**4. Targeting dihydrofolate reductase (DHFR) enzyme**

Inhibitors of DHFR are classified as either 'classical' or 'non-classical' antifolates. The 'classical' antifolates are characterized by a *p*-aminobenzoylglutamic acid side-chain in the molecule and thus closely resemble folic acid itself. Methotraxate (MTX) is the most well known drug among the 'classical' antifolates. Compounds classified as 'non-classical' inhibitors of DHFR do not possess the *p*-aminobenzoylglutamic acid side-chain but rather have a lipophilic side-chain. MTX serves as an antimetabolite, which means that it has a similar structure to that of a cell metabolite, resulting in a compound with a biological activity that is antagonistic to that of the metabolite, which in this case is folic acid (Barnhart et al., 2001; Takemura et al., 1997).

New, more lipophilic antifolates have been developed in an attempt to circumvent the mechanisms of resistance, such as decreased active transport, decreased polyglutamation, DHFR mutations and so on (Assaraf, 2007; Gangjee et al., 2006; Takemura et al., 1997). In a series of synthesized compounds for this purpose in our center the pyrimidine ring remained (figure 8) and the side-chain attachment at the position 2 was replaced with different substituent.

Fig. 8. The central structure of novel DHFR inhibitors.

Remarks in Successful Cellular Investigations for

presented in figure 9.

**5. Discussion** 

Fighting Breast Cancer Using Novel Synthetic Compounds 95

These modified antifolates differ from the traditional 'classical' analogues by increased potency, greater lipid solubility, or improved cellular uptake. Although being very effective as inhibitors, problems still remain with respect to the issue of toxicity due to the lack of selectivity (Cody et al., 2003; Graffner-Nordberg et al., 2004; McGuire, 2003). To evaluate the cytotoxic potency of these compounds, we have used the clonogenic assay. MCF-7 cells were plated in 6-well plates (200 cells/well) for 24 hours before treatment with the test compounds to allow the attachment of cells to the wells surface. Seven different concentrations of each compound, doxorubicin (as reference), and 0.5% DMSO (applied solvent to dissolve the compound) were added to the monolayer cells in triplicates. The plates were then incubated for 10 days at 37 ºC in atmosphere of 5% CO2. The media were removed after 10 days and the colonies were stained with a solution of 0.5% crystal violet in ethanol for 10 minutes and the number of colonies containing more than 50 cells was counted under microscope. The relation between the number of the colonies (as a percentage to the control containing 0.5% DMSO) and the concentrations of each compound were plotted to get survival curve of the tumor cell line and IC50 values were calculated. Cellular viability test results for some examples of this series of novel DHFR inhibitors are

Human mammary gland adenocarcinoma MCF-7 cell line (ATCC HTB-22TM) is proven to be a good breast tissue model for anticancer drugs investigations in our experiments. However, selection of a suitable cell line is only a part of a successful and meaningful *in vitro* cellular examination of potential anticancer agents. Many different factors might very much influence the final outcome of the evaluation of a medication in a cellular experiment, among them are the cell culture media and its components during the time of drug exposure and afterward, exposure time, drug solvent, volume of drug solution to be added to the cell culture media, the proper use of agonists and antagonists for the purpose of elaborations on the results and making a meaningful conclusion, methodology of cellular viability assessment, and the most important factor; the personnel who run the experiment. We are not going to extensively discuss all of these parameters and their specific influences on the final result and conclusion, but the limited examples presented in this chapter may be

The importance of a suitable protocol for the measurement of survival percentage (live versus death) of cells is underestimated in many of experiments. Selection of the method in many instances is easily a matter of facility, budget and distributing companies' advertisements in the region. However, one should notice that for many known and unknown reasons, various methods of MTT, XTT, SRB, fluorescence dye staining and so on might work or not for different experiments. The main reason might well be the cellular measurement criteria for any of these methods. One should keep in mind that although mitochondria is the heart of cellular energy system, but MTT and XTT experiments would only measure the functionality of a mitochondrial enzyme (Cody et al., 2003; Marshall et al., 1995; Scudiero et al., 1988) and would not necessarily reflect the cell viability. The same is very much true for many of staining methods e.g Annexin V which is an indication of cell membrane flip-flop that would most properly occur during the process of apoptosis (Kolodgie et al., 2003; Van Heerde et al., 2000). Both of these methods are extensively used for the measurement of the cytotoxicity of many different agents. The chemical structure of

sufficient to raise awareness for a good cellular practice.

Fig. 9. Cytotoxicity measurement of seven selected DHFR inhibitors on MCF-7 cell line resulted from clonogenic assay.

These modified antifolates differ from the traditional 'classical' analogues by increased potency, greater lipid solubility, or improved cellular uptake. Although being very effective as inhibitors, problems still remain with respect to the issue of toxicity due to the lack of selectivity (Cody et al., 2003; Graffner-Nordberg et al., 2004; McGuire, 2003). To evaluate the cytotoxic potency of these compounds, we have used the clonogenic assay. MCF-7 cells were plated in 6-well plates (200 cells/well) for 24 hours before treatment with the test compounds to allow the attachment of cells to the wells surface. Seven different concentrations of each compound, doxorubicin (as reference), and 0.5% DMSO (applied solvent to dissolve the compound) were added to the monolayer cells in triplicates. The plates were then incubated for 10 days at 37 ºC in atmosphere of 5% CO2. The media were removed after 10 days and the colonies were stained with a solution of 0.5% crystal violet in ethanol for 10 minutes and the number of colonies containing more than 50 cells was counted under microscope. The relation between the number of the colonies (as a percentage to the control containing 0.5% DMSO) and the concentrations of each compound were plotted to get survival curve of the tumor cell line and IC50 values were calculated. Cellular viability test results for some examples of this series of novel DHFR inhibitors are presented in figure 9.
