Harmine promotes periodontal ligament cell-induced tissue regeneration.

OBJECTIVE: to investigate whether harmine has a promotive effect on human periodontal ligament cells (hPDLCs)-induced tissue regeneration. MATERIALS AND METHODS: Various concentrations of harmine on hPDLCs proliferation were tested. Osteogenic and cementogenic characteristics were examined in hPDLC/rhBMP-2 and hPDLC/harmine by alizarin red S staining, real-time PCR, and Western blotting assay. The activity of harmine was investigated in an ectopic transplantation nude mouse model. RESULTS: We determined that 10 µM of harmine was the threshold concentration. hPDLC/harmine showed similar mineralized nodule formation in alizarin S staining compared to hPDLC/rhBMP-2. In real-time PCR, the highest gene expression level was observed for Runx2 in hPDLC/harmine at all time points. The level of CEMP-1 in hPDLC/harmine was higher at 7 days than hPDLCs alone. Thicker band of Runx2 in hPDLC/harmine was observed than in hPDLC/rhBMP-2 at 7 days by Western blotting. The band for CEMP-1 in hPDLC/harmine was thicker than hPDLCs alone at both 7 and 14 days. In ectopic transplantation, hPDLCs with harmine showed a comparable amount of mineralized tissue formation compared to rhBMP-2. hPDLCs with harmine or rhBMP-2 formed both bone and cementum-like tissue with Sharpey's fiber-like collagen insertion. CONCLUSION: Harmine can be a potential candidate for promoting hPDLCs-induced tissue regeneration.

Human periodontal ligament stem cells suppress T-cell proliferation via down-regulation of non-classical major histocompatibility complex-like glycoprotein CD1b on dendritic cells.

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) from the periodontal ligament tissue were recently identified as mesenchymal stem cells (MSCs). The capabilities of PDLSCs in periodontal tissue or bone regeneration have been reported, but their immunomodulatory role in T-cell immune responses via dendritic cells (DCs), known as the most potent antigen-presenting cell, has not been studied. The aim of this study is to understand the immunological function of homogeneous human STRO-1+ CD146+ PDLSCs in DC-mediated T-cell immune responses to modulate the periodontal disease process. MATERIAL AND METHODS: We utilized highly purified (> 95%) human STRO-1+ CD146+ PDLSCs and human bone marrow mesenchymal stem cells (BMSCs). Each stem cell was co-cultured with human monocyte-derived DCs in the presence of lipopolysaccharide isolated from Porphyromonas gingivalis, a major pathogenic bacterium responsible for periodontal disease, in vitro to examine the immunological effect of each stem cell on DCs and DC-mediated T-cell proliferation. RESULTS: We discovered that STRO-1+ CD146+ PDLSCs, as well as BMSCs, significantly decreased the level of non-classical major histocompatibility complex glycoprotein CD1b on DCs, resulting in defective T-cell proliferation, whereas most human leukocyte antigens and the co-stimulatory molecules CD80 and CD86 in/on DCs were not significantly affected by the presence of BMSCs or STRO-1+ CD146+ PDLSCs. CONCLUSIONS: This study unveiled an immunomodulatory role of STRO-1+ CD146+ PDLSCs in negatively regulating DC-mediated T-cell immune responses, demonstrating their potential to be utilized in promising new stem cell therapies.

Comparison of the clinical characteristics of Mycobacterium tuberculosis and nontuberculous mycobacteria patients with joint involvement.

Nontuberculous mycobacteria (NTM) joint involvement is rare. However, the incidence of NTM disease is increasing and it is difficult to distinguish NTM from Mycobacterium tuberculosis (MTB). Here, the clinical characteristics of NTM joint involvement were compared with those of MTB. Distal joint involvement and precipitating factors were significantly more frequent for NTM joint infections. Because pathologic findings of NTM and MTB were similar, microbiological investigations are needed.

Isolation and characterization of human mesenchymal stem cells from gingival connective tissue.

BACKGROUND AND OBJECTIVE: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. MATERIAL AND METHODS: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. RESULTS: GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. CONCLUSIONS: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

The influence of geometrical characteristics on the photocatalytic activity of TiO2 nanotube arrays for degradation of refractory organic pollutants in wastewater.

The effects of geometrical characteristics such as surface area (SA) and porosity of TiO2 nanotube arrays (TNAs) on its photocatalytic activity were investigated by applying variable voltages and reaction times for the anodization of Ti substrates. While larger SA of nanotubes was observed under higher applied potential, the porosity of TNAs decreased by increasing anodizing voltage. Under applied potential of 80 V, the SA of TNAs increased from 0.164 to 0.471 m2/g as anodization time increased from 1 to 5 hours, respectively. However, no significant effect on the porosity of TNAs was observed. On the other hand, both SA and porosity of TNAs, synthesized at 60 V, increased by augmenting the anodization time from 1 to 3 hours. But further increasing of anodization time to 5 hours resulted in a decreased SA of TNAs with no effect on their porosity. Accordingly, the TNAs with SA of 0.368 m2/g and porosity of 47% showed the highest photocatalytic activity for degradation of 4-chlorobenzoic acid (4CBA). Finally, the degradation of refractory model compounds such as carbamazepine and bisphenol-A was tested and more than 50% of both compounds could be degraded under UV-A irradiation (λmax=365 nm).

