MCF-7 Human Breast Carcinoma Research Articles

Novel Surfactant‐like Hypocrellin Derivatives to Achieve Simultaneous Drug Delivery in Blood Plasma and Cell Uptake

Water-soluble derivatives of hypocrellins can be safely delivered in blood plasma but lose their photodynamic activity in vivo due to poor cell uptake, while hydrophobic derivatives retaining their activity may aggregate in the blood plasma and block vascular networks. Considering both drug delivery and biological activity, surfactant-like hypocrellin B (HB) derivatives, sodium 12-2-HB-aminododecanoate (SAHB) and sodium 11,11'-5,8-HB-dimercaptoundecanoate (DMHB), were first designed and then synthesized in the current work. Both SAHB and DMHB were photoactive, generating free radicals and reactive oxygen species, as confirmed by EPR and chemical measurements. Most importantly, DMHB was not only readily soluble, allowing preparation of an intravenous injection solution at a clinically acceptable concentration, but it was also more photodynamic therapy (PDT) active to human breast carcinoma MCF-7 cells than its parent HB under irradiation. The photodynamic activity was exactly identical to the (1)O(2) quantum yield and was not reduced by the improved water solubility, suggesting an independent hydrophilicity or lipophilicity. To our knowledge, this is a new strategy that possesses general significance for converting hydrophobic photosensitizers into clinically usable PDT drugs.

Abstract 3206: A novel prodrug of the green tea polyphenol (−)-epigallocatechin-3-gallate inhibits the telomerase gene by modifying DNA methylation and histone acetylation in breast cancer cells

Abstract Epigallocatechin-3-gallate (EGCG), a major component of green tea polyphenols (GTPs), has been reported to down-regulate telomerase activity in breast cancer cells thereby increasing cellular apoptosis and inhibiting cellular proliferation. However, the major concerns with GTPs and EGCG are the bioavailability and poor stability under physiological conditions. In the present study, we have observed that treatments with EGCG and a novel pro-drug of EGCG (Pro-EGCG) dose-dependently inhibit the cellular proliferation of human breast carcinoma MCF-7 and MDA-MB-231 cells. Inhibition of telomerase has received wide attention in breast cancer prevention because of its unique expression in cancer cells and low level of expression in normal mammary epithelial cells. Both EGCG and Pro-EGCG inhibited the transcription of hTERT (human telomerase reverse transcriptase) by epigenetic modification in MCF-7 and MDA-MB-231 cells. The inhibition of hTERT expression was consistently correlated with hTERT promoter hypomethylation and deacetylation. Chromatin immunoprecipitation (ChIP) analysis of the hTERT promoter revealed that EGCG and Pro-EGCG increased the level of inactive trimethyl H3K9, but decreased active dimethyl H3K4 and acetyl H3K9. In addition, EGCG and Pro-EGCG significantly inhibited DNA methyltransferase (DNMT) activity, which might have an important role in the down-regulation of hTERT by demethylation of the CpG sites in the hTERT promoter in breast cancer cells. Bisulfite sequencing of the hTERT promoter confirmed the induced hypomethylation of the hTERT promoter by EGCG and Pro-EGCG. Down-regulation of hTERT was also mediated through significant inhibition of histone acetyltransferase (HAT), an enzyme responsible for histone acetylation, by EGCG and Pro-EGCG. Histone deacetylase (HDAC) activity was not altered by EGCG and Pro-EGCG, which reveals that EGCG- and Pro-EGCG-induced histone deacetylation was mediated through inhibition of HAT, but not by effects on HDACs in these breast cancer cells. These results show that EGCG and Pro-EGCG are inhibiting the cellular proliferation and inducing apoptosis in these breast carcinoma cells but not in normal control mammary epithelial MCF10A cells, at least in part, through epigenetic modification of telomerase expression. Further, our results also show that Pro-EGCG is more stable than EGCG and required a lesser dose than EGCG to facilitate the same cellular and epigenetic effects in breast carcinoma cells. Collectively, our data provide new insights into breast cancer prevention through epigenetic modulation of telomerase by using Pro-EGCG, a more stable form of EGCG, as a novel chemopreventive drug. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3206.

