A Single Injection of rAAV-shmTOR in Peripheral Nerve Persistently Attenuates Nerve Injury-Induced Mechanical Allodynia.

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.

scAAV2-Mediated Expression of Thioredoxin 2 and C3 Transferase Prevents Retinal Ganglion Cell Death and Lowers Intraocular Pressure in a Mouse Model of Glaucoma.

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.

mTOR inhibition as a novel gene therapeutic strategy for diabetic retinopathy.

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.

AAV expressing an mTOR-inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity.

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.

Gleditsia sinensis thorn extract inhibits human colon cancer cells: the role of ERK1/2, G2/M-phase cell cycle arrest and p53 expression.

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.

Inhibition of mTOR via an AAV-Delivered shRNA Tested in a Rat OIR Model as a Potential Antiangiogenic Gene Therapy.

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.

Inhibitory effects of the aqueous extract of Magnolia officinalis on the responses of human urinary bladder cancer 5637 cells in vitro and mouse urinary bladder tumors induced by N-Butyl-N-(4-hydroxybutyl) nitrosamine in vivo.

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.

Relationship Between Neutralizing Antibodies Against Adeno-Associated Virus in the Vitreous and Serum: Effects on Retinal Gene Therapy.

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.

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

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.

Effects of Stuffer DNA on the Suppression of Choroidal Neovascularization by a rAAV Expressing a mTOR-Inhibiting shRNA.

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.

Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell-cycle arrest via p21WAF1 expression.

Naringin, an active flavonoid found in citrus fruit extracts, has pharmacological utility. The present study identified a novel mechanism of the anticancer effects of naringin in urinary bladder cancer cells. Naringin treatment resulted in significant dose-dependent growth inhibition together with G(1)-phase cell-cycle arrest at a dose of 100 microM (the half maximal inhibitory concentration) in 5637 cells. In addition, naringin treatment strongly induced p21WAF1 expression, independent of the p53 pathway, and downregulated expression of cyclins and cyclin dependent kinases (CDKs). Moreover, treatment with naringin induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Among the pathways examined, only PD98059, an ERK-specific inhibitor, blocked naringin-dependent p21WAF1 expression. Consistently, blockade of ERK function reversed naringin-mediated inhibition of cell proliferation and decreased cell-cycle proteins. Furthermore, naringin treatment increased both Ras and Raf activation. Transfection of cells with dominant-negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, the naringin-induced reduction in cell proliferation and cell-cycle proteins also was abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E-CDK2 complexes and naringin-dependent inhibition of cell growth. Overall, these unexpected findings concerning the molecular mechanisms of naringin in 5637 cancer cells provide a theoretical basis for the therapeutic use of flavonoids to treat malignancies.

Cancer gene therapy using adeno-associated virus vectors.

Gene therapy has offered highly possible promises for treatment of cancers, as many potential therapeutic genes involved in regulation of molecular processes may be introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion, metastasis, and/or can stimulate the immune response against tumors. Therefore, viral and non-viral gene delivery systems have been developed to establish an ideal delivery vector for cancer gene therapy over the past several years. Among the currently developed virus vectors, the adeno-associated virus (AAV) vector is considered as one of those that are closest to the ideal vector mainly for genetic diseases due to the following prominent features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very low host immune response and long-term expression. Particularly, the most important attribute of AAV vectors is their safety profile in clinical trials ranging from CF to Parkinson's disease. Although adenovirus and several other oncolytic viruses have been more frequently used to develop cancer gene therapy, AAV also has many critical properties to be exploited for a cancer gene delivery vector. In this review, we will briefly summarize the basic biology of AAV and then mainly focus on recent progresses on AAV vector development and AAV-mediated therapeutic vectors for cancer gene therapy.

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2.

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.

Activation of matrix metalloproteinase-9 by TNF-alpha in human urinary bladder cancer HT1376 cells: the role of MAP kinase signaling pathways.

