RESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Piruvato Carboxilase/antagonistas & inibidores , Vitamina D/análogos & derivados , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Humanos , Estresse Oxidativo/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
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-γ-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.
Assuntos
Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Glutamina/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Vitamina D/análogos & derivados , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Linhagem Celular Transformada , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Ácido Glutâmico/metabolismo , Glutamina/análogos & derivados , Glutamina/metabolismo , Glutamina/farmacologia , Humanos , Cinética , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitamina D/farmacologia , Elemento de Resposta à Vitamina DRESUMO
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.