Human Mammary Cancer Research Articles

Characterisation and biological activities of proanthocyanidins from the barks of Pinus massonian and Acacia mearnsii

The biological activities and characterisation of proanthocyanidins (PAs) from the barks of Pinus massonian and Acacia mearnsii have been studied in our research. The free-radical scavenging activity of the PAs was measured by means of the DPPH method, and it was clear that the PA product had a strong radical scavenging ability. Furthermore, anti-tumour activities of PAs on different human cancer cells were investigated. The results indicated that PAs from the bark of A. mearnsii had better anti-tumour activities than those from the bark of P. massonian, and the PAs extracted from the ethyl acetate fraction had better anti-tumour activities than those from the water fraction. PAs from the bark of A. mearnsii in an ethyl acetate fraction (PAE) had an effective inhibition on human mammary cancer cells (MDA- MB-231) and human liver cancer cells (BEL-7402), a weak effect on human cervical cancer cells (Hela), but no effect on human lung cancer cells (A549); while the PAs from the bark of A. mearnsii in a water fraction (PAW) had weak effect on MDA- MB-231, Hela and A549, but no effect on BEL-7402. PAs from the bark of P. massonian in ethyl acetate fraction (PPE) had weak effect on Hela and BEL-7402; whereas the PAs from the bark of P. massonian in a water fraction (PPW) had a weak effect on Hela, but no effect on the other cells. PAs were characterised by HPLC- ESI-MS analysis and the PA dimers and trimers were identified, respectively.

Improved plasma stability and sustained release profile of gemcitabine via polypeptide conjugation

To enhance the stability of the anticancer drug gemcitabine (2′-deoxy-2′,2′-difluorocytidine), it was conjugated to poly- l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly- l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48 h, and reached equilibrium at 72 h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug.

Abstract B66: Aggressiveness of MMTV-Wnt-1 mammary tumors corresponds with enrichment of a CD44+/CD24− putative cancer stem cell population

Abstract MMTV-Wnt-1 transgenic mice develop heterogeneous mammary tumors that accurately recapitulate human basal-like breast cancer. Additionally, targeted alterations in the Wnt signaling pathway in mammary epithelial cells preferentially induce mammary cancers from progenitor cells. Therefore, we hypothesized that MMTV-Wnt-1 mammary tumors harbor cells with cancer stem cell characteristics. To test this hypothesis, we characterized (in vitro and in vivo) two newly generated, clonal mammary epithelial cell lines (WA4 and WG4) from a MMTV-Wnt-1 transgenic mouse tumor. The cell lines were analyzed using flow cytometry for CD44 and CD24 cell surface markers (a CD44+/CD24− profile is indicative of putative human and murine mammary cancer stem cells), mammosphere formation (low density seeding on non-adherent plates in stem-cell media), in vitro scratch assay (to measure wound healing), and by an in vitro Matrigel invasion chamber assay (to quantitate cell invasion). Finally, unsorted WA4 and WG4 cell lines were transplanted into syngeneic mammary fat pads of C57BL/6 mice (1×10^7 cells/mouse; n=5/cell line), and tumor burden was assessed after 3 weeks. Flow cytometric analysis demonstrated that the percentage of CD44+/CD24− cells was 62.2% in the WA4 line and 2.4% in the WG4 line. Serum starvation enriched the WA4 line for CD44+/CD24− cells (from 62.2% to 82.0%), without altering the composition of the WG4 line. Approximately 30% of unsorted WA4 cells, but <1% of unsorted WG4 cells, formed mammospheres. Additionally, the WA4 line showed increases in wound healing by scratch assay and significant increases in invasion (226-fold increase versus WG4 cells after 30 hours). In vivo, the WA4 cell line was associated with larger, more aggressive tumors on average (+SD) than the WG4 line (2.09 +/−0.68 g and 0.03 +/−0.02 g, respectively, p<0.0001). WA4 tumors were characterized by increased cellular proliferation, microvascular proliferation, and a basal appearance lacking ductal structures. In conclusion, MMTV-Wnt-1 transgenic mouse mammary tumors harbor a CD44+/CD24− putative cancer stem cell population, and an increased percentage of this population corresponds with a more aggressive tumor phenotype. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B66.

Somatic structural rearrangements in genetically engineered mouse mammary tumors

BackgroundHere we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer.ResultsWe show that although Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines.ConclusionsPaired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions.

