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The functionalized human serine protease granzyme B/VEGF??? targets tumor vasculature and ablates tumor growth.

ABSTRACT: The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF??? binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF??? fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF??? that correlated closely to total VEGFR-2 expression. GrB/VEGF??? localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF??? induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF??? localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF??? to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF??? showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF??? at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.

SUBMITTER: Mohamedali KA

PROVIDER: S-EPMC3921020 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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The functionalized human serine protease granzyme B/VEGF₁₂₁ targets tumor vasculature and ablates tumor growth.

Mohamedali Khalid A KA Cao Yu Y Cheung Lawrence H LH Hittelman Walter N WN Rosenblum Michael G MG

Molecular cancer therapeutics 20130715 10

The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF₁₂₁ binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF₁₂₁ fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF₁₂₁ that correlated closely to total VEGFR-2 expression. GrB/VEGF₁₂₁ localized efficiently into VEGFR-2-expressin ...[more]

PMID: 23858102

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Project description:Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

Project description:Tumor angiogenesis is characterized by abnormal vessel morphology leading to erratic and insufficient delivery of chemotherapeutics and oxygen, making the tumor core not only highly hypoxic but also unresponsive toward treatment. Such hypoxic conditions promote tumor aggressiveness, leading to the establishment of metastatic disease. Most anti-angiogenic treatments aim toward the destruction of tumor vasculature, which proves countereffective by further increasing its aggressive nature. Hence, developing drugs which target or regulate these processes might lead to a better delivery of chemotherapeutics resulting in tumor shrinkage. Plant-derived natural compounds having a bioactive ingredient, especially triterpenoids, have been known to possess anticancer properties. AECHL-1, a recently isolated novel triterpenoid with proven anticancer potential, is seemingly noncytotoxic toward HEK 293 and HUVECs. Also, cytotoxicity was absent during in vivo studies involving intraperitoneal injections with 5 µg/kg body weight AECHL-1 on SCID mice. When used at subtoxic doses, it was found to be effective in suppression of neo-vessel formation as demonstrated in the chick chorioallantoic membrane, rat aortic rings, Matrigel plugs and xenograft tumors implanted in SCID mice. Tumor vasculature from AECHL-1-treated mice showed greater mural cell coverage and relatively normalized architecture. Investigations into the molecular mechanisms responsible for these observations revealed an effect on the actin cytoskeleton of stimulated HUVECs as well as the VEGFR2-mediated MAPK pathway. AECHL-1 could effectively distinguish between stimulated and nonstimulated endothelial cells. AECHL-1 could also downregulate HIF-1α expression and VEGF secretion under hypoxic conditions, thus reducing the fears of unnecessarily aggravating tumor metastasis as a result of anti-angiogenic therapy. Results obtained from the aforementioned studies make it clear that though AECHL-1 shows promise in discouraging and pruning neo-vasculature, it may not affect existing vasculature as the doses used for the assays are significantly lower than the ones causing endothelial cell death and has potential to be considered as a candidate for therapeutic drug development.

Dataset Information

The functionalized human serine protease granzyme B/VEGF??? targets tumor vasculature and ablates tumor growth.

Publications

The functionalized human serine protease granzyme B/VEGF₁₂₁ targets tumor vasculature and ablates tumor growth.

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