RESUMEN
1,25-Dihydroxyvitamin D(3) (1,25D) induces apoptosis in MCF-7 cells via the intrinsic pathway involving bax translocation to mitochondria, cytochrome c release and reactive oxygen species (ROS) generation. Vitamin D up-regulated protein 1 (VDUP1), an apoptotic regulatory gene induced by 1,25D in HL-60 cells, is a negative regulator of thioredoxin (Trx1), a redox protein which neutralizes ROS and protects against oxidative stress induced apoptosis. Due to the involvement of oxidative stress in 1,25D mediated apoptosis, we analyzed whether VDUP1 or Trx1 are altered by 1,25D in MCF-7 cells. In contrast to HL-60 cells, VDUP1 mRNA was not up-regulated by 1,25D in MCF-7 cells, indicating that transcriptional up-regulation of this gene is not required for 1,25D mediated apoptosis. 1,25D did not affect the expression or activity of Trx1 in MCF-7 cells, however, Trx1 activity was higher in MCF-7 cells selected for resistance to 1,25D mediated apoptosis. In untreated MCF-7 cells, Trx1 was present only in the cytosol, and the majority was in the oxidized state. In 1,25D treated MCF-7 cells, Trx1 was present in both cytosol and nucleus, and the nuclear Trx1 pool was in the reduced state. Nuclear localization of Trx1 in 1,25D treated MCF-7 cells was confirmed by immunofluorescent microscopy. Although redox status is known to alter the ability of Trx1 to bind apoptosis signal regulating kinase 1 (ASK1), no changes in ASK1 transcript or protein levels were observed in 1,25D treated MCF-7 cells. Collectively, these studies indicate that although VDUP1 and ASK1 are not altered by 1,25D, changes in redox status and sub-cellular distribution of Trx1 occurs during 1,25D mediated apoptosis of MCF-7 cells.
Asunto(s)
Calcitriol/farmacología , Fracciones Subcelulares/metabolismo , Tiorredoxinas/metabolismo , Apoptosis , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Oxidación-Reducción/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Vitamin D(3) compounds offer an alternative approach to anti-hormonal therapies for human breast cancer. 1,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) acts through the nuclear Vitamin D(3) receptor (VDR), a phosphoprotein and ligand-dependent transcription factor. Our lab has shown that 1,25-(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species (ROS). TPA, a protein kinase C (PKC) activator, does not induce cytochrome c release or Bax translocation, thus demonstrating that it has no effect on mitochondria and apoptosis on its own. However, when the MCF-7(D(3)Res) cells (a Vitamin D(3)-resistant variant) are treated with 1,25-(OH)(2)D(3) in the presence of TPA, the cells displayed apoptotic morphology and redistribution of both cytochrome c and Bax. TPA pretreatment greatly enhances 1,25-(OH)(2)D(3) stimulated 24-hydroxylase luciferase activity and VDR protein expression, although transactivation is lower in the MCF-7(D(3)Res) cells compared to the parental cell line. The observation that the phorbol ester TPA sensitizes the Vitamin D(3)-resistant variant to the effects of 1,25-(OH)(2)D(3) suggests an important role for phosphorylation in dictating sensitivity to Vitamin D(3)-mediated apoptosis. This study demonstrates that the effects of 1,25-(OH)(2)D(3) on mitochondrial disruption might be sensitized through activators of PKC.