Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 1. Characterization of a tamoxifen-resistant cell line (MCF-7TamR). MCF-7TamR (▪) and parental MCF-7 (□) cells were plated in six-well plates under normal conditions (FBS), hormone-deprived conditions (HD), or hormone-deprived conditions with 10−7 mol/L tamoxifen (Tam). Cells were counted in triplicate every 2 d after treatment using a hemocytometer. Results are representative of three independent experiments.

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques:

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 2. Gene expression analysis of tamoxifen-resistant (R) and parental (M) cells. Resistant (MCF-7TamR) and parental (MCF-7) cells were plated in six-well plates under normal conditions (FBS), hormone-deprived conditions (HD), or hormone-deprived conditions with 10−7

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Gene Expression

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 3. Western blot analysis of tamoxifen-resistant (R) and parental (M) cells. Resistant and parental cells were plated in six-well plates under normal conditions (FBS), hormone-deprived conditions (HD), or hormone-deprived conditions with 10−7 mol/L tamoxifen (Tam) for 24 or 48 h. Total cell lysates were analyzed by Western blot.

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Western Blot

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 4. MCF-7TamR cells displayed a greater percentage of cells in the S phase. MCF-7 (M) and MCF-7TamR (R) cells were plated in hormone-depleted medium and treated with (A) 10−7 mol/L β-estradiol (Estrogen), (B) mock-treated with PBS (Hormone deprived), or (C) 10−7 mol/L tamoxifen (Tamoxifen). Cells were pulse-labeled for 2 h with 10 μmol/L BrdUrd and harvested via fixation in 70% ethanol for 30 min at room temperature. DNA synthesis was determined by an immunofluorescence assay with an α-BrdUrd antibody conjugated to FITC. Total DNA was visualized using DAPI and the percentage of cells in the S phase was determined by dividing the number of BrdUrd-positive cells (FITC) by the number of DAPI-positive cells. Results are representative of three independent experiments.

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Labeling, DNA Synthesis, Immunofluorescence

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 5. The growth of tamoxifen-resistant cells is dependent on ERα. A, MCF-7TamR (square) and MCF7 (circle) cells were plated in six-well plates in triplicate. Cells were treated with (open) and without (solid) 10−7 mol/L ICI. Cell growth was monitored every 2 d by direct cell counting in triplicate. B, MCF-7TamR cells were treated with 10−8 mol/L ICI (I) or mock-treated (M) for 24, 48, or 72 h. Protein expression was analyzed by Western blot. C, MCF-7TamR cells were transfected with pCMV ERα and E2F1-Luc; the corresponding control vectors pCMV and pGL3 basic were used as controls. D, MCF-7TamR cells were transfected with E2F1-Luc or with pGL3 basic (control). Following transfection, cells were treated with or without 10−7 mol/L ICI. Results represent multiple experiments done in triplicate.

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Cell Counting, Expressing, Western Blot, Transfection, Control

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 6. ERα interacts with Sp-1 in tamoxifen-resistant cells. A, MCF-7TamR cells were plated in hormone-depleted medium and treated with 10−7 mol/L tamoxifen (Tam) or mock-treated with PBS (HD). Cells were harvested for coimmunoprecipitation at 24 h after treatment. Cell lysates immunoprecipitated with α-ERα antibodies or normal rabbit IgG and immunoprecipitated complexes were separated by SDS-PAGE, and protein levels were analyzed by Western blot with antibodies against ERα and Sp-1. The input represents 10% of the total protein submitted to immunoprecipitation. MCF-7 (M) and MCF-7TamR (R) cells were hormone deprived (HD) and treated with 10−7 mol/L tamoxifen (Tam). ChIP analysis was done with α-ERα, α-Sp-1, α-ACTR, and α-CARM1, and recruitment of proteins to the (B) E2F1 and (C) Cdk2 promoters was determined using promoter-specific primers and semiquantitative RT-PCR. The band intensities of the PCR products were quantitated using Quantity One (Bio-Rad).

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Cancer Research

Article Title: Estrogen Receptor Regulates E2F1 Expression to Mediate Tamoxifen Resistance

doi: 10.1158/1541-7786.mcr-09-0395

Figure Lengend Snippet: FIGURE 7. Tamoxifen-resistant breast cancer cell growth is dependent on E2F1. MCF-7TamR cells were transfected with Ri-E2F1 (Ri-E) or Ri-Control (Ri-C) and treated with (black) or without (white) 10−7 mol/L tamoxifen. A, cell growth was monitored 3 d after transfection, and statistical values represent data from four replicate samples. B, cells were also collected for Western blot analysis to monitor protein expression.

