THE MECHANISM OF PPARN3 MEDIATED DOWN REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EPXRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR n1 4

To test the interference of regular serum on PPARγ activation by PPARγ ligands and to select an appropriate serum condition for stronger response of breast cancer cells to different drug

washed and reseeded at a density of 1.0 X 10 cells/100mm dish After 14 days of incubation, cells were stained with crystal violet Graphical representation of colony counts from colony forming assays Results denote means +/-SD computed from three experiments *, p<0.05, treated versus untreated control

3A4.4 Anti-tumor effect of 15d-PGJ2 is partially ROS/RNS-dependent

Evidence from literature has shown induction of ROS/RNS by various PPARγ ligands in different cancer cells, which in turn is linked to mitochondrial damage and subsequent cell death (Ichihara and Pelliniemi, 2007; Ito et al., 2007; Julie et al., 2008; Wang and Mak, 2011) In previous sections, we also showed that PPARγ ligands produced abundant ROS/RNS in breast cancr cells (Figure 9B), and pre-incubation with FeTPPS completely scavenged the ROS/RNS detected by DCFDA staining (Figure 10A)

As a next step in identifying the specific involvement of PPARγ ligand-induced ROS in cell survival, FeTPPS was again used to decompose all ROS/RNS generated by 15d-PGJ2 MCF-7 cells were incubated with FeTPPS for 2h prior to exposure to 15d-PGJ2 for 24h Cell viability was then assessed using crystal violet assay As shown in figure 16A, the presence of FeTPPS although not completely, but significantly rescued the cell viability loss initially induced by 5µM and 10µM of 15d-PGJ2 (Figure 16A) Given the fact that the partial rescuing effect was only observed at higher concentration of 15d-PGJ2, which induced a more potent production of ROS/RNS (Figure 9C), we infer that 15d-PGJ2-mediated cell viability loss is fractionally ROS/RNS dependent, especially at higher concentration of the drug Next we asked if FeTPPS could similarly rescue the

colonogenic ability of cells from 15d-PGJ2-induced inhibition MCF-7 cells were

pre-incubated with FeTPPS for 2h, before they were challenged with 15d-PGJ2

for 16h and then reseeded The colony forming assay showed that the presence of

FeTPPS rescued approximately 20% out of 80% loss in colony-forming ability of

15d-PGJ2-treated MCF-7 cells (Figure 16B) Taken together, these results suggest

the involvement of peroxynitrite in 15d-PGJ2-induced inhibition on cell survival

Figure 16: FeTPPS rescues the loss of cell viability and cologenic ability induced by PPARγ ligand in breast cancer cells

(A) MCF-7 (1.5X105 cells/12-well dishes) cells were exposed to 15d-PGJ2 for 24h with or without 2h pre-incubation of 50µM FeTPPS Crystal violet assay was then used to determine cell viability as described in Materials and Methods

Results denote means +/-SD computed from two experiments done in duplicate (B) MCF-7 (3 X105 cells/6-well dishes) cells were exposed to 15d-PGJ2 for 16h with or without 2h pre-incubation of 50µM FeTPPS Following that, cells were washed and reseeded at a density of 1.0 X 104 cells/100mm dish After 14 days of incubation, cells were stained with crystal violet Graphical representation of colony counts from colony forming assays Results denote means +/-SD computed from three experiments *, p<0.05, **, p<0.01, treated versus untreated control

3A4.5 Reduced NHE1 expression is responsible for PPARγ-mediated tumor effects

anti-We have thus far demonstrated that PPARγ activation leads to down-regulation of NHE1 expression, and PPARγ ligands significantly inhibited the short-term cell viability and long-term colony forming ability in various breast cancer cells Previous study from our group also showed that the expression of NHE1 was critical for tumour growth (Akram et al., 2006) Intrigued by the parallel repression of NHE1 expression and tumor growth by PPARγ activation, we asked

if the anti-tumor effect of PPARγ is dependent on NHE1 down-regulation

The first study that demonstrated the importance of NHE1 in cell proliferation was by Pouyssegur and colleagues They reported that fibroblasts lacking NHE1 activity showed markedly impaired growth in neutral and acidic pH compared to NHE1-competent cells (Pouyssegur et al., 1984) To confirm the role of NHE1 in breast cancer cell proliferation, we silenced the NHE1 gene in MDA-MB-231 cells using siRNA specific for NHE1 gene The transfected cells were replated onto 100mm dish and kept for 14 days, before the colonies formed were stained

with crystal violet Corroborating with previous findings, colony forming assay showed that cells transfected with NHE1 siRNA exhibited remarkably reduced colonogenic ability (supplementary Figure 1A of published paper) (Kumar et al., 2009)

