Search

Inhibition of angiogenesis is an effective and low toxic therapeutic avenue for the treatment of cancer patients in addition to traditional interventions. Majority of current available angiogenesis inhibitors for cancer therapies are growth factor inhibitors and small molecule tyrosine kinase inhibitors. A number of endogenous proteins and/or proteolytic fragments of extracellular matrix proteins are shown to have the activity of inhibition of angiogenesis by directly targeting endothelial cells. Structural analyses have indicated that a common structure of anti-parallel β-sheet with a highly positively charged surface presents in many of those inhibitors. This common structural feature is critical for the maintenance of their anti-angiogenic function. With this structural information, we have designed and developed a new class of anti-angiogenic proteins by integrating the short anti-parallel β-sheet forming sequences of endogenous anti-angiogenic proteins into a stable host protein, the extracellular domain-1 of cluster of differentiation 2 molecule (CD2D1). 1D 1H NMR spectra analyses indicated that the designed anti-angiogenic protein (ref to as ProAgio) folded as a β-sheet structure similar to that of the parental protein, CD2D1. ProAgio inhibited the growth of human umbilical vein cells (HUVECs) without affecting the growth of epithelial cells, suggesting a specific effect to endothelial cells. ProAgio effectively reduced endothelial tubules formed by the co-culture of HUVECs and PC3 cells on matrix gel in vitro. The designed anti-angiogenic protein was further site-specifically PEGylated in order to improve PK/PD properties and reduce immunogenicity. Examinations with PC3 xenografts showed that both ProAgio and the PEGylated ProAgio dramatically inhibited tumor growth. Immunofluorescence staining analyses of the endothelial marker CD31 indicated dramatic decreases in tumor vessels in lengths and branching points. Histological and immunofluorescence staining analyses of tissue slices of major organs indicated that there were no pathological damages to the tissue structure or disruption of normal vessels associated with the treatment of our designed anti-angiogenic agent. Overall, our studies developed a novel anti-angiogenesis agent that may have great clinical potentials. Our concept of protein design can be extended to the development of other novel protein drugs.

Platinum drugs have been the cornerstone in treating human ovarian cancer, as a result of major advances in the field of synthetic and analytical chemistry. Drugs such as cisplatin and carboplatin that are routinely used for chemotherapy, form intra- and inter-strand platinum-DNA cross links, ultimately leading to cancer cell death. But unfortunately in many cases this platinum-DNA association is also responsible for treatment resistance, leading to patient death. It has been extensively documented that inhibition of DNA repair pathways and a greater understanding of cellular reactions upon platinum drug treatment, are crucial in diminishing toxicity and improving the effectiveness of such drugs. Pyrodach-2, belonging to a class of novel platinum compounds called phosphaplatins, demonstrated enhanced cytotoxicity against ovarian cancer cells and no DNA-binding in a recently published study (Bose et. al., 2009). These observations inspired the following study, which aims at determining the anti-cancer properties of two other pyrophosphate complexes, namely pyrodach-4 and RRD4.Platinum compounds have been known to channel their effects through multiple pathways. In the first part of this study, the signaling mechanism(s) of pyrodach-4, which induced ovarian cancer cell death upon treatment, were investigated. Cell-death and apoptosis associated genes along with their products make good targets, which can be exploited in the development of drugs to treat human diseases. Using various assays such as gene expression microarray and qRT-PCR, levels of various such genes were analyzed. Interestingly pyrodach-4 utilized genes belonging to both the extrinsic and intrinsic apoptotic cell death machinery, namely Bax, Fas, PTEN and PUMA to induce maximal cisplatin-sensitive and -resistant ovarian cancer cell death at 24 hours post treatment. Further, anti-apoptotic genes such as Bcl-2 decreased in treated cells. In contrast, protein expression results for Fas and Bax proteins did not exactly mimic the results from the gene expression analyses, suggesting that post-transcriptional modifications might be at play. Additionally, findings from investigating RRD4 cytotoxicity, suggested that both the cisplatin-sensitive and resistant cell lines were sensitive to 24 hour treatment with RRD4. Combined results from the gene expression, protein expression and ELISA analyses proved that RRD4-treated cells may be differentially regulated than those treated with pyrodach-4, hinting a faster uptake process, and a distinct mechanism of action of RRD4, in comparison to pyrodach-4. Effects of platinum drugs on tumor vasculature have become the subject of increased study. In part two of this study, treatment of HUVECs with pyrodach-4 and RRD4 caused intense cell death, adding anti-angiogenesis to the repertoire of phosphaplatin anti-cancer functions. Finally, in accordance with the anti-cancer activities seen in vitro, pyrodach-4 toxicity and efficacy studies were performed, as the third part of the study. Results from the toxicity and efficacy studies demonstrated that phosphaplatins were well tolerated and performed much better in comparison to cisplatin- and carboplatin-treated mice. Collectively, data presented in this study reveal the probable pathways triggered upon pyrodach-4 and RRD4 treatments, making phosphaplatins a class of novel targeted compounds, which can be used to treat human ovarian cancer.

Beginning with the development of an embryo and throughout one's adult life angiogenesis plays an essential role for organ growth and repair. The balancing of pro-angiogenic factors to anti-angiogenic factors is what regulates the process of angiogenesis. When this balance is disrupted in one direction or the other, numerous disorders can occur including ocular and inflammatory diseases and, in particular, tumor growth and metastasis. Therefore, inhibition of angiogenesis has become an effective therapy for arresting the growth of tumors. A key regulator of angiogenesis involves vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (KDR). The binding of VEGF to KDR activates the receptor which, in turn, initiates the signaling cascade leading to cell proliferation, migration, and survival. The extracellular portion of KDR consists of seven Ig-like domains (Ig1-7), in which Ig1-3 is believed to be responsible for the ligand binding and Ig4-7 for receptor dimerization. We hypothesized that a soluble fragment of the KDR extracellular domain (KDR-Ig4-7) that lacks a tyrosine kinase domain may serve as a KDR antagonist by binding to the membrane bound KDR to form a KDR dimer thus preventing VEGF induced signal transduction. In this study various KDR proteins (KDR-Ig4-7, KDR-Ig4-6, KDR-Ig5-7 and KDR-Ig5-6) were produced, purified and characterized for their potential anti-angiogenic ability. We demonstrated that KDR-Ig4-7 was able to specifically bind to human umbilical vein endothelial cells (HUVEC), and accumulated specifically in tumors indicating its ability to target endothelial cells. Furthermore, KDR-Ig4-7 was able to inhibit HUVEC (and various breast and leukemic cancer cells) proliferation, tube formation and migration. An apoptosis assay conducted using T47-D cells revealed that the induction of apoptosis was the likely mechanism for the inhibition of cell proliferation by KDR-Ig4-7. The inhibitory effect of KDR-Ig-4-7 was at least, in part, through the blocking of ERK phosphorylation. Among all the variants tested, KDR-Ig-4-7 was the most potent inhibitor, which suggested the involvement of domains Ig4 and Ig7 in the receptor dimerization process. Moreover, we found that the inhibitory activity of KDR-Ig4-7 was enhanced in the presence of VEGF, which suggested that VEGF might induce a conformational change which favors the interaction between KDR-Ig4-7 and KDR. In our 4T1 xenograft animal studies, however, KDR-Ig4-7 treatment was not effective. In summary, our results suggest that KDR-Ig4-7 is a functional antagonist to KDR.