Vijay Kumar Kutala

Aberrations in one-carbon metabolism induce oxidative DNA damage in sporadic breast cancer

The authors investigated the role of dietary micronutrients and eight functional polymorphisms of one-carbon metabolism in modulating oxidative stress in sporadic breast cancer. PCR-restriction fragment length polymorphism (RFLP) and PCR-amplified fragment length polymorphism (AFLP) methods were used for genetic analysis in 222 sporadic breast cancer cases and 235 controls. Standardized food frequency questionnaire was used for dietary micronutrient assessment. 8-oxo-2′-deoxyguanosine (8-oxodG), folate, and estradiol were estimated using commercial ELISA kits. Reverse-phase HPLC coupled with fluorescence detector was used for plasma homocysteine analysis. Total glutathione was estimated using Ellman’s method. Reduced folate carrier 1 (RFC1) G80A and methylenetetrahydrofolate reductase (MTHFR) C677T were associated with risks of 1.34 (95% CI 1.01–1.79)- and 1.84 (95% CI 1.14–3.00)-folds, respectively, for sporadic breast cancer while cytosolic serine hydroxymethyl transferase (cSHMT) C1420T was associated with reduced risk (OR 0.71, 95% CI 0.53–0.94). Significant increase in plasma 8-oxo-2′-deoxyguanosine (Pxa0<xa00.004) and homocysteine (Pxa0<xa00.0001); and significant decrease in total glutathione (Pxa0<xa00.01) and dietary folate (Pxa0=xa00.006) was observed in cases than in controls. Oxidative DNA damage showed direct association with menopause (Pxa0=xa00.02), RFC1 G80A (Pxa0<xa00.05) and homocysteine (Pxa0<xa00.0001); and inverse association with dietary folate (Pxa0<xa00.0001), plasma folate (Pxa0<xa00.0001), cSHMT C1420T (Pxa0<xa00.05) and glutathione (Pxa0<xa00.001). To conclude, the aberrations in one-carbon metabolism induce oxidative stress in sporadic breast cancer either by affecting the folate pool or by impairing remethylation.

Epistatic interactions between loci of one-carbon metabolism modulate susceptibility to breast cancer

In view of growing body of evidence substantiating the role of aberrations in one-carbon metabolism in the pathophysiology of breast cancer and lack of studies on gene–gene interactions, we investigated the role of dietary micronutrients and eight functional polymorphisms of one-carbon metabolism in modulating the breast cancer risk in 244 case–control pairs of Indian women and explored possible gene–gene interactions using Multifactor dimensionality reduction analysis (MDR). Dietary micronutrient status was assessed using the validated Food Frequency Questionnaire. Genotyping was done for glutamate carboxypeptidase II (GCPII) C1561T, reduced folate carrier (RFC)1 G80A, cytosolic serine hydroxymethyltransferase (cSHMT) C1420T, thymidylate synthase (TYMS) 5′-UTR tandem repeat, TYMS 3′-UTR ins6/del6, methylenetetrahydrofolate reductase (MTHFR) C677T, methyltetrahydrofolate-homocysteine methyltransferase (MTR) A2756G, methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) A66G polymorphisms by using the PCR-RFLP/AFLP methods. Low dietary folate intake (Pxa0<xa00.001), RFC1 G80A (OR: 1.38, 95% CI 1.06–1.81) and MTHFR C677T (OR: 1.74 (1.11–2.73) were independently associated with the breast cancer risk whereas cSHMT C1420T conferred protection (OR: 0.72, 95% CI 0.55–0.94). MDR analysis demonstrated a significant tri-variate interaction among RFC1 80, MTHFR 677 and TYMS 5′-UTR loci (Ptrendxa0<xa00.02) with high-risk genotype combination showing inflated risk for breast cancer (OR 4.65, 95% CI 1.77–12.24). To conclude, dietary as well as genetic factors were found to influence susceptibility to breast cancer. Further, the current study highlighted the importance of multi-loci analyses over the single-locus analysis towards establishing the epistatic interactions between loci of one-carbon metabolism modulate susceptibility to the breast cancer.

