**5. Discussion**

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

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

resulted from clonogenic assay.

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 sufficient to raise awareness for a good cellular practice.

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 under investigation compound, its solvent, its cellular site of action, the exposure time, the lag time from the beginning of exposure to the start of measurement, and even the selection of cell line might dramatically alter the final survival curve. Methotrexate is a good example of MTT limitation in cytotoxicity measurement (Haber et al., 1993) and colleagues have shown that MTT protocol is not able to assess the cytotoxicity of this anticancer agent on various cells including MCF-7. Our experiments on other novel DHFR inhibitors have also proven the same conclusion when MTT results were not conclusive while the clonogenic g assay could easily provide a meaningful dose-response result. Figure 10 shows a comparison of MTT versus clonogenic assay for the measurement of methotrexate as well as some other novel DHFR inhibitors. As is shown in this figure, clonogenic assay was more successful in determining the LD50 of these compounds in MCF-7 cell line, but not the MTT protocol. Alteration of the exposure time and lag time between the addition of drugs and start of MTT assay, media components and calculation method were not helpful to provide a conclusive survival curve using this method (Data are not shown).

Fig. 10. Comparison of clonogenic versus MTT assays for the measurement of methotrexate and some other novel DHFR inhibitors.

Remarks in Successful Cellular Investigations for

**1**

the analysis of various anticancer candidates.

**95% Confidence Intervals 0.9985 to 1.398**

**Discrepancy Analysis**

0 25 50 75 100

**Location (g/ml)**

**ACHN**

**NR-CLN**

method.

**10**

24 hours 72 hours

**100**

**Precente of** 

MDCK cell.

**absorbance**

**1000**

Fighting Breast Cancer Using Novel Synthetic Compounds 97

based techniques like the flowcytometery, as well as proliferation based measurements like the clonogenic assay present good results, cell membrane integrity and mitochondrial enzyme function based assays have a significant lag time before the presenting of measurable alterations. A successful cellular repair event during this lag time may change the final conclusion dramatically. One needs to be aware of these possibilities in interpretation of cytotoxicity test results. Figure 11 represents the measurement of cisplatin cytotoxicity effect on MDCK cell line using MTT assay. As is shown in this figure, a 48 hours exposure time difference is needed to acquire a reasonable survival curve using this method.

> **MDCK MTT Assay after 1 h exposure to cisplatin**

**1 2 3 4 5 6 7 8 9 10 Cisplatin Concentration (ug/ml)**

Fig. 11. The lag time required to get a good MTT result on the cytotoxicity of cisplatin on

A discrepancy analysis to measure the cytotoxicity of many of novel anticancer drugs developed in our center under the same condition on the same cell line using two different methods of clonogenic assay and the neutral red assay, did not show agreement with a clear horizontal line and 95% confidence interval of about 1. It would further prove the importance of the selection and application of a suitable survival measurement system in

Fig. 12. A comparison of clonogenic versus neutral red assay for the measurement of cell survival after exposure to various novel anticancer drugs, using a discrepancy analysis

**NR**

 Slope Y-intercept

0 25 50 75 100 125

**CLN**


**Slope & Intercept Analysis**

Clonogenic assay is usually considered as a final answer for drugs cytotoxicity because of its long waiting time to acquire result. A minimum of five to six cellular doubling times to look at results in clonogenic assay might well overcome all cellular adventures of arrest, repair, detoxification and exertion pumps influences on drug cellular mortality which might affect the result of cross sectional measurement methods like MTT and Annexin V. Figure 7 is another example of the limitation of these type of experiments in some instances in comparison with the clonogenic assay. Clonogenic assay, however, would surprisingly fail to present a meaningful graph of cytotoxicity after exposure to some compounds.

