MCF-7 Human Mammary Carcinoma Cells Research Articles

Synthesis and Cytotoxicity Evaluation of Tyrosine-5 Fluorinated Analogues of RA-VII, An Antitumor Bicyclic Hexapeptide

AbstractRA-VII analogues fluorinated at each ε-position of Tyr-5 were designed. The synthesis of these peptide analogues commenced with the preparation of atropisomeric fluorocycloisodityrosines from protected 3-fluoro-L-tyrosyl-3-boronyl-L-tyrosine mediated by copper(II) acetate and 4-(dimethylamino)pyridine. After N-methylation, the tetrapeptide segment was coupled with the N-terminus of each fluorocycloisodityrosine to afford a hexapeptide. After removal of the C- and N-terminal protecting groups, each peptide was subjected to macrocyclization to produce an analogue. The analogue with a β-oriented fluorine atom was equipotent to RA-VII with regard to cytotoxicity toward human mammary carcinoma MCF-7 cells, and showed 2.1-fold and 1.4-fold lower cytotoxicities than RA-VII toward human promyelocytic leukemia HL-60 and human colorectal cancer HCT-116 cells, respectively, whereas the analogue with an α-oriented fluorine atom showed 7.7-fold, 6.0-fold, and 14-fold lower cytotoxicities than RA-VII toward those cells, respectively.

BIOACTIVITIES AND CHEMOPROFILING COMPARISONS OF CHENOPODIUM AMBROSIOIDES L. AND CHENOPODIUM BOTRYS L. GROWING IN KASHMIR, INDIA

Objectives: The objectives of the study were the gas chromatography-mass spectrometry (GC-MS) identification and comparison of the chemical constituents, evaluation of the antifungal and anticancer activities of two species of genus Chenopodium, for example, Chenopodium ambrosioides (Ca) and Chenopodium botrys (Cb) growing in Kashmir, Himalayan region.Methods: The hydrodistilled essential oil of Ca and Cb was subjected to GC-MS analysis and antifungal activity for minimum inhibitory concentration (MIC) determination against different human pathogenic fungal strains using broth microdilution assay in 96-well microtiter plates as per the protocol of the clinical and laboratory standards institute (2008 M27-A3). The anti-proliferative ability of the essential oils was also evaluated against the two cell lines MCF-7 (human mammary carcinoma cells) and A549 (Human lung adenocarcinoma epithelial cells).Results: A total of 34 compounds identified in Ca with α-terpinene (37.17%), isoascaridole (20.48%), and ascaridole (14.83%) as the key compounds. The key compounds of Cb were shyobunol (18.91%), and hedycaryol (9.51%), germacrene-D-4-ol (8.57%), with 65 identified compounds. Both the species were found to have comparable antifungal activities against human pathogenic fungi with MIC80 values in the range of 0.031 mg/ml– 0.256 mg/ml for Ca and 0.031 mg/ml–0.126 mg/ml in case of Cb. Maximum anti-proliferative activity was observed at 125 μg/ml concentration in A549 cell line, while as the oils inhibited the growth of MCF-7 cell line at a lower concentration of 31.25 μg/ml.Conclusion: The essential oils of Ca and Cb were found to have potent anticancer and antifungal activities and can have potential use as a natural fungicide.

BIOACTIVITIES AND CHEMOPROFILING COMPARISONS OF CHENOPODIUM AMBROSIOIDES L. AND CHENOPODIUM BOTRYS L. GROWING IN KASHMIR, INDIA

