Anwesha Chatterjee

MicroRNA-93 regulates NRF2 expression and is associated with breast carcinogenesis

MicroRNAs (miRNA) are small non-coding RNAs that regulate the expression of approximately 60% of all human genes and play important roles in disease processes. Recent studies have demonstrated a link between dysregulated expression of miRNAs and breast carcinogenesis. Long-term estrogen exposure is implicated in development of human breast cancers, yet underlying mechanisms remain elusive. We have recently demonstrated that antioxidant vitamin C (vit C) prevents estrogen-induced breast tumor development. In this study, we investigated the role of vit C in the regulation of microRNA-93 (miR-93) and its target gene(s) in a rat model of mammary carcinogenesis. Female August Copenhagen Irish (ACI) rats were treated with vit C in the presence or absence of 17β-estradiol (E2) for 8 months. We demonstrate an increased expression of the miR-93 in E2-treated mammary tissues and in human breast cell lines and vit C treatment reverted E2-mediated increase in miR-93 levels. MiRNA target prediction programs suggest one of the target genes of miR-93 to be nuclear factor erythroid 2-related factor 2 (NRF2). In contrast with miR-93 expression, NRF2 protein expression was significantly decreased in E2-treated mammary tissues, mammary tumors, and in breast cancer cell lines, and its expression was significantly increased after vit C treatment. Ectopic expression of miR-93 decreased protein expression of NRF2 and NRF2-regulated genes. Furthermore, miR-93 decreased apoptosis, increased colony formation, mammosphere formation, cell migration and DNA damage in breast epithelial cells, whereas silencing of miR-93 in these cells inhibited these carcinogenic processes. Taken together, our findings suggest an oncogenic potential of miR-93 during E2-induced breast carcinogenesis.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17β-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways.

Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer

BackgroundEstrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.MethodsFemale ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2.ResultsThe expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage.ConclusionsOur studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.

Novel Aza-resveratrol analogs: synthesis, characterization and anticancer activity against breast cancer cell lines.

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

The objective of the present study was to characterize the role of resveratrol (Res) and vitamin C (VC) in prevention of estrogen‐induced breast cancer through regulation of cap “n”collar (CNC) b‐zip transcription factors. Human breast epithelial cell line MCF‐10A was treated with 17β‐estradiol (E2) and VC or Res with or without E2. mRNA and protein expression levels of CNC b‐zip transcription factors nuclear factor erythroid 2‐related factor 1 (Nrf1), nuclear factor erythroid 2 related factor 2 (Nrf2), nuclear factor erythroid 2 related factor 3 (Nrf3), and Nrf2‐regulated antioxidant enzymes superoxide dismutase 3 (SOD3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2‐mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF‐10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17β‐Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen‐induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf‐dependent pathways.

Differential regulation of estrogen receptors α and β by 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol}, a novel resveratrol analog.

Breast cancer is the second leading cause of death among women in the United States. Estrogens have been implicated as major risk factors in the development of breast neoplasms. Recent epidemiologic studies have suggested a protective role of phytoestrogens in prevention of breast and other cancers. Resveratrol, a naturally occurring phytoestrogen found notably in red grapes, berries and peanuts, has been shown to possess potent anti-cancer properties. However, the poor efficacy of resveratrol has prevented its use in a clinical setting. In order to improve the efficacy of resveratrol, we have synthesized a small combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the growth of breast cancer cell lines. We have recently shown that one of the synthesized analogs, 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol} (HPIMBD), has better anti-cancer properties than resveratrol. The objective of this study was to investigate the differential regulation of estrogen receptors (ERs) α and β as a potential mechanism of inhibition of breast cancer by HPIMBD. Estrogen receptors α and β have been shown to have opposing roles in cellular proliferation. Estrogen receptor α mediates the proliferative responses of estrogens while ERβ plays an anti-proliferative and pro-apoptotic role. We demonstrate that HPIMBD significantly induces the expression of ERβ and inhibits the expression of ERα. HPIMBD also inhibits the protein expression levels of oncogene c-Myc and cell cycle protein cyclin D1, genes downstream to ERα and important regulators of cell cycle, and cellular proliferation. HPIMBD significantly induces protein expression levels of tumor suppressors p53 and p21 in MCF-7 cells. Additionally, HPIMBD inhibits c-Myc in an ERβ-dependent fashion in MCF-10A and ERβ1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this novel compound. Molecular docking studies confirm higher affinity for binding of HPIMBD in the ERβ cavity. Thus, HPIMBD, a novel azaresveratrol analog may inhibit the proliferation of breast cancer cells by differentially modulating the expressions of ERs α and β.

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and β in breast cancer cells

Breast cancer is a public health concern worldwide. Prolonged exposure to estrogens has been implicated in the development of breast neoplasms. Epidemiologic and experimental evidence suggest a chemopreventive role of phytoestrogens in breast cancers. Resveratrol, a naturally occurring phytoestrogen, has been shown to have potent anti-cancer properties. However, poor efficacy and bioavailability have prevented the use of resveratrol in clinics. In order to address these problems, we have synthesized a combinatorial library of azaresveratrol analogs and tested them for their ability to inhibit the proliferation of breast cancer cells. We have recently shown that 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), has better anti-cancer properties than resveratrol and any other resveratrol analog we have synthesized so far. The objective of this study was to investigate the regulation of estrogen receptors (ERs) α and β by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERβ and inhibits that of ERα. TIMBD inhibits mRNA and protein expression levels of oncogene c-Myc, and cell cycle protein cyclin D1, which are important regulators of cellular proliferation. TIMBD significantly induces protein expression levels of tumor suppressor genes p53 and p21 in MCF-7 cells. TIMBD inhibits c-Myc in an ERβ-dependent fashion in MCF-10A and ERβ1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this analog. ERβ plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBDs inhibition.

