Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.     

Design,synthesis and biological activities of antiangiogenic hypoxic cytotoxin,triazine-N-oxide derivatives

For cancer therapy, hypoxia represents an important tumor specific target. Therefore we designed and synthesized antiangiogenic hypoxic cytotoxins as 'hypoxia modifiers'. They can be activated bioreductively in hypoxic cells to kill the oxygen-deficient tumor cells selectively and prevent their re-growth. The aromatic heterocycle di-N-oxides, tirapazamine (TPZ), TX-1102, and TX-402 inhibited growth of EMT6/KU cells, SAS/neo cells, and SAS/Trp248 cells (mutant p53 gene transformant) under hypoxic condition. They also induced apoptosis selectively at a dose of 10 microM each under hypoxic condition for 5 h. Their hypoxic cytotoxicities and apoptosis inducing activities were p53-independent because the activities in SAS/neo cells were almost similar to that in SAS/Trp248 cells. In angiogenesis inhibition assay using chick embryo chorioallantoic membrane (CAM), TPZ, TX-1102, TX-402 and TX-1033 showed 40, 25, 60 and 60% inhibition of angiogenesis each at a dose of 10 microg/CAM. On the other hand, the nitrosopyrimidine, TX-1041 had neither antiangiogenic activity nor cytotoxicity. Therefore the di-N-oxide group is thought to be required for the biological activities. TX-1102 was a potent antiangiogenic hypoxic cytotoxin inducing apoptosis p53-independently.     

Systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scFv.

BACKGROUND: A long-standing goal in genetic therapy for cancer is a systemic gene delivery system that selectively targets tumor cells, including metastases. Here we describe a novel cationic immunolipoplex system that shows high in vivo gene transfer efficiency and anti- tumor efficacy when used for systemic p53 gene therapy of cancer. MATERIALS AND METHODS: A cationic immunolipoplex incorporating a biosynthetically lipid-tagged, anti-transferrin receptor single-chain antibody (TfRscFv), was designed to target tumor cells both in vitro and in vivo. A human breast cancer metastasis model was employed to evaluate the in vivo efficacy of systemically administered, TfRscFv-immunolipoplex-mediated, p53 gene therapy in combination with docetaxel. RESULTS: The TfRscFv-targeting cationic immunolipoplex had a size of 60-100 nm, showed enhanced tumor cell binding, and improved targeted gene delivery and transfection efficiencies, both in vitro and in vivo. The p53 tumor suppressor gene was not only systemically delivered by the immunolipoplex to human tumor xenografts in nude mice but also functionally expressed. In the nude mouse breast cancer metastasis model, the combination of the p53 gene delivered by the systemic administration of the TfRscFv-immunolipoplex and docetaxel resulted in significantly improved efficacy with prolonged survival. CONCLUSIONS: This is the first report using scFv-targeting immunolipoplexes for systemic gene therapy. The TfRscFv has a number of advantages over the transferrin (Tf) molecule itself: (1) scFv has a much smaller size than Tf producing a smaller immunolipoplex giving better penetration into solid tumors; (2) unlike Tf, the scFv is a recombinant protein, not a blood product; (3) large scale production and strict quality control of the recombinant scFv, as well as scFv-immunolipoplex, are feasible. The sensitization of tumors to chemotherapy by this tumor-targeted and efficient p53 gene delivery method could lower the effective dose of the drug, correspondingly lessening the severe side effects, while decreasing the possibility of recurrence. Moreover, this approach is applicable to both primary and recurrent tumors, and more significantly, metastatic disease. The TfRscFv-targeting of cationic immunolipoplexes is a promising method of tumor targeted gene delivery that can be used for systemic gene therapy of cancer with the potential to critically impact the clinical management of cancer.     

