**2. Targeting estrogen receptors**

Estrogens are known to play an important role in the regulation of the development and maintenance of the female reproductive system, in particular of the uterus, ovaries and breast. Moreover, estrogens are involved in the growth and/or function of several other tissues such as bone, liver, brain, and the cardiovascular system (Ciocca & Roig, 1995).

Remarks in Successful Cellular Investigations for

2004).

Fighting Breast Cancer Using Novel Synthetic Compounds 89

Estrogen Receptor Modulators (SERMs), which function as estrogen agonists in some tissues (bone, brain and the cardiovascular system) but as antagonists in others (uterus and breast). Estrogen action is mediated through two Estrogen Receptor (ER) subtypes, ERα and ERβ, which have distinct target tissue distributions and functional activities (Gustafsson et al., 2003; Matthews & Gustafsson, 2003; Välimaa et al., 2004). ERα is predominantly found in the uterus, bone, cardiovascular tissue, and liver and is the predominant ER expressed in breast cancer. ERβ is expressed in many tissues including prostate, breast, vascular endothelium, and ovary. The precise function of ERβ and its role in breast is not clear (Fox et al., 2008; Novelli et al., 2008). Recent studies indicate that ERβ expression may have a potential protective effect on normal cells against ERα induced hyperproliferation (Bardin et al.,

Fig. 1. Stimulation and inhibition of MCF-7 breast cancer cell line exposed to estradiol, tamoxifen and mix of these two agents for 7 days in phenol red-free RPMI media incubated

tamoxifen and other known SERMs such as ralolxifen and rasofoxifen.

Estrogen receptors can bind a variety of steroidal and non-steroidal ligands. Tamoxifen was the first SERM approved for the treatment of breast cancer (Jordan, 1988). The search for better SERMs has driven efforts to increase the chemical diversity of these compounds, especially the non-steroidal ones (Meegan & Lloyd, 2003). Figure 2 shows the structures of

in 37°C and 5% CO2 humified incubator.


Table 2. Different popular methods to measure cellular alterations after exposure to cytotoxic agents.

Figure 1 represents the general effects of estradiol (as a proliferative estrogen receptor stimulant agent) and tamoxifen (as an estrogen receptor blocking agent) on the growth curve of MCF-7 cell line. To obtain this, 50,000 cells were seeded in four series of cell culture petri dishes and incubated in phenol red-free RPMI media supplemented with 10% fetal bovine serum for 7 days. From the beginning, three different series of petri dishes were selected for the experiments; estradiol was added into the media of one series, tamoxifen was added to the media of the second series and a mix of these two agents was added to the third series of petri dishes. Cells in each perti dish were counted for seven consecutive days as the presentation of cell proliferation in control, estradiol exposed, tamoxifen exposed, and affected by both of estradiol and tamoxifen agents. As is seen in figure 1, estradiol has a significant effect to promote the growth of MCF-7 breast cancer cells compared to the control cells. MCF-7 cells, however, are arrested for at least five days before being able to start a significant proliferation after the exposure to the estrogen-blocking agent of tamoxifen. This block is effective enough to prevent the stimulating effect of estradiol when cells are exposed to both agents simultaneously. This experiment would further emphasize on the stimulating effect of estrogen receptors in breast cancer progression.

Several studies have established that estrogens are predominantly involved in the initiation and proliferation of breast cancer. Lots of efforts are now being devoted to block estrogen formation and action as an anticancer strategy (Clemons & Goss, 2001; Jensen et al., 2001; Nelson et al., 2009). This has led to the development of compounds termed Selective

Trypan blue, Phenol red, …) Cell membrane integrity (Shirazi et al., 2005;

MTT and XTT Function of mitochondrial

Bromodeoxyuridine Cellular DNA synthesis

Blotting techniques DNA, RNA and Protein

Vital dyes (Methylene blue,

Clonogenic assay,

Thymidine assay,

Flowcytometry

Light and electron microscopes

breast cancer progression.

cytotoxic agents.

**Method Measurement criteria Sample methodology** 

cell numbers Cellular proliferation (Shirazi & Eftekhari, 2004;

enzymes

synthesis machinery

Population based cell cycle analysis, Individual cell content and biophysical status

