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In previous animal model studies, we demonstrated the potential of rAAV2-sVEGFRv-1, which encodes a truncated variant of the alternatively spliced soluble version of VEGF receptor-1 (VEGFR1), as a human gene therapy for age-related macular degeneration (AMD) and diabetic retinopathy (DR). Here, we elucidate in vitro some of the mechanisms by which rAAV2-sVEGFRv-1 exerts its therapeutic effects. Human umbilical vein endothelial cells (HUVECs) were infected with rAAV2-sVEGFRv-1 or a control virus vector in the presence of members of the VEGF family to identify potential binding partners via ELISA, which showed that VEGF-A, VEGF-B, and placental growth factor (PlGF) are all ligands of its transgene product. In order to determine the effects of rAAV2-sVEGFRv-1 on cell proliferation and permeability, processes that are important to the progression AMD and DR, HUVECs were infected with the therapeutic virus vector under the stimulation of VEGF-A, the major driver of the neovascularization that characterizes the forms of these conditions most associated with vision loss. rAAV2-sVEGFRv-1 treatment, as a result, markedly reduced the extent to which these processes occurred, with the latter determined by measuring zonula occludens 1 expression. Finally, the human microglial HMC3 cell line was used to show the effects of the therapeutic virus vector upon inflammatory processes, another major contributor to angiogenic eye disease pathophysiology, with rAAV2-sVEGFRv-1 reducing therein the secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6. Combined with our previously published in vivo data, the in vitro activity of the expressed transgene here further demonstrates the great promise of rAAV2-sVEGFRv-1 as a potential human gene therapeutic for addressing angiogenic ocular conditions.
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Dependovirus , Terapia Genética , Células Endoteliales de la Vena Umbilical Humana , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Humanos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Dependovirus/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Proliferación Celular , Degeneración Macular/terapia , Degeneración Macular/genética , Degeneración Macular/metabolismo , Retinopatía Diabética/terapia , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Factor B de Crecimiento Endotelial Vascular/genética , Factor B de Crecimiento Endotelial Vascular/metabolismo , Factor de Crecimiento Placentario/genética , Factor de Crecimiento Placentario/metabolismoRESUMEN
Elevated intraocular pressure (IOP) in glaucoma causes retinal ganglion cell (RGC) loss and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. In this paper, we aimed to develop a novel gene therapy for glaucoma using an AAV2-based thioredoxin 2 (Trx2)-exoenzyme C3 transferase (C3) fusion protein expression vector (scAAV2-Trx2-C3). We evaluated the therapeutic effects of this vector in vitro and in vivo using dexamethasone (DEX)-induced glaucoma models. We found that scAAV2-Trx2-C3-treated HeLa cells had significantly reduced GTP-bound active RhoA and increased phosphor-cofilin Ser3 protein expression levels. scAAV2-Trx2-C3 was also shown to inhibit oxidative stress, fibronectin expression, and alpha-SMA expression in DEX-treated HeLa cells. NeuN immunostaining and TUNEL assay in mouse retinal tissues was performed to evaluate its neuroprotective effect upon RGCs, whereas changes in mouse IOP were monitored via rebound tonometer. The present study showed that scAAV2-Trx2-C3 can protect RGCs from degeneration and reduce IOP in a DEX-induced mouse model of glaucoma, while immunohistochemistry revealed that the expression of fibronectin and alpha-SMA was decreased after the transduction of scAAV2-Trx2-C3 in murine eye tissues. Our results suggest that AAV2-Trx2-C3 modulates the outflow resistance of the trabecular meshwork, protects retinal and other ocular tissues from oxidative damage, and may lead to the development of a gene therapeutic for glaucoma.
