MicroRNAs and gene regulation in breast cancer.

MicroRNAs (miRs) are non-protein-coding small RNAs that control the gene expression posttranscriptionally. miRs can regulate different cellular functions as well as many pathological conditions. Dysregulated miR expression profiles have been identified in different cancer types including lung cancer, breast cancer, and prostate cancer. The expression of miRs varies according to their roles in each specific cancer type. Although many miRs and their target genes have been identified in different cancers, data are still scant to validate those target genes and the mechanistic role of these miRs. The possibility of targeting cancer-associated miRs is suggested to open a new field for cancer therapy. Therapeutic strategies targeting miRs involve neutralization of the oncogenic miRs by antagomirs using locked nucleic acid oligonucleotides, miR sponges, or the restoration/overexpression of tumor-suppressing miRs that are downregulated or depleted in cancers. Although it is suggested that therapeutic applications of miRs in different pathological conditions will make a huge revolution in gene therapy, there is still an enormous challenge in employing these strategies efficiently in cancer inhibition, seemingly, due to the complexity of cancer and the current inefficient development of delivery systems for the therapeutic miRs.

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 17ß-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.

Maternal urinary phthalates and sex-specific placental mRNA levels in an urban birth cohort.

BACKGROUND: Prenatal urinary concentrations of phthalates in women participants in an urban birth cohort were associated with outcomes in their children related to neurodevelopment, autoimmune disease risk, and fat mass at 3,5,7, and 8 years of life. Placental biomarkers and outcomes at birth may offer biologic insight into these associations. This is the first study to address these associations with candidate genes from the phthalate and placenta literature, accounting for sex differences, and using absolute quantitation methods for mRNA levels. METHODS: We measured candidate mRNAs in 180 placentas sampled at birth (HSD17B1, AHR, CGA, CYP19A1, SLC27A4, PTGS2, PPARG, CYP11A1) by quantitative PCR and an absolute standard curve. We estimated associations of loge mRNA with quartiles of urinary phthalate monoesters using linear mixed models. Phthalate metabolites (N = 358) and mRNAs (N = 180) were transformed to a z-score and modeled as independent, correlated vectors in relation to large for gestational age (LGA) and gestational diabetes mellitus (GDM). RESULTS: CGA was associated with 4 out of 6 urinary phthalates. CGA was 2.0 loge units lower at the 3rd vs. 1st quartile of mono-n-butyl phthalate (MnBP) (95% confidence interval (CI): -3.5, -0.5) in male placentas, but 0.6 loge units higher (95% CI: -0.8, 1.9) in female placentas (sex interaction p = 0.01). There was an inverse association of MnBP with PPARG in male placentas (-1.1 loge units at highest vs. lowest quartile, 95% CI: -2.0, -0.1). CY19A1, CYP11A1, CGA were associated with one or more of the following in a sex-specific manner: monobenzyl phthalate (MBzP), MnBP, mono-iso-butyl phthalate (MiBP). These 3 mRNAs were lower by 1.4-fold (95% CI: -2.4, -1.0) in male GDM placentas vs. female and non-GDM placentas (p-value for interaction = 0.04). The metabolites MnBP/MiBP were 16% higher (95% CI: 0, 22) in GDM pregnancies. CONCLUSIONS: Prenatal concentrations of certain phthalates and outcomes at birth were modestly associated with molecular changes in fetal placental tissue during pregnancy. Associations were stronger in male vs. female placentas, and associations with MnBP and MiBP were stronger than other metabolites. Placental mRNAs are being pursued further as potential mediators of exposure-induced risks to the health of the child.

4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, 1 a novel resveratrol analog, differentially regulates estrogen receptors α and ß 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 ß by TIMBD in breast cancer cell lines. We demonstrate that TIMBD significantly induces the mRNA and protein expression levels of ERß 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 ERß-dependent fashion in MCF-10A and ERß1-transfected MDA-MB-231 cells, suggesting regulation of ERs as an important upstream mechanism of this analog. ERß plays a partial role in inhibition of proliferation by TIMBD while ERα overexpression does not significantly affect TIMBD's inhibition.

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 17ß-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.

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 17ß-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. 17ß-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.

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 17ß-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.

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

BACKGROUND: Estrogen 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 17ß-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. METHODS: Female 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Resveratrol and its analogs suppress HIV replication, oxidative stress, and inflammation in macrophages.