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E(2)) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E(2), and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E(2) in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E(2)-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

IntroductionWe have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.MethodsHMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).ResultsvHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.ConclusionsWe have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Inhibition of Rho-Associated Kinase Signaling Prevents Breast Cancer Metastasis to Human Bone

Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility. Aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Here, we show that ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines compared with nonmetastatic tumors and cell lines. Overexpression of ROCK confers a metastatic phenotype on the nonmetastatic MCF-7 cell line. Inhibition of ROCK activity, by either a specific ROCK inhibitor (Y27632) or ROCK-targeted small interfering RNAs, reduces cell migration and proliferation in vitro and metastasis to bone in vivo using a novel "human breast cancer metastasis to human bone" mouse model. Expression of the c-Myc-regulated miR-17-92 cluster is shown to be elevated in metastatic breast cancer cells compared with nonmetastatic cells and diminished by Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. Together, these findings suggest that augmented ROCK signaling contributes to breast cancer metastasis. The effects of ROCK on tumor cell invasion/motility and growth may derive from regulating cytoskeletal actin-myosin contraction and modulating the c-Myc pathway, including c-Myc-dependent microRNAs. Inhibition of ROCK or the pathway it stimulates, therefore, may represent a novel approach for treatment of breast cancer metastases.

Epithelial transformation by KLF4 requires Notch1 but not canonical Notch1 signaling

The transcription factors Notch1 and KLF4 specify epithelial cell fates and confer stem cell properties. Suggesting a functional relationship, each gene can act to promote or suppress tumorigenesis in a context-dependent manner, and alteration of KLF4 or Notch pathway genes in mice gives rise to similar phenotypes. Activation of a conditional allele of KLF4 in RK3E epithelial cells rapidly induces expression of Notch1 mRNA and the active, intracellular form of Notch1. KLF4-induced transformation was suppressed by knockdown of endogenous Notch1 using siRNA or an inhibitor of γ- secretase. Chromatin immunoprecipitation assay shows that KLF4 binds to the proximal Notch1 promoter in human mammary epithelial cells, and siRNA-mediated suppression of KLF4 in human mammary cancer cells results in reduced expression of Notch1. Furthermore, KLF4 and Notch1 expression are correlated in primary human breast tumors (N=89; Pearson analysis, r > 0.5, P

The Antiproliferative Effect of Kefir Cell-Free Fraction on HuT-102 Malignant T Lymphocytes

Kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, bacteria, and yeast) to pasteurized milk; it has been shown to control several cellular types of cancer, such as Sarcoma 180 in mice, Lewis lung carcinoma, and human mammary cancer. Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia, which is a fatal disease with no effective treatment. The current study aims at investigating the effect of a cell-free fraction of kefir on HuT-102 cells, which are HTLV-1–positive malignant T-lymphocytes. Cells were incubated with different kefir concentrations: the cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir cell-free fraction on the proliferation of HuT-102 cells was then assessed. The levels of transforming growth factor (TGF)-α mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction. Finally, the growth inhibitory effects of kefir on cell cycle progression and/or apoptosis were assessed by flow cytometry. The maximum cytotoxicity recorded at 80 μg/μL for 48 hours was only 43%. The percent reduction in proliferation was very significant, dose and time dependent, and reached 98% upon 60-μg/μL treatment for 24 hours. Kefir cell-free fraction caused the downregulation of TGF-α, which is a cytokine that induces the proliferation and replication of cells. Finally, a marked increase in cell cycle distribution was noted in the pre-G1 phase. In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1–positive malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

Melatonin modulates microfilament phenotypes in epithelial cells: implications for adhesion and inhibition of cancer cell migration.

Cell migration and adhesion are cytoskeleton- dependent functions that play a key role in epithelial physiology. Specialized epithelial cells in water transport have specific microfilament rearrangements that make these cells adopt a polyhedral shape, forming a sealed monolayer which functions as permeability barrier. Also, specific polarized microfilament phenotypes are formed at the front and the rear of migratory epithelial cells. In pathological processes such as cancer, increased migration occurs in invasive cells driven by the formation of polarized and differential microfilament phenotypes. Melatonin, the main product secreted by the pineal gland during dark phase of the photoperiod, acts as a cytoskeletal modulator in normal and cancer cells. In this paper we will summarize evidence supporting that melatonin acts as a microfilament modulator in epithelial MDCK cells, and we will describe its effects on cytoskeleton organization involved in the mechanism by which melatonin synchronizes water transport. In addition, we will review recent data that indicate that melatonin is able to switch microfilament phenotypes in MCF-7 human mammary cancer cells, from invasive migratory cells to dormant microfilament phenotypes that occur in non- migratory cells. Moreover, we will discuss the implications of the cytoskeleton as therapeutic target for cancer cells.

