Resveratrol Analogs: Potential Chemopreventive Agents in Breast Cancer

Title from PDF of title page, viewed on June 3, 2015Dissertation advisor: Hari K. BhatVitaIncludes bibliographic references (pages 157-187)Thesis (Ph.D.)--School of Pharmacy and School of Biological Sciences. University of Missouri--Kansas City, 2014Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Estrogens have been added to the list of known human carcinogens. Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism mediated oxidative stress in estrogen-induced breast carcinogenesis. It was recently demonstrated from our laboratory that antioxidants vitamin C or butylated hydroxyanisole (BHA) strongly inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of present study was to characterize the role of CNC b-zip transcription factors in antioxidant-mediated prevention of breast cancer. The present study was based on the central hypothesis that 17β-estradiol mediates carcinogenic insult in the cellular environment by producing reactive oxygen species (ROS) / oxidative stress during its metabolism to quinones. This oxidative stress can be controlled by production of phase-II detoxifying antioxidant enzymes. In order to test this hypothesis we examined the cellular levels of different antioxidant enzymes after treatment with E2. We then investigated the molecular mechanism(s) and pathways iv involved in E2-induced breast carcinogenesis. To address the question if antioxidant agents can reverse E2-induced oxidative stress and ultimately mediate chemoprevention of breast cancer, we checked several different cellular antioxidant enzymes’ levels and dissected out possible molecular pathways involved after treatment with naturally occurring well studied antioxidants like resveratrol (Res), resveratrol analogs (TIMBD and HPIMBD) and Vitamin C. Resveratrol has been shown to reduce primary tumor growth of xenografts in a nude mouse model. But its clinical applications in prevention of breast cancer are limited because of its lower efficacy in in vivo systems. Thus, to improve the anticancer and antioxidant efficacy of Res and to use it as a successful agent targeting breast cancer, pharmacologically active resveratrol analogs have been synthesized. Our newly synthesized Res-analog compounds: 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD) and 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1, 2-diol} (HPIMBD), have effectively inhibited the growth of breast cancer cells and have no/minimal cytotoxicity towards normal cells. To further delineate the mechanisms responsible for higher growth inhibitory potency of TIMBD and HPIMBD, we have aimed at finding out its antioxidant potentials. In this current work we have shown the contribution of TIMBD and HPIMBD in providing antioxidant defense in human breast epithelial cells. In our previous studies we have shown that estrogen-induced breast carcinogenesis is initiated by down-regulation of expression of the antioxidant enzymes, superoxide dismutase 3 (SOD3, also known as extracellular superoxide dismutase) and NAD(P)H:Quinone Oxidoreductase 1 (NQO1), via a nuclear factor erythroid 2-related factor 2 (Nrf2)- dependent signaling pathway. In this present study, we have shown that a new and potent resveratrol analogs TIMBD and HPIMBD, synthesized by our collaborating team functions to inhibit E2-dependent breast cancer development by up-regulation of the expression of SOD3 and NQO1 via this same Nrf2-dependent pathway. We have also provided evidence how TIMBD and HPIMBD mediate antioxidant defense through regulation of CNC-bzip transcription factors other than Nrf2; nuclear factor erythroid 2- related factor 1 (Nrf1) and nuclear factor erythroid 2-related factor 3 (Nrf3). We also investigated the potential roles of Res-analogs in prevention of epithelialmesenchymal transition (EMT). An epithelial-mesenchymal transition can be defined as a biological process which mediates a phenotypical change in the polarized epithelial cells to mesenchymal cells. Epithelial cells, which interact with basement membrane, undergo different complex biochemical and molecular changes to become mesenchymal cells. The process of EMT increases a cell’s migratory and invasive properties. It is well documented that the process of EMT plays a very critical role in cancer metastasis. The effects of Res-analogs on EMT and the migration of human breast cancer cell lines were studied. We found that Res-analogs significantly increased epithelial marker E-cadherin expression and down-regulated matrix metalloproteases (MMPs) and expression of mesenchymal markers, such as snail, slug, zeb1/2. In present studies, we have demonstrated the potential of Res-analogs in prevention of EMT these studies suggest that our novel Res-analogs may have the potential to be therapeutic agents for breast cancer chemoprevention. In order to dissect out the possible molecular mechanism of Res-analogs on the suppression of EMT and breast cancer cell metastasis, we found a critical involvement of β-catenin. The expression and nuclear translocation of β-catenin was significantly down-regulated with Res-analogs implicating that these analogs may prevent breast cancer cell metastasis involving β-catenin pathway. We have also tested the ability of these Res-analogs to inhibit the proliferation of 5 breast cancer cell lines and 3 non-neoplastic breast epithelial cell lines and compared their inhibition potential with Res. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell proliferation was carried out in human breast cancer and non-neoplastic breast epithelial cell lines. The breast cancer cell lines tested were MCF-7, T47D, MDA-MB-231, MDA-MB-468 and BT-20. The non-neoplastic breast cell lines tested were MCF-10A, MCF-10F and HMEC. Of all the analogs tested, two analogs, 4- hydroxyphenyl-immino-methylbenzene-1,2-diol (HPIMBD) and TIMBD showed higher potency for inhibiting the proliferation of breast cancer cells compared to Res. Furthermore, TIMBD or HPIMBD showed higher potency for inhibiting the growth of triple negative breast cancer cells (MDA-MB-231, MDA-MB-468 and BT-20) compared to estrogen receptor positive breast cancer cells MCF-7 and T47D. There was neither inhibition nor proliferation by TIMBD or HPIMBD of non-neoplastic breast epithelial celllines. TIMBD and HPIMBD induced Beclin-1 and LC3-II which suggest autophagy mediated inhibition of cell growth. Beclin-1 is known to be suppressed in breast cancers and it’s over expression is reported to inhibit breast cancer. Results from our studies demonstrate that Res-analogs TIMBD and HPIMBD are better than Res in inhibiting specifically breast cancer cell growth and shows higher potency for inhibiting the growth of triple negative breast cancer cells by inducing autophagy with an earlier onset for triple negative breast cancer cells. Therefore, TIMBD and HPIMBD may be better chemotherapeutic agents than Res against breast cancer and more specifically against triple negative breast cancer cell growth, a cancer type prevalent in minority African American population with poor prognosis.General introduction -- General materials and methods -- Identification and characterization of resveratrol analogs in inhibiting breast cancer cell growth and possible mechanism of action of chemoprevention -- Determination of the mechanism of inhibition of E2-induced breast cancer by res-analogs through regulation of cnc-b zip transcription factors (NRFS) -- Determination of the role of res-analogs in epithelial mesenchymal transition (EMT) -- Novel res-analogs: TIMBD and HPIMBD demonstrate cytotoxicity towards breast cancer cells by a mechanism involving an early onset autophagy -- Appendi