**1. Introduction**

84 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

Sandri, M. T., Johansson, H., Colleoni, M., Zorzino, L., Passerini, R., Orlando, L., & Viale, G.

Sorlie, T., Perou, C. M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M.

Sotiriou, C., Neo, S. Y., McShane, L. M., Korn, E. L., Long, P. M., Jazaeri, A., Martiat, P., Fox,

Tamimi, R. M., Baer, H. J., Marotti, J., Galan, M., Galaburda, L., Fu, Y., Deitz, A. C.,

van 't Veer, L. J., Dai, H., van de Vijver, M. J., He, Y. D., Hart, A. A., Mao, M., Peterse, H. L.,

carcinoma. *Anticancer Res,* Vol. 24, pp.1261-66.

*Sci U S A,* Vol. 100, pp.10393-98.

*Cancer Res,* Vol. 10, pp.R67.

implications. *Proc Natl Acad Sci U S A,* Vol. 98, pp.10869-74.

S., Hortobagyi, G. N., & Pusztai, L. (2005). Breast cancer molecular subtypes respond differently to preoperative chemotherapy. *Clin Cancer Res,* Vol. 11:5678-85.

(2004). Serum levels of HER2 ECD can determine the response rate to low dose oral cyclophosphamide and methotrexate in patients with advanced stage breast

B., van de Rijn, M., Jeffrey, S. S., Thorsen, T., Quist, H., Matese, J. C., Brown, P. O., Botstein, D., Eystein Lonning, P., & Borresen-Dale, A. L. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical

S. B., Harris, A. L., & Liu, E. T. (2003). Breast cancer classification and prognosis based on gene expression profiles from a population-based study. *Proc Natl Acad* 

Connolly, J. L., Schnitt, S. J., Colditz, G. A., & Collins, L. C. (2008). Comparison of molecular phenotypes of ductal carcinoma in situ and invasive breast cancer. *Breast* 

van der Kooy, K., Marton, M. J., Witteveen, A. T., Schreiber, G. J., Kerkhoven, R. M., Roberts, C., Linsley, P. S., Bernards, R., & Friend, S. H. (2002). Gene expression profiling predicts clinical outcome of breast cancer. *Nature,* Vol. 415, pp.530-36. van de Vijver, M. J., He, Y. D., van't Veer, L. J., Dai, H., Hart, A. A., Voskuil, D. W.,

Schreiber, G. J., Peterse, J. L., Roberts, C., Marton, M. J., Parrish, M., Atsma, D., Witteveen, A., Glas, A., Delahaye, L., van der Velde, T., Bartelink, H., Rodenhuis, S., Rutgers, E. T., Friend, S. H., & Bernards, R. (2002). A gene-expression signature as a predictor of survival in breast cancer. *N Engl J Med*, Vol. 347, pp.1999-2009.

Breast cancer is one of the most life threatening risks in women's life. In spite of considerable progress in its understanding and challenges, treatment is not yet the correct word to apply on this disease and losing life is the most foreseeing adventure in many patients. Although new gene therapy based approaches are looking for the cure of breast malignant cells, but using cytotoxic agents is currently the main chemotherapy approach to fight this problem. Effective chemotherapy treatment of breast cancer requires targeting the pathways that support the cell growth and proliferation. A good *in vitro* investigational model is essential to understand the process of carcinogenesis, risk and hazard mechanism of carcinogens, protection from carcinogens, mode of action and efficacy of novel and even in practice chemotherapeutic agents. The main part for any of these laboratory models is suitable cell lines to properly address the problem and goal of investigation.

Estrogen Receptor (ER) is considered to cause different growth responses in ER-positive, normal, preneoplastic and neoplastic cells (DuMond et al., 2001; Roy & Cai, 2002; Welshons et al., 2003). One of the most significant researches in cancer treatment has been based on designing and studying the ER-antagonism effects of molecules on cells. This is important to select suitable cell lines for *in vitro* drug discoveries studies. Table 1 shows a list of epithelial breast cell lines with different expression in estrogen receptor.

Intracellular enzymes responsible for the different consequences of receptors stimulations and signaling cascades are also under big considerations in fighting breast cancer cells. Dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase) is an example of pivotal importance in biochemistry and medicinal chemistry. DHFR catalyzes the reduction of folate or 7,8-dihydrofolate to tetrahydrofolate and intimately couples with thymidylate synthase (TS). Reduced folates are carriers of one-


<sup>\*</sup> Afshin Zarghi3, Farzad Kobarfard3, Rezvan Zendehdel1, Maryam Nakhjavani1, Sara Arfaiee3, Tannaz Zebardast3, Shohreh Mohebi3, Nassim Anjidani1, Azadeh Ashtarinezhad1 and Shahram Shoeibi4

*<sup>1</sup>Department of Pharmaco-Toxicology, SBMU Pharmacy School, Tehran, Iran 3Department of Medicinal Chemistry, SBMU Pharmacy School, Tehran, Iran 4Food and Drug Organization, Ministry of Health Treatment and Medical Education, Tehran, Iran*

Remarks in Successful Cellular Investigations for

**growth media** 

medium & 10% FBS

**Cell line Suitable** 

HCC2157 ACL-4

'thymineless' death.