Abstract 4878: Bioactive dietary supplements reactivate ER expression in ER-negative breast cancer cells by epigenetic mechanisms involving active chromatin modification

Abstract Breast cancer is the second leading cause of cancer-related death among women in the United States. One of the important classifications of breast tumors is based on the presence or absence of the estrogen receptor (ER). While the majority of breast cancers are ER-positive, approximately 30% are ER-negative. Patients with ER-positive breast cancer receive hormonal therapy using either tamoxifen or aromatase inhibitors, and have a better prognosis. However, treatment of patients with ER-negative tumor is challenging due to the poor response to hormonal therapies in the absence ER expression. Many studies have demonstrated the chemopreventive properties of green tea polyphenols (GTPs) and sulforaphane (SFN) through inhibition of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activity, respectively. In the present study, we have observed that GTPs and SFN dose-dependently inhibit the cellular proliferation in both ER-positive and ER-negative human breast carcinoma MCF-7 and MDA-MB-231 cells, respectively. Treatment of ER-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ER mRNA expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ER reactivation at 3 days in MDA-MB-231 cells. The reactivation of ER expression was consistently correlated with ER promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ER promoter revealed that GTPs and SFN decreased the level of dimethyl H3K9, but increased the acetylation by increasing the level of acetyl H3, acetyl H4 and acetyl H3K9. In addition, a combination of GTPs and SFN significantly inhibited DNMT and HDAC activity, which might have an important role in the reactivation of ER expression in MDA-MB-231 cells. The combination of GTPs- and SFN-mediated inhibition of DNMT activity and increase of acetylation underscores the importance of DNMTs and HDACs in maintaining the repressive environment at target genes such as ER. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that HDAC inhibitors combined with demethylating agents are a promising strategy for the epigenetic reactivation of ER expression for the effective treatment of ER-negative breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4878.

Abstract 2485: Inhibitory effects of the water extract of Nelumbo Nucifera leaf on human breast carcinoma MCF-7 cells in vitro and in vivo

Abstract Over the passed decade, advances in understanding carcinogenesis have made possible the identification of the candidates of chemopreventive agents to be developed in targeting the key molecules. Lotus (Nelumbo nucifera Gaertn) is widely distributed in central and southern Taiwan. Its leaves are becoming popular as an ingredient of healthy beverages. Nelumbo nucifera Leaf extract (NLE) has been reported to exhibit different therapeutic effects, such as anti-oxidant and reduction of risks for cardiovascular disease. This study was aimed to investigate the antitumor effect of NLE on human MCF-7 cell line in vitro and in vivo. Qualitative analysis showed that the NLE contains several compounds using nine kinds of standard polyphenols by high-performance liquid chromatography (HPLC) method. The polyphenols present in identified compound of NLE were mainly gallic acid, rutin and quercetin. The MTT data showed no significant inhibition on growth of MCF-7 cells following NLE treatment, whereas flow cytometry revealed that the cell cycle was arrested at G0/G1 phase with a dose of 3 mg/ml in MCF-7 cells (approximately 86.54%). With western blotting, we found that treatment with NLE particularly induced p53 phosphorylation and p21 expression, and down-regulated expression of cyclins (cyclin D, cyclin E) and cyclin-dependent kinases (cdk2, cdk4) in a dose-dependent manner. The increased p21 also correlated with the decrease of cyclin D/cdk4 and cyclin E/cdk2 complexes, which was responsible for the high protein expression of Rb/E2F. Inactivation of fatty acid synthase (FAS) was shown in vitro and in vivo, implying that FAS was a potent regulator as well of NLE- mediated anti-carcinogenesis. The tumoricidal effect of NLE was further measured using a xenograft nude mice model. Treatment with NLE (0.5 and 1%) effectively reduced the tumor volume and tumor weight in the nude mice inoculated with MCF-7 cells when compared to the control tissue samples. These results confirmed that cell cycle arrest is sufficient to elicit tumor regression after NLE treatment. In conclusion, our studies indicate that NLE offers an anti-tumor activity and potentially can act as a chemotherapeutic agent. 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 2485.