The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in tumor invasion and metastasis. In this study, the factors and signaling pathways that are involved in the regulation of the MMP-9 expression were examined in urinary bladder cancer HT1376 cells. Tumor necrosis factor-alpha (TNF-alpha) stimulated the secretion of MMP-9 in HT1376 cells, as shown by zymography and immunoblot analysis. At the level of transcription, TNF-alpha also stimulated 5'-flanking promoter activity of MMP-9. Transcription factor NF-kappaB, AP-1 and Sp-1 binding sites were identified by a gel shift assay to be cis-elements for TNF-alpha activation of the MMP-9 promoter. TNF-alpha activates multiple signaling pathways in HT1376 cells, including the extracellular signal-regulated kinase (ERK1/2), p38 MAP kinase and JNK pathways. Chemical inhibitors, which specifically inhibit each of these TNF-alpha-activated pathways, were used to examine the signaling pathways involved in TNF-alpha-mediated MMP-9 expression. The ERK1/2 inhibitor, U0126 and the p38 MAP kinase inhibitor, SB203580, significantly down-regulated TNF-alpha-induced MMP-9 expression and promoter activity. The transactivation of TNF-alpha-stimulated NF-kappaB, AP-1 and Sp-1 were inhibited by U0126 and SB203580 treatment. In conclusion, the findings of the present study indicate that TNF-alpha induces MMP-9 expression in HT1376 cells by activating transcription factors, which are involved in the ERK1/2- and p38 MAP kinase-mediated control of MMP-9 regulation, namely, NF-kappaB, AP-1 and Sp-1.

Intravitreal Injection of AAV Expressing Soluble VEGF Receptor-1 Variant Induces Anti-VEGF Activity and Suppresses Choroidal Neovascularization.

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.

Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model.

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.

STP-A11, an oncoprotein of Herpesvirus saimiri augments both NF-kappaB and AP-1 transcription activity through TRAF6.

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer.

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound- healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Efficient gene expression by self-complementary adeno-associated virus serotype 2 and 5 in various human cancer cells.

The feasibility of various self-complementary AAV (scAAV) serotypes as efficient gene delivery vehicles in human cancer cells was evaluated. To dissect the transduction characteristics, we infected a variety of human cancer cells with scAAV1-6 or scAAV8 expressing GFP. scAAV2 led to the best transduction efficiency with nearly complete transgene expression at 1000 MOI in most cancer cells, regardless of cell/tissue origins. scAAV5 could also induce effective gene expression, even though gene transfer potency by scAAV5 was poorer than that by scAAV2. Substantial portion of transgene expression lasted over a month following gene delivery by both scAAV 2 and scAAV5, indicating that long-term gene expression can occur. Moreover, co-infection of scAAV2 and scAAV5 can induce simultaneous transgene expressions introduced via each vector. Thus, the current study provide evidence that scAAV2 and scAAV5 vectors are excellent gene transfer tools in a wide variety of human cancer cells, independently driving persistent transgene expression.

Aqueous extract of Magnolia officinalis mediates proliferative capacity, p21WAF1 expression and TNF-alpha-induced NF-kappaB activity in human urinary bladder cancer 5637 cells; involvement of p38 MAP kinase.

Magnolia officinalis is a commonly used herb in East Asian countries and has multiple pharmacological effects. Although Magnolia officinalis has a variety of pharmacological effects on certain cancer cell types, the molecular mechanisms on urinary bladder cancer are unclear. An aqueous extract of M. officinalis inhibited cell proliferation in cultured human urinary bladder cancer 5637 cells. Inhibition of proliferation was associated with G1 cell cycle arrest. Treatment with M. officinalis extract blocked the cell cycle in the G1 phase, down-regulated the expression of cyclins and CDKs and up-regulated the expressions of p21WAF1 and p27 Kip1, which are CDK inhibitors. In addition, M. officinalis extract induced a marked activation of p38 MAP kinase and JNK. SB203580, a p38 MAP kinase specific inhibitor, blocked the expression of M. officinalis extract-dependent p38 MAP kinase and p21WAF1. Blockage of the p38 MAPK kinase function reversed M. officinalis extract-induced cell proliferation. These data demonstrate that M. officinalis extract-induced cell growth inhibition appears to be linked to the activation of p38 MAP kinase through p21WAF1 expression. Moreover, treatment of 5637 cells with M. officinalis extract suppressed constitutive and TNF-alpha-induced-nuclear factor-kappa B (NF-kappaB) activation. Furthermore, the transactivation of TNF-alpha-stimulated NF-kappaB was inhibited by SB203580 treatment. Collectively, these results suggest that the p38 MAP kinase pathway contributes, at least partially, to the anti-cancer activity of M. officinalis extract in human urinary bladder tumor 5637 cells.