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Glaucoma , Presión Intraocular , Humanos , Ratones , Animales , Células Ganglionares de la Retina/metabolismo , Fibronectinas/metabolismo , Tiorredoxinas/metabolismo , Células HeLa , Transferasas/metabolismo , Glaucoma/genética , Glaucoma/terapia , Glaucoma/metabolismo , Modelos Animales de EnfermedadRESUMEN
Activation of mammalian target of rapamycin (mTOR) has been known as one of the contributing factors in nociceptive sensitization after peripheral injury. Its activation followed by the phosphorylation of downstream effectors causes hyperexcitability of primary sensory neurons in the dorsal root ganglion. We investigated whether a single injection of rAAV-shmTOR would effectively downregulate both complexes of mTOR in the long-term and glial activation as well. Male SD rats were categorized into shmTOR (n = 29), shCON (n = 23), SNI (n = 13), and Normal (n = 8) groups. Treatment groups were injected with rAAV-shmTOR or rAAV-shCON, respectively. DRG tissues and sciatic nerve were harvested for Western blot and immunohistochemical analyses. Peripheral sensitization was gradually attenuated in the shmTOR group, and it reached a peak on PID 21. Western blot analysis showed that both p-mTORC1 and p-mTORC2 were downregulated in the DRG compared to shCON and SNI groups. We also found decreased expression of phosphorylated p38 and microglial activation in the DRG. We first attempted a therapeutic strategy for neuropathic pain with a low dose of AAV injection by interfering with the mTOR signaling pathway, suggesting its potential application in pain treatment.
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Neuralgia , Traumatismos del Sistema Nervioso , Ratas , Masculino , Animales , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Ratas Sprague-Dawley , Neuralgia/etiología , Neuralgia/terapia , Neuralgia/metabolismo , Nervio Ciático/metabolismo , Traumatismos del Sistema Nervioso/metabolismo , Sirolimus , Serina-Treonina Quinasas TOR/metabolismo , Ganglios Espinales/metabolismo , MamíferosRESUMEN
In addition to laser photocoagulation, therapeutic interventions for diabetic retinopathy (DR) have heretofore consisted of anti-VEGF drugs, which, besides drawbacks inherent to the treatments themselves, are limited in scope and may not fully address the condition's complex pathophysiology. This is because DR is a multifactorial condition, meaning a gene therapy focused on a target with broader effects, such as the mechanistic target of rapamycin (mTOR), may prove to be the solution in overcoming these concerns. Having previously demonstrated the potential of a mTOR-inhibiting shRNA packaged in a recombinant adeno-associated virus to address a variety of angiogenic retinal diseases, here we explore the effects of rAAV2-shmTOR-SD in a streptozotocin-induced diabetic mouse model. Delivered via intravitreal injection, the therapeutic efficacy of the virus vector upon early DR processes was examined. rAAV2-shmTOR-SD effectively transduced mouse retinas and therein downregulated mTOR expression, which was elevated in sham-treated and control shRNA-injected (rAAV2-shCon-SD) control groups. mTOR inhibition additionally led to marked reductions in pericyte loss, acellular capillary formation, vascular permeability, and retinal cell layer thinning, processes that contribute to DR progression. Immunohistochemistry showed that rAAV2-shmTOR-SD decreased ganglion cell loss and pathogenic Müller cell activation and proliferation, while also having anti-apoptotic activity, with these effects suggesting the therapeutic virus vector may be neuroprotective. Taken together, these results build upon our previous work to demonstrate the broad ability of rAAV2-shmTOR-SD to address aspects of DR pathophysiology further evidencing its potential as a human gene therapeutic strategy for DR.
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Diabetes Mellitus , Retinopatía Diabética , Animales , Dependovirus/genética , Diabetes Mellitus/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/terapia , Vectores Genéticos/genética , Ratones , ARN Interferente Pequeño/metabolismo , Retina/patología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Expanding on previous demonstrations of the therapeutic effects of adeno-associated virus (AAV) carrying small-hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)-stimulated endothelial cells were treated with AAV2-shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2-shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2-shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2-shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.
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Dependovirus , Factor A de Crecimiento Endotelial Vascular , Dependovirus/genética , Dependovirus/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , ARN Interferente Pequeño/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Purpose: Recent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR). Methods: Sprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition. Results: Compared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders. Conclusions: Taken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.
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Dependovirus/genética , Técnicas de Silenciamiento del Gen/métodos , Terapia Genética/métodos , Neovascularización Retiniana/terapia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Ratas Sprague-DawleyRESUMEN
Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3' UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.
