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p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

Yang JY, Ha SA, Yang YS, Kim JW - BMC Cancer (2010)

Bottom Line: The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms.YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype.Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, College of Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea.

ABSTRACT

Background: Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.

Methods: Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.

Results: YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.

Conclusion: Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

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Effect of different concentration of doxorubicin treatments on the survival of MCF-7 breast cancer cell lines. A and C: Viability of MCF-7 cells in the presence of a series of concentrations of doxorubicin (0.01-1000 nM) for two weeks, the percent viability was measured following the standard protocols (non-radioactive cell proliferation assay kit, Promega). B: Control MCF-7 cells; black circlet (●), doxorubicin-treated cells; white circlet (○). Time dependence effect response of doxorubicin (10 nM) on cellular growth rate of MCF-7 cells was assayed by comparing the each value with control. For the counting of colony number, cells were stained with 1% methylene blue for 20 m and washed with water. Each data point is the average of at least three independent experiments performed.
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Figure 1: Effect of different concentration of doxorubicin treatments on the survival of MCF-7 breast cancer cell lines. A and C: Viability of MCF-7 cells in the presence of a series of concentrations of doxorubicin (0.01-1000 nM) for two weeks, the percent viability was measured following the standard protocols (non-radioactive cell proliferation assay kit, Promega). B: Control MCF-7 cells; black circlet (●), doxorubicin-treated cells; white circlet (○). Time dependence effect response of doxorubicin (10 nM) on cellular growth rate of MCF-7 cells was assayed by comparing the each value with control. For the counting of colony number, cells were stained with 1% methylene blue for 20 m and washed with water. Each data point is the average of at least three independent experiments performed.

Mentions: Human breast MCF-7 carcinoma cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin (Gibco) in standard culture conditions (95% air-5% CO2, 37°C). The cells were inspected on a daily basis with inverted microscope and documented with digital camera (Leica, DM IRB/DC300). 100 μl MCF-7 cells (2 × 104 cells/ml) were distributed into each well of 96 well plates (Becton Dickinson, USA) and allowed to adhere 18 hours and further incubated for two weeks with increasing concentrations (0.01--1000 nM) of doxorubicin hydrochloride (Duchefa), to a final volume of 200 μl per well, then MTS reagent (The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit, Promega) was added into each well and incubated for 2 h before reading at a wavelength of 490 nm. A 50% growth inhibition (IC50) values for doxorubicin were calculated from dose-response curves obtained from the three independent experiments (Figure 1A and 1C). We generated doxorubicin resistant MCF-7 cell clones following the single-step selection procedure with the optimized low dose-effect of doxorubicin (10-20 nM). Then, the resistant clones were maintained in the medium with or without doxorubicin. The resistant phenotype of isolated clones, after the single-step selection, was re-examined 24 weeks later and confirmed to be stable. Time dependent effect of doxorubicin, on cellular growth rate of MCF-7 cells, was examined by incubating ~1000 cells with 10 nM doxorubicin for 15 days (with 3 day intervals) and the ratios of viable cell percent were calculated by comparing the control with the values of each days. After trypsinization, cells were collected by centrifugation, then resuspended in PBS buffer. The cell suspension was mixed with an equal volume of trypan blue solution (0.4%). Each sample was counted in triplicates with hemacytometer. Stained (dead) and unstained (viable) cells were counted with an inverted microscope.


p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

Yang JY, Ha SA, Yang YS, Kim JW - BMC Cancer (2010)

Effect of different concentration of doxorubicin treatments on the survival of MCF-7 breast cancer cell lines. A and C: Viability of MCF-7 cells in the presence of a series of concentrations of doxorubicin (0.01-1000 nM) for two weeks, the percent viability was measured following the standard protocols (non-radioactive cell proliferation assay kit, Promega). B: Control MCF-7 cells; black circlet (●), doxorubicin-treated cells; white circlet (○). Time dependence effect response of doxorubicin (10 nM) on cellular growth rate of MCF-7 cells was assayed by comparing the each value with control. For the counting of colony number, cells were stained with 1% methylene blue for 20 m and washed with water. Each data point is the average of at least three independent experiments performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913965&req=5

Figure 1: Effect of different concentration of doxorubicin treatments on the survival of MCF-7 breast cancer cell lines. A and C: Viability of MCF-7 cells in the presence of a series of concentrations of doxorubicin (0.01-1000 nM) for two weeks, the percent viability was measured following the standard protocols (non-radioactive cell proliferation assay kit, Promega). B: Control MCF-7 cells; black circlet (●), doxorubicin-treated cells; white circlet (○). Time dependence effect response of doxorubicin (10 nM) on cellular growth rate of MCF-7 cells was assayed by comparing the each value with control. For the counting of colony number, cells were stained with 1% methylene blue for 20 m and washed with water. Each data point is the average of at least three independent experiments performed.
Mentions: Human breast MCF-7 carcinoma cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin (Gibco) in standard culture conditions (95% air-5% CO2, 37°C). The cells were inspected on a daily basis with inverted microscope and documented with digital camera (Leica, DM IRB/DC300). 100 μl MCF-7 cells (2 × 104 cells/ml) were distributed into each well of 96 well plates (Becton Dickinson, USA) and allowed to adhere 18 hours and further incubated for two weeks with increasing concentrations (0.01--1000 nM) of doxorubicin hydrochloride (Duchefa), to a final volume of 200 μl per well, then MTS reagent (The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit, Promega) was added into each well and incubated for 2 h before reading at a wavelength of 490 nm. A 50% growth inhibition (IC50) values for doxorubicin were calculated from dose-response curves obtained from the three independent experiments (Figure 1A and 1C). We generated doxorubicin resistant MCF-7 cell clones following the single-step selection procedure with the optimized low dose-effect of doxorubicin (10-20 nM). Then, the resistant clones were maintained in the medium with or without doxorubicin. The resistant phenotype of isolated clones, after the single-step selection, was re-examined 24 weeks later and confirmed to be stable. Time dependent effect of doxorubicin, on cellular growth rate of MCF-7 cells, was examined by incubating ~1000 cells with 10 nM doxorubicin for 15 days (with 3 day intervals) and the ratios of viable cell percent were calculated by comparing the control with the values of each days. After trypsinization, cells were collected by centrifugation, then resuspended in PBS buffer. The cell suspension was mixed with an equal volume of trypan blue solution (0.4%). Each sample was counted in triplicates with hemacytometer. Stained (dead) and unstained (viable) cells were counted with an inverted microscope.

Bottom Line: The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms.YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype.Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, College of Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea.

ABSTRACT

Background: Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.

Methods: Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.

Results: YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.

Conclusion: Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

Show MeSH
Related in: MedlinePlus