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Pit-1 inhibits BRCA1 and sensitizes human breast tumors to cisplatin and vitamin D treatment.

Seoane S, Arias E, Sigueiro R, Sendon-Lago J, Martinez-Ordoñez A, Castelao E, Eiró N, Garcia-Caballero T, Macia M, Lopez-Lopez R, Maestro M, Vizoso F, Mouriño A, Perez-Fernandez R - Oncotarget (2015)

Bottom Line: Administration of 1α, 25-dihydroxy-3-epi-vitamin D3 (3-Epi, an endogenous low calcemic vitamin D metabolite) reduced Pit-1 expression, and synergized with cisplatin, thus, decreasing cell proliferation and apoptosis in vitro, and reducing tumor growth in vivo.In addition, fifteen primary cultures of human breast tumors showed significantly decreased proliferation when treated with 3-Epi+cisplatin, compared to cisplatin alone.This response positively correlated with Pit-1 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Santiago de Compostela, Santiago de Compostela 15782, Spain.

ABSTRACT
The POU class 1 homeobox 1 (POU1F1, also known as Pit-1), pertaining to the Pit-Oct-Unc (POU) family of transcription factors, has been related to tumor growth and metastasis in breast. However, its role in response to breast cancer therapy is unknown. We found that Pit-1 down-regulated DNA-damage and repair genes, and specifically inhibited BRCA1 gene expression, sensitizing breast cancer cells to DNA-damage agents. Administration of 1α, 25-dihydroxy-3-epi-vitamin D3 (3-Epi, an endogenous low calcemic vitamin D metabolite) reduced Pit-1 expression, and synergized with cisplatin, thus, decreasing cell proliferation and apoptosis in vitro, and reducing tumor growth in vivo. In addition, fifteen primary cultures of human breast tumors showed significantly decreased proliferation when treated with 3-Epi+cisplatin, compared to cisplatin alone. This response positively correlated with Pit-1 levels. Our findings demonstrate that high levels of Pit-1 and reduced BRCA1 levels increase breast cancer cell susceptibility to 3-Epi+cisplatin therapy.

No MeSH data available.


Related in: MedlinePlus

3-Epi enhances cisplatin effect in Pit-1-sensitized breast cancer cellsA. Pit-1 overexpressing MCF-7 cells (MCF-7/Pit-1) treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin were analyzed for DNA-damage in a comet assay. DNA-damage induced by each treatment is indicated by average tail length. Data are represented as mean ± SD. A representative image is shown in B. Scale bar: 10 μm. B. Immunofluorescence of p-H2AX protein, as marker of DNA-damage, after treatment with ethanol, 3-Epi, cisplatin, and 3-Epi+cisplatin in MCF-7/Pit-1 cells. Scale bar: 10 μm. Cisplatin and 3-Epi+cisplatin modify DNA-damage phosphorylated proteins. C. Immunoblot analysis of extracts in the conditions described above was done for the DNA-damage phosphorylated (p) proteins, p-ATMSer1981, p-ATRSer428, p-Chk1Ser296, p-Chk2Thr68, p-BRCA1Ser988, p-p53Ser15, p-RbSer139, and p-H2AXSer139. GAPDH was used as loading control. Values correspond to a densitometric analysis. D. Immunoblot analysis of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1Ser296, p-H2AXSer139, and GAPDH (used as loading control) in MCF-7 and MCF-7/Pit-1 cells treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin. Values correspond to a densitometric analysis.
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Figure 6: 3-Epi enhances cisplatin effect in Pit-1-sensitized breast cancer cellsA. Pit-1 overexpressing MCF-7 cells (MCF-7/Pit-1) treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin were analyzed for DNA-damage in a comet assay. DNA-damage induced by each treatment is indicated by average tail length. Data are represented as mean ± SD. A representative image is shown in B. Scale bar: 10 μm. B. Immunofluorescence of p-H2AX protein, as marker of DNA-damage, after treatment with ethanol, 3-Epi, cisplatin, and 3-Epi+cisplatin in MCF-7/Pit-1 cells. Scale bar: 10 μm. Cisplatin and 3-Epi+cisplatin modify DNA-damage phosphorylated proteins. C. Immunoblot analysis of extracts in the conditions described above was done for the DNA-damage phosphorylated (p) proteins, p-ATMSer1981, p-ATRSer428, p-Chk1Ser296, p-Chk2Thr68, p-BRCA1Ser988, p-p53Ser15, p-RbSer139, and p-H2AXSer139. GAPDH was used as loading control. Values correspond to a densitometric analysis. D. Immunoblot analysis of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1Ser296, p-H2AXSer139, and GAPDH (used as loading control) in MCF-7 and MCF-7/Pit-1 cells treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin. Values correspond to a densitometric analysis.