Objectives: The objectives of the study were the gas chromatography-mass spectrometry (GC-MS) identification and comparison of the chemical constituents, evaluation of the antifungal and anticancer activities of two species of genus Chenopodium, for example, Chenopodium ambrosioides (Ca) and Chenopodium botrys (Cb) growing in Kashmir, Himalayan region.Methods: The hydrodistilled essential oil of Ca and Cb was subjected to GC-MS analysis and antifungal activity for minimum inhibitory concentration (MIC) determination against different human pathogenic fungal strains using broth microdilution assay in 96-well microtiter plates as per the protocol of the clinical and laboratory standards institute (2008 M27-A3). The anti-proliferative ability of the essential oils was also evaluated against the two cell lines MCF-7 (human mammary carcinoma cells) and A549 (Human lung adenocarcinoma epithelial cells).Results: A total of 34 compounds identified in Ca with α-terpinene (37.17%), isoascaridole (20.48%), and ascaridole (14.83%) as the key compounds. The key compounds of Cb were shyobunol (18.91%), and hedycaryol (9.51%), germacrene-D-4-ol (8.57%), with 65 identified compounds. Both the species were found to have comparable antifungal activities against human pathogenic fungi with MIC80 values in the range of 0.031 mg/ml– 0.256 mg/ml for Ca and 0.031 mg/ml–0.126 mg/ml in case of Cb. Maximum anti-proliferative activity was observed at 125 μg/ml concentration in A549 cell line, while as the oils inhibited the growth of MCF-7 cell line at a lower concentration of 31.25 μg/ml.Conclusion: The essential oils of Ca and Cb were found to have potent anticancer and antifungal activities and can have potential use as a natural fungicide.

The combined effects of all-trans-retinoic acid and docosahexaenoic acid on the induction of apoptosis in human breast cancer MCF-7 cells.

Breast cancer is one of the most women's cancers in the worldwide. In vivo and in vitro studies showed that all-trans-retinoic acid (ATRA) and docosahexaenoic acid (DHA) can modulate differentiation and apoptosis in both cancer and immune cells. Nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) activation in the presence of their ligands, plays a critical role in the proliferation, differentiation, and apoptosis of normal cells. We hypothesized that ATRA and DHA, as ligands of RARs and RXRs respectively, may have synergistic effects on the induction of apoptosis in MCF-7 human mammary carcinoma cell lines. MCF-7 cells were seeded in a 24-well plate at 3 × 105 cells per well. The cells were treated with 5 µM ATRA, 30 DHA, and various combinations of them over a 3-day trial. Apoptosis was measured by Annexin V-FITC kit and flow cytometery. Our results showed that the combination treatment of ATRA and DHA (5 µM and 30 µM and half dose at 2.5 µM and 15 µM, respectively) in a dose-dependent manner induced apoptosis rate in MCF-7 cells significantly more than single treatment of ATRA or DHA, as compared to control group (P < 0.05). We conclude that the combination of ATRA and DHA at the well-balanced proportion may be effective in cancer cell apoptosis. Further studies provide details about the potential synergistically effects of combination treatment of ATRA and DHA in growth inhibition and differentiation of human mammary cancer cells.

Frequent infection of human cancer xenografts with murine endogenous retroviruses in vivo.

Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infected with XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effects on xenograft growth potential with significant consequences for interpretation of many xenograft studies.

Using a sequence of estrogen response elements as a DNA aptamer for estrogen receptors in vitro.

Estrogen receptors (ERs) are overexpressed in approximately 70% of breast cancer cases, and they play an important role in tumorigenesis. ERs are strong predictive factors for measuring responses to hormonal therapies. Aptamers are short and single stranded oligonucleotides that are able to recognize target molecules with high affinity. In the present study, we selected and synthesized an oligonucleotide, which has a similar sequence to estrogen response element in the Xenopus Vitellogenin A2 gene. The synthesized oligonucleotide was evaluated by using immunostaining of paraffin-embedded breast cancer tissues and treating MCF-7 human mammary carcinoma cell line in vitro. We found that the synthesized oligonucleotide had a high binding affinity to ER similar to estradiol. Using a specific anti-ER antibody as a standard control, we showed that the synthesized oligonucleotide specifically recognized and immunostained tumor cells of breast cancer without cross-reaction with normal tissues. The overall agreement of ER detection between the anti-ER antibody and the ER aptamer was 97.1% (kappa value=0.943; 95% CI=0.879-1.006; p<0.002). Similar to tamoxifen or fulvestrant, the oligonucleotide also had an inhibitory effect on cell proliferation of MCF-7 cell line in a dose- and time-dependent fashion but had no cytotoxic effect on human normal mammary epithelial cells. Therefore, the synthesized oligonucleotide may be used as an aptamer for immunostaining of paraffin-embedded tissue sections for breast cancer diagnosis, as well as a potential ER antagonist in the treatment of breast cancer.

Identification of lipidomic biomarkers for coexposure to subtoxic doses of benzo[a]pyrene and cadmium: the toxicological cascade biomarker approach.