Abstract 5219: Relationship between intratumoral drug distribution and tumor microenvironment

Abstract Purpose: Anticancer agents released from nanocarriers have to reach cancer cells in order to demonstrate their cytotoxic activity. The distribution pattern of anticancer agents can be affected by the tumor microenvironment which varies among different types of tumors. The purpose of this study is to delineate the relationship between drug transport and tumor microenvironmental factors, such as extracellular matrix (ECM) integrity. Such studies provide insight into the factors that hinder the accessibility of cargo molecules to cancer cells. Hypothesis: In tumors with a “permissive” ECM, small molecules are likely to diffuse farther after release from nanocarriers, while they are likely to be hindered in the tumors with denser ECM. Non-invasive monitoring of drug release and subsequent intratumoral distribution of the released drug can be visualized using a dual MR contrast technique capable of distinguishing between released and intact molecules in vivo. Methods: GdDTPA was used as a surrogate tracer for a water-soluble, small molecular-weight drug. Liposomes were used as the model nanocarrier in this study. Xenografts derived from the highly-invasive and metastatic MDA-MB-231 human breast carcinoma cell line, and weakly-metastatic MCF-7 human breast carcinoma cells were used. Liposomes encapsulating GdDTPA, iron particles, and rhodamine (Lip-Gd/Fe/Rho) were prepared using a sonication method, followed by extrusion. Orthotopic xenografts were grown in female SCID mice by injecting 3×106 of cancer cells into the mammary fat pad of the animal. All MR studies were carried out on a horizontal bore Bruker Biospec 9.4T spectrometer. MR images were acquired before, 30 min, and 23 hrs post-i.v. administration of Lip-Gd/Fe/Rho. To identify iron particles, a 3D fast low-angle shot (FLASH) sequence was used, while a 3D fast spin echo (RARE: Rapid Acquisition with Relaxation Enhancement) sequence with an effective echo time of 50 ms and five different repetition times was acquired to track GdDTPA released from liposomes. After completion of the MRI study, tumors were excised, fixed and were cut into 10μm sections for imaging with second harmonic generation multiphoton microscopy. Region of interest analysis were carried out on the MRI data. Results/Discussion: In vivo MRI results demonstrated that a decrease in T1 of the tumors was observed following i.v. administration of the Lip-Gd/Fe/Rho, indicating successful delivery and degradation of liposomes in the tumor. The comparison of differences in liposome delivery and its correlation with collagen-I density in the ECM of MDA-MB-231 and MCF-7 tumors is currently underway. Our results demonstrate the feasibility if using complementary imaging techniques to characterize the factors affecting intratumoral distribution of drug molecules after a release from nanocarriers. Acknowledgment: Supported by NIH grant R21 EB008162 (YK). 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 5219.

Synthesis, antiproliferative activity evaluation and structure–activity relationships of novel aromatic urea and amide analogues of N-phenyl- N′-(2-chloroethyl)ureas