Mentions: To assess if 3-Epi+cisplatin produced more DNA damage than cisplatin alone, DNA fragmentation and DNA damage-induced proteins were evaluated. By comet assay we showed that 3-Epi+cisplatin treatment in MCF-7/Pit-1 cells induced a significantly (P < 0.05) longer trailing tail, indicating increased DNA fragmentation (Figure 6A). Immunofluorescence analysis of p-H2AX showed increased DNA damage after 3-Epi+cisplatin as compared to cisplatin alone (Figure 6B). Proteins involved in DNA damage and repair were also evaluated by Western blot in MCF-7/Pit-1 cells. Figure 6C shows high basal phosphorylation levels for ATM, BRCA1, and p53, as well as increased ATM, ATR, Chk1, Chk2, BRCA1, p53, Rb, and H2AX phosphorylation in cisplatin-treated cells. Treatment with 3-Epi+cisplatin increased p-Chk1 and p-H2AX proteins, with respect to cisplatin alone. To further compare the effect of treatments on MCF-7/Pit-1 (overexpressing Pit-1) and wild MCF-7 cells, a Western blot of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1, and p-H2AX proteins was also carried out. Figure 6D shows that high levels of Pit-1 potentiate the effect of 3-Epi+cisplatin treatment.


Pit-1 inhibits BRCA1 and sensitizes human breast tumors to cisplatin and vitamin D treatment.

Seoane S, Arias E, Sigueiro R, Sendon-Lago J, Martinez-Ordoñez A, Castelao E, Eiró N, Garcia-Caballero T, Macia M, Lopez-Lopez R, Maestro M, Vizoso F, Mouriño A, Perez-Fernandez R - Oncotarget (2015)

