Meena Katdare

Inhibition of proliferation and modulation of estradiol metabolism: novel mechanisms for breast cancer prevention by the phytochemical indole-3-carbinol.

Abstract Aberrant proliferation is an early-occurring intermediate event in carcinogenesis whose inhibition may represent preventive intervention. Indole-3-carbinol (13C), a glucosinolate metabolite from cruciferous vegetables, inhibits organ site carcinogenesis in rodent models. Clinically relevant biochemical and cellular mechanisms for the anticarcinogenic effects of 13C, however, remain unclear. Experiments were conducted on reduction mammoplasty derived 184-B5 cells initiated with chemical carcinogen (184-B5/BP) or with oncogene (184-B5/HER), and on mammary-carcinoma-derived MDA-MD-231 cells to examine whether (i) 13C inhibits aberrant proliferation in initiated and transformed cells, and (ii) inhibition of aberrant proliferation is associated with altered cell-cycle progression, estradiol (E2) metabolism, and apoptosis. Aberrant proliferation in 184-B5/BP, 184-B5/HER, and MDA-MB-231 cells was evident by a 55%-67% decrease in the ratio of quiescent (Q = GO) to proliferative (P = S + M) phase of the cell cycle, a 72%-90% decrease in apoptosis, and a 76%-106% increase in anchorage-dependent growth. These cells also exhibited a 88%-90% decrease in the ratio of C2 to C16α-hydroxylation products of E2. Treatment of 184-B5/ BP, 184-B5/HER, and MDA-MB-231 cells to cytostatic dose of 50 μM13C resulted in an 137%-210% increase in Q/P 13C ratio, a 4- to 18-fold increase in E2, metabolite ratio, a 2-fold increase in cellular apoptosis, and a 54%-61% inhibition of growth. The preventive efficacy of 13C on human mammary carcinogenesis may be due in part to its ability to regulate cell-cycle progression, increase the formation of antiproliferative E2 metabolite, and induce cellular apoptosis.

Selenium Decreases Thyroid Cancer Cell Growth by Increasing Expression of GADD153 and GADD34

Selenium (Se) supplementation is reported to decrease the incidence and total mortality of cancer. Whereas in vitro and in vivo studies have shown a decrease in prostate, lung, and liver cancers, this has not been shown in thyroid cancer. ARO (anaplastic), NPA (BRAF positive papillary), WRO (BRAF negative papillary), and FRO (follicular) cells treated with 150 μM seleno-l-methionine (SM) were assessed for viability at 24, 48, and 72 h. Treated FRO cells were examined for cell cycle using flow cytometry, for apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and for gene expression using microarray. Genes identified as upregulated were confirmed by real-time PCR (RT-PCR) and proteins by Western blot analysis. SM treatment significantly decreased the proliferation of all cell lines. TUNEL assay showed no evidence of apoptosis, and flow cytometry showed a significant cell-cycle arrest in S (271% increase, P= 0.006) and G2/M (61% increase, P= 0.002) compared to control. Microarray revealed 21 differentially expressed genes with greater than twofold change. A relative overexpression of growth arrest and DNA damage inducible (GADD)34 and GADD153 in treated cells was confirmed with RT-PCR and Western blot. SM inhibits thyroid cancer cell proliferation through a time dependent upregulation of the GADD family of genes and arrest in S and G2/M phases of the cell cycle. This is the first report of selenium induced inhibition of thyroid cancer cell growth.

Prevention of mammary preneoplastic transformation by naturally-occurring tumor inhibitors

Aberrant hyperproliferation (AH) is a late occurring post-initiational event that precedes mammary tumorigenesis in vivo. Experiments on the spontaneously immortalized, non-tumorigenic murine mammary epithelial C57/MG and MMEC cells were designed to validate AH as an in vitro cellular marker for preneoplastic transformation. Colony forming efficiency (% CFE) in anchorage-independent conditions of growth represented the quantitative parameter for AH. C57/MG and MMEC cells, upon treatment with chemical carcinogens or transfection with oncogenes, exhibited at least a 60-300-fold increase in AH relative to that seen in appropriate untreated controls. Transplantation of mammary epithelial cells initiated either by chemical carcinogens or by oncogenes into mammary fat pads of syngeneic mice produced rapidly growing tumors at the transplant site within 4-6 weeks. The tumor-derived T1/Pr1 and myc3/Pr1 cell lines (positive controls) exhibited at least an 800-900-fold increase in AH. Treatment of initiated cells with naturally occurring tumor inhibitors eicosapentaenoic acid (EPA), indole-3-carbinol (I3C), (-)epigallocatechin gallate (EGCG), squalene (SQE), and perillyl alcohol (PA) at non-toxic doses, resulted in a 70-99% inhibition of AH, depending on the initiator and the chemopreventive test compound. Upregulation of AH in initiated mammary epithelial cells in vitro prior to tumorigenesis in vivo, and persistent inhibition of AH by diverse naturally occurring tumor inhibitors, provides evidence for AH as a cellular surrogate endpoint for induction and modulation of mammary neoplastic transformation.