Tumor cell-mediated neovascularization and lymphangiogenesis contrive tumor progression and cancer metastasis

Robust neovascularization and lymphangiogenesis have been found in a variety of aggressive and metastatic tumors. Endothelial sprouting angiogenesis is generally considered to be the major mechanism by which new vasculature forms in tumors. However, increasing evidence shows that tumor vasculature is not solely composed of endothelial cells (ECs). Some tumor cells acquire processes similar to embryonic vasculogenesis and produce new vasculature through vasculogenic mimicry, trans-differentiation of tumor cells into tumor ECs, and tumor cell–EC vascular co-option. In addition, tumor cells secrete various vasculogenic factors that induce sprouting angiogenesis and lymphangiogenesis. Vasculogenic tumor cells actively participate in the formation of vascular cancer stem cell niche and a premetastatic niche. Therefore, tumor cell-mediated neovascularization and lymphangiogenesis are closely associated with tumor progression, cancer metastasis, and poor prognosis. Vasculogenic tumor cells have emerged as key players in tumor neovascularization and lymphangiogenesis and play pivotal roles in tumor progression and cancer metastasis. However, the mechanisms underlying tumor cell-mediated vascularity as they relate to tumor progression and cancer metastasis remain unclear. Increasing data have shown that various intrinsic and extrinsic factors activate oncogenes and vasculogenic genes, enhance vasculogenic signaling pathways, and trigger tumor neovascularization and lymphangiogenesis. Collectively, tumor cells are the instigators of neovascularization. Therefore, targeting vasculogenic tumor cells, genes, and signaling pathways will open new avenues for anti-tumor vasculogenic and metastatic drug discovery. Dual targeting of endothelial sprouting angiogenesis and tumor cell-mediated neovascularization and lymphangiogenesis may overcome current clinical problems with anti-angiogenic therapy, resulting in significantly improved anti-angiogenesis and anti-cancer therapies.     

Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.     

Lipocalin 2 antagonizes the proangiogenic action of ras in transformed cells

Lipocalin 2 is an iron-binding secreted protein that converts embryonic kidney mesenchyme to epithelia. Previously, we reported that lipocalin 2 could revert 4T1-ras-transformed mesenchymal tumor cells to a more epithelial phenotype, increase E-cadherin expression, and suppress cell invasiveness in vitro and in vivo, indicating that lipocalin 2 is a metastasis suppressor. Here, we show that lipocalin 2 can suppress the ras-induced expression of vascular endothelial growth factor in 4T1 cells via down-regulation of ras mitogen-activated protein kinase and ras phosphatidylinositol-3-kinase signaling. In addition, the expression of thrombospondin-1 (an antiangiogenic molecule) was increased in tumors formed by 4T1-ras cells into which lipocalin 2 was stably introduced. Tumor angiogenesis, assessed via an intradermal tumor angiogenesis assay, was also suppressed by lipocalin 2. We also show that caveolin-1 is a critical mediator of this activity. These data provide new insights into the action of lipocalin 2 and raise the possibility that the administration of lipocalin 2 may be useful for inhibiting tumor angiogenesis, in addition to suppressing tumor metastasis, in cancers which show ras activation.     

The Novel Fusion Proteins,GnRH-p53 and GnRHIII-p53, Expression and Their Anti-Tumor Effect

p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing to the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain mutation or deletion of p53. Gonadotrophin-releasing hormone (GnRH), as the ligand of Gonadotrophin-releasing hormone receptor (GnRH-R), was used to deliver p53 into tumor cells. The p53 fusion proteins GnRH-p53 and GnRH iii-p53 were expressed and their targeted anti-tumor effects were determined. GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of the growth of H1299 cells in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.     