Tamoxifen synergizes with 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, novel azaresveratrol analogs, in inhibiting the proliferation of breast cancer cells

We have recently shown that 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD) and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), novel analogs of resveratrol (Res), selectively inhibited the proliferation of breast cancer cells. In the current study, we tested HPIMBD and TIMBD individually in combination with tamoxifen (Tam) for inhibition of growth of breast cancer cells. Tamoxifen was first tested on non-neoplastic breast epithelial cell lines and its dose that does not inhibit their growth was determined. A combination of this low dose of Tam with either of the Res analogs HPIMBD or TIMBD, resulted in synergistic inhibition of proliferation of breast cancer cells. Both estrogen receptor (ER)-positive and negative breast cancer cell lines responded to the combination. The combination resulted in a substantial decrease in IC50 values of Res analogs in all breast cancer cell lines tested. Mechanistic studies showed a synergistic increase in apoptosis and autophagy genes (beclin-1 and LC3BII/I) with the combination in ER-negative MDA-MB-231 cells. In ER-positive MCF-7 and T47D cells, the mechanism of synergy was found to be inhibition of expression of ERα and oncogene c-Myc. The combination treatment had a synergistic effect in inhibiting the colony forming and spheroid forming ability of cancer cells. Taken together, our findings indicate that a combination of Tam and Res analogs HPIMBD or TIMBD represents a novel approach to enhancing the use of Tam in therapy for breast cancers. Considering the urgent need for novel therapeutic strategies to treat ER-negative breast cancers and overcoming resistance in ER-positive cancers, this combinatorial approach is worthy of continued investigation.

Antioxidant activities of novel resveratrol analogs in breast cancer

The objective of the present study was to characterize the role of novel resveratrol (Res) analogs: 4‐(E)‐{(4‐hydroxyphenylimino)‐methylbenzene, 1, 2‐diol} (HPIMBD) and 4‐(E)‐{(p‐tolylimino)‐methylbenzene‐1,2‐diol} (TIMBD) as potent antioxidants against breast cancer. Non‐neoplastic breast epithelial cell lines MCF‐10A and MCF‐10F were treated with 17β‐estradiol (E2), Res, HPIMBD, and TIMBD for up to 72 h. mRNA and protein levels of antioxidant genes, superoxide dismutase 3 (SOD3) and N‐quinoneoxidoreductase‐1 (NQO1) and transcription factors, nuclear factor erythroid 2‐related factor (Nrf) 1, 2 and 3 were quantified after the above treatments. Generation of reactive oxygen species (ROS) was measured by CM‐H2‐DCFDA and oxidative‐DNA damage was determined by measuring 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG). HPIMBD and TIMBD scavenged cellular ROS production, attenuated oxidative DNA damage, increased mRNA and protein expression levels of SOD3 and NQO1 and activated Nrf signaling pathway. Our studies demonstrate that HPIMBD and TIMBD have the potential as novel antioxidants to prevent development of breast cancer.

Abstract 4648: Differential regulation of estrogen metabolizing CYP1A1 and CYP1B1 enzymes by novel resveratrol analogs in breast cancer cells

Evidence from epidemiological and experimental studies suggest an association between diets rich in phytoestrogens and reduced risk of breast cancer. Resveratrol, a bioflavonoid, found naturally in grapes, fruits and nuts, has been shown to be a potent chemopreventive agent in vitro. However, poor bioavailability, rapid metabolism and poor specificity limit its use as the preferred anticancer agent. In an attempt to improve the anticancer properties of resveratrol, we synthesized several analogs of resveratrol with appropriate chemical modifications. Of the compounds that were screened for anticancer activity towards breast cancer cells, two analogs, namely, HPIMBD and TIMBD showed better potency and selectivity in inhibiting the growth of breast cancer cell lines while they did not significantly affect the growth of non-neoplastic breast epithelial cell lines. In this study, we investigated the ability of resveratrol and resveratrol analogs in the regulation of estrogen metabolizing genes, cytochrome P450 (Cyp) 1A1, 1B1 and their upstream regulator AhR in breast cancer and non-neoplastic breast epithelial cells. Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines T47D and MDA-MB-231 were treated with resveratrol, HPIMBD or TIMBD. From cells in culture, mRNA and protein was isolated and expression analyses of CYP1A1, CYP1B1 and AhR carried out by PCR and western blot analyses. Unlike resveratrol, HPIMBD and TIMBD significantly induced the mRNA and protein expression levels of CYP1A1 and AhR while they inhibited that of CYP1B1. Also, HPIMBD and TIMBD induced the expression of CYP1A1 in an AhR-dependent fashion while they inhibited CYP1B1 expression of CYP1B1 by a proteosomal degradation pathway. Cyp1A1 preferentially metabolizes estradiol to 2-hydroxyestradiol and Cyp1B1 to 4-hydroxyestradiol. While, 2-hydroxyestradiol and its methylated product are suggested to be chemo-protective, 4-hydroxyestradiol is regarded as a carcinogenic metabolite of estradiol. Taken together, our findings indicate that novel resveratrol analogs HPIMBD and TIMBD differentially regulate the expressions of CYP1A1 and CYP1B1 in breast cancer cells that may have implications in chemoprevention of breast cancer by these analogs. Note: This abstract was not presented at the meeting. Citation Format: Amruta Mukund Ronghe, Anwesha Chatterjee, Subhash Padhye, Hari K. Bhat. Differential regulation of estrogen metabolizing CYP1A1 and CYP1B1 enzymes by novel resveratrol analogs in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2015-4648