J Breast Cancer.  2006 Dec;9(4):293-300. 10.4048/jbc.2006.9.4.293.

Peroxisome proliferator-activated receptor gamma activator inhibits cell growth of MDA-MB-231 breast cancer cells through induction of apoptosis

• Jo EJ
• Youn HJ
• Jung SH

• KMID: 2286600
• DOI: http://doi.org/10.4048/jbc.2006.9.4.293

Abstract

PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) has become a potential target for the prevention and treatment of human cancers. PPARgamma ligands inhibit cell proliferation of estrogen receptoralpha(ERalpha)-positive breast cancer cells. However, it has recently been shown that ERalpha-negatively inhibits PPARgamma signaling in breast cancer cells, indicating that PPARgamma ligand may be more useful for treating ERalpha-negative breast cancer cells compared to ERalpha-positive breast cancer cells. In this study, we attempted to elucidate the role of PPARg in ERalpha-negative breast cancer cells.
METHODS
The effect of PPARgamma ligand on the growth of MDA-MB-231 cells was measured by MTT assay and flow cytometric analysis. TUNEL staining and Hoechst 33342 fluorescent staining were used to observe the effects of PPARgamma ligand on cell apoptosis. The regulatory proteins of the cell cycle were measured by Western blot.
RESULTS
The treatment of MDA-MB-231 human breast cancer cells with the PPARgamma ligand, trgoglitazone, was shown to induce inhibition of cell growth in a dose-dependent manner. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. The apoptotic effect by troglitazone demonstrated that apoptotic cells were elevated from 2.5-fold of the control level at 10 mM, to 3.1-fold at 50micrometer and to 3.5-fold at 75 mM of troglitazone. Moreover, troglitazone treatment dose-dependently caused a marked decrease in the pRb, cyclin D1, cyclin D2, cyclin D3, cdk2, Cdk4 and Cdk6 expressions and there was a significant increase in the p21 and p27 expressions.
CONCLUSION
These results indicate that trgoglitazone induces cell-cycle G1 arrest and apoptosis in ERalpha-negative MDA-MB-231 breast cancer cells. Collectively, this paper shows that PPARgamma ligand is an important player as a member of the chemotherapeutic candidates for treating ERalpha-negative breast cancer.

Keyword

ER-negative MDA-MB-231 breast cancer cells; PPARgamma; Apoptosis; Cell

MeSH Terms

Apoptosis*
Blotting, Western
Breast Neoplasms*
Breast*
Cell Cycle
Cell Proliferation
Cyclin D1
Cyclin D2
Cyclin D3
Estrogens
Humans
In Situ Nick-End Labeling
Ligands
Peroxisomes*
PPAR gamma*
Cyclin D1
Cyclin D2
Cyclin D3
Estrogens
Ligands
PPAR gamma

Figure

• 

  Fig 1 Troglitazone induces PPARγ up-regulation in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were treated with the indicated concentrations of troglitazone for 48h. Cells were lysed by lysis buffer and the amounts of PPARγ were measured by Western blot. β-actin was used as a loading control. The analysis of electrophoretic band was performed with the LAS-1000 (Fujifilm, Japan). Datas are the means of four separate experiments (bars, SE). p-values are determined using Student's t-test (*p<0.002 versus zero concentration).

• 

  Fig 2 MTT assay showing effect of three PPARg agonists, rosiglitazone, ciglitazone, and troglitazone on the growth of MDA-MB-231 breast cancer cells. MDA-MB-231 breast cancer cells were treated with treated with the indicated concentrations of PPARg agonists for 48h. Cell growth of MDA-MB-231 breast cancer cells was determined by MTT assay. Datas are the means of four separate experiments (bars, SE). p-values are determined using Student's t-test (* p<0.004 versus zero concentration).

• 

  Fig 3 Flow cytometry analysis demonstrating the effect of troglitazone on the cell cycle of MDA-MB-231 breast cancer cells. MDA-MB-231 breast cancer cells treated with troglitazone for 48h at various concentrations. The troglitazone induced the G1 phase proportion and decreased the S phase proportion of MDA-MB-231 breast cancer cells in a analyzed by flow cytometry.

• 

  Fig 4 Dose-dependent effect of troglitazone on cell cyclins, p21, p27, and pRb in MDA-MB-231 breast cancer cells. Cells were treated with the indicated concentrations of troglitazone for 48 h. Cell extracts were separated by SDS/PAGE, followed by Western blot. β-actin was used as a loading control. The analysis of electrophoretic band was performed with the LAS-1000 (Fujifilm, Japan).

• 

  Fig 5 Induction of apoptosis with troglitazone in MDA-MB-231 breast cancer cells. Detection of apoptosis in breast cancer cell was carried out using a DNA fragmentation assay based on TUNEL staining. MDA-MB-231 breast cancer cells were cultured on 6 well plates and treated with troglitazone for 48h at various concentrations. Positive nuclei stained brown, and negative nuclei stained blue. Error bars, SE; n=3 in each group (*p<0.005 versus zero concentration).

• 

  Fig 6 Fluorescence micrographs of MDA-MB-231 cells stained with Hoechst 33342 (C×400). MDA-MB-231 breast cancer cells were labeled with Hoechst 33342. The cells were examined by fluorescence microscopy. Untreated cells (a) kept a normal chromatin pattern and showed no change in a nuclear morphology. Cells treated with troglitazone, 10 µM (b), 50 µM (c), and 75 µM (d) demonstrated typical findings of apoptosis, marked by nuclear condensation and fragmentation.

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