Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives.
2019; 9 (1): 19176
Mammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis. Sirt6 activation protects against metabolic and aging-related diseases, and Sirt6 inhibition is considered a cancer therapy. Available Sirt6 modulators show insufficient potency and specificity, and even partially contradictory Sirt6 effects were reported for the plant flavone quercetin. To understand Sirt6 modulation by quercetin-based compounds, we analysed their binding and activity effects on Sirt6 and other Sirtuin isoforms and solved crystal structures of compound complexes with Sirt6 and Sirt2. We find that quercetin activates Sirt6 via the isoform-specific binding site for pyrrolo[1,2-a]quinoxalines. Its inhibitory effect on other isoforms is based on an alternative binding site at the active site entrance. Based on these insights, we identified isoquercetin as a ligand that can discriminate both sites and thus activates Sirt6 with increased specificity. Furthermore, we find that quercetin derivatives that inhibit rather than activate Sirt6 exploit the same general Sirt6 binding site as the activators, identifying it as a versatile allosteric site for Sirt6 modulation. Our results thus provide a structural basis for Sirtuin effects of quercetin-related compounds and helpful insights for Sirt6-targeted drug development.
View details for DOI 10.1038/s41598-019-55654-1
View details for PubMedID 31844103
The epigenetic regulator SIRT7 guards against mammalian cellular senescence induced by ribosomal DNA instability.
The Journal of biological chemistry
In the yeast Saccharomyces cerevisiae, genomic instability in rDNA repeat sequences is an underlying cause of cell aging and is suppressed by the chromatin-silencing factor Sir2. In humans, rDNA instability is observed in cancers and premature aging syndromes, but its underlying mechanisms and functional consequences remain unclear. Here, we uncovered a pivotal role of sirtuin 7 (SIRT7), a mammalian Sir2 homolog, in guarding against rDNA instability and show that this function of SIRT7 protects against senescence in primary human cells. We found that, mechanistically, SIRT7 is required for association of SNF2H (also called Smarca5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A, member 5), a component of the nucleolar heterochromatin-silencing complex NoRC, with rDNA sequences. Defective rDNA-heterochromatin silencing in SIRT7-deficient cells unleashed rDNA instability, with excision and loss of rDNA gene copies, which in turn induced acute senescence. Mounting evidence indicates that accumulation of senescent cells significantly contributes to tissue dysfunction in aging-related pathologies. Our findings identify rDNA instability as a driver of mammalian cellular senescence and implicate SIRT7-dependent heterochromatin silencing in protecting against this process.
View details for PubMedID 29728458
Inhibition of pyruvate carboxylase by 1 alpha,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression
2017; 411: 171?81
Maintaining reductive-oxidative (redox) balance is an essential feature in breast cancer cell survival, with cellular metabolism playing an integral role in maintaining redox balance through its supply of reduced NADPH. In the present studies, the effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on redox balance was investigated in early stages of breast cancer. Treatment with 1,25(OH)2D promoted oxidative stress in MCF10A-ras and MCF10A-ErbB2 breast epithelial cells, as measured by the decreased ratios of NADPH/NADP+ and reduced to oxidized glutathione (GSH/GSSG). The mRNA and protein expression of the enzyme pyruvate carboxylase (PC) was downregulated with 1,25(OH)2D treatment, suggesting a potential mechanism. Genetic depletion of PC in MCF10A-ras cells resulted in a decreased ratio of NADPH/NADP+ and GSH/GSSG, with 1,25(OH)2D treatment having no further effect. Mutation analysis confirmed the presence and functionality of a vitamin D response element in the PC gene promoter region. Collectively, these results provide evidence that 1,25(OH)2D promotes oxidative stress in early breast cancer progression through transcriptional downregulation of PC.
View details for DOI 10.1016/j.canlet.2017.09.045
View details for Web of Science ID 000416299700020
View details for PubMedID 29024812
View details for PubMedCentralID PMC5763507
SIRT6: Novel Mechanisms and Links to Aging and Disease.
Trends in endocrinology and metabolism
2017; 28 (3): 168-185
SIRT6, a member of the Sirtuin family of NAD(+)-dependent enzymes, has established roles in chromatin signaling and genome maintenance. Through these functions, SIRT6 protects against aging-associated pathologies including metabolic disease and cancer, and can promote longevity in mice. Research from the past few years revealed that SIRT6 is a complex enzyme with multiple substrates and catalytic activities, and uncovered novel SIRT6 functions in the maintenance of organismal health span. Here, we review these new discoveries and models of SIRT6 biology in four areas: heterochromatin stabilization and silencing; stem cell biology; cancer initiation and progression; and regulation of metabolic homeostasis. We discuss the possible implications of these findings for therapeutic interventions in aging and aging-related disease processes.
View details for DOI 10.1016/j.tem.2016.10.002
View details for PubMedID 27836583
View details for PubMedCentralID PMC5326594
1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2016; 163: 147?56
Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23±2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-(13)C5] glutamine tracer kinetics, into the TCA cycle by 31±0.2% and 17±0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-?-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast cancer.