Mitochondrial-Targeted Curcuminoids: A Strategy to Enhance Bioavailability and Anticancer Efficacy of Curcumin

Although the anti-cancer effects of curcumin has been shown in various cancer cell types, in vitro, pre-clinical and clinical studies showed only a limited efficacy, even at high doses. This is presumably due to low bioavailability in both plasma and tissues, particularly due to poor intracellular accumulation. A variety of methods have been developed to achieve the selective targeting of drugs to cells and mitochondrion. We used a novel approach by conjugation of curcumin to lipophilic triphenylphosphonium (TPP) cation to facilitate delivery of curcumin to mitochondria. TPP is selectively taken up by mitochondria driven by the membrane potential by several hundred folds. In this study, three mitocurcuminoids (mitocurcuminoids-1, 2, and 3) were successfully synthesized by tagging TPP to curcumin at different positions. ESI-MS analysis showed significantly higher uptake of the mitocurcuminoids in mitochondria as compared to curcumin in MCF-7 breast cancer cells. All three mitocurcuminoids exhibited significant cytotoxicity to MCF-7, MDA-MB-231, SKNSH, DU-145, and HeLa cancer cells with minimal effect on normal mammary epithelial cells (MCF-10A). The IC50 was much lower for mitocurcuminoids when compared to curcumin. The mitocurcuminoids induced significant ROS generation, a drop in ΔØm, cell-cycle arrest and apoptosis. They inhibited Akt and STAT3 phosphorylation and increased ERK phosphorylation. Mitocurcuminoids also showed upregulation of pro-apoptotic BNIP3 expression. In conclusion, the results of this study indicated that mitocurcuminoids show substantial promise for further development as a potential agent for the treatment of various cancers.

Oxidative stress in coronary artery disease: epigenetic perspective.

The association between oxidative stress and coronary artery disease (CAD) is well documented. However, the role of epigenetic factors contributing to oxidative stress is relatively unexplored. In this study, we aimed to explore the impact of DNA methylation profile in BCL2/E1B adenovirus interacting protein 3 (BNIP3), extracellular superoxide dismutase (EC-SOD) and glutathione-S-transferase P1 (GSTP1) on the oxidative stress in CAD. Further, the contribution of folate pathway genetic polymorphisms in regulating epigenome was elucidated. The expression of BNIP3, EC-SOD, and GSTP1 were studied by using Maxima@SYBR-green based real-time qPCR approach in peripheral blood samples. Combined bisulfite restriction analysis and methylation-specific PCR were used to study promoter CpG island methylation. Further, the effect of homocysteine on BNIP3 gene expression was studied in human aortic endothelial cells in vitro. CAD cases exhibited upregulation of BNIP3, downregulation of EC-SOD and GSTP1. Hypomethylation of BNIP3 and hypermethylation of EC-SOD were observed in CAD cases. The expression of BNIP3 was positively correlated with homocysteine, MDA, protein carbonyls, and methylene tetrahydrofolate reductase C677T, while showing inverse association with cytosolic serine hydroxymethyl transferase C1420T. The expressions of EC-SOD and GSTP1 showed positive association with thymidylate synthase (TYMS) 2R3R, while inverse association with MDA, protein carbonyls, and methionine synthase reductase (MTRR) A66G. In vitro analysis showed homocysteine-dependent upregulation of BNIP3. The results of this study suggest that the aberrations in one-carbon metabolism appear to induce altered gene expression of EC-SOD, GSTP1, and BNIP3, and thus contribute to the increased oxidative stress and increased susceptibility to CAD.

Cross-talk between one-carbon metabolism and xenobiotic metabolism: implications on oxidative DNA damage and susceptibility to breast cancer.