Objectives: HIV suppression in brain viral reservoirs, especially macrophages, and microglia is critical to suppress HIV neuropathogenesis and subsequently HIV-associated neurocognitive disorders (HAND). Since most antiretroviral therapy (ART) drugs do not achieve optimal therapeutic concentrations in the brain and can cause neurotoxicity, an alternative/adjuvant therapy is needed to suppress HIV neuropathogenesis. In this study, our objectives were to examine the anti-HIV, antioxidant, and anti-inflammatory potential of resveratrol (RES) and its synthetic analogs 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD) and 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol} (HPIMBD) in HIV-infected macrophages. Methods: We used HIV replication (viral load), oxidative stress (reactive oxygen species and antioxidant enzymes), and inflammatory response (pro- and anti-inflammatory cytokines/chemokines) assays to achieve the objectives of the study. Results: Our results showed that RES and its analogs HPIMBD and TIMBD at 25 µM concentration significantly decrease HIV replication in both primary monocyte-derived macrophages and U1-differentiated macrophages. Moreover, RES and its analogs do not induce any cytotoxicity for up to 3 days in these cells. Further, treatment with RES and TIMBD (25 µM) also reduced the levels of reactive oxygen species without affecting the expression of antioxidant enzymes, SOD1, and catalase in U1 macrophages. Besides, RES and HPIMBD treatment inhibited the proinflammatory cytokines and chemokines in U1 macrophages, which was associated with decreased levels of anti-inflammatory cytokines. Importantly, our western blot experiments show that RES also decreases cellular proinflammatory cytokine IL-1ß, which is usually elevated in both myeloid and neuronal cells upon HIV infection. Conclusions: Taken together, our results suggest that RES and/or its analogs are important adjuvants that may be used not only to suppress HIV but also oxidative stress and inflammation in brain viral reservoirs.

Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer.

Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidative stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17ß-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female August Copenhagen Irish (ACI) rats were treated with E2, vitamin C, vitamin C + E2, BHA and BHA + E2 for up to 8 months. Superoxide dismutase 3 (SOD3) was suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased DNA damage, colony and mammosphere formation, and migration in SOD3 knocked down MCF-10A cells, and nuclear translocation of SOD3 in vitamin C-treated mammary tissues and in MCF-10A cells suggest protective role of SOD3 against DNA damage and mammary carcinogenesis. Our studies further demonstrate that SOD3, but not SOD2 and SOD1, is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidative stress and in the prevention of estrogen-mediated breast cancer.

Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer.

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. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17ß-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Time course studies indicate that NQO1 levels tend to increase after 4 months of E2 treatment but decrease on chronic exposure to E2 for 8 months. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2.

Nutraceuticals in HIV and COVID-19-Related Neurological Complications: Opportunity to Use Extracellular Vesicles as Drug Delivery Modality.

People living with HIV/AIDS (PLWHA) are at an increased risk of severe and critical COVID-19 infection. There is a steady increase in neurological complications associated with COVID-19 infection, exacerbating HIV-associated neurocognitive disorders (HAND) in PLWHA. Nutraceuticals, such as phytochemicals from medicinal plants and dietary supplements, have been used as adjunct therapies for many disease conditions, including viral infections. Appropriate use of these adjunct therapies with antiviral proprieties may be beneficial in treating and/or prophylaxis of neurological complications associated with these co-infections. However, most of these nutraceuticals have poor bioavailability and cannot cross the blood-brain barrier (BBB). To overcome this challenge, extracellular vesicles (EVs), biological nanovesicles, can be used. Due to their intrinsic features of biocompatibility, stability, and their ability to cross BBB, as well as inherent homing capabilities, EVs hold immense promise for therapeutic drug delivery to the brain. Therefore, in this review, we summarize the potential role of different nutraceuticals in reducing HIV- and COVID-19-associated neurological complications and the use of EVs as nutraceutical/drug delivery vehicles to treat HIV, COVID-19, and other brain disorders.

Effect of alcohol on drug efflux protein and drug metabolic enzymes in U937 macrophages.