Seven subsets of aromatic urea and amide analogues of N-phenyl- N′-(2-chloroethyl)ureas (CEU) have been synthesized by nucleophilic addition of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlorobutanoyl chloride, and acryloyl chloride, respectively, to selected anilines or benzylamines to afford 3-chloropropylureas ( 1, CPU), 2-chloroacetylureas ( 2, CAU), ethylureas ( 3, EU), 2-chloroacetamides ( 4, CA), 3-chloropropionamides ( 5, CPA), 4-chlorobutyramides ( 6, CBA) and acrylamides ( 7, Acr). The molecular structure of these compounds has been confirmed by IR, 1H and 13C NMR, and MS spectra and their purity also confirmed by HPLC. The CEU analogues were evaluated for their antiproliferative activity against three human tumor cell lines, namely human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7. CAU ( 2c to 2g), CA ( 4a to 4d, 4f and 4g), CPA ( 5a) and Acr ( 7a and 7b) had IC 50 ranging from 1.4 to 25 μM. CAU, CA, CPA and Acr exhibited interesting antiproliferative activity through mechanism(s) of action unrelated to the acylation of glutamic acid at position 198 on β-tubulin that is characterizing CEU.

Lead identification of conformationally restricted benzoxepin type combretastatin analogs: synthesis, antiproliferative activity, and tubulin effects

We have synthesized a series of polymethoxylated rigid analogs of combretastatin A-4 which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products. The compounds display antiproliferative activity when evaluated against the MCF-7 and MDA human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepine (11g) was found to be the most potent product when evaluated against the MCF-7 breast cancer cell line. A brief computational study of the structure–activity relationship for the synthesized compounds is presented. These 4,5-diarylbenzoxepins are identified as potentially useful scaffolds for the further development of antitumor agents which target tubulin.

The effect of siRNA-Egr-1 and camptothecin on growth and chemosensitivity of breast cancer cell lines

We have examined the effects of a siRNA targeting the Egr-1, alone or in combination with the breast cancer therapeutic camptothecin (Cpt), in suppressing breast cancer cell survival and anchorage-independent growth in the breast cancer cell lines SK-BR3 and MCF-7. In mammary and lung tumors, as well as most normal tissues, Egr-1 expression is low, suggesting a possible relation between the low levels of Egr-1 and the development of mammary neoplasias. However, analyses of the expression of Egr-1 in breast carcinoma cells, SK-BR3 and MCF-7 demonstrated a relatively high expression of the endogenous Egr-1 in these cells. To investigate the effect of the blocking of the endogenous Egr-1 in breast cancer cells, we used small interfering RNA (siRNA) against Egr-1 alone or in combination with Cpt, and expected that the cell sensitivity to chemotherapeutic drug would increase, when blocked with the Egr-1 gene and treated with Cpt. Thus, we performed in vitro experiment to clarify the effect of Egr-1 on tumor cell lines growth. We made control and siRNA-Egr-1 using vector plasmids and then transfected SK-BR3 and MCF-7 cells. After treating the cells with siRNA-Egr-1, the cell lines were assayed with Cpt to confirm the effect of Egr-1 siRNA using the cell expression of mRNA and protein, proliferation assay and anchorage activity with soft agar. Human SK-BR3 and MCF-7 breast carcinoma cell growth and capacity of anchorage transfected with siRNA-Egr-1 or treated with cpt was slower than that of the control group. This effect was increased when the cells were given simultaneously siRNA and camptothecin. The results strongly suggest that siRNA-Egr-1 alone or in combination with camptothecin could be a potent antineoplastic agent in suppressing the growth of breast tumor despite the known role of Egr-1 as a tumor-suppressor in several other types of human cancers.

Synthesis, characterization, antibacterial and antiproliferative activities of monodisperse chitosan- based silver nanoparticles

Silver nanoparticles (AgNPs) have been known to have inhibitory and bactericidal effects. In this paper we investigated: (i) the synthesis and kinetics of finely dispersed AgNPs in a biocompatible chitosan-derived polysaccharide solution; (ii) the antibacterial activities toward both gram− ( Escherichia coli and Pseudomonas aeruginosa) and gram+ bacteria ( Lactobacillus fermentum, Staphylococcus aureus and Baciilus subtilis) and yeast ( Candida albicans) of the obtained AgNPs; and (iii) in vitro cytotoxicity against HepG2 (hepatocellular carcinoma), Lu (human lung carcinoma), KB (human epidermic carcinoma), MCF-7 (human breast carcinoma) cancer cell lines. The samples demonstrated considerably high antibacterial and antiproliferative activities against above bacterial strains and cell lines, which can be explained on the basis of finely and homogenously dispersed nanoparticles.