The aim of this case–control study is to explore the role of aberrations in xenobiotic metabolism in inducing oxidative DNA damage and altering the susceptibility to breast cancer. Cytochrome P4501A1 (CYP1A1) m1 (OR: 1.41, 95% CI 1.08–1.84), CYP1A1 m4 (OR: 5.13, 95% CI 2.68–9.81), Catecholamine-O-methyl transferase (COMT) H108L (OR: 1.49, 95% CI 1.16–1.92), and glutathione S-transferase (GST) T1 null (OR: 1.68, 95% CI 1.09–2.59) variants showed association with breast cancer risk. Reduced folate carrier 1 (RFC1) 80A/CYP1A1 m1/CYP1A1 m4 and RFC1 80A/thymidylate synthase (TYMS) 5′-UTR 2R/methionine synthase (MTR) 2756G/COMT 108L genetic combinations were found to inflate breast cancer risk under the conditions of low dietary folate (345xa0±xa0110 vs. 379xa0±xa0139xa0μg/day) and low plasma folate (6.81xa0±xa01.25 vs. 7.09xa0±xa01.26xa0ng/ml) by increasing plasma 8-oxo-2′-deoxyguanosine (8-oxodG). This increase in 8-oxodG is attributed to low methionine (49.38xa0±xa023.74 vs. 53.90xa0±xa023.85xa0μmol/l); low glutathione (378xa0±xa0242 vs. 501xa0±xa0126xa0μmol/l) and GSTT1 null variant; and hypermethylation of CpG island of extracellular-superoxide dismutase (EC-SOD) (92.78xa0±xa011.49 vs. 80.45xa0±xa09.86%), which impair O-methylation of catechol estrogens to methoxy estrogens, conjugation of glutathione to semiquinones/quinones and free radical scavenging respectively. Our results suggest cross-talk between one-carbon metabolism and xenobiotic metabolism influencing oxidative DNA damage and susceptibility to breast cancer.

Association of aberrations in one-carbon metabolism with molecular phenotype and grade of breast cancer

We have earlier demonstrated the role of aberrant one‐carbon metabolism in the etiology of breast cancer. In the current study, we examine the clinical utility of these factors in predicting the subtype of breast cancer and as indicators of disease progression. Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) and PCR–amplified fragment length polymorphism (AFLP) approaches were used for genetic analysis. Plasma folate and homocysteine were measured using Axsym folate kit and reverse phase HPLC, respectively. Multiple linear regression models were used to test the predictability of disease progression. Luminal A subtype was associated with late age of onset, higher body mass index and lack of family history of breast cancer. Thymidylate synthase (TYMS) 5′‐UTR 28u2009bp tandem repeat (OR: 2.09, 95% CI: 1.05–4.16) and methylene tetrahydrofolate reductase (MTHFR) C677T (OR: 4.10, 95% CI: 1.40–11.95) were strongly associated with Luminal B. Reduced folate carrier (RFC1) G80A (OR: 2.92, 95% CI: 1.22–6.97) and methionine synthase (MTR) A2756G (OR: 4.71, 95% CI: 1.66–13.31) polymorphisms were associated with LuminA‐HH subtype while MTHFR C677T showed association with HER‐enriched (OR: 30.41, 95% CI: 6.47–142.91). Cytosolic serine hydroxymethyltransferase (cSHMT) conferred protection against basal‐like breast cancer (OR: 0.47, 95% CI: 0.22–0.98). HER‐enriched and basal‐like subtypes showed positive association with familial breast cancer and inverse association with plasma folate. Hyperhomocysteinemia was observed in Luminal B and basal‐like subtypes. Multiple linear regression models of aberrant one‐carbon metabolism were found to be moderate predictors of breast cancer grade (area under the receiver operating characteristic curve, Cu2009=u20090.72, 95% CI: 0.58–0.87, Pu2009=u20090.008). To conclude, aberrations in one‐carbon metabolism predict the subtype of breast cancer and disease progression.