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PURPOSE: We determine the prevalence of neutralizing antibodies (NAbs) to adeno-associated virus (AAV) in the vitreous humor and serum of patients with vitreoretinal diseases and investigate the relationship between NAb titers in the vitreous humor and serum. METHODS: We analyzed NAbs to AAV serotypes 2, 5, 8, and 9 via in vitro neutralization in the vitreous humor and serum from 32 patients requiring vitrectomy for vitreoretinal diseases. The blood-retinal barrier (BRB) was evaluated for integrity based on preoperative examinations, with vitreous hemorrhage (VH) on funduscopy or dye leakage on fluorescein angiography observed indicating disruption. RESULTS: NAb levels were much lower in the vitreous humor than in the serum regardless of serotype. Patients with VH had higher levels of NAbs in the vitreous humor than those without VH. The NAb ratio (ratio between NAb titers in the serum and vitreous humor) was much lower in patients with epiretinal membrane with than in those without leakage. A significantly lower NAb ratio was noticed in patients with than in those without BRB disruptions. CONCLUSIONS: The NAb ratio between levels in serum and vitreous humor varies according to the condition of the BRB. Therefore, in addition to measuring the serum NAb level, physicians should examine BRB integrity when planning retinal gene therapy. TRANSLATIONAL RELEVANCE: This study provides substantial basis for retinal gene therapy using AAVs and how maintenance of BRB integrity in target diseases should be considered.
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Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency.
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Purpose: With anti-VEGF-based treatments for wet AMD requiring frequent injections, it is often burdensome to both patients and healthcare providers. To explore its possibility as a desirable alternative, we investigated the therapeutic potential of a recombinant adeno-associated virus 2 expressing a soluble variant of VEGF receptor-1 (rAAV2-sVEGFRv-1) in a laser-induced choroidal neovascularization (CNV) model, as CNV is a defining feature of AMD progression. Methods: C57/B6 mice were intravitreally administered with rAAV2-sVEGFRv-1, rAAV2-GFP, or clinically used bevacizumab after CNV lesions were induced via laser photocoagulation. Immunostaining was performed with phalloidin and CD31 to measure CNV extensiveness, F4/80 and CD11b for inflammatory cell infiltration, and pan-cytokeratin to visualize fibrotic progression. Results: rAAV2-sVEGFRv-1 (5.0 × 107 viral genomes) possesses antiangiogenic, anti-inflammatory, and antifibrotic properties. rAAV2-sVEGFRv-1 was demonstrated to significantly decrease retinal CNV lesion size (1336 ± 186) when compared to rAAV2-GFP-treated (2949 ± 437, P = 0.0043), mock-treated (3075 ± 265, P = 0.0013), and bevacizumab-treated models (995 ± 234). Infiltration by inflammatory cells significantly decreased with rAAV2-sVEGFRv-1 administration, while groups treated with rAAV2-GFP did not. Additionally, antiapoptotic activity was observed via TUNEL assay in rAAV2-sVEGFRv-1 (16.0 ± 3.6) and rAAV2-GFP (46.0 ± 7.5, P = 0.003). Overall, the rAAV2-sVEGFRv-1 viral vector was positively comparable to bevacizumab, indicating it as effective as approved therapeutics. Conclusions: The ability of a low dose of rAAV2-sVEGFRv-1 to exert a therapeutically relevant anti-VEGF effect in a CNV model is demonstrated, and strongly suggests gene therapy as an effective and convenient treatment for sustained VEGF suppression.
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Neovascularización Coroidal/terapia , Terapia Genética , Parvovirinae/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Western Blotting , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/metabolismo , Dependovirus , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inyecciones Intravítreas , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.
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Choroidal neovascularization (CNV) is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD) and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF), the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR) has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA), which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA) after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV) delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV.