Negative Growth Regulation of Oncogene‐transformed Human Breast Epithelial Cells by Phytochemicals: Role of Apoptosis

Evidence from epidemiological investigations and from experiments on animal models supports the concept that natural phytochemicals present in fresh fruits, vegetables, and grain products may offer protection against human organ-site carcinogenesis.1–5 Such phytochemicals as phytoalexins, terpenoids, flavanoids, and retinoids, either as single agents or as adjuvants, may represent valuable lead compounds for preventive/therapeutic intervention. Clinically relevant mechanistic evidence for the efficacy of phytochemicals, at present, is largely dependent on extrapolation and is therefore equivocal. Human tissue–derived in vitro models and mechanistic end point biomarkers provide an innovative approach for reducing a need for extrapolation of basic research data for their clinical relevance. Our earlier studies have sought to develop reliable models from explant and cell cultures of noncancerous breast tissue, wherein induction and modulation of chemical carcinogenor oncogene-induced preneoplastic transformation is quantified at molecular, biochemical, and cellular levels using a spectrum of mechanistic biomarker assays.6–11 In the multistep process of human breast carcinogenesis, transformation of target epithelial tissue from the terminal duct lobular unit (TDLU) to simple hyperplasia, atypical hyperplasia, lobular carcinoma in situ, and ductal carcinoma in situ exhibits progressively increased risk for developing aggressive breast cancer.12 Furthermore, the comedo type of ductal carcinoma in situ that lacks estrogen receptor (ER) frequently overexpresses HER-2/neu, mutant p53, and epidermal growth factor receptor (EGFR). These lesions are at higher risk for developing drug-resistant, invasive breast cancer.13–15 A cell culture model exhibiting these characteristics represents a valuable experimental system for clinically relevant translational research. To this end, efforts are focused to establish epithelial cell cultures from reduction mammoplasty-derived normal breast tissue, and proliferative disease without atypia, atypical

Chemopreventive Agents Inhibit Aberrant Proliferation of the Aneuploid Phenotype in a Colon Epithelial Cell Line Established from Apc 1638N [+/−] Mouse

Loss of function of the adenomatous polyposis coli (APC) tumor suppressor gene predisposes for familial adenomatous polyposis (FAP) syndrome. The Apc gene knockout mice exhibit accelerated intestinal carcinogensis modifiable by diverse pharmacological agents. Present experiments utilized the Apc[+/−] 1638N COL colon epithelial cell line (origin: histologically normal colon) as the model. Retinoid receptor modulator 9‐cis‐retinoic acid (9‐cis‐RA), ornithine decarboxylase inhibitor difluoromethyl ornithine (DFMO), and nonselective cyclooxygenase inhibitor sulindac (SUL) represented the chemopreventive test compounds. Population doubling, cell cycle progression, and anchorage‐independent growth provided mechanistic end points for chemopreventive efficacy. Treatment of 1638N COL cells with 9‐cis‐RA, DFMO and SUL produced a dose‐dependent cytostatic growth arrest by decreasing the number of population doublings and altering aneuploid G0/G1: S+G2/M ratio. The clonally expanded 1638N‐Cl1 cells selected for anchorage‐independent growth exhibited decreased anchorage‐independent colony formation in response to treatment with the three test compounds. Susceptibility of preneoplastic 1638N COL cells to mechanistically distinct chemopreventive agents validates a unique epithelial cell culture model for FAP syndrome, and facilitates investigations on Apc regulated colon carcinogenesis and cancer prevention.

The nutritional herb Epimedium grandiflorum inhibits the growth in a model for the Luminal A molecular subtype of breast cancer

The Luminal A subtype of breast cancer expresses the estrogen receptor (ER)-α and progesterone receptor (PR), but not the human epidermal growth factor receptor (HER)-2 oncogene. This subtype of breast cancer responds to endocrine therapy involving the use of selective estrogen receptor modulators and/or inhibitors of estrogen biosynthesis. However, these therapeutic agents are frequently associated with long-term systemic toxicity and acquired tumor resistance, emphasizing the need to identify non-toxic alternative treatments for chemo-endocrine therapy responsive breast cancer. The present study utilized the human mammary carcinoma-derived, ER+/PR+/HER-2- MCF-7 cell line as a model of the Luminal A subtype of breast cancer to examine the growth inhibitory effect of the Chinese nutritional herb Epimedium grandiflorum (EG) and determine the mechanisms underlying this effect. MCF-7 cells maintained in a serum-depleted culture medium retained their ability to grow in response to 17β-estradiol (E2). Treatment of the MCF-7 cells with EG resulted in dose-dependent inhibition of E2-promoted growth. Mechanistically, EG inhibited E2-promoted cell cycle progression through G1 stage arrest and modulated the cellular metabolism of E2, increasing the formation of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Long-term treatment of MCF-7 cells with EG inhibited E2-promoted anchorage independent growth, a surrogate in vitro biomarker of tumorigenesis. In conclusion, the results of the present study demonstrate the growth inhibitory effects of EG on MCF-7 cells and identified clinically relevant mechanistic leads for its anti-tumorigenic efficacy.