View details for DOI 10.1016/j.jsbmb.2016.04.022
View details for Web of Science ID 000383824800018
View details for PubMedID 27154413
View details for PubMedCentralID PMC5012911
SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2016; 23 (5): 434-440
Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing has been observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover an essential role of the human SIRT6 enzyme in pericentric transcriptional silencing, and we show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, residue K18 of histone H3 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, depletion of these transcripts through RNA interference rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and regulation of acetylated H3K18 at heterochromatin, and demonstrate the pathogenic role of deregulated pericentric transcription in aging- and cancer-related cellular dysfunction.
View details for DOI 10.1038/nsmb.3202
View details for Web of Science ID 000375633100015
View details for PubMedID 27043296
Altered Glucose Metabolism in Harvey-ras Transformed MCF10A Cells
2015; 54 (2): 111-120
Metabolic reprogramming that alters the utilization of glucose including the "Warburg effect" is critical in the development of a tumorigenic phenotype. However, the effects of the Harvey-ras (H-ras) oncogene on cellular energy metabolism during mammary carcinogenesis are not known. The purpose of this study was to determine the effect of H-ras transformation on glucose metabolism using the untransformed MCF10A and H-ras oncogene transfected (MCF10A-ras) human breast epithelial cells, a model for early breast cancer progression. We measured the metabolite fluxes at the cell membrane by a selective micro-biosensor, [(13)C6 ]glucose flux by (13)C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of glucose metabolism enzymes by quantitative PCR. Results from these studies indicated that MCF10A-ras cells exhibited enhanced glycolytic activity and lactate production, decreased glucose flux through the tricarboxylic acid (TCA) cycle, as well as an increase in the utilization of glucose in the pentose phosphate pathway (PPP). These results provide evidence for a role of H-ras oncogene in the metabolic reprogramming of MCF10A cells during early mammary carcinogenesis.
View details for DOI 10.1002/mc.22079
View details for Web of Science ID 000347279800004
View details for PubMedID 24000146
Maternal exercise during pregnancy reduces risk of mammary tumorigenesis in rat offspring
EUROPEAN JOURNAL OF CANCER PREVENTION
2014; 23 (6): 502-505
Breast cancer is the most common cancer among women. Emerging research indicates that modifying lifestyle factors during pregnancy may convey long-term health benefits to offspring. This study was designed to determine whether maternal exercise during pregnancy leads to reduced mammary tumorigenesis in female offspring. Pregnant rats were randomly assigned to exercised and sedentary groups, with the exercised group having free access to a running wheel and the sedentary group housed with a locked wheel during pregnancy. Female pups from exercised or sedentary dams were weaned at 21 days of age and fed a high fat diet without access to a running wheel. At 6 weeks, all pups were injected with the carcinogen N-methyl-N-nitrosourea. Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared with pups from sedentary dams (100%). Neither tumor latency nor histological grade differed between the two groups. These data are the first to demonstrate that exercise during pregnancy potentiates reduced tumorigenesis in offspring. This study provides an important foundation towards developing more effective modes of behavior modification for cancer prevention.
View details for DOI 10.1097/CEJ.0000000000000029
View details for Web of Science ID 000342896600002
View details for PubMedID 24950432
- 1,25-Dihydroxyvitamin D regulation of glucose metabolism in Harvey-ras transformed MCF10A human breast epithelial cells JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 2013; 138: 79-89
1 alpha, 25-Dihydroxyvitamin D regulates hypoxia-inducible factor-1 alpha in untransformed and Harvey-ras transfected breast epithelial cells
2010; 298 (2): 159-166
The purpose of this study was to determine the mechanism by which 1?, 25-dihydroxyvitamin D (1,25(OH)(2)D) alters hypoxia-inducible factor-1? (HIF-1?) protein in untransformed and Harvey-ras (H-ras) oncogene transfected MCF10A breast epithelial cells. Treatment with 1,25(OH)(2)D (10nM) increased both mRNA (2.55±0.6-fold vs. vehicle, p=0.03) and protein levels (2.37±0.3-fold vs. vehicle, p<0.0001) of HIF-1? in MCF10A cells in 12h, which remained elevated at 24h. However, in H-ras transfected MCF10A cells, 1,25(OH)(2)D treatment increased HIF-1? protein level (2.08±0.38-fold vs. vehicle, p=0.05) at 12h, with no change in mRNA level and HIF-1? protein level returned to baseline after 24h. A transcription inhibitor prevented the 1,25(OH)(2)D induction of HIF-1? protein and mRNA levels in MCF10A cells, but failed to alter the induction of HIF-1? protein level in H-ras transfected MCF10A cells. On the other hand, inhibition of proteasomal degradation prevented the 1,25(OH)(2)D-induced HIF-1? protein level in H-ras transfected MCF10A but not in MCF10A cells. These results support that 1,25(OH)(2)D regulates HIF-1? protein level via transcriptional regulation in MCF10A cells in contrast to through proteosomal degradation with the presence of H-ras oncogene in MCF10A cells.
View details for DOI 10.1016/j.canlet.2010.06.014
View details for Web of Science ID 000284300000003
View details for PubMedID 20655141