The search for model bioassay systems indicating activation of different toxicological signaling pathways is one of the paramount goals of modern toxicology. Especially coexposure scenarios need to be investigated with respect to synergistic and interdependent effects for the activation of toxicological signaling pathways. The present study introduces an experimental in vitro model system for nontoxic and low-dose coexposures of human mammary carcinoma MCF-7 cells against polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP) and heavy metals such as cadmium. For the first time, a multivariate model that identifies 18 metabolic biomarkers has been shown to be sufficient to separate BP-treated cells from coexposed or control cells. A "toxicological pathway color code model" is introduced to visualize the results. Different biomarker subsets can be associated with specific HER2 signaling steps. A tiered cascade biomarker approach is proposed that could be used to identify profiles associated with tumorigenic potency of environmental toxicants in coexposure scenarios, including possible synergistic or additive effects.

Comparative Efficacy of Extracts from Lycium Barbarum Bark and Fruit on Estrogen Receptor Positive Human Mammary Carcinoma MCF-7 Cells

Chemo-endocrine therapy for estrogen receptor positive (ER+) breast cancer exhibits acquired tumor resistance. Herbal medicines provide integrative support for breast cancer patients. Present study compared the efficacy of aqueous extracts from Lycium barbarum bark (LBB) and Lycium barbarum fruit (LBF) on ER+ MCF-7 cells. Cellular growth and 17ß-estradiol (E2) metabolism quantified the efficacy. MCF-7 cells maintained in serum depleted medium+ E2 exhibited increased anchorage-dependent and anchorage-independent growth. LBB exhibited greater potency than LBF (95% reduction in IC50). LBB produced a 6.8-fold increase, 40% decrease, and a 3.7-fold increase in 2-hydroxyestrone (2-OHE1), 16α-hydroxyestrone (16α-OHE1), and estriol (E3) formation. The corresponding values for LBF were 3.9, 33, and 10.5. LBB produced a16.3-fold and a twofold increase in 2-OHE1:16α-OHE1 and E3:16α-OHE1 ratios, whereas LBF produced a sixfold and a 2.9-fold increase. The efficacy of LBB is due to increased 2-OHE1 formation, whereas that of LBF is due to accelerated conversion of 16α-OHE1 to E3. Specific growth inhibitory profiles of LBB and LBF may be due to their distinct chemical composition and their complementary actions on E2 metabolism. This study validates a mechanistic approach to identify efficacious herbal extracts for clinical ER+ breast cancer.

Effects of triclocarban on the transcription of estrogen, androgen and aryl hydrocarbon receptor responsive genes in human breast cancer cells

Triclocarban (TCC) is an antimicrobial agent that is used in detergents, soaps and other personal hygiene products. Similarly to triclosan the widespread use of TCC has raised concerns about its endocrine potential. In luciferase-based reporter assays TCC has been shown to enhance estrogenic and androgenic activities following cellular coexposure with estrogen or dihydrotestosterone, respectively. The present study demonstrates that although coexposure with TCC enhances the estrogenic and androgenic readout of luciferase-based reporter cell lines such as HeLa9908 and MDA-kb2, it fails to act as a xenoandrogen on transcriptional level, nor does it induce cell proliferation in the estrogen sensitive E-screen. In addition TCC did not alter the expression of estrogen responsive genes in human mammary carcinoma MCF-7 cells exposed to 17β-estradiol, bisphenol A, butylparaben or genistein.However, TCC was shown to interfere with the regulon of the aryl hydrocarbon receptor (AhR) as TCC showed a costimulatory effect on transcription of CYP1A1 and CYP1B1, effectively lowering the transcriptional threshold for both genes in the presence of estrogens. It thus seems, that while the induction of the respective luciferase reporter assays by TCC is an unspecific false positive signal caused by luciferase stabilisation, TCC has the potential to interfere with the regulatory crosstalk of the estrogen receptor (ER) and the AhR regulon.

