Figure 1.
Chemical structure and cytotoxicity assay.
(A) Structure of PTER and PTER-ITC conjugate. (B) Cytotoxicity induced by increasing doses of PTER-ITC and (C) PTER in breast cancer cells as determined by MTT assay. (D) Effect of GW9662 on survival of MCF-7 and (E) MDA-MB-231 cells alone and in the presence of PTER-ITC and PTER. Data are shown as mean ± SEM of three independent experiments. * and # indicate statistically significant differences with respect to controls (vehicle-treated) and only PTER-ITC treated groups, respectively. p<0.05.
Figure 2.
PTER-ITC upregulates PPARγ and PTEN expression levels.
(A) PPARγ expression in three breast cancer cell lines as determined by RT-PCR (left) and immunoblot analysis (right). (B) Effect of PTER-ITC, PTER and rosiglitazone on PPARγ and PTEN mRNA expression as determined by RT-PCR in MCF-7 and (C) MDA-MB-231 cells. (D) Effect of PTER-ITC, PTER and rosiglitazone on PPARγ and PTEN protein expression as determined by immunoblot analysis in MCF-7 and (E) MDA-MB-231 cells. Histogram (right panel in each figure) shows relative band intensities normalized to the corresponding β-actin level. Data are expressed as x-fold increase relative to control; values shown as mean ± SEM of three independent experiments. * and # indicate statistically significant differences with respect to controls for PPARγ and PTEN proteins, respectively. p<0.05; ROSI, rosiglitazone.
Figure 3.
Induction of PPARγ expression in response to different treatments.
(A) Immunofluorescence analysis to detect PPARγ protein in MCF-7 and (B) MDA-MB-231 breast cancer cells after treatment with GW9662, PTER-ITC, PTER and rosiglitazone. Figures show one representative experiment of three performed. Magnification, 200×.
Figure 4.
PTER-ITC alters PPAR activity and induces differentiation of MCF-7 cells.
(A) Effect of PTER-ITC and PTER on the activity of various PPAR in MCF-7 cells, as determined by transactivation assay. Data are expressed as a percentage of PPAR activity relative to the respective control. Values shown as mean ± SEM of three independent experiments. * indicates significant difference relative to vehicle-treated control; p<0.05. (B) Effect of PPARβ inhibitor (GSK0660) and (C) PPARγ inhibitor (GW9662) and activator (rosiglitazone) on PTER-ITC-induced transactivation of PPAR. Cells were transfected with pcMX-PPARβ/γ plasmids, together with PPRE-tk-luc and Renilla plasmids (18 h). Cells were then pre-treated with GSK0660/GW9662 (4 h), followed by PTER-ITC/rosiglitazone treatment (24 h). Data are expressed as percentages of PPARβ/γ activity relative to the vehicle-treated control ( = 100). Values are shown as mean ± SEM of three independent experiments. *, # and ## indicate statistically significant difference compared to respective controls, only PTER-ITC (either 10 or 20 µM) and rosiglitazone-treated groups, respectively; p<0.05. ns, not significant. (D) Oil Red O staining showing lipid accumulation in MCF-7 cells treated with different doses of PTER-ITC and rosiglitazone (10 µM), observed by light microscopy (200x). Histogram (right) shows spectrophotometric estimation of intracellular neutral lipids. Values shown as mean ± SEM of two independent experiments. * indicates significant difference relative to vehicle-treated controls; p<0.05.
Figure 5.
Analysis of PTER-ITC docking pattern with PPARγ.
(A) Mode of binding of resveratrol, PTER and PTER-ITC to PPARγ. Note the distinct orientations of the ligands. The broad range of ligand binding ability of PPARγ can be explained in part by the large T-shaped ligand binding area, which permits ligands to adopt distinct orientations (figures generated with PyMOL molecular graphics system). (B) Interaction of PTER-ITC within the ligand-binding pocket. Residues H323 and Y327 of protein chain A are involved in hydrogen bond formation with N3 of the ligand. Yellow dashed lines indicate bonding; interacting residues are labeled. (C) Ligand interaction plot showing different hydrophobic and two hydrogen bond interactions of PTER-ITC with PPARγ. Hydrogen bonds are indicated by green dashed lines, with their respective distances.