Hyul-Tong-Ryung suppresses PMA-induced MMP-9 expression by inhibiting AP-1-mediated gene expression via ERK1/2 signaling pathway in MCF-7 human breast cancer cells

Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase1/2 (ERK1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase1/2 (ERK 1/2) signaling pathway.

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines - human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells - were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.

Anti-Tumoral Activity of a Short Decapeptide Fragment of the Alzheimer’s Aβ Peptide

The inhibition of angiogenesis is regarded as a promising avenue for cancer treatment. Although some antiangiogenic compounds are in the process of development and testing, these often prove ineffective in vivo, therefore the search for new inhibitors is critical. We have recently identified a ten amino acid fragment of the Alzheimer Abeta peptide that is anti-angiogenic both in vitro and in vivo. In the present study, we investigated the antitumoral potential of this decapeptide using human MCF-7 breast carcinoma xenografts nude mice. We observed that this decapeptide was able to suppress MCF-7 tumor growth more potently than the antiestrogen tamoxifen. Inhibition of tumor vascularization as determined by PECAM-1 immunostaining and decreased tumor cell proliferation as determined by Ki67 immunostaining were observed following treatment with the Abeta fragment. In vitro, this peptide had no direct impact on MCF-7 tumor cell proliferation and survival suggesting that the inhibition of tumor growth and tumor cell proliferation observed in vivo is related to the antiangiogenic activity of the peptide. Taken together these data suggest that this short Abeta derivative peptide may constitute a new antitumoral agent.

Synthesis, molecular structure, and validation of metalloprobes for assessment of MDR1 P-glycoprotein-mediated functional transport

The human genome is known to consist of 49 ATP-binding cassette (ABC) transporter genes. Among these ABC proteins, overexpression of multidrug resistance (MDR1) P-glycoprotein (Pgp/ABCB1) is the best characterized barrier to successful chemotherapeutic treatments, impacts pharmacokinetics of numerous recognized drugs, and is also quickly emerging as an important target in the development of neurodegenerative diseases. Therefore, there exists an urgent need to seek radiopharmaceuticals, incorporated with generator-produced radionuclides to assist their widespread deployment, for noninvasive assessment of Pgp-mediated functional transport activity in vivo. gallium(III) complexes (5a and 5b) possessing octahedral geometry were synthesized, analytically characterized, and evaluated for their potential to serve as probes of Pgp-mediated functional transport activity in cellulo and in vivo. While unlabeled compounds (5a and 5b) were examined via cell cytotoxicity assays, the (67)Ga-labeled counterparts (6a and 6b) were evaluated via cell transport studies and quantitative biodistribution studies in mdr1a/1b((-/-)) gene-deleted mice and their wild-type (WT) counterparts. cytotoxicity data of 5a and 5b displayed profiles modified by the expression of Pgp in drug-resistant cells. (67)Ga-metalloprobes (6a and 6b) showed high accumulation in human epidermal carcinoma drug-sensitive KB-3-1 cells (Pgp-), human breast carcinoma MCF-7 (Pgp-) cells; an inhibitor (LY335979, 1 microM) induced accumulation in multidrug resistant (MDR, Pgp+) KB-8-5, KB-8-5-11 cells, and stably transfected MCF-7/MDR1 cells, thus demonstrating their ability to interrogate Pgp-mediated functional transport activity in cellulo. In mdr1a/1b((-/-)) gene-deleted mice, the (67)Ga-metalloprobe (6b) showed 8-fold greater brain uptake and retention compared with WT counterparts and no significant difference in blood pharmacokinetics. molecular imaging of the functional transport activity of MDR1 Pgp (ABCB1) with (67/68)Ga-metalloprobes could enable non-invasive monitoring of the blood-brain barrier, tumors, and tissues in vivo.