Cellular morphology and structural features

Figure 1 represents the general effects of estradiol (as a proliferative estrogen receptor stimulant agent) and tamoxifen (as an estrogen receptor blocking agent) on the growth curve of MCF-7 cell line. To obtain this, 50,000 cells were seeded in four series of cell culture petri dishes and incubated in phenol red-free RPMI media supplemented with 10% fetal bovine serum for 7 days. From the beginning, three different series of petri dishes were selected for the experiments; estradiol was added into the media of one series, tamoxifen was added to the media of the second series and a mix of these two agents was added to the third series of petri dishes. Cells in each perti dish were counted for seven consecutive days as the presentation of cell proliferation in control, estradiol exposed, tamoxifen exposed, and affected by both of estradiol and tamoxifen agents. As is seen in figure 1, estradiol has a significant effect to promote the growth of MCF-7 breast cancer cells compared to the control cells. MCF-7 cells, however, are arrested for at least five days before being able to start a significant proliferation after the exposure to the estrogen-blocking agent of tamoxifen. This block is effective enough to prevent the stimulating effect of estradiol when cells are exposed to both agents simultaneously. This experiment would further emphasize on the stimulating effect of estrogen receptors in

Several studies have established that estrogens are predominantly involved in the initiation and proliferation of breast cancer. Lots of efforts are now being devoted to block estrogen formation and action as an anticancer strategy (Clemons & Goss, 2001; Jensen et al., 2001; Nelson et al., 2009). This has led to the development of compounds termed Selective

Table 2. Different popular methods to measure cellular alterations after exposure to

**references** 

Shokrzadeh et al., 2006)

Shirazi et al., 1996)

(Shirazi et al., 2004; Tamaddon et al., 2007)

(Hammers et al., 2002; Maghni et al., 1999; Raaphorst et al., 1998; Yokochi & Gilbert, 2007)

(Ko et al., 1993; Singh et al., 2008; Skliris et al., 2002)

(Lukyanova et al., 2009; Niknafs & Shirazi, 2002; Skliris et al., 2002;)

(Lukyanova et al., 2009; Russo et al., 1977; Vic et al., 1982)

Estrogen Receptor Modulators (SERMs), which function as estrogen agonists in some tissues (bone, brain and the cardiovascular system) but as antagonists in others (uterus and breast). Estrogen action is mediated through two Estrogen Receptor (ER) subtypes, ERα and ERβ, which have distinct target tissue distributions and functional activities (Gustafsson et al., 2003; Matthews & Gustafsson, 2003; Välimaa et al., 2004). ERα is predominantly found in the uterus, bone, cardiovascular tissue, and liver and is the predominant ER expressed in breast cancer. ERβ is expressed in many tissues including prostate, breast, vascular endothelium, and ovary. The precise function of ERβ and its role in breast is not clear (Fox et al., 2008; Novelli et al., 2008). Recent studies indicate that ERβ expression may have a potential protective effect on normal cells against ERα induced hyperproliferation (Bardin et al., 2004).

Fig. 1. Stimulation and inhibition of MCF-7 breast cancer cell line exposed to estradiol, tamoxifen and mix of these two agents for 7 days in phenol red-free RPMI media incubated in 37°C and 5% CO2 humified incubator.

Estrogen receptors can bind a variety of steroidal and non-steroidal ligands. Tamoxifen was the first SERM approved for the treatment of breast cancer (Jordan, 1988). The search for better SERMs has driven efforts to increase the chemical diversity of these compounds, especially the non-steroidal ones (Meegan & Lloyd, 2003). Figure 2 shows the structures of tamoxifen and other known SERMs such as ralolxifen and rasofoxifen.

Remarks in Successful Cellular Investigations for

these compounds on MCF-7 cell line.

**-0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75**

**Log Concentrations ( molar)**

**-0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75**

**Log Concentrations ( molar)**

at the end of this chapter.

**3. Targeting COX-2 enzyme** 

comparable antiproliferative effects on cancer cells.

Therefore compounds a to d selectively bind to ERα.

(Zhu et al., 2006).

**0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9**

**0.5**

**1.0**

**Absorption**

**1.5**

**Absorption**

Fighting Breast Cancer Using Novel Synthetic Compounds 91

absorbance for each well was measured at 540 nm. A comparison of absorbance in each well containing different concentrations of each compound to the control wells could easily represent the number of live cells in that well as a result of the cell mitochondrial function

The results of anti-proliferative MTT assays of compounds a to d on MCF-7 breast cancer cells are shown in the graphs below (Figure 4). Start point (time 0) is shifted in each set of figures for a better clarification of the shape and trends of graphs in case of compounds a, b and d. These graphs show the comparative cytotoxic and antiproliferative effects of all of

> **0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6**

**0.5**

**1.0**

**Absorption**

Fig. 4. Cytotoxic effects of tamoxifen and compounds a to d on MCF-7 cell line presents a

Estrogen receptor binding studies were carried out for the compounds with ERα and ERβ using a fluorescence polarization procedure to prove the stimulatory and inhibitory mechanism being through the estrogenic receptors (data not shown). The compounds were active on ERα at nanomolar concentrations and on ERβ at micromolar concentrations.