The role of aldo‐keto reductase 1C3 (AKR1C3)‐mediated prostaglandin D2 (PGD2) metabolism in keloids

Keloids are progressively expanding scars, mostly prevalent in individuals of African descent. Previous data identified increased mast cell number and activation state in keloids suggesting a role in disease progression. The major eicosanoid secreted by mast cells is prostaglandin D2 (PGD2), a relatively unstable pro‐inflammatory mediator which can be spontaneously converted to 15‐deoxy‐(Delta12,14)‐prostaglandin J2(15d‐PGJ2) or enzymatically metabolized to 9α,11β‐PGF2 by aldo‐keto reductase 1C3 (AKR1C3). In this work, we investigated the possible role of PGD2 and its metabolites in keloids using CRL1762 keloid fibroblasts (KF) and immunohistochemical staining. Our data suggested approximately 3‐fold increase of tryptase‐positive mast cell count in keloids compared with normal skin. Furthermore, AKR1C3 was overexpressed in the fibrotic area of keloids while relatively weak staining detected in normal skin. Metabolism of PGD2 to 9α,11β‐PGF2 by both, KF and normal fibroblasts, was dependent on AKR1C3 as this reaction was attenuated in the presence of the AKR1C3 inhibitor, 2′‐hydroxyflavanone, or in cells with decreased AKR1C3 expression. 15d‐PGJ2, but not the other tested PGs, inhibited KF proliferation, attenuated KF‐mediated collagen gel contraction and increased caspase‐3 activation. In addition, treatment with 15d‐PGJ2 activated P38‐MAPK, induced reactive oxygen species and upregulated superoxide dismutase‐1 (SOD‐1). Finally, inhibition of P38‐MAPK further augmented 15d‐PGJ2‐induced caspase‐3 cleavage and attenuated its effect on SOD‐1 transcription. This work suggests that localized dual inhibition of AKR1C3 and P38‐MAPK may inhibit keloid progression. Inhibiting AKR1C3 activity may generate oxidative environment due to redirection of PGD2 metabolism towards 15d‐PGJ2 while inhibition of P38‐MAPK will sensitize keloid cells to ROS‐induced apoptosis.

Oral melanoma in a gravid, HIV-positive woman

A 30-year-old gravid, African-American womanwith multidrug-resistant HIV based on genotypetesting presented to the Department of Dermatologywith a 10-year history of asymptomatic hyperpigmen-tationofthegingivaandlip,whichhaddarkenedoverthe course of her current pregnancy. Physical exam-ination found an asymmetric, ill-defined, 3-cm darkbrown and black plaque with irregular bordersinvolving the vermillion and mucosal lip, gingiva,and hard palate (Fig 1). There was no cervicaladenopathy.Initialpunchbiopsyfromtherightuppermucosal lip found a melanoma in situ involving thelateral margins. Subsequent incisional biopsy foundinvasive mucosal lentiginous melanoma with aBreslowdepthof1.45mm(Fig2).Pertinentlaboratoryfindings included a decreased CD4 count of 177 cellsper cubic millimeter anda viral load of624copies permilliliter.Aftermultidisciplinaryconsensuswithotolar-yngology and surgical oncology, the patient under-went wide resection with 1-cm margins after delivery.Sentinellymphnodebiopsyresultswerenegative.Thepatient is disease free 21 months after resection andhas required no further adjuvant treatment.

Abstract B45: TRAIL sensitivity in triple-negative breast cancer cell lines from different ethnic backgrounds