Effect of Modulation of hnRNP L Levels on the Decay of bcl-2 mRNA in MCF-7 Cells

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.

Grape Seed Proanthocyanidins Ameliorate Doxorubicin-Induced Cardiotoxicity

Doxorubicin (Dox) is one of the most widely used and successful chemotherapeutic antitumor drugs. Its clinical application is highly limited due to its cumulative dose-related cardiotoxicity. Proposed mechanisms include the generation of reactive oxygen species (ROS)-mediated oxidative stress. Therefore, reducing oxidative stress should be protective against Dox-induced cardiotoxicity. To determine whether antioxidant, grape seed proanthocyanidin extract (GSPE) attenuates Dox-induced ROS generation and protects cardiomyocytes from Dox-induced oxidant injury, cultured primary cardiomyocytes were treated with doxorubicin (Dox, 10 microM) alone or GSPE (50 microg/ml) with Dox (10 microM) for 24 hours. Dox increased intracellular ROS production as measured by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, induced significant cell death as assessed by propidium iodide, and declined the redox ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) and disrupted mitochondrial membrane potential as determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethlbenzimidazole-carbocyanide iodine (JC-1). Analysis of agarose gel electrophoresis revealed Dox-induced nuclear DNA damage with the ladder like fragmentation. GSPE treatment suppressed those alterations. Electron Spin Resonance (ESR) spectroscopy data also showed that GSPE strongly scavenged hydroxyl radical, superoxide and DPPH radicals. Together, these findings indicate that GSPE in combination with Dox has protective effect against Dox-induced toxicity in cardiomyocytes, which may be in part attributed to its antioxidative activity. Importantly, flow cytometric analysis demonstrated that co-treatment of Dox and GSPE did not decrease the proliferation-inhibitory effect of Dox in MCF-7 human breast carcinoma cells. Thus, GSPE may be a promising adjuvant to prevent cardiotoxicity without interfering with antineoplastic activity during chemotherapeutic treatment with Dox.

Estrogenic activities of two synthetic pyrethroids and their metabolites

Synthetic pyrethroids (SPs) are among the most common pesticides in current use, and so far, several SPs have been assessed for their potential estrogenicities by various methods. Previous studies have shown that the estrogenicities partly come from their metabolites. Although considerable information is available with respect to the metabolism and environmental degradation of SPs, little is known about the estrogenicities of the metabolites. In this study, permethrin (PM) and β-cypermethrin (CP), as well as their metabolites (3-phenoxybenzoic alcohol (PBCOH), 3-phenoxybenzaldehyde (PBCHO) and 3-phenoxybenzoic acid (PBCOOH) were evaluated for their estrogenic activities in the MCF-7 human breast carcinoma cell line. In the MCF-7 cell proliferation assay, PM and CP exhibited significant estrogenic activities at 10 −7 mol/L, comparable to 17β-estradiol (E 2) of 10 −9 mol/L, with the relative proliferative effect ratios of 55.4% and 56.3%, respectively. The real-time quantitative polymerase chain reaction (qRT-PCR) results confirmed the estrogenicities of PM and CP with significant alteration of pS2 and ERα mRNA levels observed at 10 −6 mol/L. For the three major metabolites, PBCOH and PBCOOH exhibited estrogenic activities in all assays, while no significant estrogenic responses was observed for PBCHO compared to the vehicle control. In particular, PBCOH had even slightly stronger estrogenic activity than its parent compounds, indicating that metabolism may be one of the reasons for the estrogenicities of the SPs. Given the widespread use of SPs, the toxicological effects of parent compounds and their metabolites should be taken into consideration in the risk assessment of SPs.