A cyclized peptide derived from α fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

0002 Adiponectin differentially affects gene expression in human mammary epithelial cells and breast cancer cells

0002 Adiponectin differentially affects gene expression in human mammary epithelial cells and breast cancer cells

Low Levels of Both Xanthine Dehydrogenase and Cellular Retinol Binding Protein Are Responsible for Retinoic Acid Deficiency in Malignant Human Mammary Epithelial Cells

The seeming impairment of retinoid metabolism in human breast tumor cells has been attributed to the lower expression of cellular retinol binding proteins (CRBPs), of alcohol/retinol dehydrogenases, or aldehyde/retinaldehyde dehydrogenases. In a previous study we indicated that xanthine dehydrogenase (XDH) is able to oxidize actively both all-trans-retinol (t-ROL) bound to the CRBP (holo-CRBP) and all-trans-retinaldehyde (t-RAL) to all-trans-retinoic acid (t-RA) in human mammary epithelial cells (HMEC). Since both XDH and CRBP are required for the biosynthesis of t-RA, we have inspected their bioavailability in both estrogen-responsive and nonresponsive human mammary epithelial cancer cells. The XDH activity, as assessed in the crude and purified extracts of both MCF7 and MDA-MB 231 cells by measuring the substrate t-RAL (that unlike t-ROL does not need CRBP), was 6 to 10 times lower than that previously encountered in normal HMEC. In addition, CRBP expression was absent in either cell line. Based on this preliminary evidence, we propose here that the low levels of XDH activity and the associated absence of CRBP in both MCF7 and MDA-MB 231 human breast cancer cells might be responsible for the retinoic acid deficiency observed in these cell model systems. This defect may be the crux of the impairment to stem cell differentiation and, hence, may be primarily implicated in human mammary carcinogenesis.

Low levels of both xanthine dehydrogenase and cellular retinol binding protein are responsible of the retinoic acid deficiency in malignant human mammary epithelial cells.

Abstract Abstract #4156 The elucidation of the molecular mechanisms underpinning cell differentiation in mammary epithelium is of broad interest and may help understanding the inability of cancer cells to differentiate. In the differentiation of epithelial cells the pivotal role of retinoic acid (RA) was demonstrated early and its deficiency in human mammary cancer cells has been ascribed to the absence of the cellular retinol binding proteins (CRBPs) and the resulting inactivity of the retinol/retinaldehyde dehydrogenase (1,2). We have previously indicated that the enzyme xanthine dehydrogenase (XDH) is able to oxidize actively both all-trans-retinol (t-ROL), bound to the retinol binding protein (CRBP), and all-trans-retinaldehyde (t-RAL) to all-trans-retinoic acid (t-RA) in non-malignant human mammary epithelial cells (HMEC) (3). Since both XDH and CRBP are required for the biosynthesis of t-RA, we have inspected their activity and bioavailability in estrogen-responsive and nonresponsive human mammary epithelial cancer cells. In both MCF7 e MDA-MB 231 mammary tumor cells, retinol oxidation by XDH to RA could not be detected. The XDH activity, as measured in the crude and purified extracts of both cell lines by assaying the substrate t-RAL (that unlike t-ROL does not need CRBP binding) was six to ten times lower than that previously encountered in normal mammary epithelial cells HMEC. In addition, CRBP expression was absent in either cell line. We have also examined the effects of estrogen on both XDH activity and expression in normal and malignant mammary epithelial cells. The exposure of both MCF7 and MDA-MB231 cells to estradiol concentrations ranging from 10 pM up to 100 nM resulted in a marked inhibitory effect on the XDH-driven oxidation of retinaldehyde, with a 34% to 86% reduction with respect to untreated control cell cultures. Furthermore, the XDH protein content, as determined by immunoassay, showed a consistent decrease of 40% with respect to control at 100 nM estradiol. Based on this combined evidence, we propose here that the low levels of XDH activity and the concurrent absence of CRBP in both MCF7 and MDA-MB 231 human breast cancer cells may be responsible for the retinoic acid deficiency observed in these cell model systems. In addition, our data support the concept that estrogen controls negatively retinoic acid bioavailability in mammary tumor cells through the inhibition of XDH enzyme activity and expression. In this context, the impairment of RA biosynthesis may be crucial to disrupt stem cell differentiation and, hence, to promote mammary carcinogenesis and tumor progression.
 References
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The antineoplasic effect of molecular iodine on human mammary cancer involves the activation of apoptotic pathways and the inhibition of angiogenesis.

Abstract Abstract #3082 Breast cancer is the most frequent malignant neoplasia in women. One factor thought to influence the incidence of breast cancer is diet. Japanese women, who have a high dietary intake of iodine (5280 vs 209 µg/day in western population) due to ingestion of seaweed, have the lowest incidence of breast cancer in the world. In normal mammary gland, iodine deficiency is involved in dysplasias, which are reversible with I2 but not with iodide administration. Recent data generated in our laboratory showed that continuous treatment with I2, but not iodide, has a potent antineoplastic effect on tumoral progression in N-methyl-N-nitrosourea-treated virgin rats (50-70%). Also I2 treatment in MCF-7 human breast cancer cells, but not in normal mammary cells (MCF-12F), shows a significant apoptotic effect induced by activation of Bax-caspases and AIF-PARP-1 pathways. Based on these findings, a study of women diagnosed with cancer (mammography and tru-cut biopsy) was initiated. Patients were divided into placebo or I2 (5 mg/day) groups for 2 to 5 weeks before surgery. One day before beginning the I2 treatment and the day of the surgery, urine and blood samples were collected. The day before surgery, a second mammography was obtained. In the tumor samples proliferation (PCNA), apoptosis (TUNEL), and vasculature area (CD-31) were analyzed by immunohistochemistry. Bax, Bcl2, p53, and VEGF proteins were analyzed by Western blot. Circulating TSH and T3 levels were measured by RIA and urinary iodide by HPLC. The results showed that although tumor size did not differ between groups, a significantly lower proliferation and increased apoptotic rate were observed in tumors from women supplemented with iodine. In these tumors the ratio Bax/Bcl2 increased, p53 levels were unchanged, and VEGF levels and vasculature area diminished significantly. Serum TSH and T3 showed no changes in any group, indicating that I2 treatment had not compromised the thyroid status. We propose that I2 supplements should be considered for use in clinical trials of breast cancer therapies. We thank Dr. Jorge Álvarez-Aguirre2,3, Dr. Alonso Gallegos-Corona2, Lic. Concepción Correa-Tinajero3, Alejandro Núñez-Nolasco2,3, and Silvia Serrato-Águila2. This work was partially supported by: PAPIIT-UNAM IN201207, and CONACyT 44976-M and 85952. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3082.

Lack of noggin expression by cancer cells is a determinant of the osteoblast response in bone metastases: Ruth Schwaninger a, Cyrill A. Rentsch a, Antoinette Wetterwald a, Geertje van derHorst c, Rutger L. van Bezooijen b,d, Gabri van der Pluijm b,c, Clemens W.G.M. Löwik b, Karin Ackermann e, Walter Pyerin e, Freddie C. Hamdy f, George N. Thalmann a, Marco

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

SERS-based plasmonic nanobiosensing in single living cells.

In this paper, we describe the development and application of a pH-sensitive plasmonics-active fiber-optic nanoprobe suitable for intracellular bioanalysis in single living human cells using surface-enhanced Raman scattering (SERS) detection. The effectiveness and usefulness of SERS-based fiber-optic nanoprobes are illustrated by measurements of intracellular pH in HMEC-15/hTERT immortalized "normal" human mammary epithelial cells and PC-3 human prostate cancer cells. The results indicate that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor cellular microenvironments at the single cell level.

Antitumor effect of substituted quinolines in breast cancer cells

AbstractCancer is caused in part by the disruption in cell homeostasis, affecting the ability to respond to extracellular signals, triggering some intracellular events that affect gap junctional intercellular communication (GJIC). Cancer cells have reduced or altered GJIC capacity. One feasible approach to reduce growth of cancer cells is to enhance/alter the GJIC. The capability of cells to communicate through the gap junction is negatively related to their growth activity. A computational docking study showed that a new class of substituted quinolines designated as PQs had a relatively high binding to gap junction protein, connexin 43. Thus, PQs were used in this study to assess their effect on human breast cancer cells. Scrape load/dye transfer and colony growth assays were performed to measure GJIC and determine the effect of the PQ compounds on colony formation in human normal mammary epithelial cells (HMEC) and breast cancer cells, respectively. PQs had a significant antitumor effect in human breast cancer cells compared with control without treatment or normal mammary epithelial cells. PQ1 (200 nM) showed a 30% increase in the GJIC in T47D cells; however, there was no effect of PQ treatment on GJIC in normal mammary epithelial cells. In addition to an increase in GJIC, 80–95% growth attenuation was observed by PQ1 in colony growth assay. Moreover, an increase in caspase 3 with PQ‐treated cells was observed, suggesting a possible involvement in apoptosis. PQ1‐treated animals showed a significant decrease in xenograft tumor growth of T47D cells in nude mice compared with control or tamoxifen‐treated animals. The results show that PQ1 has a promising role in exerting antitumor activity in human breast cancer cells. Treatment with PQ1 in T47D cells caused an increase in GJIC activity and active caspase 3, and a decrease in colony growth and cell viability. Drug Dev Res 69:526–534, 2008. © 2008 Wiley‐Liss, Inc.