Introduction: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can kill tumor cells while leaving normal cells unharmed. However, many breast and ovarian cancer cells are resistant to TRAIL-mediated apoptosis. Breast cancers can be divided into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or HER-2 amplification (referred to as basal or triple-negative breast cancer). Due to the lack of specific target there is no targeted therapy for triple negative breast cancer. Recently, we demonstrated that a set of triple negative breast cancer cell lines with mesenchymal features are sensitive to TRAIL. Still TRAIL has very little or no effect on the triple negative cells with epithelial characteristics. We are investigating if this is true for the cell lines derived from different ethnic group. Method: Both epithelial (viz., MDA-MB-468, from African American and HCC1937 from non Hispanic white) and mesenchymal cell lines (viz., MDA-MB-157 from African American, and MDA-MB-231 from non Hispanic white patient) were selected for this study. All of the cell lines were grown in MEM medium supplemented with 10% FCS with or without 1ug/ml TRAIL for 16h. Percentage of growth inhibition was determined by MTS assay. Correlation between growth inhibition and apoptosis were established by mitochondrial membrane depolarization, caspase assay, and DNA fragmentation. Result: Both of the epithelial triple negative cells were non responsive to TRAIL. Only some (10.5% for MB-468 and 18.6% for HCC1937) growth inhibition was observed even with highest concentration (5ug/ml) of TRAIL irrespective to the ethnicity differences. The effect of TRAIL in these cell lines was not dose dependant. On the other hand, both cell lines with mesenchymal feature from both ethnicities were highly responsive to TRAIL. More than 70% (71.5% for MB-157 and 75.2% for MB-231) of growth inhibition was achieved with TRAIL at 1ug/ml concentration and the effect was dose dependent. In epithelial cells both the TRAIL-receptor-1 (TR-1) and TRAIL receptor-2 (TR-2) expression were very low. In MB-231 silencing of only TR-2 reduces the effect of TRAIL but not TR-1. Conclusions: Though cells from non Hispanic white patients were a little more sensitive to TRAIL than cells from African American patient, the difference was minor and it was not statistically significant. Low level or TR-2 expression in epithelial cell lines may contribute to TRAIL resistant to these cell lines. We think TRAIL could be used either alone or in combination to treat breast cancer with mesenchymal phenotype. Appropriate combination could be searched for TRAIL therapy for breast cancer with epithelial phenotype. We are investigating additional cell lines derived from different racial or ethnic groups to determine if there is any correlation between ethnicity and TRAIL sensitivity and underlying possible molecular mechanism. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B45.

Abstract 1754: Transforming growth factor beta as potential prognostic biomarker of breast cancer: Evidence of ‘TGF-β switch’

Purpose: Transforming growth factor-beta (TGF-β 1 , β 2 , β 3 ) and its receptors, potent inhibitors of normal epithelial cell proliferation also play a dual role from suppressor to promoter during later stages of breast neoplasia and metastases. No clinical studies have reported the levels of circulatory and tumor tissue TGF-β/TβR/SMAD markers to correlate TGF-β pathway in breast cancer progression. Multifactorial analysis is likely to offer a better predictability and identify patients likely to benefit from anti TGF-β strategies. Experimental Design: This prospective study was designed to evaluate prognostic value of TGF-β, TβR and SMAD in 120 randomly enrolled pre-therapeutic breast cancer patients from Gujarat Cancer & Research Institute, India. Circulating TGF-β levels (β 1 , β 2 , β 3 ) were evaluated from patients with known clinicopathologic prognosticators compared to 87 healthy individuals. Gene expression of TGF-β (1, 2, 3), their cognate receptors (TβR-I, II, III) and signaling mediators SMAD (3, 4, 7) from breast tumors and non-involved tissue were determined by Real Time PCR. Kaplan-Meier Survival analysis with a median follow-up of 49 months was performed. Comparisons were made between early and advanced breast cancer to correlate with ‘TGF-β switch’. Results: Circulatory TGF-β 1 was higher in advanced stage patients than early stage. Higher levels of TGF-β 1 correspond with patients in category of >40-≤60 age, premenopausal, grade-I tumors, ductal carcinomas, moderate + poor differentiated tumors, lymphatic permeation, lymphocytic infiltration, absence of vascular permeation, necrosis and stromal involvement. Reduced overall survival was associated with high TGF-β 1 . TGF-β 1 expression was higher in advanced (31.6%) than early stages (16.7%). TGF-β 2 mRNA was higher in 47.6% early and 39.5% advanced stage. Serum TGF-β 3 was higher in patients than control. TGF-β 3 was downregulated in 97.6% early and 92.1% advanced tumors. TβR-I downregulation of early 85.71%, decreased to 76.32% in advanced stage. TβR-I upregulation correlated reduction in overall survival. TβR-II was downregulated in early tumors and 6.58% advanced tumors showed upregulation. Advanced tumor patients with lower TβR-II relapsed faster than early stage. Notably, TβR-III was downregulated in 30.95% early tumors while 69.05% exhibited an upregulation. TβR-III upregulation was associated with reduced RFS in advanced than early stage patients. All SMAD were downregulated in 88% patients. ERα was upregulated in 44.5% and ERβ downregulated in 97.3% patients. TGF-β 2 showed 1.49 fold upregulation in ER negative tumors. Conclusions: The present comprehensive study is the first clinical report of Indian breast cancer patients, indicating the dual role of TGF-β and suggests its potential as a prognostic marker for breast cancer. [Support: ICMR, GCS to ST, Fulbright-Nehru India DPR Fellowship #15094403 to HD] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1754.