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Retinal transduction by intravitreally administered adeno-associated viral (AAV) vector is previously known to be extremely limited to the neural retina except AAV2 capsid type. Recently, we showed that prior laser photocoagulation enhances retinal transduction of intravitreally administered AAV vectors, including the outer retina and retinal pigment epithelium (RPE). Here, by performing short-pulse laser pretreatment on the mouse retina, we demonstrate RPE cells transduced by three different capsid types of AAV vectors, AAV2, AAV5, and AAV8, using RPE wholemounts. For all capsid types, laser pretreatment effectively induced the transduction of RPE cells in and around the laser site.
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Proteínas de la Cápside/genética , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Enfermedades de la Retina/genética , Enfermedades de la Retina/terapia , Epitelio Pigmentado de la Retina/fisiología , Animales , Coagulación con Láser , Ratones , Células Fotorreceptoras de Vertebrados/fisiología , Neuronas Retinianas/fisiología , Transducción Genética , Visión OcularRESUMEN
Since tenascin C is a factor expressed highly in the tumor-associated matrix, it would be a desirable first step for targeting the tumor-specific microenvironment. In fact, a high level of tenascin C expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors.
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Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Tenascina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Células HCT116 , Células HT29 , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recombinant adeno-associated virus serotype 5 (rAAV5) is considered to be a promising gene transfer vehicle. However, preferential gene delivery to the tumor remains a requirement for cancer treatment. We generated rAAV5 mutants bearing tumor marker-binding peptides and analyzed their properties as viral vectors, as well as their transduction efficiencies and preferential antitumoral potencies. All of the mutants were successfully produced. Transduction analyses showed that rAAV5 mutants harboring tumor-homing peptides, including RGD and TnC, transduced human cancer cells expressing corresponding receptors on their surfaces. RGDS peptides and TnC antibodies significantly suppressed transduction by rAAV5-RGD and rAAV5-TnC. Cytotoxicity was evident upon transfer of HSV-TK to cells by re-targeted rAAV5. These results provide evidence that rAAV5 vectors, genetically armed with tumor-targeting ligands, preferentially infect human cancer cells harboring the corresponding receptors, thereby inducing antitumoral effects. Further optimization of rAAV5 mutant viruses should thus facilitate practical exploitation of these vectors for gene-based cancer treatment.
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The thorns of Gleditsia sinensis are used as a medicinal herb in China and Korea. However, the mechanisms responsible for the antitumor effects of the water extract of Gleditsia sinensis thorns (WEGS) remain unknown. HCT116 cells treated with the WEGS at a dose of 800 µg/mL (IC50) showed a significant decrease in cell growth and an increase in cell cycle arrest during the G2/M-phase. G2/M-phase arrest was correlated with increased p53 levels and down-regulation of the check-point proteins, cyclinB1, Cdc2 and Cdc25c. In addition, treatment with WEGS induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAP kinase and JNK (c-Jun N-terminal kinases). Moreover, inhibition of ERK by treatment of cells with the ERK-specific inhibitor PD98059 blocked WEGS-mediated p53 expression. Similarly, blockage of ERK function in the WEGS-treated cells reversed cell-growth inhibition and decreased cell cycle proteins. Finally, in vivo WEGS treatment significantly inhibited the growth of HCT116 tumor cell xenografts in nude mice with no negative side effects, including loss of body weight. These results describe the molecular mechanisms whereby the WEGS might inhibit proliferation of colon cancer both in vitro and in vivo, suggesting that WEGS has potential as an anticancer agent for the treatment of malignancies.
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Ciclo Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Gleditsia/química , Extractos Vegetales/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Medicamentos Herbarios Chinos/farmacología , Flavonoides/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Programmed cell death 4 (PDCD4), a protein that binds to eukaryotic initiation factor 4A (eIF4A), inhibits the initiation of translation. Although a number of tumor suppressors target transcription, Pdcd4 is the first suppressor targeting protein translation, and has also been suggested to function as a tumor suppressor gene in human cancer. The majority of tumor suppressors are mutationally inactivated, but the expression of Pdcd4 is downregulated with progression in a number of human cancer sites, including the lung. METHODS: An aerosol of lentivirus-shRNA Pdcd4 was delivered into A/J mice, through a nose-only inhalation system twice a week for 1 month. RESULTS AND CONCLUSIONS: Downregulated Pdcd4 resulted in increase levels of antiapoptotic and uPA-regulated proteins. We also found that downregulated Pdcd4 induced the mTOR/p70S6K pathway and cell-cycle proteins. Our results suggest that Pdcd4 may perform a critical function in the regulation of lung cancer cell proliferation.
