Investigation of mechanisms of chemically-induced breast cancer as targets for anticancer drugs

Breast cancer is the second cause of death among female cancers and its incidence is increasing rapidly all over the world. It was reported that endogen estrogens and xenoestrogens play a role in the initiation and progression of breast cancer via two complementary pathways; by increasing cell proliferation via binding to estrogen receptor and by inducing oxidative DNA damage as a result of metabolism of estrogens to their reactive quinone metabolites. Melatonin, a natural indolic hormone, is known to be beneficial in breast cancer via modulation of estrogen receptor mediated effects and via inhibiting local biosynthesis of estrogens by aromatase inhibition. Furthermore melatonin is reported to play a role as a potent adjuvant agent to increase effectiveness and to reduce side effects of the conventional breast cancer therapy. However short half-life and low bioavailibility of melatonin limit its therapeutic use. Therefore the present thesis is aimed to investigate the potential effects of melatonin analogues on the selected targets that are involved in the chemical-induced breast carcinogenesis. Our selected target proteins were are estrogen receptor, aromatase and CYP1B1 enzymes. The inhibitory activies of the compounds on estrogen induced cell proliferation as a consequence of their ER antagonist effects were investigated. Inhibitory effects of the compounds on both aromatase, enzyme responsible for local estrogen biosynthesis, and CYP1B1, enzyme catalysing biotransformation of chemicals/estrogens to their reactive metabolites, were investigated. Furthermore short-term and long-term cytotoxicity of the compounds were evaluted in both non-tumorigenic breast epithelial cell line (MCF-10A) and two different breast cancer cell lines (MCF-7 BUS and MDA-MB231). We found that some of the compounds have ER antagonist, aromatase inhibitor and/or CYP1B1 inhibitory activitiy; some of them were effective against more than one target. Some of the compounds that were found to be effective on selected targets caused cytotoxicity on human breast cancer cell lines without having cytotoxic effect on non-tumorogenic breast cancer cell line which indicates that these molecules can be promising candidates for breast cancer therapy without causing harm to the healthy cells. Some of the tested indol derivatives were found to inhibit formation of colonies dramatically in long term cytotoxicity studies in MCF-7 cells which suggests an important role for them as novel drug candidates. It was also found that some compounds are more cytotoxic to ER + (MCF-7 BUS) cells than ER-(MDA-MB-231) breast cancer cells implying the hormon related mode of action of their cytotoxic activity. On the other hand for some compounds, there were no differences between their cytotoxic effects on ER+ or ER- cell lines suggesting that off-target mechanisms may be involved in their cytotoxicity. Although effective compounds are not as potent as current therapy agents, they were much more effective than their parent compound, melatonin, on all three selected targets. We suggest that these compounds could be promising candidates with lower side-effects in the prevention and treatment of breast cancer as an adjuvant therapeutical agent. The structure-activity relationships obtained from this study may provide data for further studies to develope more potent molecules and alternatives to current therapies. In the present thesis, target identification, selection and validation of method, medium/high throughput screening on selected target, molecular modelling studies to provide predictive data, early phases of the efficacy and toxicity determination were conducted to identify a hit/lead among tested indole derivative melatonin analogs as in the preclinical stages of drug discovery studies. Keywords: Breast cancer; ER antagonist; Aromatase inhibitor; CYP1B1 inhibitor, Melatonin analogues