Table 1.
Hydrogen bonds and hydrophobic interactions between ligand and PPAR-γ ligand binding domain (LBD).
Figure 6.
PTER-ITC induces PPARγ-dependent apoptosis in breast cancer cells.
(A) Representative FACS analysis of cells using annexin V as marker. Histogram (right) shows the apoptosis rate induced by PTER-ITC alone and in the presence of GW9662. Values are mean ± SEM from three independent experiments. * and # indicate statistically significant differences compared to vehicle-treated control and only PTER-ITC treated groups, respectively; p<0.05. (B) Apoptosis induced by PTER-ITC alone and in the presence of GW9662, visualized by fluorescence microscopy using DNA-binding fluorochrome DAPI in MCF-7 and MDA-MB-231 breast cancer cells. Figures show a representative experiment of three performed. Magnification, 200×. Arrows indicate the formation of apoptotic bodies.
Figure 7.
PTER-ITC induces caspase-dependent apoptosis in breast cancer cells.
(A) Effects of 10 and 20 µM PTER-ITC on caspase-8, -9 and -3/7 activities in MCF-7 and MDA-MB-231 cells. Results are the mean ± SEM of three independent experiments. * indicates statistically significant difference relative to respective controls; p<0.05. (B) Effect of caspase inhibitors on PTER-ITC-induced apoptosis in MDA-MB-231 cells. Data shown as mean ± SEM of three independent experiments. * and # indicate statistically significant difference with respect to control and only PTER-ITC-treated cells, respectively; p<0.05.
Figure 8.
PTER-ITC alters PPARγ activity through p38 MAPK and JNK pathways.
(A) PTER-ITC induces PPARγ expression through p38 MAPK and JNK pathways in MCF-7 and MDA-MB-231 cells. The experiment was performed in duplicate and yielded similar results. Histogram (bottom) shows relative band intensities normalized to the corresponding β-actin level, where the vehicle-treated group = 1. (B) Effects of p38 MAPK and JNK inhibitors on PTER-ITC-induced apoptosis in MCF-7 and MDA-MB-231 cells. Data are shown as mean ± SEM of three independent experiments. * and # indicate statistically significant difference with respect to vehicle-treated control and only PTER-ITC treated cells, respectively; p<0.05.
Figure 9.
PTER-ITC induces apoptosis by targeting PPARγ-related proteins.
Immunoblot analysis for apoptotic markers and PPARγ-regulated genes in response to PTER-ITC and GW9662 treatment in (A) MCF-7 and (B) MDA-MB-231 cells. Cells were pre-treated with 10 µM GW9662 (1 h) before treatment with 10 and 20 µM PTER-ITC (24 h). Whole-cell extracts were resolved by SDS-PAGE and probed with indicated antibodies. Expression levels of samples were normalized to the corresponding β-actin levels. Histogram (right panels in each figure) show data expressed as x-fold change relative to control; bars show mean ± SEM of three independent experiments. a, b and c indicate significant levels of differences with respect to control, 10 and 20 µM only PTER-ITC-treated groups, respectively, for each protein. p<0.05. (C) Effect of PPARγ siRNA on PTER-ITC-induced apoptosis of MCF-7 and (D) MDA-MB-231 cells. Both cells were transfected with PPARγ siRNA (final concentration 100 nM). After 24 h, cells were treated with 20 µM PTER-ITC and incubated (24 h). Levels of PPARγ-related proteins were detected in cell lysates by immunoblot analysis. Histogram (right panel in each figure) shows relative band intensities normalized to the corresponding β-actin level. Data are expressed as x-fold change relative to control; bars show mean ± SEM of three independent experiments. a and b indicate significant differences with respect to vehicle-treated control and only 20 µM PTER-ITC-treated groups, respectively; p<0.05.
Figure 10.
Possible mode of action of PTER-ITC-induced apoptosis and cell growth inhibition in MCF-7 cells.
PTER-ITC activates p38 MAPK and JNK, which in turn up regulate PPARγ expression and receptor activity. PPARγ decreases survivin expression and up regulates PTEN expression, both of which increase caspase-9 activity, leading to increased caspase-3/7 activity, which finally results in cell death.