Figure 13: Effects of PPARγ ligands on breast cancer cell morphology and cell viability... C MCF-7 1.5X105 cells/12-well dishes cells were treated with 15d-PGJ2, for 24h, before crystal
Trang 1Figure 13: Effects of PPARγ ligands on breast cancer cell morphology and cell viability
(A) MCF-7 (3 X105 cells/6-well dishes) and MDA-MB-231 (2 X 105 cells/6-well dishes) cells were treated with 15d-PGJ2, for 24h, and pictures were taken to show
Trang 2the morphology of the cells (B) MCF-7 (3 X10 cells/6-well dishes) cells were treated with ciglitazone for 24h before pictures were taken to show the morphology of the cells (C) MCF-7 (1.5X105 cells/12-well dishes) cells were treated with 15d-PGJ2, for 24h, before crystal violet assay was used to determine cell viability as described in Materials and Methods Results denote means +/-SD computed from two experiments done in duplicate *, p<0.05, **, p<0.01, treated versus untreated control
3A4.2 PPARγ ligands inhibit colony formation by breast cancer cells
Previous study showed that PPARγ ligands were capable of reducing colony forming ability of ovarian cancer cells (Vignati et al., 2006) Together with the data obtained from short-term viability assay, we were stimulated to question if PPARγ ligands could equally inhibit the cologenic ability of breast cancer cells
To this end, MCF-7 cells were exposed to 15d-PGJ2 or ciglitazone for 16h, before equal number of cells were replated onto 100mm dish and maintained for 14 days After crystal violet staining, we observed a dose-dependent reduction in the number of colonies formed in both cells treated with 15d-PGJ2 or ciglitazone (Figure 14A, B) As the colony forming assay is used to detect cells that preserved progeny-producing ability after drug treatment (Franken et al., 2006), the result
Trang 3reduction in the number as well as the size of the colonies in both breast cancer cell lines (Figure 14C) It is noteworthy that 15d-PGJ2 resulted in different extent
of inhibition in the colony-froming ability in all three cell lines Treatment with 15d-PGJ2 caused a 95% decrease in the number of colonies formed in
MDA-MB-231 cells, a 83% reduction in MCF-7 cells and only a 36% down-regulation in T47D cells (Figure 14D) Interesetingly, the extent of inhibition on cologenic ability of different breast cancer cells mirror-imaged the level of PPARγ receptor
in these cell lines The three breast cancer cell lines express different endogenous level of PPARγ receptor T47D cells that express lowest level of PPARγ showed the least sensitivity to 15d-PGJ2, while 15d-PGJ2 resulted in the maximal reduction in the number of colonies formed by MDA-MB-231 cells expressing the highest level of PPARγ receptor
Trang 4Figure 14: PPARγ agonists reduce colony-forming ability of breast cancer cells
Trang 53A4.3 Anti-tumor effect of 15d-PGJ 2 is PPARγ-dependent
The correlation between the PPARγ receptor level and the sensitivity to PPARγ ligands in different breast cancer cell lines strongly implies a regualtory link between PPARγ activity and its anti-tumor effects To further cofirm that the tumoricidal property of 15d-PGJ2 is a functional consequence of PPARγ activity,
we used the PPARγ inhibitor GW9662 As indicated previously, GW9662 functions as a PPARγ antagonist by modifying the cysteine of PPARγ ligand binding region and it was effective in blocking the PPRE luciferase reporter activity induced by 15d-PGJ2 (Figure 6A) Capitalizing on the antagonistic property of GW9662, we pre-incubated MCF-7 cells with GW9662 before exposure to 15d-PGJ2 As shown in the long-term conloy forming assay, GW9662 protected the MCF-7 cells from the significant reduction in the number of colonies formed after treatment with 15d-PGJ2 (Figure 15A) Based on this reversal in long-term colongenic ability by PPARγ antagonism and the correlative data from cells lines expressing varying intrinsic levels of PPARγ receptor, we concluded that 15d-PGJ2 induces a PPARγ-dependent anti-tumor effect in breast cancer cells