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Proteínas Reguladoras de la Apoptosis/administración & dosificación , Regulación hacia Abajo , Pulmón/metabolismo , ARN Interferente Pequeño/administración & dosificación , Proteínas de Unión al ARN/administración & dosificación , Administración por Inhalación , Administración Intranasal , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Ciclo Celular , Proliferación Celular , Vectores Genéticos , Humanos , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos A , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The thorns of Gleditsia sinensis have traditionally been used in the treatment of several diseases, which includes their use as anti-tumor agents, but there has been no scientific evidence of this anti-tumor effect. However, the present study has identified a novel mechanism for the anti-tumor effect of Gleditsia sinensis thorns in the treatment of colon cancer. Treatment with the ethanol extract of Gleditsia sinensis thorns (EEGS) resulted in significant growth inhibition together with G2/M-phase cell cycle arrest at a dose of 600 microg/ml (IC50) in HCT116 cells. In addition, treatment with EEGS induced p27 expression and down-regulated expression of cyclins and cyclin-dependent kinases. Moreover, EEGS treatment induced phosphorylation of extracellular signal-regulated kinases (ERK), p38 MAP kinase and JNK (c-Jun N-terminal kinases). Among the pathways examined, only PD98059 (ERK-specific inhibitor) abolished EEGS-dependent p27 expression. Similarly, suppression of ERK function reversed EEGS-mediated cell proliferation inhibition and decreased cell cycle proteins. In addition, tumor necrosis factor-alpha (TNF-alpha)-induced matrix metalloproteinase-9 (MMP-9) expression was inhibited by EEGS treatment via decreased transcriptional activity of both activator protein-1 (AP-1) and nuclear factor-kappaB. Finally, EEGS treatment significantly reduced tumor sizes in HCT116 cell-xenografted tumor tissues, which was associated with the changed levels of ERK phosphorylation, p27 and MMP-9 expression. Overall, these results have identified a novel molecular mechanism for EEGS in the treatment of colon cancer and might provide a theoretical basis for the potential therapeutic use of EEGS in the treatment of malignancies.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Etanol/farmacología , Gleditsia/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Extractos Vegetales/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This study investigated the anticancer activity of Magnolia officinalis on urinary bladder cancer in vitro and in vivo, and elucidated the mechanism of its activity. An aqueous extract of M. officinalis inhibited cell viability and DNA synthesis in cultured human urinary bladder cancer 5637 cells. Inhibition of proliferation was the result of apoptotic induction, because FACS analyses of 5637 cells treated with M. officinalis showed a sub-G1 phase accumulation. M. officinalis extract also increased cytoplasmic DNA-histone complex dose-dependently. These inhibitory effects were associated with the upregulation of proapoptotic molecules Bax, cytochrome c and caspase 3. Treatment of 5637 cells with M. officinalis extract suppressed the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9, as revealed by zymographic and immunoblot analyses. When M. officinalis extract was given to mice simultaneously with the carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine, which induces urinary bladder tumors, the size of the induced tumors was smaller. Finally, histological data indicated that the histological grade of carcinoma and the depth of invasion were dramatically decreased by treatment with M. officinalis extract in mice with N-butyl-N-(4-hydroxybutyl) nitrosamine-induced urinary bladder tumors. In conclusion, the findings showed that M. officinalis extract exhibited potential chemopreventive activity against urinary bladder tumor in vitro and in vivo.
Asunto(s)
Anticarcinógenos/farmacología , Butilhidroxibutilnitrosamina/toxicidad , Carcinoma de Células Transicionales/tratamiento farmacológico , Magnolia/química , Extractos Vegetales/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocromos c/metabolismo , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Neoplasias de la Vejiga Urinaria/inducido químicamente , Proteína X Asociada a bcl-2/metabolismoRESUMEN
For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.