There are many different mechanisms which might cause these differences in the result of the viability measurement using different methodologies. Cellular target of the test compound and the cellular repair system are two of the most possible explanation. Rosenberg confusion about the effects of electric field on the cells resulted in cisplatin identification and later use as a very important and most used anticancer drug in many different kinds of malignancies including the breast cancer (Rosenberg et al., 1969). Cells in Rosenberg set up did not die, rather changed shape and remained alive for a long time (Rosenberg, 1985, 1977). Cisplatin, like many other anti-mitotic agents, does not kill cells right after exposure. Its principle mechanism of action is on the DNA and thus while stopping DNA synthesis and cell proliferation, won't affect the mitochondrial action and cell membrane integrity. That is why, while the thymidine assay and cell cycle progression

under investigation compound, its solvent, its cellular site of action, the exposure time, the lag time from the beginning of exposure to the start of measurement, and even the selection of cell line might dramatically alter the final survival curve. Methotrexate is a good example of MTT limitation in cytotoxicity measurement (Haber et al., 1993) and colleagues have shown that MTT protocol is not able to assess the cytotoxicity of this anticancer agent on various cells including MCF-7. Our experiments on other novel DHFR inhibitors have also proven the same conclusion when MTT results were not conclusive while the clonogenic g assay could easily provide a meaningful dose-response result. Figure 10 shows a comparison of MTT versus clonogenic assay for the measurement of methotrexate as well as some other novel DHFR inhibitors. As is shown in this figure, clonogenic assay was more successful in determining the LD50 of these compounds in MCF-7 cell line, but not the MTT protocol. Alteration of the exposure time and lag time between the addition of drugs and start of MTT assay, media components and calculation method were not helpful to provide

Fig. 10. Comparison of clonogenic versus MTT assays for the measurement of methotrexate

Clonogenic assay is usually considered as a final answer for drugs cytotoxicity because of its long waiting time to acquire result. A minimum of five to six cellular doubling times to look at results in clonogenic assay might well overcome all cellular adventures of arrest, repair, detoxification and exertion pumps influences on drug cellular mortality which might affect the result of cross sectional measurement methods like MTT and Annexin V. Figure 7 is another example of the limitation of these type of experiments in some instances in comparison with the clonogenic assay. Clonogenic assay, however, would surprisingly fail

There are many different mechanisms which might cause these differences in the result of the viability measurement using different methodologies. Cellular target of the test compound and the cellular repair system are two of the most possible explanation. Rosenberg confusion about the effects of electric field on the cells resulted in cisplatin identification and later use as a very important and most used anticancer drug in many different kinds of malignancies including the breast cancer (Rosenberg et al., 1969). Cells in Rosenberg set up did not die, rather changed shape and remained alive for a long time (Rosenberg, 1985, 1977). Cisplatin, like many other anti-mitotic agents, does not kill cells right after exposure. Its principle mechanism of action is on the DNA and thus while stopping DNA synthesis and cell proliferation, won't affect the mitochondrial action and cell membrane integrity. That is why, while the thymidine assay and cell cycle progression

to present a meaningful graph of cytotoxicity after exposure to some compounds.

a conclusive survival curve using this method (Data are not shown).

and some other novel DHFR inhibitors.

based techniques like the flowcytometery, as well as proliferation based measurements like the clonogenic assay present good results, cell membrane integrity and mitochondrial enzyme function based assays have a significant lag time before the presenting of measurable alterations. A successful cellular repair event during this lag time may change the final conclusion dramatically. One needs to be aware of these possibilities in interpretation of cytotoxicity test results. Figure 11 represents the measurement of cisplatin cytotoxicity effect on MDCK cell line using MTT assay. As is shown in this figure, a 48 hours exposure time difference is needed to acquire a reasonable survival curve using this method.

Fig. 11. The lag time required to get a good MTT result on the cytotoxicity of cisplatin on MDCK cell.

A discrepancy analysis to measure the cytotoxicity of many of novel anticancer drugs developed in our center under the same condition on the same cell line using two different methods of clonogenic assay and the neutral red assay, did not show agreement with a clear horizontal line and 95% confidence interval of about 1. It would further prove the importance of the selection and application of a suitable survival measurement system in the analysis of various anticancer candidates.

Fig. 12. A comparison of clonogenic versus neutral red assay for the measurement of cell survival after exposure to various novel anticancer drugs, using a discrepancy analysis method.

Remarks in Successful Cellular Investigations for

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