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Xanthopterin‐induced apoptosis in leukemic cell lines

The pteridines xanthopterin (2‐amino‐4,6(3H,5H)‐pteridinedione) and isoxanthopterin (2‐amino‐4,7(3H,8H)‐pteridinedione) exhibit anticancer effects in mice and cytotoxicity in confluent human mammary carcinoma MCF7 cells and other transformed cell lines (Lord et al, Cancer Lett. 2005, 222, 119). We wished to study the effects of these pteridines in additional cell lines in an attempt to understand the mechanism of their cytotoxicity. We now report that xanthopterin shows a dose‐dependent cytotoxic effect on Jurkat cells as shown by the MTT assay for cellular viability (Malich et al, Toxicology 1997, 124, 179). Xanthopterin and, to a much less extent, isoxanthopterin induce apoptotic and necrotic‐like morphology in Jurkat T, 769‐P and EL‐4, but not K‐562, cell lines. Dose response curves and cytofluorometric analysis show that the two pteridines in a time and concentration‐dependent manner decrease Jurkat cell viability by inducing caspase 6 activity and rapid cellular disorganization, nuclear condensation, and fragmentation. This suggests that xanthopterin may have some potential as an apoptosis‐inducing agent in leukemia cells (Korhonen et al 2005 Current Drug Targets – Inflammation and Allergy 4, 471).

The Effects of n-3 Fatty Acids and Bexarotene on Breast Cancer Cell Progression

Breast cancer cell growth can be inhibited in vivo by retinoid X receptor (RXR) specific retinoids. In both animal and cell culture studies, omega-3 fatty acids share growth regulatory effects similar to those of RXR specific retinoids (rexinoids). One synthetic rexinoid, bexarotene (LCD 1069, Targretin), is used clinically to treat cancer patients. Of concern is that some patients are unable to tolerate high doses of such treatment drugs. We hypothesized that n-3 fatty acids and bexarotene may work synergistically to slow breast cancer cell growth. To test our hypothesis, we used MCF-7 human mammary carcinoma cells and an in vitro cell culture model. We investigated the relationship between the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) alone and in conjunction with bexarotene in slowing MCF-7 cell growth. Following a 72 hr incubation with the respective treatments, bexarotene enhanced cell growth (p < 0.05) while DHA showed a strong growth inhibitory effect which was not enhanced by the addition of bexarotene (p < 0.05). EPA alone was not effective in altering cell growth (p < 0.05). Interestingly, when combined with bexarotene, EPA was more effective at slowing cell growth than when cells received EPA alone. Thus, select omega-3 fatty acids alone are more effective than bexarotene in slowing MCF-7 cell progression. However, the use of the RXR-selective retinoids may enhance the growth regulatory mechanisms of the fatty acid EPA.

Abstract 5473: Antiproliferative and antimetastasic activity of a novel vasopressin V2 receptor peptide agonist in a breast cancer model

Abstract Desmopressin (1-deamino-8-D-arginine vasopressin) is a synthetic analog of vasopressin with antimetastatic properties. The molecule is a well known and safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor, which is expressed in endothelium and also in some tumor cells. Previous data revealed that perioperative administration of desmopressin can minimize the spread and survival of residual cancer cells. This cyclic nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity and half life. Such positions belong to the disulphide loop (between positions 1-6), which have a major interaction with its receptor. In this work, we evaluated the in vitro biological activity of a novel analog designated VQ (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) in MCF7 human mammary carcinoma cells, as well as its in vivo antitumor action in combination with chemotherapy using the syngeneic F3II mammary carcinoma model in Balb/c mice. Because V2 receptor signaling has been associated with cytostatic effects, we have examined peptide ability to modify in vitro cell proliferation of MCF7 cells expressing V2 receptor. Incubation with VQ analog (100-1500 nM) during 72 h significantly reduced cell proliferation (p&amp;lt;0.001) showing a better performance than desmopressin at higher doses of 500-1500 nM (p&amp;lt;0.05). Inhibition of cell proliferation was associated with an increase of intracellular cAMP levels and this was correlated with PKA activation in MCF7 cells. A similar antiproliferative profile was obtained with loop tetrapeptides (aminoacid positions 2-5) at low doses (&amp;lt;500 nM), although cytostatic activity of tetrapeptides was lost at high concentrations. Three weekly i.v. doses of VQ (0.3 µg/kg/dose) reduced by 40% the subcutaneous tumor volume in mice implanted with F3II mammary carcinoma cells, while VQ treatment plus weekly i.p. cycles of carmustine (20 mg/kg/cycle) was even more effective (p&amp;lt;0.001). Furthermore, VQ treatment, either in combination or not with carmustine cycles, prevented lung metastatic progression in tumor-bearing mice. VQ also inhibited F3II lung colonization in the experimental metastasis assay. Our results indicate that the novel cyclic peptide analog VQ can exert a remarkable antitumor and antimetastatic effect in a preclinical model of breast carcinoma. Loop conformation of the peptide seems to be essential for interaction with V2 receptor and for the biological activity of the compound. 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 5473.