Article Snippet: Resistant cells were characterized by using a cell proliferation assay under three conditions: (a) normal or FBS condition, (b) hormone-deprived condition using a medium containing 10% charcoal-dextran stripped FBS (10% CDS-DMEM, Hyclone), and (c) tamoxifen (10−7 mol/L, Tocris)–treated condition.

Techniques: Transfection, Control, Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: lncRNA CYTOR promotes tamoxifen resistance in breast cancer cells via sponging miR-125a-5p

doi: 10.3892/ijmm.2019.4428

Figure Lengend Snippet: Characterization of tamoxifen resistant MCF7 cells. (A) The cell proliferation assay indicated that the growth rate of MCF7-P was similar to that of MCF7/TAM1 and MCF7/TAM2. (B) Different from MCF7-P, the growth of MCF7/TAM1 and MCF7/TAM2 did not rely on the addition of E2. (C) The mRNA levels of ER target genes (PgR and pS2) were decreased in MCF7/TAM1 and MCF7/TAM2 cells compared with parental cells. (D) MCF7/TAM1 and MCF7/TAM2 were relatively insensitive towards tamoxifen treatment compared with MCF7-P cells. (E) Western blotting showed that the protein expression of TAZ and p-ERK1/2 was increased in MCF7/TAM1 and MCF7/TAM2 compared with MCF7-P, whereas the expression of ER and ERK1/2 was not altered. (F) Quantification analysis of TAZ, p-ERK1/2, ER and ERK1/2 expression in E was presented. *** P<0.001. MCF7-P, parental MCF7 cells; p-ERK1/2, phosphorylated-extracellular signal regulated kinase 1/2; MCF7/TAM1, tamoxifen-resistant MCF7 subline1; MCF7/TAM2, tamoxifen-resistant MCF7 subline2; ER, estrogen receptor.

Article Snippet: MCF7 cells were exposed to tamoxifen (1 μ M; Selleck Chemicals) for over 12 months and two colonies were picked and cultured as MCF7/TAM1 and MCF7/TAM2 cells.

Techniques: Proliferation Assay, Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: lncRNA CYTOR promotes tamoxifen resistance in breast cancer cells via sponging miR-125a-5p

doi: 10.3892/ijmm.2019.4428

Figure Lengend Snippet: lncRNA CYTOR promoted tamoxifen resistance in breast cancer cells. (A) Screen of breast cancer-associated lncRNA (from LncRNADisease database) showed that several lncRNAs were differentially expressed between MCF7-P and MCF7/TAM1. (B) Screen of breast cancer-associated lncRNA (from LncRNADisease database) showed that several lncRNAs were differentially expressed between MCF7-P and MCF7/TAM2. (C) CYTOR siRNA decreased CYTOR expression in MCF7/TAM1 cells and MCF7/TAM2 cells. (D) Silencing of CYTOR re-sensitized MCF7/TAM1 cells to tamoxifen treatment. (E) Silencing of CYTOR re-sensitized MCF7/TAM2 cells to tamoxifen treatment. (F) Silencing of CYTOR showed no effect on the proliferation of MCF7/TAM1 without treatment of tamoxifen. (G) Silencing of CYTOR showed no effect on the proliferation of MCF7/TAM2 without treatment of tamoxifen. * P<0.05, *** P<0.001. CYTOR, cytoskeleton regulator RNA; lnc, long noncoding; si, small interfering; RT-q, reverse transcription; MCF7/TAM1, tamoxifen-resistant MCF7 subline1; MCF7/TAM2, tamoxifen-resistant MCF7 subline2.

Article Snippet: MCF7 cells were exposed to tamoxifen (1 μ M; Selleck Chemicals) for over 12 months and two colonies were picked and cultured as MCF7/TAM1 and MCF7/TAM2 cells.

