Determining the Intracellular Localization and Efficacy of Novel Anticancer Agents in Human Breast Cancer Cell Lines Through the Use of Fluorescent Microscopy

Breast cancer is the second most frequently diagnosed cancer and it ranks second among cancer deaths in women. The anthracycline antibiotic doxorubicin, more commonly known as Adriamycin, is a drug widely used in breast cancer chemotherapy. The mechanism of action of this particular drug is still somewhat unclear, although it is thought to interrupt the progression of a known DNA unwinding enzyme, Topoisomerase II. Another drug used in breast cancer is the anthracenedione, Mitoxantrone. This drug is used in the treatment of metastatic breast cancer. Mitoxantrone is also a Topoisomerase II inhibitor. In some cases, patients become resistant to chemotherapies or have malignancies that recur. These recurrences are sometimes due to cells that are resistant to the current chemotherapeutic tactic being applied. These cells that resistant express P-glycoprotein. P-glycoprotein is an ATP-dependent efflux pump that has evolved to remove harmful substances from cells. In the case of chemotherapy failure, it is a good chance that patients exhibit an overproduction of P-glycoprotein in their tumor cells. This leads to a population of cells that has the unique ability to rid itself of drug and express multi-drug resistance.Although these chemotherapeutic agents are effective anticancer drugs their efficacy is limited by drug-induced cardiotoxicity and therapy must be stopped once a patient has received a cumulative dose of 550mg/m2. Researchers have sought other equally effective Topoisomerase II inhibitors that lack the cardiotoxicity. The development of 9-aza-anthrapyrazoles has led to drugs that are not only active against breast cancer, but are non-cardiotoxic. Furthermore, an interesting observation has been made in which compounds containing two terminal tertiary amine side chains, as seen in the compounds BBR3378 and BBR3409, are active in PGP expressing MCF-7 cells. The purpose of this thesis is to determine the potency of these novel cancer drugs and also to determine intracellular drug distribution in cells. The two colorimetric assays that were used during this project were the MTT cell proliferation assay and a trypan blue exclusion assay.