Resveratrol Analogs: Potential Chemopreventive Agents in Breast Cancer
Abstract
Epidemiological 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
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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.
Table of Contents
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 -- Appendix
Degree
Ph.D.