Techniques: Expressing, Reverse Transcription

Journal: International Journal of Molecular Medicine

Article Title: lncRNA CYTOR promotes tamoxifen resistance in breast cancer cells via sponging miR-125a-5p

doi: 10.3892/ijmm.2019.4428

Figure Lengend Snippet: SRF is upregulated and acts as a target gene of miR-125a-5p in tamoxifen resistant MCF7 cells. (A) There was a putative binding site for miR-125a-5p on the 3′UTR of SRF. (B) Transfection of recombinant SRF increased SRF protein expression in MCF7 cells. (C) Quantification analysis of SRF protein expression in B was presented. (D) Overexpression of SRF decreased sensitivity of MCF7 cells towards tamoxifen treatment. (E) RT-qPCR showed that SRF was overexpressed in MCF7/TAM1 and MCF7/TAM2 compared with parental cells. (F) Western blotting showed that SRF protein levels were increased in MCF7/TAM1 and MCF7/TAM2. (G) Quantification analysis of SRF protein expression in F was presented. (H) RT-qPCR suggested that SRF mRNA levels were decreased towards miR-125a-5p overexpression in MCF7/TAM1 and MCF7/TAM2 cells. ** P<0.01 and *** P<0.001. SRF is upregulated and acts as a target gene of miR-125a-5p in tamoxifen resistant MCF7 cells. (I) Western blotting showed that SRF protein levels were decreased towards miR-125a-5p overexpression in MCF7/TAM1 and MCF7/TAM2 cells. (J) Quantification analysis of SRF protein expression in I was presented. (K) Dual luciferase reporter assay showed that SRF was a target gene of miR-125a-5p in MCF7/TAM1 cells. ** P<0.01. SRF, serum response factor; RT-q, reverse transcription-quantitative; UTR, untranslated region; MCF7/TAM1, tamoxifen-resistant MCF7 subline1; MCF7/TAM2, tamoxifen-resistant MCF7 subline2; NC, negative control; miR, microRNA; WTT, wild-type; Mut, mutant.

Article Snippet: MCF7 cells were exposed to tamoxifen (1 μ M; Selleck Chemicals) for over 12 months and two colonies were picked and cultured as MCF7/TAM1 and MCF7/TAM2 cells.

Techniques: Binding Assay, Transfection, Recombinant, Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Reverse Transcription, Negative Control, Mutagenesis

Journal: International Journal of Molecular Medicine

Article Title: lncRNA CYTOR promotes tamoxifen resistance in breast cancer cells via sponging miR-125a-5p

doi: 10.3892/ijmm.2019.4428

Figure Lengend Snippet: CYTOR regulates tamoxifen sensitivity via targeting SRF. (A) Transfection of recombinant SRF increased SRF protein expression in MCF7/TAM1 and MCF7/TAM2 cells. (B) Quantification analysis of SRF protein expression in A was exhibited. (C) In MCF7/TAM1 and MCF7/TAM2 cells, silencing of CYTOR decreased TAZ and p-ERK1/2 expression, which was reversed upon transfection of recombinant SRF. (D) Quantification of protein expression in C was exhibited. (E) Overexpression of SRF attenuated CYTOR siRNA induced elevation of tamoxifen sensitivity in MCF7/TAM1 cells. (F) Overexpression of SRF attenuated CYTOR siRNA induced elevation of tamoxifen sensitivity in MCF7/TAM2 cells. * P<0.05; ** P<0.01 and *** P<0.001. SRF, serum response factor; CYTOR, cytoskeleton regulator RNA; si, small interfering; MCF7/TAM1, tamoxifen-resistant MCF7 subline1; MCF7/TAM2, tamoxifen-resistant MCF7 subline2; p-ERK 1/2, phosphorylated-extracellular protein kinase 1/2.

Article Snippet: MCF7 cells were exposed to tamoxifen (1 μ M; Selleck Chemicals) for over 12 months and two colonies were picked and cultured as MCF7/TAM1 and MCF7/TAM2 cells.

Techniques: Transfection, Recombinant, Expressing, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: lncRNA CYTOR promotes tamoxifen resistance in breast cancer cells via sponging miR-125a-5p

doi: 10.3892/ijmm.2019.4428

Figure Lengend Snippet: CYTOR expression is increased in tamoxifen non-responder of patients with breast cancer. (A) RT-qPCR indicated that CYTOR expression was increased in tumor tissues compared with normal tissues from patients with breast cancer. (B) The CYTOR expression was increased in tumor tissues from tamoxifen resistant patients compared with those from tamoxifen untreated patients. (C) RT-qPCR showed that SRF levels were increased in tumor tissues compared with normal tissues from patients with breast cancer. (D) The Pearson correlation analysis showed a significant positive correlation between CYTOR expression and SRF mRNA levels. *** P<0.001. SRF, serum response factor; RT-q, reverse transcription-quantitative; CYTOR, cytoskeleton regulator RNA.

Article Snippet: MCF7 cells were exposed to tamoxifen (1 μ M; Selleck Chemicals) for over 12 months and two colonies were picked and cultured as MCF7/TAM1 and MCF7/TAM2 cells.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription