Andrea L. George

Endothelial progenitor cell biology in disease and tissue regeneration

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent.

Estrogen Induced Metastatic Modulators MMP-2 and MMP-9 Are Targets of 3,3′-Diindolylmethane in Thyroid Cancer

Background Thyroid cancer is the most common endocrine related cancer with increasing incidences during the past five years. Current treatments for thyroid cancer, such as surgery or radioactive iodine therapy, often require patients to be on lifelong thyroid hormone replacement therapy and given the significant recurrence rates of thyroid cancer, new preventive modalities are needed. The present study investigates the property of a natural dietary compound found in cruciferous vegetables, 3,3′-diindolylmethane (DIM), to target the metastatic phenotype of thyroid cancer cells through a functional estrogen receptor. Methodology/Principal Findings Thyroid cancer cell lines were treated with estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We observed that DIM inhibits estrogen mediated increase in thyroid cell migration, adhesion and invasion, which is also supported by ER-α downregulation (siRNA) studies. Western blot and zymography analyses provided direct evidence for this DIM mediated inhibition of E2 enhanced metastasis associated events by virtue of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9. Conclusion/Significance Our data reports for the first time that DIM displays anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer cell proliferation and in vitro metastasis associated events, namely adhesion, migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to promote and enhance metastasis, were determined to be targets of DIM. This anti-estrogen like property of DIM may lead to the development of a novel preventive and/or therapeutic dietary supplement for thyroid cancer patients by targeting progression of the disease.

Synthetic Toll Like Receptor-4 (TLR-4) Agonist Peptides as a Novel Class of Adjuvants

Background Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. Methodology/Principal Findings We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant. Conclusions/Significance Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.

Estrogen activity as a preventive and therapeutic target in thyroid cancer

Thyroid cancer is the most common endocrine-related cancer with increasing incidences during the last five years. Interestingly, according to the American Thyroid Association, the incidences of thyroid proliferative diseases occur four to five times more in women than in men with the risk of developing thyroid disorders being one in every eight females. Several epidemiological studies have suggested a possible correlation between incidences of thyroid malignancies and hormones but the precise contribution of estrogen in thyroid proliferative disease initiation, and progression is not well understood. This review is an attempt to define the phenotypic and genotypic modulatory effects of estrogen on thyroid proliferative diseases. The significance and relevance of expression of estrogen receptors, α and β, in normal and malignant thyroid tissues and their effects on different molecular pathways involved in growth and function of the thyroid gland are discussed. These novel findings open up areas of developing alternative therapeutic treatments and preventive approaches which employ the use of antiestrogen to treat thyroid malignancies.

Hypoxia and estrogen are functionally equivalent in breast cancer-endothelial cell interdependence

BackgroundRapid breast tumor development relies on formation of new vasculature to supply the growing malignancy with oxygenated blood. Previously we found that estrogen aided in this neovasculogenesis via recruitment of bone marrow derived endothelial progenitor cells (BM-EPCs), leading to increased vessel formation and vascular endothelial growth factor (VEGF) production in vivo. However, the cellular mechanism of this induction and the signaling pathways involved need elucidation.ResultsUsing the murine mammary cell line TG1-1 we observed estrogen (E2) lead to an up regulation of hypoxia inducible factor-1 (HIF-1), an effect abrogated by the anti-estrogen Fulvestrant and the HIF-1 inhibitor YC-1 (3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole) suggesting the interchangeability of hypoxia and estrogen mediated effects. Estrogen modulation of HIF-1 and subsequent effects on endothelial cells is dependent on the Akt/PI3K pathway and protein synthesis as validated by the use of the inhibitors wortmannin and cycloheximide which abrogated estrogen’s effects respectively. Estrogen treated TG1-1 cells secreted higher levels of VEGF which were comparable to secreted levels from cells grown under hypoxic conditions. Soluble factors in conditioned media from E2 treated breast cancer cells also lead to migration and tube formation of human umbilical vein endothelial cells (HUVEC) in vitro.ConclusionsOur data provide evidence that estrogen signaling mediates the tumor vasculogenic process required for breast cancer progression and involves a key regulator of the hypoxia signaling pathway. Further, hypoxia and estrogen are interchangeable as both similarly modulate epithelial-endothelial cell interaction.