Aquaporin 1-expressing MCF-7 mammary carcinoma cells show enhanced migration in vitro

Recent studies have demonstrated that aquaporin (AQP) expression facilitates cell migration and promotes angiogenesis and neutrophil motility. Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we investigated the expression of AQP in MCF-7 human mammary carcinoma cells and characterized its function in cell migration. Reverse Transcription–Polymerase Chain Reaction, Immunoblot and Immunofluorescence analysis demonstrated two populations of MCF-7 cell clones with low (AQP1L) and high (AQP1H) AQP1 expression and the AQP1 protein expression patterns in the plasma membrane of MCF-7 cells. MCF-7 cell clones (AQP1L and AQP1H) with low and about two-fold higher osmotic water permeability were identified by functional assays with corresponding low and high AQP1 expression. Cell migration rate was remarkably higher in AQP1H cells as compared to AQP1L cells, assessed by wound healing and transwell migration assays. Adenoviral-mediated mRNA and protein expression of AQP1 in AQP1L cells increased their water permeability and migration rate to the level similar to AQP1H cells. The results provided direct evidence that aquaporin-mediated plasma membrane water permeability played an important role in mammary carcinoma cell migration and may be associated with mammary carcinoma invasion and metastasis.

Specific glycosphingolipids mediate epithelial-to-mesenchymal transition of human and mouse epithelial cell lines

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-beta, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-beta is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-beta treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-beta-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).

Estrogen and progesterone interactive effects in postconfluent MCF-7 cell culture

17β-Estradiol (E2) stimulates morphological differentiation of an MCF-7 human mammary carcinoma cell line resulting in the development of multicellular rounded nodules (foci) above the epithelial monolayer. Examining the combined effect of progesterone (P4) and E2 on foci formation we detected P4-dependent foci enlargement and phenotypic modification. Notably, P4 dose-dependently potentiated lower dose E2-induced increases in foci numbers. We detected P4-dependent changes in cytoskeleton protein expression levels and accelerated cell division. P4 alone or in combination with E2 additively modified the expression of adhesion proteins and stimulated expression of tropomyosin (Tm). Antiprogestin and antiestrogen pretreatment abrogated P4-dependent increases in foci number and stimulation of Tm expression, indicating involvement of both E2 and P4 receptor signaling. Novel aspects of endocrine-regulated changes in microfilament and adhesion protein composition are discussed in association with tumorigenesis and metastatic capability in breast carcinoma cells.

The influence of temperature on antiproliferative effects, cellular uptake and DNA platination of the clinically employed Pt(II)-drugs

Cellular uptake of a drug is one of the most important factors influencing its pharmacodynamics and pharmacokinetics. Our laboratory has previously studied platinum uptake following cisplatin, carboplatin and oxaliplatin treatment at sub-lethal doses of selected tumour cell lines. Here we report on the influence of temperature on dose-dependent antiproliferative effects, cellular uptake and DNA platination of these platinum-based drugs tested on MCF-7 human mammary carcinoma cell line. Inductively coupled plasma-mass spectrometry (ICP-MS) technique has been chosen to perform Pt determinations on cells treated with drug concentrations similar with those usually found in vivo in human plasma. The high sensitivity and analytical rapidity of this technique made possible to carry out a very large amount of Pt determinations (about 300) necessary for this study. Hyperthermia (43°C) proved a synergistic effect with cisplatin on cell growth inhibition, while only an additive effect was demonstrated for carboplatin and oxaliplatin. This behaviour might be explained by the higher DNA platination ratio between data at 43 and 37°C of cisplatin with respect to those of carboplatin and oxaliplatin.

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

BackgroundThe sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer.MethodsNIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with pre-existing pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies).ResultsIn this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray.ConclusionThus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer.

Inhibition of human cytochrome p450 1b1 further clarifies its role in the activation of dibenzo[a,l]pyrene in cells in culture

Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo[a,l]pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3' ,5'-tetramethoxy-stilbene (TMS). Results from DBP-DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP-DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture.

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.