Raj K. Tiwari

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.

Metastatic Phenotype Is Regulated by Estrogen in Thyroid Cells

BACKGROUND Over 200 million people worldwide are affected by thyroid proliferative diseases, including cancer, adenoma, and goiter, annually. The incidences of thyroid malignancies are three to four times higher in women, suggesting the possible involvement of estrogen. Based on this observed sex bias, we hypothesize that estrogen modulates the growth and metastatic propensity of thyroid cancer cells. METHODS In this study, two thyroid cell lines (Nthy-ori 3-1 and BCPAP) were evaluated for the presence of estrogen receptor (ER) by Western blot analysis and estrogen responsiveness by using a cell proliferation assay. In addition, the effect of estradiol (E(2)) on modulation of metastatic phenotype was determined by using in vitro adhesion, migration, and invasion assays. RESULTS Thyroid cells expressed a functionally active ER-alpha and ER-beta as evidenced by 50-150% enhancement of proliferation in the presence of E(2). E(2) also enhanced adhesion, migration, and invasion of thyroid cells in an in vitro experimental model system that, based on our results, is modulated by beta-catenin. CONCLUSION Our data provide evidence that the higher incidence of thyroid cancer in women is potentially attributed to the presence of a functional ER that participates in cellular processes contributing to enhanced mitogenic, migratory, and invasive properties of thyroid cells. These findings will enable and foster the possible development of antiestrogenic therapy targeting invasion and migration, thus affecting metastatic propensity.

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.

Targeting the immune system in cancer.

The concept of cancer immunotherapy provides a fresh perspective as it is not associated with many of the drawbacks of conventional therapies such as chemotherapy, radiotherapy and surgery. When fully activated the immune system has immense potential as is evident from mis-matched transplanted organs undergoing rapid immunological attack and rejection. However, the development of immune strategies for cancer therapy has been associated with challenges of their own. Early attempts at cancer vaccination were carried out in an empirical manner that did not always lead to reproducibility. This led to a search of tumor associated antigens with the belief that specific targeting of these antigens would lead to successful tumor elimination. Active vaccination with TAA peptides or passive vaccination with specific lymphocytes against these TAAs did not however demonstrate encouraging results in clinical trials. This was mainly because of the lack of an activating immune response which is required for continuous stimulation of lymphocytes and also because of the selection of tumor escape variants that did not express the particular TAA. On the positive side, attempts at characterizing TAAs illuminated the molecular changes that attribute a malignant phenotype to cancer cells. Attempts at cytokine therapy were also met with challenges of high systemic toxicity and a lack of specific lymphocyte activation. It was therefore realized that an ideal vaccinating agent should be able to combine the effects of both these therapeutic strategies, i.e., it should be able to induce an innate immune response which can be tailored to a tumor specific adaptive immune response. By this, the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. Animal experiments with these therapeutic modalities have demonstrated exciting results, although their evaluation in clinical trials has not indicated exceptional tumor protection in a large percentage of the patients. These observations only further underscore the multivariate and dynamic nature of the immune system and the many ways in which tumor cells modulate themselves and their surroundings to escape immune surveillance. Assessment of successful therapeutic intervention will require periodic evaluations of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients. With the development of advanced genetic technologies and continuous identification of tumor antigens, the field of cancer immunotherapy is progressing at an exciting pace giving us hope for the advent of effective treatment modalities that will prolong tumor free survival and enhance the quality of life in patients with malignant disease.

17β-Estradiol Mobilizes Bone Marrow–Derived Endothelial Progenitor Cells to Tumors

Neovascularization is critical for tumor growth and development. The cellular mediators for this process are yet to be defined. We discovered that bone marrow-derived endothelial progenitor cells (BM-EPC), having the phenotype (CD133+, CD34+, VEGFR-2+), initiate neovascularization in response to TG1-1 mammary cells implanted in the inguinal mammary gland of Tie-2 GFP transgenic mice. The fluorescence tag allowed for tracing the migration of green fluorescent protein-tagged endothelial progenitor cells to tumor tissues. We discovered that 17-beta estradiol supplementation of ovariectomized mice significantly enhanced BM-EPC-induced neovascularization and secretion of angiogenic factors within the tumor microenvironment. Cell-based system analyses showed that estrogen-stimulated BM-EPCs secreted paracrine factors which enhanced TG1-1 cell proliferation and migration. Furthermore, TG1-1 cell medium supplemented with estrogen-induced BM-EPC mediated tubulogenesis, which was an experimental in vivo representation of the neovasculature. Our data provide evidence of BM-EPC mammary tumor cell interactions and identify a novel cellular mediator of tumor progression that can be exploited clinically.

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.

Abrogation of Estrogen-Mediated Cellular and Biochemical Effects by Indole-3-Carbinol

The use of naturally occurring phytoantiestrogens for prevention and therapy of breast cancer is an alternative to synthetic antiestrogens. We have been examining the mechanism of action of the antiestrogen indole-3-carbinol (I3C), a constituent of compounds present in cruciferous vegetables. I3C abrogates the cell-proliferative effect of 17β-estradiol (E2), as observed in several different estradiol-responsive breast cancer cell lines and isolated cell clones. Modulation of E2 activity by I3C, in part, was by the induction of the 2-hydroxylation pathway, one of the two competing hydroxylation pathways of estrone conversion that resulted in the formation of metabolites with antiestrogenic properties. I3C-mediated induction of the 2-hydroxylation pathway correlated with a selective induction of cytochrome P-450 1A1 by I3C in E2-responsive human breast cancer cells. Induction of neither the 2-hydroxylation pathway nor cytochrome P-450 1A1 was observed in estrogen-nonresponsive human breast cancer cells. This selective effect warranted a further search for biochemical targets of I3C related to E2 function. To this end, we observed that E2-mediated phosphorylation of the estrogen receptor is inhibited by I3C. Our results are consistent with the hypothesis that I3C exerts its antiestrogenic effect by intervention in the E2-estrogen receptor signal transduction pathways and by alterations in E2 metabolism that resulted in the formation of metabolites with antiestrogenic activity.

Differences in Glycosylation Patterns of Heat Shock Protein, gp96: Implications for Prostate Cancer Prevention

Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.

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.

Computational peptide dissection of Melan-a/MART-1 oncoprotein antigenicity

We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the hosts proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.