Streptochlorin, a marine natural product, inhibits NF-kappaB activation and suppresses angiogenesis in vitro

Angiogenesis is an essential step in tumor progress and metastasis. Accordingly, small molecules that inhibit angiogenesis would appear to be a promising way to cure angiogenesis-related diseases, including cancer. In the present study, we report that streptochlorin, a small molecule from marine actinomycete, exhibits a potent antiangiogenic activity. The compound potently inhibited endothelial cell invasion and tube formation stimulated with vascular endothelial cell growth factor (VEGF) at low micromolar concentrations where it showed no cytotoxicity to the cells. In addition, streptochlorin inhibited TNF-alpha-induced NF-kappaB activation in the newly developed cell-based reporter gene assay. These data demonstrate that streptochlorin is a new inhibitor of NF-kappaB activation and can be a basis for the development of novel anti-angiogenic agents.     

p32 promotes melanoma progression and metastasis by targeting EMT markers,Akt/PKB pathway,and tumor microenvironment

Melanoma originates from melanin-producing cells called melanocytes. Melanoma poses a great risk because of its rapid ability to spread and invade new organs. Cellular metastasis involves alteration in the gene expression profile and their transformation from epithelial to mesenchymal state. Despite of several advances, metastatic melanoma being a key cause of therapy failure and mortality remains poorly understood. p32 has been found to be involved in various physiological and pathophysiological conditions. However, the role of p32 in melanoma progression and metastasis remains underexplored. Here, we identify the role of p32 in the malignancy of both murine and human melanoma. p32 knockdown leads to reduced cell proliferation, migration, and invasion in murine and human melanoma cells. Furthermore, p32 promotes in vitro tumorigenesis, inducing oncogenes and EMT markers. Mechanistically, we show p32 regulates tumorigenic and metastatic properties through the Akt/PKB signaling pathway in both murine and human melanoma. Furthermore, p32 silencing attenuates melanoma tumor progression and lung metastasis in vivo, modulating the tumor microenvironment by inhibiting the angiogenesis, infiltration of macrophages, and leukocytes in mice. Taken together, our findings identify that p32 drives melanoma progression, metastasis, and regulates the tumor microenvironment. p32 can be a target of a novel therapeutic approach in the regulation of melanoma progression and metastasis.Subject terms: Lung cancer, Cell migration     

Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality (1). Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released (2-5). Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect (6). Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells (7). In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.(8) Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells (9). The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes (10). In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells (11). Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.     

The absence of p53 promotes metastasis in a novel somatic mouse model for hepatocellular carcinoma

We have generated a mouse model for hepatocellular carcinoma using somatic delivery of oncogene-bearing avian retroviral vectors to the liver cells of mice expressing the viral receptor TVA under the control of the albumin gene promoter (Alb-TVA mice). Viruses encoding mouse polyoma virus middle T antigen (PyMT) induced tumors, which can be visualized with magnetic resonance imaging, in 65% of TVA-positive animals. While these tumors can exceed 10 mm in diameter, they do not invade locally or metastasize to the lungs. Delivery of PyMT-expressing viruses to Alb-TVA mice lacking an intact p53 gene does not increase tumor incidence. However, the resulting tumors are poorly differentiated, invasive, and metastatic to the lungs. Gene expression microarrays identified over 100 genes that are differentially expressed between tumors found in p53 wild-type and p53 null mice. Some of these genes, such as cathepsin E and Igf2, have been previously implicated in tumor cell migration and invasion. Tumors induced in p53 null, TVA transgenic mice by PyMT mutants with changes in specific tyrosine residues fail to form metastases, indicating that metastasis is dependent on both the oncogene and the absence of p53.     

Regulation of tumor angiogenesis by thrombospondin-1

Angiogenesis plays a critical role in the growth and metastasis of tumors. Thrombospondin-1 (TSP-1) is a potent angiogenesis inhibitor, and down-regulation of TSP-1 has been suggested to alter tumor growth by modulating angiogenesis in a variety of tumor types. Expression of TSP-1 is up-regulated by the tumor suppressor gene, p53, and down-regulated by oncogenes such as Myc and Ras. TSP-1 inhibits angiogenesis by inhibiting endothelial cell migration and proliferation and by inducing apoptosis. In addition, activation of transforming growth factor beta (TGF-beta) by TSP-1 plays a crucial role in the regulation of tumor progression. An understanding of the molecular basis of TSP-1-mediated inhibition of angiogenesis and tumor progression will aid in the development of novel therapeutics for the treatment of cancer.     

