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Effects of adiponectin on breast cancer cell growth and signaling.

Grossmann ME, Nkhata KJ, Mizuno NK, Ray A, Cleary MP - Br. J. Cancer (2008)

Bottom Line: In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth.In vitro, MDA-ERalpha7 cells were growth inhibited by globular Acrp30 while the parental cells were not.This inhibition appeared to be due to blockage of JNK2 signalling.

View Article: PubMed Central - PubMed

Affiliation: Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.

ABSTRACT
Obesity is a risk factor for postmenopausal breast cancer. Adiponectin/Acrp30 is lower in obese individuals and may be negatively regulating breast cancer growth. Here we determined that five breast cancer cell lines, MDA-MB-231, MDA-MB-361, MCF-7, T47D, and SK-BR-3, expressed one or both of the Acrp30 receptors. In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth. Oestrogen receptor (ER) positive MCF-7 and T47D cells were inhibited at lower Acrp30 concentrations than ER-negative SK-BR-3 cells. Growth inhibition may be related to apoptosis since PARP cleavage was increased by Acrp30 in the ER-positive cell lines. To investigate the role of ER in the response of breast cancer cells to Acrp30, we established the MDA-ERalpha7 cell line by insertion of ER-alpha into ER-alpha-negative MDA-MB-231 cells. This line readily formed tumours in athymic mice and was responsive to oestradiol in vivo. In vitro, MDA-ERalpha7 cells were growth inhibited by globular Acrp30 while the parental cells were not. This inhibition appeared to be due to blockage of JNK2 signalling. These results provide information on how obesity may influence breast cancer cell proliferation and establish a new model to examine interactions between ER and Acrp30.

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Growth curves for breast cancer cell lines in response to increasing concentrations of Acrp30. (A) MCF-7, (B) T47-D, (C) MDA-MB-361, (D) SK-BR-3 and (E) MDA-MB-231 cells. The concentration of Acrp30 in ng ml−1 is shown below each graph. Each point represents three or more wells. *Indicates significantly different from 0 ng ml−1 as defined by ANOVA p=0.0002, Dunnett's multiple comparison post-test P<0.01.
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fig1: Growth curves for breast cancer cell lines in response to increasing concentrations of Acrp30. (A) MCF-7, (B) T47-D, (C) MDA-MB-361, (D) SK-BR-3 and (E) MDA-MB-231 cells. The concentration of Acrp30 in ng ml−1 is shown below each graph. Each point represents three or more wells. *Indicates significantly different from 0 ng ml−1 as defined by ANOVA p=0.0002, Dunnett's multiple comparison post-test P<0.01.

Mentions: For the assays shown in Figure 1 on the MCF-7, T47D, MDA-MB-361, SK-BR-3, and MDA-MB-231, cells were harvested and counted using a coulter counter, plated at a density of 5 × 103 cells per well in 96-well plates, and allowed to attach overnight in an incubator. The following day, complete medium was removed from the wells, replaced with serum-free medium, and the cells returned to the incubator for 18–24 h (to allow for cell cycle synchronisation). Treatment with Acrp30 was then performed and the cells incubated for 48 h, at which time a cell proliferation assay was performed. For growth assays shown in Figure 4 comparing the MDA-wt and MDA-ERα7 cells 5 × 103 cells per well were placed in 96-well plates. The following morning the media was replaced with serum-free L-15 with or without other factors as described in the figure legend. For the experiments in Figures 2 and 5, cells were plated at 5 × 105 in 6-well plates. The following day the media was replaced with various amounts of Acrp30 (RND Systems, Minneapolis, MN, USA) or gAcrp30 (Cell Sciences, Canton, MA, USA) or as described for each experiment. After 24 h of treatment the cells were either harvested or treated for various times with FCS. The growth assay was performed using 10 μl of CCK-8 reagent from the Cell Counting Kit-8 as per manufacturer's instructions (Dojindo Laboratories, Japan). In this assay a formazan dye is generated by the activity of dehydrogenases in cells that is directly proportional to the number of living cells. The plates were then incubated for 1.5 to 3 h depending on the cell line in a CO2 incubator, after which the plates were read on an ELISA reader at 450 nm. A standard curve was included on each plate to allow for estimation of approximate cell numbers in the treated wells, as was appropriate with negative and/or positive controls.