Tumor-Mediated Suppression of Myeloid to Dendritic Cell Differentiation Via Down Regulation of Protein Kinase C βII Expression

Cancer generates a state of immune suppression that contributes to tumor out-growth and the escape phase of the immune editing of nascent tumors. One hallmark of tumor mediated immune suppression is a decreased number of dendritic cells (DC) and an accumulation of immature myeloid cells (myeloid derived suppressor cells, MDSC) that are themselves directly immunosuppressive. Impairment of DC differentiation is mediated by numerous tumor derived factors (VEGF, GM-CSF, IL-6, and M-CSF) that activate Stat3. The mechanism by which Stat3 signaling subsequently inhibits DC differentiation has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor à DC differentiation and that knock down of PKC βII expression blocks DC differentiation. This leads us to hypothesize that tumor derived factor activation of Stat3 may inhibit DC differentiation by down regulating PKC βII expression. To test our hypothesis, we utilized the hematopoietic progenitor-like cell line KG1. In response to the phorbol ester PMA, KG1 differentiates into a DC-like cell (KG1-DC). KG1 cultured in media conditioned by a human (MCF-7) or murine (DA3) mammary cancer cell line had a 48% (DA3) and 51% (MCF-7) reduction in PKC βII protein levels. Additionally, tumor conditioned media treatment significantly decreased PKC βII mRNA transcript levels (38-fold reduction compared to untreated, p<0.01). According to our proposed model, decreased PKC βII expression should inhibit DC differentiation. Consistent with this, KG1 cultured in MCF-7 conditioned media and then differentiated to KG1-DC elicited significantly less allogeneic T cell proliferation (a key measure of DC function) than KG1-DC generated in control media (p<0.01). To test the role of Stat3 activity on PKC βII expression, we have generated a series of clones stably expressing wild type, constitutive active, and dominant negative Stat3 constructs in K562, a second DC progenitor-like cell line. Compared to the parental cell line, PKC βII mRNA transcript levels were significantly reduced (>10-fold) in clones stably expressing the constitutive active Stat3 construct (p<0.1). Clones expressing the dominant negative or wild type Stat3 constructs have PKC βII mRNA transcript levels comparable to or greater than the parental cell line (no difference to 2.5 fold increase). Similarly, PKC βII protein levels in clones stably expressing the constitutive active construct were reduced 75–95% compared to the parental cell line, while PKC βII protein levels in clones expressing the dominant negative construct were increased by 57–110%. We have found that culture of a myeloid progenitor cell line in tumor conditioned media significantly decreases PKC βII mRNA transcript levels and protein levels, suggesting that tumor conditioned media decreases PKC βII gene expression. Decreased PKC βII expression was accompanied by impairment in DC differentiation, as measured by a significant decrease in the ability of KG1-DC to stimulate allogeneic T cell proliferation. Expression of a constitutive active Stat3 construct significantly decreased PKC βII transcript levels in K562, a DC progenitor-like cell line. Taken together, these observations support the hypothesis that tumor derived factors inhibit DC differentiation by down regulation of PKC βII expression via Stat3 hyperactivation.

Regulation of Estrogen Receptor-mediated Long Range Transcription via Evolutionarily Conserved Distal Response Elements

Nuclear signaling by estrogens rapidly induces the global recruitment of estrogen receptors (ERs) to thousands of highly specific locations in the genome. Here, we have examined whether ER binding sites that are located distal from the transcription start sites of estrogen target genes are functionally relevant. Similar to ER binding sites near the proximal promoter region, ER binding sites located at distal locations are occupied by ERs after estrogen stimulation. And, like proximal bound ERs, ERs occupied at distal sites can recruit coactivators and the RNA polymerase transcription machinery and mediate specific structural changes to chromatin. Furthermore, ERs occupied at the distal sites are capable of communicating with ERs bound at the promoter region, possibly via long range chromosome looping. In functional analysis, disruption of the response elements in the distal ER binding sites abrogated ER binding and significantly reduced transcriptional response. Finally, sequence comparison of the response elements at the distal sites suggests a high level of conservation across different species. Together, our data indicate that distal ER binding sites are bona fide transcriptional enhancers that are involved in long range chromosomal interaction, transcription complex formation, and distinct structural modifications of chromatin across large genomic spans.