Shu-Gan-Liang-Xue Decoction, a Chinese herbal formula, down-regulates the expression of steroid sulfatase genes in human breast carcinoma MCF-7 cells

Ethnopharmacological relevance Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula, has been used to ameliorate hot flushes symptom in breast cancer patients for decades. Aim of the study Steroid sulfatase (STS) has a crucial role in regulating the estrogen biosynthesis within breast tumors. We aimed to investigate whether SGLXD could modulate STS expression in human breast carcinoma MCF-7 cells. Materials and methods By semi-quantitative/quantitative reverse transcription-PCR, we investigated the transcript level of STS in MCF-7 cells treated with various concentrations of SGLXD. By using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells and dual luciferase reporter (DLR) based bioluminescent measurements, we evaluated the enzymatic activity of STS after SGLXD treatment. Results By RT-PCR and real time PCR, the mRNA level of STS was decreased by SGLXD treatment, in the dose-dependent manner, compared to negative control ( p < 0.01). By DLR assay, different concentrations of SGLXD significantly inhibited the enzymatic activity of STS in MCF-7 cells dose-dependently ( p < 0.05). Conclusions SGLXD could act as a selective estrogen enzyme modulator by down-regulating the STS expression in MCF-7 cells.

Role of Glutathione Transferases in the Mechanism of Brostallicin Activation

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.

The pyrethroid metabolites 3-phenoxybenzoic acid and 3-phenoxybenzyl alcohol do not exhibit estrogenic activity in the MCF-7 human breast carcinoma cell line or Sprague–Dawley rats

Synthetic pyrethroids are one of the most frequently and widely used class of insecticides, primarily because they have a higher insect to mammalian toxicity ratio than organochlorines or organophosphates. The basic structure of pyrethroids can be characterized as an acid joined to an alcohol by an ester bond. Pyrethroid degradation occurs through either oxidation at one or more sites located in the alcohol or acid moieties or hydrolysis at the central ester bond, the latter reaction being important for mammalian metabolism of most pyrethroids. The primary alcohol liberated from the ester cleavage is hydroxylated to 3-phenoxybenzyl alcohol, which for most pyrethroids is then oxidized to 3-phenoxybenzoic acid. These products may then be conjugated with amino acids, sulfates, sugars, or sugar acids. In vitro studies have suggested that some of the pyrethroids may have estrogenic activity. Interestingly, the chemical structure of specific pyrethroid metabolites indicates that they may be more likely to interact with the estrogen receptor than the parent compounds. Two of the pyrethroid metabolites, 3-phenoxybenzoic acid (3PBA) and 3-phenoxybenzyl alcohol (3PBalc) have been reported to have endocrine activity using a yeast based assay. 3PBAlc exhibited estrogenic activity with reported EC 50s of 6.67 × 10 −6 and 2 × 10 −5 while 3PBAcid exhibited anti-estrogenic activity with a calculated IC 50 of 6.5 × 10 −5. To determine if the metabolites were able to cause the same effects in a mammalian system, the estrogen-dependent cell line, MCF-7, was utilized. Cells were treated with 1.0, 10.0 or 100.0 μM concentrations of each metabolite and cytotoxicity was assessed. The two lowest concentrations of both metabolites did not induce cell death and even appeared to increase proliferation over that of the control cells. However, when cellular proliferation was measured using a Coulter counter neither metabolite stimulated proliferation (1.0 nM, 10.0 nM, or 10.0 μM) or induced an estrogen receptor α/ERE-controlled luciferase reporter in the MCF-7 cells. Following the in vitro screenings, the metabolites were then evaluated for estrogenic activity in vivo using the uterotrophic assay in Sprague–Dawley rats. Animals were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once daily for 3 days. Neither metabolite had any effect on uterine wet weight, body weight, or organ weight. Lastly, in order to determine if either metabolite was able to alter the onset of puberty, immature female rats were orally gavaged (10.0, 5.0, and 1.0 mg/kg) once a day with the metabolites beginning 1 day post-weaning until the onset of puberty as evidenced by vaginal opening (VO). Again, neither metabolite had any effect on the onset of VO.