in a number of diseases including Alzheimer disease, Parkinson disease, atherosclerosis, alcoholic liver disease, diabetes, and pre‐malignant inflammation.8, 10, 20 Recent studies have shown that levels of HNE and lipid peroxidation derived DNA adducts were increased in hepatocellular carcinoma and thyroid carcinoma.21
In this study, calf thymus DNA (10 μg/mL) was incubated with increasing concentration from 100 μmol/L to 1 mmol/L HNE at 37°C for 24 hours. Double‐stranded calf thymus DNA was chosen for the study because it is analogous to human genomic DNA. The damage to calf thymus DNA was caused by formation of adducts with 4‐HNE. The observed damage might be due to free radical generation in this process. The increase in hyperchromicities of DNA was observed for the increasing concentration of 4‐HNE as compared to the native calf thymus DNA. The hyperchromicity can be attributed to the single‐stranded breaks, destabilization of hydrogen bonds, and modification of nitrogenous bases which result in the destruction of the chromophoric groups that attack on sugar‐phosphate back bone. A decrease in the fluorescence intensity may be a result of the structural perturbations in DNA helix and generation of strand breaks due to modification by HNE.
Previous studies have demonstrated presence of auto‐antibodies in cancer patients19, 22 and it has been suggested that the molecular changes responsible for the autoimmune reaction in cancer sera may be related to the oncogenic process.23, 24
To analyze the role of modified DNA in eliciting immune response in cancer patients, the randomly picked 79 cases of breast, head and neck, lung, and cervical cancer patients were selected for immunological studies. Sera from cancer patients were obtained and screened for presence/prevalence of antibodies against the modified DNA. Sera from age‐ and sex‐matched normal healthy individuals served as control. The auto‐antibodies showed appreciably higher binding to modified DNA as compared to the native analog. Appreciably higher inhibition in the binding of these auto‐antibodies was observed with HNE‐DNA as compared to the native form.
Our results are in line with earlier reports wherein antibodies, both poly‐ and mono‐clonal, have been generated against RCS‐modified DNA bases16, 25, 26 These specific RCS‐DNA antibodies have been successfully used to measure carbonyl stress in human and animal studies following oxidative and peroxidative insult.10, 16, 27
A test based on the demonstration of antibodies to tumor antigen in sera of patients, as described here, could be of great importance for early diagnosis of cancer because of the prolonged time course of carcinogenesis and the possibility that a very small tumor or a subtle biochemical change in the cell might be able to produce a detectable level of auto‐antibody in response to chemical carcinogens, before the emergence of the tumor phenotype.28 Biomarkers could prove vital for the identification of early cancer and hence reduce morbidity and mortality of the disease.
5. Conclusion
The results indicate that preferential recognition of HNE‐modified DNA as compared to native DNA by circulating antibodies in cancer patients. The results obtained here point toward the role of 4‐HNE causing DNA damage to the extent of neo‐epitopes generation. The production of the neo‐epitopes on the modified DNA may be recognized as foreign by the immune system, specifically B cells and T helper cells. Although the mechanism of exposure of double‐stranded DNA to the immune system is still unclear, but one of the speculation is that, in cancer the process of the apoptosis may become defective. This may lead to either an increase in cell death and/or a decrease in dead cell clearance. Thus, the 4‐HNE‐modified DNA might be available for its interaction with the immune system. It may initiate the generation of the auto‐antibodies against them. The formation of auto‐antibodies will take effect only after the formation of modified DNA antigen‐specific B cells. The data, though preliminary, may go a long way in establishing these antibodies as biological marker for cancer diagnosis as well as to track the prognosis during the course of therapy. A large prospective and multicentric study is also required to establish the role of the antibodies to the DNA modified by HNE‐system in the development and progression of cancer.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for financial assistance through the research group project No. RGP‐VPP‐175.
Faisal M, Shahab U, Alatar AA, and Ahmad S. Preferential recognition of auto‐antibodies against 4‐hydroxynonenal modified DNA in the cancer patients. J Clin Lab Anal. 2017;31:e22130 10.1002/jcla.22130
Contributor Information
Abdulrahman A. Alatar, Email: aalatar@ksu.edu.sa.
Saheem Ahmad, Email: ahmadsaheem@gmail.com.
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