Tamoxifen (TAM) is a non-steroidal anti-oestrogen drug, which has led to an increase in both disease-free and overall survival of breast cancer patients after primary surgery(Reference Rutqvist, Johansson, Signomklao, Johansson, Fornande and Wilking1). A complicating factor is the relapse in breast cancer patients during tamoxifen therapy and in this subset of patients, treatment is only palliative and the recurrent breast cancer is incurable(Reference van Dalen, Heering and Barak2). Endometrial cancer and other serious side-effects of therapy have been reported in tamoxifen-treated patients and TAM-induced DNA adducts were found in leucocyte DNA from breast cancer patients(Reference Hemminki, Rajaniemi, Koskinen and Hansson3).
Poly(ADP-ribose) polymerase (PARP) is a highly conserved, abundant protein, with three functional domains identified within the PARP polypeptide by limited proteolysis(Reference Kameshita, Matsuda, Taniguchi and Shizuta4). PARP protein and associated poly(ADP-ribosyl)ation reactions are thought to play a number of roles in different biological processes such as DNA repair, recombination, apoptosis, p53 function and maintenance of genomic stability(Reference D'Amours, Desnoyers, D'Silva and Poirier5).
In recent years, changes in the status of DNA methylation, known as epigenetic alterations, have turned out to be one of the most common molecular alterations in human neoplasia including breast cancer(Reference Jones and Baylin6). Muller et al. (Reference Muller, Widschwendter, Fiegl, Ivarsson, Goebel, Perkmann, Marth and Widschwendter7) detected the prognostic value for RASSF1A methylation in pre-therapeutic sera of patients with breast cancer. The present study has been designed to evaluate the effect of nutritional supplementation of co-enzyme Q10, riboflavin and niacin (CoRN) on modulation of DNA repair enzyme PARP and RASSF1A DNA methylation pattern in breast cancer patients undergoing tamoxifen therapy.
Materials and methods
Study patients
Patients were recruited from the Medical Oncology Department of the Government Royapettah Hospital, Chennai, India, via their physicians according to the process approved by the Institutional Human Ethical Review Board to conduct a single blinded study. They were aged between 43 and 70 years with histopathology-confirmed breast cancer. Patients with diabetes mellitus, renal and hepatic diseases were excluded from the study.
Study design
Forty-two socio-economically and age-matched disease-free, healthy controls were recruited in group I. Two different groups of patients were recruited for the study in groups II and III – group II: eighty-four untreated breast cancer patients; group III: eighty-four breast cancer patients treated for more than 1 year with TAM; group IV and V – group III patients followed up for 45 d (group IV) and 90 d (group V) after supplementation with co-enzyme Q10 (100 mg Kaneka®Q10; Kaneka Corporation, Osaka, Japan), riboflavin (10 mg; Madras Pharmaceuticals, Chennai, India) and niacin (50 mg; Madras Pharmaceuticals, Chennai, India) one dosage per d along with TAM (10 mg Nolvadex®, AstraZeneca, Bangalore, India) twice per d. Subjects were advised to maintain their usual diet during the study period and were advised not to take any other medication other than those required during the study.
Serum and DNA isolation
Serum was collected from 2 ml blood in a serum separator tube (Vacutainer; Becton Dickinson, Rutherford, NJ, USA) by centrifugation after clotting at 3000 rpm for 10 min. High molecular weight genomic DNA was isolated from whole blood by conventional phenol–chloroform and ethanol extraction(Reference Sambrook, Fritsch and Maniatis8).
Methylation-specific PCR
RASSF1A DNA methylation: bisulphite modification
Bisulfite modification was done by following the method of Herman et al. (Reference Herman, Graff, Myöhänen, Nelkin and Baylin9). Briefly, 1 μg DNA was denatured by incubating with 5·5 μl NaOH (2 m) for 10 min at 37°C. Subsequently, 30 μl hydroquinone (10 mm; Sigma) and 520 μl sodium bisulphite (3 m; Sigma) at pH 5, freshly prepared, were added and mixed. The DNA was overlaid with four drops of mineral oil and the sample was incubated at 50°C for 16 h. Bisulphite-modified DNA was purified using a Wizard DNA CleanUp System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
Methylation-specific PCR
CpG islands in RASSF1A genes were detected by methylation-specific PCR following the method of Fackler et al. (Reference Fackler, McVeigh, Evron, Garrett, Mehrotra, Polyak, Sukumar and Argani10). Forward and reverse primers were synthesized which corresponds to the predicted sequence of methylated or unmethylated genomic DNA after sodium bisulphite treatment. For the reaction, 1 μl sodium bisulphite-treated DNA was added to 24 μl reaction buffer (1·25 mm-dNTP, 16·6 mm-(NH4)2SO4, 67 mm-2-amino-2-hydroxymethyl-1,3-propanediol (Tris), pH 8·8, 6·7 mm-MgCl2, 10 mm-β-mercaptoethanol, 0·1 % DMSO and 1·25 U RedTaq; Sigma, St. Louis, MO, USA) containing 100 ng each of forward and reverse primers specific to the unmethylated and methylated DNA sequences. Conditions were 94°C for 5 min, followed by thirty-five cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 45 s, with a final extension cycle of 72°C for 5 min. The PCR products were resolved by electrophoresis in a 2 % agarose gel and the ethidium bromide-stained PCR products were imaged with the Eagle Eye II Video System (Stratagene, La Jolla, CA, USA). RASSF1A unmethylated DNA (180 bp): forward: 5′-GGT TGT ATT TGG TTG GAG TG; reverse: 5′-CTA CAA ACC TTT ACA CAC AAC A. RASSF1A methylated DNA (160 bp): forward: 5′-GTT GGT ATT CGT TGG GCG C; reverse: 5′-GCA CCA CGT ATA CGT AAC G.
Analysis of poly(ADP-ribose) polymerase by immunoblotting
Serum protein concentration was estimated by the method of Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall11). Protein (50 mg) was boiled with sample solubilizing buffer for 5 min and separated on 10 % SDS–PAGE by the method of Laemmli(Reference Laemmli12). The gel was transferred using blotting buffer (25 mm-Tris, 192 mm-glycine and 10 % methanol) on to a nitrocellulose membrane (Hybond C+; Amersham Life Sciences) at 30 V for 3 h. The membrane was incubated with 1:1000 anti-PARP primary antibody (Calbiochem International, CA, USA) for 3 h. After extensive washing the membrane was incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bangalore GeneI, Chennai, India; 1:500 dilution). The bands were detected using the DAB/hydrogen peroxide chromogen system (6 mg 3,3′-diaminobenzidine dihydrochloride and 30 mg nickel chloride in 10 ml 50 mm-Tris–HCl, pH 7·5 containing 10 μl H2O2).
Statistical analysis
Statistical analysis was performed with one-way ANOVA followed by least significant difference test using the Statistical Package for Social Science version 10.0 (SPSS, Chicago, IL, USA). Values are expressed as means and standard deviations.
Results
Protein expression of PARP (116 kDa) protein was determined by immunoblot using anti-PARP antibody. Fig. 1 shows the protein expression pattern in different study groups. In group IV and V patients there was a significant increase (P < 0·01) in PARP concentration compared to groups I, II and III. In group I controls no RASSF1A DNA methylation pattern was found, but all the eighty-four patients in group II had methylated RASSF1A DNA pattern. In group III patients RASSF1A DNA methylation was found in thirty out of eighty-four patients. Methylation pattern changed in twenty of these patients in group IV and group V leading to disappearance of RASSF1A DNA methylation. Fig. 2 shows a representative methylation pattern of RASSF1A in breast cancer patients. Lane 5 shows the methylated RASSF1A DNA and the disappearance of the methylated pattern in lanes 6 and 7 after CoRN supplementation.
Poly(ADP-ribose) polymerase (PARP) expression levels in different study subjects. (A) Lane 1, molecular-weight marker; lane 2, control subjects (group I); lane 3, untreated breast cancer patients (group II); lane 4, breast cancer patients treated with tamoxifen (group III); lane 5, breast cancer patients treated with tamoxifen + co-enzyme Q10, riboflavin and niacin (CoRN) (45 d) (group IV); lane 6, breast cancer patients treated with tamoxifen + CoRN (90 d) (group IV). (B) Values are means with their standard deviations depicted by vertical bars. Comparisons were made between: a, group I and group II; b, group I and III; c, group II and III; d, group III and IV; e, group III and V; f, group I and V. Statistical significance is expressed as: *P < 0·05, **P < 0·01, ***P < 0·001.
Methylation-specific PCR of RASSF1A pattern in the study subjects. Lane 1, DNA marker; lane 2, breast cancer cell line MCF-7 used as a positive control showing methylated DNA; lane 3, normal lymphocyte DNA used as positive control for unmethylated DNA; lane 4, untreated breast cancer patients; lane 5, breast cancer patients treated with tamoxifen; lane 6, breast cancer patients treated with tamoxifen + co-enzyme Q10, riboflavin and niacin (CoRN) (45 d); lane 7, breast cancer patients treated with tamoxifen + CoRN (90 d). 160 bp, Methylated DNA; 180 bp, unmethylated DNA.