3-Epi enhances cisplatin effect in Pit-1-sensitized breast cancer cellsA. Pit-1 overexpressing MCF-7 cells (MCF-7/Pit-1) treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin were analyzed for DNA-damage in a comet assay. DNA-damage induced by each treatment is indicated by average tail length. Data are represented as mean ± SD. A representative image is shown in B. Scale bar: 10 μm. B. Immunofluorescence of p-H2AX protein, as marker of DNA-damage, after treatment with ethanol, 3-Epi, cisplatin, and 3-Epi+cisplatin in MCF-7/Pit-1 cells. Scale bar: 10 μm. Cisplatin and 3-Epi+cisplatin modify DNA-damage phosphorylated proteins. C. Immunoblot analysis of extracts in the conditions described above was done for the DNA-damage phosphorylated (p) proteins, p-ATMSer1981, p-ATRSer428, p-Chk1Ser296, p-Chk2Thr68, p-BRCA1Ser988, p-p53Ser15, p-RbSer139, and p-H2AXSer139. GAPDH was used as loading control. Values correspond to a densitometric analysis. D. Immunoblot analysis of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1Ser296, p-H2AXSer139, and GAPDH (used as loading control) in MCF-7 and MCF-7/Pit-1 cells treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin. Values correspond to a densitometric analysis.
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: 3-Epi enhances cisplatin effect in Pit-1-sensitized breast cancer cellsA. Pit-1 overexpressing MCF-7 cells (MCF-7/Pit-1) treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin were analyzed for DNA-damage in a comet assay. DNA-damage induced by each treatment is indicated by average tail length. Data are represented as mean ± SD. A representative image is shown in B. Scale bar: 10 μm. B. Immunofluorescence of p-H2AX protein, as marker of DNA-damage, after treatment with ethanol, 3-Epi, cisplatin, and 3-Epi+cisplatin in MCF-7/Pit-1 cells. Scale bar: 10 μm. Cisplatin and 3-Epi+cisplatin modify DNA-damage phosphorylated proteins. C. Immunoblot analysis of extracts in the conditions described above was done for the DNA-damage phosphorylated (p) proteins, p-ATMSer1981, p-ATRSer428, p-Chk1Ser296, p-Chk2Thr68, p-BRCA1Ser988, p-p53Ser15, p-RbSer139, and p-H2AXSer139. GAPDH was used as loading control. Values correspond to a densitometric analysis. D. Immunoblot analysis of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1Ser296, p-H2AXSer139, and GAPDH (used as loading control) in MCF-7 and MCF-7/Pit-1 cells treated with ethanol, 100 nM of 3-Epi, 5 μM of cisplatin, and 100 nM of 3-Epi + 5 μM of cisplatin. Values correspond to a densitometric analysis.
Mentions: To assess if 3-Epi+cisplatin produced more DNA damage than cisplatin alone, DNA fragmentation and DNA damage-induced proteins were evaluated. By comet assay we showed that 3-Epi+cisplatin treatment in MCF-7/Pit-1 cells induced a significantly (P < 0.05) longer trailing tail, indicating increased DNA fragmentation (Figure 6A). Immunofluorescence analysis of p-H2AX showed increased DNA damage after 3-Epi+cisplatin as compared to cisplatin alone (Figure 6B). Proteins involved in DNA damage and repair were also evaluated by Western blot in MCF-7/Pit-1 cells. Figure 6C shows high basal phosphorylation levels for ATM, BRCA1, and p53, as well as increased ATM, ATR, Chk1, Chk2, BRCA1, p53, Rb, and H2AX phosphorylation in cisplatin-treated cells. Treatment with 3-Epi+cisplatin increased p-Chk1 and p-H2AX proteins, with respect to cisplatin alone. To further compare the effect of treatments on MCF-7/Pit-1 (overexpressing Pit-1) and wild MCF-7 cells, a Western blot of Bcl-2, cleaved PARP, caspase 3 active, p-Chk1, and p-H2AX proteins was also carried out. Figure 6D shows that high levels of Pit-1 potentiate the effect of 3-Epi+cisplatin treatment.

Bottom Line: Administration of 1α, 25-dihydroxy-3-epi-vitamin D3 (3-Epi, an endogenous low calcemic vitamin D metabolite) reduced Pit-1 expression, and synergized with cisplatin, thus, decreasing cell proliferation and apoptosis in vitro, and reducing tumor growth in vivo.In addition, fifteen primary cultures of human breast tumors showed significantly decreased proliferation when treated with 3-Epi+cisplatin, compared to cisplatin alone.This response positively correlated with Pit-1 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Santiago de Compostela, Santiago de Compostela 15782, Spain.

ABSTRACT
The POU class 1 homeobox 1 (POU1F1, also known as Pit-1), pertaining to the Pit-Oct-Unc (POU) family of transcription factors, has been related to tumor growth and metastasis in breast. However, its role in response to breast cancer therapy is unknown. We found that Pit-1 down-regulated DNA-damage and repair genes, and specifically inhibited BRCA1 gene expression, sensitizing breast cancer cells to DNA-damage agents. Administration of 1α, 25-dihydroxy-3-epi-vitamin D3 (3-Epi, an endogenous low calcemic vitamin D metabolite) reduced Pit-1 expression, and synergized with cisplatin, thus, decreasing cell proliferation and apoptosis in vitro, and reducing tumor growth in vivo. In addition, fifteen primary cultures of human breast tumors showed significantly decreased proliferation when treated with 3-Epi+cisplatin, compared to cisplatin alone. This response positively correlated with Pit-1 levels. Our findings demonstrate that high levels of Pit-1 and reduced BRCA1 levels increase breast cancer cell susceptibility to 3-Epi+cisplatin therapy.

No MeSH data available.


Related in: MedlinePlus