7 research outputs found
Molecular evolution of breast cancer cells leads to a chemoresistant phenotype and down-regulation of miR-200c.
<p>A) Molecular Evolution Assay. The epithelial breast cancer cell line BT474 was sequentially treated with chemotherapy. Cells were treated with 50 nM doxorubicin for 72 hours. Subsequently, medium was replaced by fresh medium until cells recovered and reached a confluency of 80%. Finally, cells were splitted for RNA isolation, cell lysis (protein), cytotoxicity assays and the next treatment round. R0 represents the untreated control cell line, whereas R1, R2, R3 and R4 represents BT474 cells that are treated for one, two, three and four times, respectively. B) Susceptibility to doxorubicin treatment. BT474 cells of R1 to R4 were treated with 0.1 and 10 µM doxorubicin for 72 hours. A CellTiter Glo assay was carried out to determine cell viability. Results are indicated as percentage of viable cells normalized to mock treated cells. C) Cell morphology of untreated and treated BT474 cells. Microscopic pictures (phase contrast) were taken from untreated BT474 cells (R0) and from doxorubicin treated and recovered cells of R4. D) Epithelial and mesenchymal marker expression in BT474 cells of R0, R2 and R4 of the Molecular Evolution Assay. E-Cadherin and Vimentin protein levels were determined by western blot analysis. Actin was used as loading control. E) miR-200c expression in BT474 cells that have undergone molecular evolution. Quantitative RT-PCR was performed to analyze miR-200c levels in BT474 cells of R1 to R4. miR-200c expression was thereby normalized to miR-191. Results are depicted as fold expression compared to the untreated control cell line (R0). Experiments were done in triplicates. For statistical analysis a student’s t-test was performed. (*p<0.05; **p<0.01; ***p<0.001).</p
The protein expression of TrkB and Bmi1 is regulated during the rounds of the Molecular Evolution Assay in BT474 cells.
<p>Total cell lysates of BT474 cells derived from R0, R2 and R4 of the Molecular Evolution Assay were subjected to western blot analysis to determine protein expression of A) TrkB isoforms, B) p-Akt, C) Bmi1 and D) p53. Actin was used as loading control.</p
TrkB and Bmi1 protein expression is hampered by overexpression of miR-200c in MDA-MB 436 cells.
<p>Total cell lysates of pre-miR-200c (pre) and scrambled control (scr) transfected MDA-MB 436 cells were subjected to western blot analysis and quantitative RT-PCR to determine expression of A) TrkB (gp145 and gp95) protein, B) TrkB mRNA C) p-Akt protein either after treatment with 0.5 µM doxorubicin for 24 hours (right panel) or untreated (left panel), D) Bmi1 protein, E) Bmi1 mRNA and F) p53 protein. α-Tubulin or Actin was used as loading control. Western blot quantification of three independent experiments was carried out by analyzing the relative intensities (rel. int.) of TrkB or Bmi1 normalized to the rel. int. of α-Tubulin or Actin using ImageJ software. For quantitative RT-PCR TrkB and Bmi1 expressions were normalized to GAPDH as reference and presented as ratio. A student’s t-test was performed to assess statistical significance. (ns = not significant; *p<0.05) DXR = doxorubicin.</p
Characterization of the epithelial and the mesenchymal breast cancer cell line BT474 and MDA-MB 436.
<p>A) miR-200c levels in a panel of mesenchymal (MDA-MB 436, MDA-MB 231, MDA-MB 157) and epithelial (MCF-7, MDA-MB 468, BT474) breast cancer cell lines. Quantitative RT-PCR was performed to assess the levels of miR-200c. Expression was normalized to miR-191 and presented as ratio. B) Epithelial and mesenchymal phenotype of BT474 and MDA-MB 436 cells. To confirm the different phenotypes, microscopic pictures were taken from both cell lines (phase contrast, left panel). Furthermore, immunofluorescence and laser scanning microscopy was carried out to determine E-Cadherin (red, middle panel) and Vimentin (red, right panel) expression. Nuclei were stained with DAPI (blue). C) Dose reponse curves and IC<sub>50</sub> values for doxorubicin. BT474 and MDA-MB 436 cells were treated with different concentrations of doxorubicin for 72 hours. Subsequently, a CellTiter Glo assay was performed to determine the percentage of viable cells (normalized to mock treated cells). IC<sub>50</sub> values are shown in the table. Experiments were done in triplicates. For statistical analysis a student’s t-test was performed. (***p<0.001).</p
Model of miR-200c regulated chemoresistance.
<p>Treatment of epithelial cells expressing miR-200c with doxorubicin leads to clonal evolution of miR-200c low expressing and mesenchymal-like cells. In consequence, a variety of miR-200c target genes are up-regulated. Besides the induction of EMT by the up-regulation of the E-Cadherin (CDH1) repressors Zeb1 and Zeb2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050469#pone.0050469-Tryndyak1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050469#pone.0050469-Li1" target="_blank">[49]</a>, this loss of miR-200c can cause an elevation of resistance factors like TrkB and Bmi1 resulting in enhanced cell survival. This leads to the activation of anti-apoptotic pathways like the phosphorylation of Akt or the degradation of p53, which can be further modulated by a complex crosstalk.</p
Overexpression of miR-200c in MDA-MB 436 cells increases susceptibility to doxorubicin.
<p>A) miR-200c levels after overexpression. After three consecutive transfections with either pre-miR-200c or scrambled control, MDA-MB 436 cells were harvested for RNA isolation and quantitative RT-PCR. miR-200c expression was normalized to miR-191 and presented as ratio. B) Cell morphology. Micrographs (phase contrast) of MDA-MB 436 cells were taken after three consecutive transfections with either pre-miR-200c or scrambled control. C) Susceptibility to doxorubicin treatment. With pre-miR-200c or scrambled control transfected MDA-MB 436 and BT474 cells were treated with 1, 5 and 10 µM doxorubicin for 72 hours. Cell viability was determined by a CellTiter Glo assay. Experiments were done in triplicates with at least two biological replicates. For statistical analysis a student’s t-test was performed. (ns = not significant; *p<0.05; **p<0.01; ***p<0.001).</p
Inhibition of miR-200c in BT474 cells causes chemoresistance to doxorubicin treatment.
<p>A) miR-200c expression after inhibiton. 24 hours after transfection with either miR-200c inhibitor or scrambled control, BT474 cells were harvested for RNA isolation and quantitative RT-PCR. miR-200c expression was normalized to miR-191 and presented as ratio. B) Cell morphology. Micrographs (phase contrast) of BT474 cells were taken 24 hours after transfection with either miR-200c inhibitor or scrambled control. C) Susceptibility to doxorubicin treatment. BT474 and MDA-MB 436 cells, transfected with inhibitor or scrambled control, were treated with 1, 10 and 20 µM doxorubicin for 72 hours. Cell viability was analyzed using CellTiter Glo. Experiments were done in triplicates with at least two biological replicates. For statistical analysis a student’s t-test was performed. (ns = not significant; *p<0.05; **p<0.01; ***p<0.001).</p