Association of TLR4 (D299G, T399I), TLR9 −1486T>C, TIRAP S180L and TNF-α promoter (−1031, −863, −857) polymorphisms with risk for systemic lupus erythematosus among South Indians

The rationale of this case-control study was to explore the association of Toll-like receptor 4 (TLR4) D299G, TLR4 T399I, TLR9 −1486 T>C, TIR-domain-containing adaptor protein (TIRAP) S180u2009L and tumor necrosis-α (TNF-α) promoter polymorphisms with susceptibility and phenotypic heterogeneity of systemic lupus erythematosus (SLE). PCR-RFLP, real-time PCR was used for the genetic analysis and expression studies and ELISA was used for the determination of specific autoantibodies. TLR4 D299G was associated with the risk for SLE (OR: 1.57, 95% CI: 1.08–2.28), while the TNF-α (−1031, −863, −857) CCC haplotype conferred protection. TLR4 and TIRAP polymorphisms were associated with reduced expression of HLA-DR. The presence of TLR4 and TLR9 polymorphisms increases the MHC2TA expression, while TIRAP polymorphism was associated with reduced expression. TLR4 D299u2009G showed an inverse association with pulmonary hypertension. TLR 4 T399I and TLR9 −1486u2009T>C showed a positive association with seizures and photosensitivity, respectively. TIRAP S180u2009L showed a positive association with alopecia and malar rashes, while an inverse association with psychosis was observed. TLR4 T399I (ru2009=u20090.14, pu2009=u20090.05) and TIRAP S180u2009L (ru2009=u20090.15, pu2009=u20090.03) showed a positive association with anti-Ro antibodies. On the other hand, TLR9 −1486u2009T>C showed an inverse association with anti-La antibodies (ru2009=u2009−0.20, pu2009=u20090.006). To conclude, TLR4 D299G increases the risk for SLE, while TNF-α CCC haplotype reduces the risk for SLE. All these polymorphisms contribute toward phenotypic heterogeneity. TLR4 T399I, TLR9 −1486u2009T>C and TIRAP S180u2009L influence specific autoantibody production in SLE.

Oxidative Stress is Associated with Genetic Polymorphisms in One-Carbon Metabolism in Coronary Artery Disease

In view of growing body of evidence favouring the association of aberrations in one-carbon metabolism and oxidative stress in the aetiology of coronary artery disease (CAD), we investigated the risk associated with polymorphisms regulating the folate uptake and transport such as the glutamate carboxypeptidase II (GCPII) C1561T, reduced folate carrier 1 (RFC1) G80A and cytosolic serine hydroxymethyltransferase (cSHMT) C1420T. We further evaluated the impact of seven putatively functional polymorphisms of this pathway on oxidative stress markers. Genotyping was performed on 288 CAD cases and 266 healthy controls along with the dietary folate assessment. GCPII C1561T polymorphism was found to be an independent risk factor (OR 2.71, 95% CI 1.47–4.98) for CAD, whereas cSHMT C1420T conferred protection (OR 0.51, 95% CI 0.37–0.70). Oxidative stress markers like the plasma levels of malondialdehyde, protein carbonyls and 8-oxo-deoxyguanosine were significantly increased and total glutathione was significantly decreased in CAD cases. Elevated oxidative stress was observed in subjects carrying GCPII 1561T and MTRR 66A-variant alleles and low oxidative stress was observed in the subjects carrying cSHMT 1420T and TYMS 5′-UTR 2R allele. GCPII C1561T, MTHFR C677T and MTRR A66G polymorphisms were observed to influence the homocysteine levels (Pxa0<xa00.05). SHMT and TYMS variants were found to decrease oxidative stress by increasing the folate pool (rxa0=xa00.38, Pxa0=xa00.003) and also by increasing the antioxidant status (rxa0=xa00.28, Pxa0=xa00.03). Influence of dietary folate status was not observed. Overall, this study revealed elevated oxidative stress that was associated with the aberrations in one-carbon metabolism which could possibly influence the CAD risk.