After confirming the importance of NHE1 in colony forming ability of MCF-7 cells, we wanted to investigate if NHE1 is involved in the PPARγ ligand-induced reduction in cell viability and colony forming ability To this end, MDA-MB-231 cells, which express lower intrinsic level of NHE1, were co-transfected with a construct encoding green fluorescent protein in addition to the vector encoding functional NHE1 protein or the parent empty vector Based on the assumption that the transfection effieciency was similar for the fluorescent protein and NHE1 plasmid, we surmised that the green fluorescent cells represented cells transiently overexpressing NHE1 The morphological changes were then studied under fluorescent mircroscopy It was observed that cells transfected with the empty vector showed formation of vacuoles after treatment of 15d-PGJ2 On the other hand, cells that over-expressed NHE1 still maintianed a viable morphology upon exposure to15d-PGJ2 (Figure 2B of published paper) (Kumar et al., 2009)

The long-term colony-forming ability of NHE1-overexpressing MDA-MB-231 cells was also assessed using colony forming assay MDA-MB-231 cells were co-transfected with plasmid encoding full length NHE1 gene or empty vector in addition to a vector conferring hygromycin resistance The transfected cells were trypsinized and reseeded onto 100mm dish after 16h exposure to 15d-PGJ2 The selection of NHE1-overexpressing cells was then achieved by including

hygromycin in the culturing medium for 14 days, before the colonies were stained with crystal violet As expected, in cells transfected with empty vector, 15d-PGJ2 induced a significant reduction in the number of colonies formed (Figure 2A of published paper) (Kumar et al., 2009) However, overexpression of NHE1 protein

in MDA-MB-231 cells successfully diminished the loss in colonogenic ability initially induced by 15d-PGJ2 Given that overexpression of NHE1 can both prevent the vacuole formation and loss in colony-forming ability brought about by 15d-PGJ2, we concluded that repression of NHE1 expression by PPARγ ligand is involved in the anti-tumor effects of PPARγ ligands

REGULATION

It was reported that various nuclear receptors can interact and modulate each other’s activity at different gene promoters We identified the close proximity of putative ERE and PPRE on NHE1 promoter, suggesting high possibility of interaction between their corresponding transcription factors Henceforth, in this section, we aimed to examine the cross regulation of ERα and PPARγ on their control of gene expression

3B.1 ESTROGEN BLOCKS THE EFFECT OF PPARγ ON NHE1

3B1.1 Regular serum versus dextran stripped serum condition

Traditionally, most of the studies on transcription factors such as ERα and PPARγ are carried out in charcoal stripped Foetal Bovine Serum (CS-FBS) The reason for treating serum with charcoal/dextran-treated is to remove various steroids, growth factors and hormones present, which may affect the response of cells or animals to drugs and complicate the result CS-FBS is prepared by treatment with proprietary charcoal/dextran before it is passed through 3 sequential 0.1μm pore-sized rated filters Besides removing potential compounding factors, charcoal treatment was also shown to reduce lot-to-lot serum variability and ensure more consistent effect on human marrow cells (Murate et al., 1988)

To test the interference of regular serum on PPARγ activation by PPARγ ligands and to select an appropriate serum condition for stronger response of breast cancer cells to different drugs, we examined the down-regulation of NHE1 protein by 15d-PGJ2 in both regular and charcoal-stripped serum conditions using Western blot Briefly, MCF-7 cells were plated in RPMI supplemented with 10% regular FBS or with 10% CS-FBS After 48h, the cells were deprived and exposed to 15d-PGJ2 in phenol-free RPMI for 24h before they were harvested for Western blot analysis As expected, PPARγ ligand was able to dose-dependently decrease NHE1 protein expression in both serum conditions (Figure 17A) Interestingly, the down-regulation of NHE1 protein was much attenuated in regular serum condition compared to that CS-FBS setting This result strongly implies the presence of interfering components in regular serum that blocked the down-regulation of NHE1 protein by 15d-PGJ2 Hence, RPMI supplemented wtih CS-FBS instead of regular serum was demonstrated to be a more justifiable condition

in our system Furthermore, it was found that mitogenic signals such as insulin, epidermal growth factors and thrombin in serum might drive the transcription of NHE1 (Besson et al., 1998) These findings could explain the reduced extent of NHE1 down-regulation by PPARγ activation in regular serum condition From component assays comparison table published by Hyclone, it was revealed that the level of prostaglandin in FBS was reduced from 443pg/ml to 210pg/ml upon charcoal/dextran treatment This is particularly important in our system to ensure the removal of prostaglandin which may lead to constitutive activation PPARγ, and thus masking the effects of exogenous PPARγ ligands added