BACKGROUND: ATP-binding cassette (ABC) proteins and cytochrome P450 (CYP) enzymes regulate the bioavailability of HIV-1 antiretroviral therapeutic drugs, non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). They are also involved in regulating, and responding to, oxidative stress in various tissues and organs including liver. This study is designed to assess the effect of alcohol on the ABCC1 and CYP enzymes involved in the metabolism of NNRTIs and PIs (CYP2B6, CYP2D6, and CYP3A4) and oxidative stress (CYP1A1, CYP2A6, and CYP2E1) in U937 macrophages. The U937 cell line has been utilized as an in vitro model of human macrophages. METHODS: The expression levels of the ABCC1 and CYP enzymes in U937 macrophages were characterized in terms of mRNA quantification, protein analysis, and assays for functional activity. In addition, oxidative stress was monitored by measuring the activities of oxidative stress marker enzymes and production of reactive oxygen species (ROS). RESULTS: The order of mRNA expression in U937 macrophages was ABCC1 ∼ CYP2A6 > CYP3A4 ∼ CYP2E1 ∼ CYP1A1 > CYP2D6 > CYP2B6. Alcohol (100 mM) increased the mRNA levels of ABCC1 and CYP2A6 (200%), CYP2B6 and CYP3A4 (150%), and CYP2E1 (400%) compared with the control. Alcohol caused significant upregulation of ABCC1, CYP2A6, CYP2E1, and CYP3A4 proteins (50 to 85%) and showed >50% increase in the specific activity of CYP2A6 and CYP3A4 in U937 macrophages. Furthermore, alcohol increased the production of ROS and significantly enhanced the activity of oxidative stress marker enzymes, superoxide dismutase, and catalase in U937 macrophages. CONCLUSIONS: Our study showed that alcohol causes increases in the genetic and functional expressions of ABCC1 and CYP enzymes in U937 macrophages. This study has clinical implications in alcoholic HIV-1 individuals, because alcohol consumption is reported to reduce the therapeutic efficacy of NNRTIs and PIs and increases oxidative stress.

Dietary quercetin exacerbates the development of estrogen-induced breast tumors in female ACI rats.

Phytoestrogens are plant compounds that structurally mimic the endogenous estrogen 17beta-estradiol (E(2)). Despite intense investigation, the net effect of phytoestrogen exposure on the breast remains unclear. The objective of the current study was to examine the effects of quercetin on E(2)-induced breast cancer in vivo. Female ACI rats were given quercetin (2.5 g/kg food) for 8 months. Animals were monitored weekly for palpable tumors, and at the end of the experiment, rats were euthanized, breast tumor and different tissues excised so that they could be examined for histopathologic changes, estrogen metabolic activity and oxidant stress. Quercetin alone did not induce mammary tumors in female ACI rats. However, in rats implanted with E(2) pellets, co-exposure to quercetin did not protect rats from E(2)-induced breast tumor development with 100% of the animals developing breast tumors within 8 months of treatment. No changes in serum quercetin levels were observed in quercetin and quercetin+E(2)-treated groups at the end of the experiment. Tumor latency was significantly decreased among rats from the quercetin+E(2) group relative to those in the E(2) group. Catechol-O-methyltransferase (COMT) activity was significantly downregulated in quercetin-exposed mammary tissue. Analysis of 8-isoprostane F(2alpha) (8-iso-PGF(2alpha)) levels as a marker of oxidant stress showed that quercetin did not decrease E(2)-induced oxidant stress. These results indicate that quercetin (2.5 g/kg food) does not confer protection against breast cancer, does not inhibit E(2)-induced oxidant stress and may exacerbate breast carcinogenesis in E(2)-treated ACI rats. Inhibition of COMT activity by quercetin may expose breast cells chronically to E(2) and catechol estrogens. This would permit longer exposure times to the carcinogenic metabolites of E(2) and chronic exposure to oxidant stress as a result of metabolic redox cycling to estrogen metabolites, and thus quercetin may exacerbate E(2)-induced breast tumors in female ACI rats.

Vitamin C and alpha-naphthoflavone prevent estrogen-induced mammary tumors and decrease oxidative stress in female ACI rats.

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17beta-estradiol (E(2)), the antioxidant vitamin C, the estrogen metabolic inhibitor alpha-naphthoflavone (ANF), or cotreated with E(2) + vitamin C or E(2) + ANF for up to 8 months. E(2) (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E(2), E(2) + vitamin C and E(2) + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E(2)-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F(2alpha) formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E(2) and decreased formation of the highly carcinogenic 4-OHE(2). These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E(2) metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Antioxidant butylated hydroxyanisole inhibits estrogen-induced breast carcinogenesis in female ACI rats.

Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen-induced cancer have not been fully elucidated. Both estrogen receptor (ER)-mediated proliferative processes and ER-independent generation of oxidative stress are suggested to play important roles in estrogen-induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17beta-estradiol (E(2)), butylated hydroxyanisole (BHA), or a combination of E(2) + BHA for up to 240 days. Cotreatment of rats with E(2) + BHA reduced estrogen-induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E(2) where tumor incidence was 82%. Proliferative changes in the breast tissue of E(2) + BHA-treated animals were similar to those observed in E(2)-treated animals. Tissue levels of 8-isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E(2) + BHA and compared to activity levels found in E(2)-treated animals and respective age-matched controls. Cotreatment with BHA inhibited E(2)-mediated increases in 8-isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen-mediated oxidant stress plays a critical role in the development of estrogen-dependent breast cancers and BHA inhibits E(2)-dependent breast carcinogenesis by decreasing oxidant stress.

Placental biomarkers of phthalate effects on mRNA transcription: application in epidemiologic research.

BACKGROUND: CYP19 and PPARgamma are two genes expressed in the placental trophoblast that are important to placental function and are disrupted by phthalate exposure in other cell types. Measurement of the mRNA of these two genes in human placental tissue by quantitative real-time polymerase chain reaction (qPCR) offers a source of potential biomarkers for use in epidemiologic research. We report on methodologic challenges to be considered in study design. METHODS: We anonymously collected 10 full-term placentas and, for each, sampled placental villi at 12 sites in the chorionic plate representing the inner (closer to the cord insertion site) and outer regions. Each sample was analyzed for the expression of two candidate genes, aromatase (CYP19) and peroxisome proliferator activated receptor protein gamma (PPARgamma) and three potential internal controls: cyclophilin (CYC), 18S rRNA (18S), and total RNA. Between and within placenta variability was estimated using variance component analysis. Associations of expression levels with sampling characteristics were estimated using mixed effects models. RESULTS: We identified large within-placenta variability in both transcripts (>90% of total variance) that was minimized to <20% of total variance by using 18S as an internal control and by modelling the means by inner and outer regions. 18S rRNA was the most appropriate internal control based on within and between placenta variability estimates and low correlations of 18S mRNA with target gene mRNA. Gene expression did not differ significantly by delivery method. We observed decreases in the expression of both transcripts over the 25 minute period after delivery (CYP19 p-value for trend = 0.009 and PPARgamma (p-value for trend = 0.002). Using histologic methods, we confirmed that our samples were comprised predominantly of villous tissue of the fetal placenta with minimal contamination of maternally derived cell types. CONCLUSION: qPCR-derived biomarkers of placental CYP19 and PPARgamma gene expression show high within-placental variability. Sampling scheme, selection of an appropriate internal control and the timing of sample collection relative to delivery can be optimized to minimize within-placenta and other sources of underlying, non-etiologic variability.

Medicinal plants and cancer chemoprevention.

Cancer is the second leading cause of death worldwide. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. A number of undesired side effects sometimes occur during chemotherapy. Natural therapies, such as the use of plant-derived products in cancer treatment, may reduce adverse side effects. Currently, a few plant products are being used to treat cancer. However, a myriad of many plant products exist that have shown very promising anti-cancer properties in vitro, but have yet to be evaluated in humans. Further study is required to determine the efficacy of these plant products in treating cancers in humans. This review will focus on the various plant-derived chemical compounds that have, in recent years, shown promise as anticancer agents and will outline their potential mechanism of action.

Phytoestrogens and breast cancer prevention: possible mechanisms of action.

OBJECTIVE: Phytoestrogens display an array of pharmacologic properties, and in recent years investigation of their potential as anticancer agents has increased dramatically. In this article we review the published literature related to phytoestrogens and breast cancer as well as suggest the possible mechanisms that may underlie the relationship between phytoestrogens and breast cancer. DATA SOURCES: Electronic searches on phytoestrogens and breast cancer were performed on MEDLINE and EMBASE in June 2007. No date restriction was placed on the electronic search. DATA EXTRACTION: We focused on experimental data from published studies that examined the characteristics of phytoestrogens using in vivo or in vitro models. We also include human intervention studies in this review. DATA SYNTHESIS: We evaluated evidence regarding the possible mechanisms of phytoestrogen action. Discussions of these mechanisms were organized into those activities related to the estrogen receptor, cell growth and proliferation, tumor development, signaling pathways, and estrogen-metabolizing enzymes. CONCLUSIONS: We suggest that despite numerous investigations, the mechanisms of phytoestrogen action in breast cancer have yet to be elucidated. It remains uncertain whether these plant compounds are chemoprotective or whether they may produce adverse outcomes related to breast carcinogenesis.