Effects of adiponectin on breast cancer cell growth and signaling.

Grossmann ME, Nkhata KJ, Mizuno NK, Ray A, Cleary MP - Br. J. Cancer (2008)

Growth curves for breast cancer cell lines in response to increasing concentrations of Acrp30. (A) MCF-7, (B) T47-D, (C) MDA-MB-361, (D) SK-BR-3 and (E) MDA-MB-231 cells. The concentration of Acrp30 in ng ml−1 is shown below each graph. Each point represents three or more wells. *Indicates significantly different from 0 ng ml−1 as defined by ANOVA p=0.0002, Dunnett's multiple comparison post-test P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361440&req=5

fig1: Growth curves for breast cancer cell lines in response to increasing concentrations of Acrp30. (A) MCF-7, (B) T47-D, (C) MDA-MB-361, (D) SK-BR-3 and (E) MDA-MB-231 cells. The concentration of Acrp30 in ng ml−1 is shown below each graph. Each point represents three or more wells. *Indicates significantly different from 0 ng ml−1 as defined by ANOVA p=0.0002, Dunnett's multiple comparison post-test P<0.01.
Mentions: For the assays shown in Figure 1 on the MCF-7, T47D, MDA-MB-361, SK-BR-3, and MDA-MB-231, cells were harvested and counted using a coulter counter, plated at a density of 5 × 103 cells per well in 96-well plates, and allowed to attach overnight in an incubator. The following day, complete medium was removed from the wells, replaced with serum-free medium, and the cells returned to the incubator for 18–24 h (to allow for cell cycle synchronisation). Treatment with Acrp30 was then performed and the cells incubated for 48 h, at which time a cell proliferation assay was performed. For growth assays shown in Figure 4 comparing the MDA-wt and MDA-ERα7 cells 5 × 103 cells per well were placed in 96-well plates. The following morning the media was replaced with serum-free L-15 with or without other factors as described in the figure legend. For the experiments in Figures 2 and 5, cells were plated at 5 × 105 in 6-well plates. The following day the media was replaced with various amounts of Acrp30 (RND Systems, Minneapolis, MN, USA) or gAcrp30 (Cell Sciences, Canton, MA, USA) or as described for each experiment. After 24 h of treatment the cells were either harvested or treated for various times with FCS. The growth assay was performed using 10 μl of CCK-8 reagent from the Cell Counting Kit-8 as per manufacturer's instructions (Dojindo Laboratories, Japan). In this assay a formazan dye is generated by the activity of dehydrogenases in cells that is directly proportional to the number of living cells. The plates were then incubated for 1.5 to 3 h depending on the cell line in a CO2 incubator, after which the plates were read on an ELISA reader at 450 nm. A standard curve was included on each plate to allow for estimation of approximate cell numbers in the treated wells, as was appropriate with negative and/or positive controls.

Bottom Line: In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth.In vitro, MDA-ERalpha7 cells were growth inhibited by globular Acrp30 while the parental cells were not.This inhibition appeared to be due to blockage of JNK2 signalling.

View Article: PubMed Central - PubMed

Affiliation: Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.

ABSTRACT
Obesity is a risk factor for postmenopausal breast cancer. Adiponectin/Acrp30 is lower in obese individuals and may be negatively regulating breast cancer growth. Here we determined that five breast cancer cell lines, MDA-MB-231, MDA-MB-361, MCF-7, T47D, and SK-BR-3, expressed one or both of the Acrp30 receptors. In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth. Oestrogen receptor (ER) positive MCF-7 and T47D cells were inhibited at lower Acrp30 concentrations than ER-negative SK-BR-3 cells. Growth inhibition may be related to apoptosis since PARP cleavage was increased by Acrp30 in the ER-positive cell lines. To investigate the role of ER in the response of breast cancer cells to Acrp30, we established the MDA-ERalpha7 cell line by insertion of ER-alpha into ER-alpha-negative MDA-MB-231 cells. This line readily formed tumours in athymic mice and was responsive to oestradiol in vivo. In vitro, MDA-ERalpha7 cells were growth inhibited by globular Acrp30 while the parental cells were not. This inhibition appeared to be due to blockage of JNK2 signalling. These results provide information on how obesity may influence breast cancer cell proliferation and establish a new model to examine interactions between ER and Acrp30.

Show MeSH
Related in: MedlinePlus