Fighting Breast Cancer Using Novel Synthetic Compounds 87

carbon fragments; hence they are important cofactors in the biosynthesis of nucleic acids and amino acids. The inhibition of DHFR or TS activity in the absence of salvage leads to

There are some other enzymes which came into special consideration in cancer development, particularly in the breast cancer. Cyclooxygenase-2 is an example that over expresses in several epithelial tumors including breast cancer. Preclinical evidence favors an anti tumor role for COX inhibitors in breast cancer because there is a clear relationship between tissue prostaglandin levels in human breast tumors and the development of metastasis and patient survival (Arun & Goss, 2004). Selective COX-2 inhibitors can prevent mammary tumors from developing cancer in experimental animals. Celecoxib (a COX-2 inhibitor) has proven to minimize the progression of carcinogen-induced mammary tumors (Arun et al., 2001). A good cell line to clearly address alterations in above mentioned

A trustable measurement approach to detect results of the application of underinvestigation agents on cells is very much important. Different methods have been applied to investigate cell alterations and ultimately cell death resulted from cancer chemotherapy and cytotoxic agents. Each of them has advantages and disadvantages in different situations and for different purposes. Misuse of any of these methods for the detection of the cytotoxicity of different agents on different cell lines is one of the main problems of many publications for years. These techniques usually look at the viability, morphology and/or biochemical function of various cellular functions. Table 2 lists some of the most popular

A precise and accurate investigation is one that selects the best possible measurement method on the best possible cell line in the most optimal situation for the best possible conclusion. Cellular investigations to look for new anti-breast cancer agents rely on these bases. MCF-7 proves to be a suitable model cell line for breast cancer investigations worldwide. This is a well known breast cancer cell line derived from a 69 years old Caucasian female. MCF-7 cell line presents most of characteristics of differentiated mammary epithelium tissues including those of expressing estradiol and estrogenic receptors features (Brandes & Hermonat, 1983). Here, we are summarizing some of our results using this cell line to search for novel anti-breast cancer agents, with emphasis and

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).


Table 1. List of breast cell lines with different expression in estrogen receptor.

systems is also critical for challenging breast cancer cells *in vitro*.

conclusive remarks on the good laboratory practice.

**2. Targeting estrogen receptors** 

methods used to measure the cytotoxicity of agents in cellular experiments.

**Kinds of receptor Oncogene considerations** 

Positive for expression of Her2-neu , Positive for expression of p53


Estrogen receptor















negative,

**Kinds of receptor Oncogene considerations** 


p53

her2/neu + (over expressed)

Negative for expression of Her2-neu, positive for expression of p53

Her2/neu, positive for expression of p53

Positive for expression of Her2 neu, positive for expression of

Negative for expression of Her2/neu, positive for expression of p53

Negative for expression of Her2/neu, positive for expression of p53

The cells exhibit a 20 fold amplification of the HER-2/neu oncogene sequence

Negative for expression of

Positive for expression of

Negative for expression of

Positive for expression of p53

Negative for expression of p53

Negative for expression of p53

BRCA1 (mutated, insertion C

at nucleotide 5382), Negative for expression of

Negative for expression

Her2/neu,

Her2/neu,

Her2-neu,

Her2-neu,

of p53

**Cell line Suitable** 

ZR-75-1 RPMI-1640

MCF-7 DMEM:F12

UACC-3199 Leibovitz's

HCC1954 RPMI-1640

HCC1500 RPMI-1640

HCC70 RPMI-1640

HCC1008 DMEM:F12

HCC1143 RPMI-1640

HCC38 RPMI-1640

UACC-893 Leibovitz's

HCC1395 RPMI-1640

HCC1937 RPMI-1640

HCC1419

HCC1806 & HCC1599

& HCC202 **growth media** 

& 10% FBS

& 10% FBS

L-15 & 10%

& 10% FBS

& 10% FBS

& 10% FBS

& 10% FBS

& 10% FBS

& 10% FBS

L-15 & 10%

& 10% FBS

RPMI-1640 & 10% FBS

RPMI-1640 & 10% FBS

& 10% FBS

FBS

FBS


Table 1. List of breast cell lines with different expression in estrogen receptor.

carbon fragments; hence they are important cofactors in the biosynthesis of nucleic acids and amino acids. The inhibition of DHFR or TS activity in the absence of salvage leads to 'thymineless' death.

There are some other enzymes which came into special consideration in cancer development, particularly in the breast cancer. Cyclooxygenase-2 is an example that over expresses in several epithelial tumors including breast cancer. Preclinical evidence favors an anti tumor role for COX inhibitors in breast cancer because there is a clear relationship between tissue prostaglandin levels in human breast tumors and the development of metastasis and patient survival (Arun & Goss, 2004). Selective COX-2 inhibitors can prevent mammary tumors from developing cancer in experimental animals. Celecoxib (a COX-2 inhibitor) has proven to minimize the progression of carcinogen-induced mammary tumors (Arun et al., 2001). A good cell line to clearly address alterations in above mentioned systems is also critical for challenging breast cancer cells *in vitro*.

A trustable measurement approach to detect results of the application of underinvestigation agents on cells is very much important. Different methods have been applied to investigate cell alterations and ultimately cell death resulted from cancer chemotherapy and cytotoxic agents. Each of them has advantages and disadvantages in different situations and for different purposes. Misuse of any of these methods for the detection of the cytotoxicity of different agents on different cell lines is one of the main problems of many publications for years. These techniques usually look at the viability, morphology and/or biochemical function of various cellular functions. Table 2 lists some of the most popular methods used to measure the cytotoxicity of agents in cellular experiments.

A precise and accurate investigation is one that selects the best possible measurement method on the best possible cell line in the most optimal situation for the best possible conclusion. Cellular investigations to look for new anti-breast cancer agents rely on these bases. MCF-7 proves to be a suitable model cell line for breast cancer investigations worldwide. This is a well known breast cancer cell line derived from a 69 years old Caucasian female. MCF-7 cell line presents most of characteristics of differentiated mammary epithelium tissues including those of expressing estradiol and estrogenic receptors features (Brandes & Hermonat, 1983). Here, we are summarizing some of our results using this cell line to search for novel anti-breast cancer agents, with emphasis and conclusive remarks on the good laboratory practice.