Estrogen-Mediated Angiogenesis in Thyroid Tumor Microenvironment Is Mediated Through VEGF Signaling Pathways

OBJECTIVES To explore the induction of a proangiogenic phenotype in endothelial cells in the thyroid tumor microenvironment by estrogen-treated thyroid cancer cells and to define the role of vascular endothelial growth factor (VEGF) in this interaction. DESIGN Cell-based in vitro systems analysis. SUBJECTS Thyroid tumor cell lines (BCPAP [papillary thyroid cancer] and ML-1 [follicular thyroid cancer]) were cultured with estradiol with and without an estrogen receptor (ER) inhibitor (fulvestrant or ICI) and used to treat human umbilical vein endothelial cells (HUVECs). INTERVENTIONS Immunofluorescence was used to confirm the presence of ERα and ERβ in BCPAP cells. Conditioned medium was then used to evaluate the induction of HUVEC tubulogenesis and migration. Secretion of VEGF in this medium was evaluated by Western blot analysis. The expression of phosphoinositide 3-kinase (PI3K), the initiator of a proangiogenic pathway, was evaluated with Western blot analysis of HUVEC lysates. The subsequent effects of an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody were also observed. RESULTS Estrogen receptor α and ERβ are expressed in thyroid cancer cells. Estrogen-stimulated ML-1 cells secreted an increased amount of VEGF likely as a result of ER signaling. In contact with this environment, HUVECs demonstrate enhanced tubulogenesis and migration. Western blot analysis documented estrogen-mediated upregulation of PI3K in HUVECs. These effects were mitigated by an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody. CONCLUSIONS Our data provide evidence that estrogen can induce a proangiogenic endothelial cell phenotype in the thyroid tumor microenvironment through ER and VEGF signaling. Our findings suggest that the effect of antiestrogenic therapy targeting tumor angiogenesis can be enhanced through VEGF inhibition.

Ex vivo derived primary melanoma cells: implications for immunotherapeutic vaccines.

Transformation of the pigment producing melanocytes into melanoma is a complex multi-step process involving the enhanced expression of various antigens considered as immunotherapeutic targets. Significant progress in melanoma research has been made over the years and has resulted in the identification of various antigens over expressed in melanoma as well as advances in immunotherapeutic treatments, which focus on modulating the immune systems response to melanoma. Despite these advances, incidences of melanoma are still on the rise thus warranting additional research in identifying new therapeutic treatments. Our focus is on developing a multivalent immunotherapeutic vaccine that targets various melanoma associated antigens. The approach focuses on the use of five primary patient derived melanoma cells (MEL-2, MEL-V, 3MM, KFM, and GLM-2, which have been characterized in this study. These cells express differential amounts of various melanoma associated antigens such as MART-1, gp100 (Pmel17), MAGE-A1 and tyrosinase as well a cell surface antigens essential for melanoma cell metastasis, such as CD146 and CD71. In addition these cells display differential in vitro migratory and invasive properties as well as have the ability to form solid tumors when implanted into BALB/c nude mice. The retention of the innate phenotype of these primary patient derived cells together with the expression of a multitude repertoire of melanoma associated antigens offers a novel opportunity to target melanoma so as to avoid immune evasion.

Identification of PSA peptide mimotopes using phage display peptide library

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.

Molecular target based combinational therapeutic approaches in thyroid cancer

BackgroundThyroid cancer, as with other types of cancer, is dependent on angiogenesis for its continued growth and development. Interestingly, estrogen has been shown to contribute to thyroid cancer aggressiveness in vitro, which is in full support of the observed increased incidence of thyroid cancer in women over men. Provided that estrogen has been observed to contribute to increased angiogenesis of estrogen responsive breast cancer, it is conceivable to speculate that estrogen also contributes to angiogenesis of estrogen responsive thyroid cancer.MethodsIn this study, three human thyroid cancer cells (B-CPAP, CGTH-W-1, ML-1) were treated with estrogen alone or estrogen and anti-estrogens (fulvestrant and 3,3′-diindolylmethane, a natural dietary compound) for 24 hours. The cell culture media was then added to human umbilical vein endothelial cell (HUVECs) and assayed for angiogenesis associated events. Vascular endothelial growth factor (VEGF) levels were also quantified in the conditioned media so as to evaluate if it is a key player involved in these observations.ResultsConditioned medium from estrogen treated thyroid cancer cells enhanced phenotypical changes (proliferation, migration and tubulogenesis) of endothelial cells typically observed during angiogenesis. These phenotypic changes observed in HUVECs were determined to be modulated by estrogen induced secretion of VEGF by the cancer cells. Lastly, we show that VEGF secretion was inhibited by the anti-estrogens, fulvestrant and 3,3′-diindolylmethane, which resulted in diminished angiogenesis associated events in HUVECs.ConclusionOur data establishes estrogen as being a key regulator of VEGF secretion/expression in thyroid cells which enhances the process of angiogenesis in thyroid cancer. These findings also suggest the clinical utility of anti-estrogens as anti-angiogenic compounds to be used as a therapeutic means to treat thyroid cancer. We also observed that 3,3′-diindolylmethane is a promising naturally occurring anti-estrogen which can be used as a part of therapeutic regimen to treat thyroid cancer.

Identification of peptide mimotopes of gp96 using single-chain antibody library

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96–peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund’s adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4+ and CD8+) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.