Association of seven functional polymorphisms of one-carbon metabolic pathway with total plasma homocysteine levels and susceptibility to Parkinson's disease among South Indians

This study from South India was performed to ascertain the impact of seven functional polymorphisms of one-carbon metabolic pathway on total plasma homocysteine levels and susceptibility to PD. A total of 151 cases of Parkinsons disease and 416 healthy controls were analyzed for fasting plasma homocysteine levels by reverse phase HPLC. PCR-RFLP approaches were used to analyze glutamate carboxypeptidase II (GCPII) 1561 C>T, reduced folate carrier 1 (RFC1) 80 G>A, cytosolic serine hydroxymethyl transferase (cSHMT) 1420 C>T, methylene tetrahydrofolate reductase (MTHFR) 677 C>T, methionine synthase (MTR) 2756 A>G and methionine synthase reductase (MTRR) 66 A>G polymorphisms. PCR-AFLP was used for the analysis of thymidylate synthase (TYMS) 5-UTR 28bp tandem repeat. PD cases exhibited elevated plasma homocysteine levels compared to controls (men: 28.8 ± 6.9 vs. 16.4 ± 8.8 μmol/L; women: 25.4 ± 5.3 vs. 11.2 ± 5.1μmol/L). Homocysteine levels showed positive correlation with male gender (r=0.39, p<0.0001) and MTRR 66 A>G (r=0.31, p<0.0001) whereas an inverse correlation was observed with cSHMT 1420 C>T polymorphism. MTRR 66 A>G polymorphism showed independent risk for PD (OR: 3.42, 95% CI: 2.35-4.98) whereas cSHMT 1420 C>T conferred protection against PD (OR: 0.11, 95% CI: 0.07-0.17). Multifactor dimensionality reduction analysis showed synergistic interactions between MTHFR 677 C>T and MTRR 66 A>G, whereas cSHMT 1420 C>T exhibited counteracting interactions in altering susceptibility to PD. To conclude, PD cases exhibited hyperhomocysteinemia and MTRR 66 A>G and cSHMT 1420 C>T gene variants were shown to modulate PD risk by altering the homocysteine levels.

Impact of Hyperhomocysteinemia on Breast Cancer Initiation and Progression: Epigenetic Perspective

AbstractOur recent study showing association of hyperhomocysteinemia and hypomethioninemia in breast cancer and other studies indicating association of hyperhomocysteinemia with metastasis and development of drug resistance in breast cancer cells treated with homocysteine lead us to hypothesize that homocysteine might modulate the expression of certain tumor suppressors, i.e., RASSF1, RARβ1, CNND1, BRCA1, and p21, and might influence prognostic markers such as BNIP3 by inducing epigenetic alteration. To demonstrate this hypothesis, we have treated MCF-7 and MDA-MB-231 cells with different doses of homocysteine and observed dose-dependent inhibition of BRCA1 and RASSF1, respectively. In breast cancer tissues, we observed the following expression pattern: BNIP3xa0>xa0BRCA1xa0>xa0RARβ1xa0>xa0CCND1xa0>xa0p21xa0>xa0RASSF1. Hyperhomocysteinemia was positively associated with BRAC1 hypermethylation both in breast cancer tissue and corresponding peripheral blood. Peripheral blood CpG island methylation of BRCA1 in all types of breast cancer and methylation of RASSF1 in ER/PR-negative breast cancers showed positive correlation with total plasma homocysteine. The methylation of RASSF1 and BRCA1 was associated with breast cancer initiation as well as progression, while BRCA1 methylation was associated with DNA damage. Vitamin B12 showed inverse association with the methylation at both the loci. RFC1 G80A and cSHMT C1420T variants showed positive association with methylation at both the loci. Genetic variants influencing remethylation step were associated positively with BRCA1 methylation and inversely with RASSF1 methylation. GCPII C1561T variant showed inverse association with BRCA1 methylation. We found good correlation of BRAC1 (rxa0=xa00.90) and RASSF1 (0.92) methylation pattern between the breast cancer tissue and the corresponding peripheral blood. To conclude, elevated homocysteine influences methionine dependency phenotype of breast cancer cells and is associated with breast cancer progression by epigenetic modulation of RASSF1 and BRCA1n.