Synthetic enzyme inhibitor: A novel targeting ligand for nanotherapeutic drug delivery inhibiting tumor growth without systemic toxicity

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
Nanomedicine: nanotechnology, biology, and medicine (Impact Factor: 6.16). 03/2011; 7(6):665-73. DOI: 10.1016/j.nano.2011.03.001
Source: PubMed


Unresolved problems associated with ligand-targeting of liposomal nanoparticles (NPs) to solid tumors include variable target receptor expression due to genetic heterogeneity and insufficient target specificity, leading to systemic toxicities. This study addresses these issues by developing a novel ligand-targeting strategy for liposomal NPs using RR-11a, a synthetic enzyme inhibitor of Legumain, an asparaginyl endopeptidase. Cell-surface expression of Legumain is driven by hypoxic stress, a hallmark of solid tumors. Legumain-targeted RR-11a-coupled NPs revealed high ligand-receptor affinity, enhanced solid-tumor penetration and uptake by tumor cells. Treatment of tumor-bearing mice with RR-11a-coupled NPs encapsulating doxorubicin resulted in improved tumor selectivity and drug sensitivity, leading to complete inhibition of tumor growth. These antitumor effects were achieved while eliminating systemic drug toxicity. Therefore, synthetic enzyme inhibitors, such as RR-11a, represent a new class of compounds that can be used for highly specific ligand-targeting of NPs to solid tumors. FROM THE CLINICAL EDITOR: This study addresses the problems associated with ligand-targeting of liposomal nanoparticles to solid tumors with variable target receptor expression. A novel and efficacious targeting strategy has been developed towards a synthetic enzyme inhibitor of Legumain. The authors demonstrate successful tumor growth inhibiting effect while eliminating systemic drug toxicity in an animal model using this strategy.

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Available from: Ze Liu, Jan 30, 2015
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    • "Furthermore, legumain has been demonstrated to activate proMMP-2, which may partially explain the observed association between legumain expression and metastatic potential [18]. The strict substrate specificity combined with over-expression in various tumor types has motivated exploitation of legumain as a pro-drug activator in cancer treatment, for instance by adding a cleavable peptide chain to doxorubicin or auristatin [10], [19] and targeting of drug compounds using a legumain enzyme inhibitor [20]. Other known biological functions of legumain include autophagic-lysosomal processing of hepatocellular proteins [21], processing of antigens for MHC class II presentation [22], and maturation in Toll-like receptor signaling [23]. "
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    ABSTRACT: The cysteine protease legumain is involved in several biological and pathological processes, and the protease has been found over-expressed and associated with an invasive and metastatic phenotype in a number of solid tumors. Consequently, legumain has been proposed as a prognostic marker for certain cancers, and a potential therapeutic target. Nevertheless, details on how legumain advances malignant progression along with regulation of its proteolytic activity are unclear. In the present work, legumain expression was examined in colorectal cancer cell lines. Substantial differences in amounts of pro- and active legumain forms, along with distinct intracellular distribution patterns, were observed in HCT116 and SW620 cells and corresponding subcutaneous xenografts. Legumain is thought to be located and processed towards its active form primarily in the endo-lysosomes; however, the subcellular distribution remains largely unexplored. By analyzing subcellular fractions, a proteolytically active form of legumain was found in the nucleus of both cell lines, in addition to the canonical endo-lysosomal residency. In situ analyses of legumain expression and activity confirmed the endo-lysosomal and nuclear localizations in cultured cells and, importantly, also in sections from xenografts and biopsies from colorectal cancer patients. In the HCT116 and SW620 cell lines nuclear legumain was found to make up approximately 13% and 17% of the total legumain, respectively. In similarity with previous studies on nuclear variants of related cysteine proteases, legumain was shown to process histone H3.1. The discovery of nuclear localized legumain launches an entirely novel arena of legumain biology and functions in cancer.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "To overcome this problem, a targeted nanoparticle (tNP) delivery method was previously developed in our laboratory that allows for safe in vivo Dox delivery to the tumor microenvironment with little or no toxic side effects [19]. Such nanoparticles specifically target cells expressing legumain in their plasma membranes, which is a common property of the tumor microenvironment, especially under hypoxic conditions [19,23,29,36]. However, before a combination therapy of IMD-0354 plus Dox could be critically tested in animal models, legumain expression and tNP drug delivery had to be assessed in vitro. "
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    ABSTRACT: Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease.
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    • "Therefore, in this study, HC was employed to suppress STAT3 activity, and re-educate TAMs to change from M2 to M1. Moreover, we followed the Legumain-targeting strategy used in our previous studies [14] and used RR-11a-coupled liposomal nanoparticles (NPs) encapsulating HC to reduce nonspecific accumulation in the reticuloendothelial system and to enhance targeting capability in solid tumors[14,15]. "
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    ABSTRACT: Tumor-associated macrophages (TAMs) are essential cellular components within tumor microenvironment (TME). TAMs are educated by TME to transform to M2 polarized population, showing a M2-like phenotype, IL-10(high), IL-12(low), TGF-β(high). STAT3 signaling triggers crosstalk between tumor cells and TAMs, and is crucial for the regulation of malignant progression. In our study, legumain-targeting liposomal nanoparticles (NPs) encapsulating HC were employed to suppress STAT3 activity and "re-educate" TAMs, and to investigate the effects of suppression of tumor progression in vivo. The results showed that TAMs treated by HC encapsuled NPs could switch to M1-like phenotype, IL-10(low), IL-12(high), TGF-β(low), and the "re-educated" macrophages (M1-like macrophages) considerably demonstrated opposite effect of M2-like macrophages, especially the induction of 4T1 cells migration and invasion in vitro, and suppression of tumor growth, angiogenesis and metastasis in vivo. These data indicated that inhibition of STAT3 activity of TAMs by HC-NPs was able to reverse their phenotype and could regulate their crosstalk between tumor cells and TAMs in order to suppress tumor progression.
    Full-text · Article · Jun 2013 · PLoS ONE
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