Discussion
Cancer patients are usually exposed to high levels of DNA-damaging agents. In addition, they may have compromised nutritional status due to the disease process itself (cachexia) or due to the side-effects of TAM and chemotherapy (nausea, vomiting, impaired gastrointestinal function)(Reference Hemminki, Rajaniemi, Koskinen and Hansson3). The purpose of the present study was to determine whether CoRN supplementation alters the extent of DNA damage and alters RASSF1A methylation.
The protein expression of PARP in group IV and V patients was found to be increased compared to the other groups, which proves the up-regulation of PARP repair enzyme by CoRN supplementation. Niacin is the dietary precursor for the synthesis of NAD+, which is the sole substrate for the nuclear enzyme PARP. PARP binds to, and is specifically activated by, DNA single- and double-strand breaks, representing one of the earliest responses to DNA damage in the cell. Upon activation, PARP synthesizes poly(ADP-ribose) (pADPr) from NAD+, on itself and on a number of acceptor proteins involved in the maintenance of chromatin architecture and DNA metabolism(Reference Boyonoski, Spronck, Jacobs, Shah, Poirier and Kirkland13). Riboflavin captures reactive metabolites like TAM and carcinogens to form a complex and thereby prevents formation of DNA adducts, prevents DNA methylation and maintains genomic stability(Reference Perumal, Shanthi and Sachdanandam14, Reference Pangrekar, Krishnaswamy and Jagadeesa15). Earlier reports have shown that riboflavin deficiency enhances the induction of the three repair-associated enzymes by carcinogens, and that riboflavin supplementation suppresses this phenomenon(Reference Webster, Gawde and Bhattacharya16). This is possibly because of the protection riboflavin provides against DNA damage, by increasing the levels of PARP. Co-enzyme Q10 has been found to increase the DNA repair rate by protecting the cells against further oxidative DNA damage(Reference Migliore, Molinu, Naccarati, Mancuso, Rocchi and Siciliano17, Reference Tomasetti, Alleva, Borghi and Collins18) and has been found to enhance the DNA repair enzyme PARP activity(Reference Tomasetti, Alleva, Borghi and Collins18).
Earlier studies in our laboratory have proved the beneficial effect of CoRN supplementation; CoRN has been found to enhance the expression of the tumour suppressor gene MnSOD. It has been found to restore lipid peroxide levels and activities of the enzymic and non-enzymic antioxidants to normal levels(Reference Perumal, Shanthi and Sachdanandam19). Cofactors of CoRN have been found to inhibit host energy loss by increasing the gluconeogenesis pathway, thereby preventing cancer cachexia(Reference Perumal, Shanthi and Sachdanandam14). CoRN supplementation to breast cancer patients has been found to counteract the TAM-induced hyperlipidaemia to normal levels(Reference Yuvaraj, Premkumar, Vijayasarathy, Gangadaran and Sachdanandam20), reduce the tumour marker levels of CEA and CA 15-3(Reference Premkumar, Yuvaraj, Vijayasarathy, Gangadaran and Sachdanandam21) and decrease the levels of serum cytokines IL-1β, IL-6, IL-8, TNF-α and vascular endothelial growth factor(Reference Premkumar, Yuvaraj, Vijayasarathy, Gangadaran and Sachdanandam22). In the present study, we found disappearance of RASSF1A DNA methylation in the serum of ten patients treated with CoRN. Serum RASSF1A DNA methylation is an easy means of detecting the treatment prognosis for patients undergoing adjuvant TAM treatment as it could help in early detection of recurrence in these patients(Reference Fiegl, Millinger, Mueller-Holzner, Marth, Ensinger, Berger, Klocker, Goebel and Widschwendter23). Disappearance of RASSF1A methylation during supplement therapy with CoRN in these patients may help in preventing the occurrence of recurrence.
Acknowledgements
The authors would like to thank the pharmaceutical companies Kaneka Corporation, Japan (co-enzyme Q10) and Madras Pharmaceuticals, India (riboflavin and niacin) for their kind gift of tablets for this study. We would like to thank our clinical collaborators Dr K. Vijayasarathy and Dr S. G. D. Gangadaran, Department of Medical Oncology, Government Royapettah Hospital, Chennai, India. No conflicts of interest exist for this paper.