Functional significance of metastasis-inducing S100A4(Mts1) in tumor-stroma interplay

Causal implication of S100A4 in inducing metastases was convincingly shown previously. However, the mechanisms that associate S100A4 with tumor progression are not well understood. S100A4 protein, as a typical member of the S100 family, exhibits dual, intracellular and extracellular, functions. This work is focused on the extracellular function of S100A4, in particular its involvement in tumor-stroma interplay in VMR (mouse adenocarcinoma cell line) tumor cells, which exhibit stroma-dependent metastatic phenotype. We demonstrated the reciprocal influence of tumor and stroma cells where tumor cells stimulate S100A4 secretion from fibroblasts in culture. In turn, extracellular S100A4 modifies the cytoskeleton and focal adhesions and triggers several other events in tumor cells. We found stabilization of the tumor suppressor protein p53 and modulation of its function. In particular, extracellular S100A4 down-regulates the pro-apoptotic bax and the angiogenesis inhibitor thrombospondin-1 genes. For the first time, we demonstrate here that the S100A4 protein added to the extracellular space strongly stimulates proteolytic activity of VMR cells. This activity most probably is associated with matrix metalloproteinases and, in particular, with matrix metalloproteinase-13. Finally, the application of the recombinant S100A4 protein confers stroma-independent metastatic phenotype on VMR tumor cells. In conclusion, our results indicate that metastasis-inducing S100A4 protein plays a pivotal role in the tumor-stroma environment. S100A4 released either by tumor or stroma cells triggers pro-metastatic cascades in tumor cells.     

Oncolytic poxviruses

The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns several directions: virus attenuation, insertion of immunomodulatory protein genes, and anti-tumor protein genes. Thymidine kinase and viral growth factor genes make the greatest contribution to the virus attenuation as their inactivation results in the virus inability to replicate in non-dividing cells, thereby contributing to increased selectivity with respect to tumor cells. Among the immunomodulatory proteins, interleukins 2, 12, and granulocyte-macrophage colony-stimulating factor proved to be most promising for oncolytic virotherapy. An attempt to use p53 protein gene expressed by vaccinia virus for addressed apoptosis of tumor cells was reported. The use of the double and triple viral recombinants carrying genes of multidirectional action seems to be most promising. Encouraging results were obtained using vaccinia virus in the oncotherapy with prodrugs and angiogenesis inhibitors. At present, two poxviral strains are undergoing Phase III clinical trials as anti-tumor preparations in the USA.     

抑制血管生成治疗肿瘤的策略和展望

新生血管生成是绝大多数肿瘤得以生长和转移的必要前提。所以 ,通过抑制肿瘤血管生成来抑制肿瘤是非常有前途的一种方法 ,有望发展成为一种新型的癌症疗法。主要可以分为两大类 :一是通过抑制促血管生成信号或扩大抑制血管生成因子的作用来干扰肿瘤新生血管的形成过程 ,这领域的广泛研究已经发现了一系列促血管生成因子及其抑制剂和血管生成抑制因子 ;二是利用肿瘤血管与正常血管的差别来携带杀伤性药物直接特异性破坏已形成的肿瘤血管 ;另外 ,内皮细胞及其前体细胞制成疫苗也可起到直接杀伤作用。到目前为止 ,虽然很多抑制肿瘤血管的药物已经被用于临床试验 ,但结果往往不尽如人意 ,从长远来看 ,需要更有效的治疗方法。包括抗血管基因治疗策略 ,靶向药物导入系统的研究 ,以及抗血管生成药物和免疫疗法、化疗和放射治疗的联合应用都在探讨中。随着肿瘤模型评估系统的发展 ,抗血管治疗肿瘤的方法在不久的将来一定会广泛进入临床应用。