Interestingly, clonogenic assays on MCF-7 cell line after exposure to these compounds fail to present solid and reliable growth inhibitory effects. Figure 5 shows some graphs resulted from the same exposure strategy of above-mentioned compounds on MCF-7, but using the clonogenic methodology to compare the results. A clear weakness is evident in these graphs preventing from any conclusive interpretation of results. We will further discuss this finding

There is considerable evidence to suggest that prostaglandins play an important role in the development and growth of cancer. The enzyme cyclooxygenase (COX) catalyses the

**1.5**

**2.0**

**Absorption**

**-0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75**

**Log Concentrations ( molar)**

**-0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75**

**Log Concentrations ( molar)**

Tamoxifen compound b

Tamoxifen compound d

Tamoxifen compound a

Tamoxifen compound c

Fig. 2. Chemical structures of some known Selective Estrogen Receptor Modulators (SERMs).

Structure-activity relationship (SAR) studies and molecular modeling studies center lead to the design of novel structures containing 1,2,3-triarylpropenone scaffold to act as potential SERMs and anti breast cancer agents with a unique structure as is shown in Figure 3.

Fig. 3. The general model of 1,2,3-triarylpropenone scaffold as a novel potential SERMs and anti breast cancer agents**.** 

The compounds a to d have been synthesized and undergone biological evaluations in an *in vitro* cellular system using MCF-7 breast cancer cell line as the model. The anti-proliferative activities of these compounds were determined using MTT assay. To do so, a ten thousands cells were seeded in phenol red-free RPMI-1640 medium supplemented with 10% FBS in each well of 96-well micro culture plates and incubated for 24 hours at 37 ºC in a 5% CO2 incubator. Different concentrations of each compound were added to the wells with respective vehicle control for 72 hours. Media were then removed and MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well. Formazon crystals were dissolved in 200 µL of DMSO after 4.5 hours incubation and the dye

S

Fig. 2. Chemical structures of some known Selective Estrogen Receptor Modulators

SERMs and anti breast cancer agents with a unique structure as is shown in Figure 3.

O

Raloxifen

Structure-activity relationship (SAR) studies and molecular modeling studies center lead to the design of novel structures containing 1,2,3-triarylpropenone scaffold to act as potential

Fig. 3. The general model of 1,2,3-triarylpropenone scaffold as a novel potential SERMs and

The compounds a to d have been synthesized and undergone biological evaluations in an *in vitro* cellular system using MCF-7 breast cancer cell line as the model. The anti-proliferative activities of these compounds were determined using MTT assay. To do so, a ten thousands cells were seeded in phenol red-free RPMI-1640 medium supplemented with 10% FBS in each well of 96-well micro culture plates and incubated for 24 hours at 37 ºC in a 5% CO2 incubator. Different concentrations of each compound were added to the wells with respective vehicle control for 72 hours. Media were then removed and MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well. Formazon crystals were dissolved in 200 µL of DMSO after 4.5 hours incubation and the dye

OH

R: alkoxy heterocycle groups X: different chemical groups

HO

O

Lasofoxifene

N

O

Tamoxifen

HO

R

X

anti breast cancer agents**.** 

O

Componds a-d

O

N

N

(SERMs).

absorbance for each well was measured at 540 nm. A comparison of absorbance in each well containing different concentrations of each compound to the control wells could easily represent the number of live cells in that well as a result of the cell mitochondrial function (Zhu et al., 2006).

The results of anti-proliferative MTT assays of compounds a to d on MCF-7 breast cancer cells are shown in the graphs below (Figure 4). Start point (time 0) is shifted in each set of figures for a better clarification of the shape and trends of graphs in case of compounds a, b and d. These graphs show the comparative cytotoxic and antiproliferative effects of all of these compounds on MCF-7 cell line.

Fig. 4. Cytotoxic effects of tamoxifen and compounds a to d on MCF-7 cell line presents a comparable antiproliferative effects on cancer cells.

Estrogen receptor binding studies were carried out for the compounds with ERα and ERβ using a fluorescence polarization procedure to prove the stimulatory and inhibitory mechanism being through the estrogenic receptors (data not shown). The compounds were active on ERα at nanomolar concentrations and on ERβ at micromolar concentrations. Therefore compounds a to d selectively bind to ERα.

Interestingly, clonogenic assays on MCF-7 cell line after exposure to these compounds fail to present solid and reliable growth inhibitory effects. Figure 5 shows some graphs resulted from the same exposure strategy of above-mentioned compounds on MCF-7, but using the clonogenic methodology to compare the results. A clear weakness is evident in these graphs preventing from any conclusive interpretation of results. We will further discuss this finding at the end of this chapter.