Other than the above mentioned components that could potentially modulate the effect of PPARγ ligand on NHE1 expression, 17β-estradiol was found to be present at higher level in regular FBS compared to CS-FBS Moreover, activated estrogen receptor was shown to bind to PPRE of PTEN and negatively interfere with PPARγ-driven transcriptional activation of PTEN (Bonofiglio et al., 2005).To further validate the different activity of estrogen receptor in regular and

CS serum conditions, the ER positive breast cancer cells, MCF-7 were transfected

with 3X ERE-TATA-Luc together with Renilla plasmid as a control for

transfection efficiency Luciferase reporter assay was then performed on transfected MCF-7 cells which were exposed to different doses of 15d-PGJ2 in RMPI media supplemented with regular FBS or CS-FBS As expected, ERE activity was significantly higher in cells grown in regular serum (Figure 17B), showing that the higher estrogen level in regular serum was able to transactivate estrogen receptor in MCF-7 It is also interesting to note that 15d-PGJ2 does-

dependently suppressed the ERE reporter activity in both serum conditions, although the extent of down-regulation was much significant in CS-FBS This result is in accordance with previous report that PPARγ agonists could disrupt ERα signalling in MCF-7 cells (Lecomte et al., 2008)

After confirming the different levels of ERα transcriptional activity in two serum conditions, we subsequently went on to investigate the ability of ERα to bind to

its response element in regular and CS FBS The in vitro binding of ERα to

classical ERE was assessed using Noshift Transcription Factor assay as described

in the Materials and Methods In agreement with the ERE reporter activity data, the efficiency of binding by ERα to ERE was 53% in charcoal stripped serum compared to that in regular serum (Figure 17C)

Taken together, these data strongly indicated the constitutive activation of estrogen receptor in regular serum condition In order to examine the true effect of PPARγ and ERα activation on NHE1 expression, all experiments were performed

in RPMI supplemented in 10% charcoal-stripped FBS The cells were also deprived in phenol-free RPMI without serum overnight to remove any remaining interfering factors, which might affect the activation of these nuclear receptors Individual experiments that were done in different serum settings are otherwise stated

Figure 17: Regular serum versus charcoal/dextran-treated serum

(A) MCF-7 cells were seeded in medium containing 10% regular serum at 2.5 X105 cells/6-well dishes or in 10% charcoal/dextran treated serum at 3.0 X105cells/6-well dishes for 48h, before they were exposed to increasing doses of 15d-PGJ2 After 24h, NHE1 protein expression was then determined by Western blot,

using β-actin as the loading control (B) MCF7 cells (7.5 X 10 cells/12-well dishes) were co-transfected with 3µg of reporter plasmid 3XERE-luc and 0.3µg of renilla as described in Materials and Methods 24h after transfection, cells were kept in medium containing either 10% regular serum or 10% charcoal/dextran treated serum for another 24h, before they were exposed to increasing doses of 15d-PGJ2 for 3h The activity of ERα was then determined using luciferase assay and the result was calculated as luciferase RLU/renilla/µg total Data represents the average +/- SD of two experiments done in duplicate (C) MCF-7 cells were seeded in medium containing 10% regular serum at 1.5 X106 cells/100mm dish or

in 10% charcoal/dextran treated serum at 1.78 X106 cells/100mm dish for 48h, before they were harvested for nuclear-cytosol fractionation as described in Materials and Methods The NoShift™ Transcription Factor Assay was utilized for determination of endogenous ERα binding to classical ERE as described in Materials and Methods Results denote means +/-SD computed from two experiments done in duplicate *, p<0.05, treated versus untreated control

3B1.2 Estrogen blocks PPARγ-mediated down-regulation of NHE1 in CS serum condition

As mentioned above, it was reported that activated estrogen receptor can bind to PPRE of PTEN and negatively interfere with PPARγ-driven transcriptional activation of PTEN (Bonofiglio et al., 2005) Based on the observation that the extent of NHE1 down-regulation of by PPARγ ligand was more pronounced in charcoal-stripped serum condition compared to regular serum condition, and the fact that the level of 17β-estradiol is much higher in regular serum than in CS-FBS, we surmised that the higher amount of E2 blocked the down-regulation of NHE1 expression by PPARγ ligand in regular serum condition To assess whether estrogen was responsible for the inhibitory effect on PPARγ-mediated reduction

of NHE1 expression in regular FBS, we re-introduced 17β-estradiol to deprived MCF-7 before addition of 15d-PGJ2 Interestingly, the down-regulation