Effect of humoral factors from hPDLSCs on the biologic activity of hABCs.

OBJECTIVE: The human periodontal ligament stem cells (hPDLSCs) and human alveolar bone-derived stromal cells (hABCs) seem to be closely involved in the maintenance of alveolar bone in an anatomically indirect manner; however, there is little study on this matter. Therefore, the effect of hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was evaluated, focusing on the humoral factors released by hPDLSCs. MATERIALS AND METHODS: Human periodontal ligament stem cells and hABCs were isolated and characterized. hPDLSCs were indirectly cocultured to observe the in vitro effect of humoral factors released from hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs. Human gingival fibroblasts (hGFs) were utilized as positive control. RESULTS: Isolated cells demonstrated the presence of stem cells within. Indirect coculture of hPDLSCs greatly inhibited osteoclastogenesis by hABCs. Osteogenesis/adipogenesis of hABCs was also inhibited by indirect coculture with hPDLSC. The magnitude of regulatory effect from hPDLSCs was significantly greater than that of hGFs. CONCLUSIONS: Humoral factors released from hPDLSCs seemed to modulate the differentiation of hABCs, and the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was all inhibited, suggesting the potential role of hPDLSCs in the maintenance of the alveolar bone.

Induction of bone formation by Escherichia coli-expressed recombinant human bone morphogenetic protein-2 using block-type macroporous biphasic calcium phosphate in orthotopic and ectopic rat models.

BACKGROUND AND OBJECTIVE: The potential of the Escherichia coli-expressed recombinant human bone morphogenetic protein-2 (ErhBMP-2) to support new bone formation/maturation using a block-type of macroporous biphasic calcium phosphate (bMBCP) carrier was evaluated in an orthotopic and ectopic rat model. MATERIAL AND METHODS: Critical-size (Φ 8 mm) calvarial defects and subcutaneous pockets in 32 Sprague-Dawley rats received implants of rhBMP-2 (2.5 µg) in a bMBCP carrier or bMBCP alone (control). Implant sites were evaluated using histological and histometric analysis following 2- and 8-wk healing intervals (eight animals/group/interval). RESULTS: ErhBMP-2/bMBCP supported significantly greater bone formation at 2 and 8 wk (10.8% and 25.4%, respectively) than the control at 2 and 8 wk (5.3% and 14.0%, respectively) in calvarial defects (p < 0.01). Bone formation was only observed for the ErhBMP-2/bMBCP ectopic sites and was significantly greater at 8 wk (7.5%) than at 2 wk (4.5%) (p < 0.01). Appositional and endochondral bone formation was usually associated with a significant increase in fatty marrow at 8 wk. The bMBCP carrier showed no evidence of bioresorption. CONCLUSION: ErhBMP-2/bMBCP induced significant bone formation in both calvarial and ectopic sites. Further study appears to be required to evaluate the relevance of the bMBCP carrier.

Osteoconductivity of Porous Titanium Structure on Implants in Osteoporosis.

In compromised bone conditions such as osteoporosis, developments of the implant surface are necessary to secure the stability of implants. This study investigated the effect of the surface porous titanium structure (PS) on the osseointegration of implants in osteoporotic bone. Bilateral ovariectomy (OVX) was performed in 4 female beagle dogs to induce osteoporosis for 32 wk. Success of induction was based on the evaluation of bone mineral density by Hounsfield units (HU) in computed tomography images. Posterior teeth in both mandibles were extracted 1 wk after OVX, and a total of 30 implants (15 implants in each group) were placed after 32 wk of osteoporosis induction. The control group implant underwent resorbable blast media (RBM) surface treatment, whereas the test group underwent RBM surface treatment in the coronal two-thirds and a PS added to the apical 3-mm portion. HU values in the mandibular trabecular bone, lumbar, and femoral head significantly decreased 32 wk after OVX, confirming osteoporotic condition after induction. Resonance frequency analysis and removal torque test showed comparable values between the 2 groups at 4 wk after implant placement. The surface topography of the implant after removal showed hard tissue integration at the PS in the test group. Bone-to-implant contact length was greater in the apical portion of the test group, although statistical significance was not found between the groups. Interthread bone area in the apical portion of the test group showed a significant increase compared to the control group (control: 0.059 ± 0.041 mm2, test: 0.121 ± 0.060 mm2, P = 0.028) with the histological feature of bone ingrowth at the PS. The findings of the study demonstrated that the surface PS could improve osteoconductivity in the osteoporotic trabecular bone by bone ingrowth at the pore space, thereby enhancing the osseointegration and stability of the implants.

STAT3 activation in endothelial cells is important for tumor metastasis via increased cell adhesion molecule expression.

Metastasis is a life-threatening feature of cancer and is primarily responsible for cancer patient mortality. Cross talk between tumor cells and endothelium is important for tumor progression and metastasis. However, very little is known about the mechanisms by which endothelial cells (ECs) that are close to tumor cells, respond to the tumor cells during tumor progression and metastasis. In this study, we exploited the use of EC-specific signal transducer activator of transcription 3 (STAT3) knockout mice to investigate the role of STAT3 in ECs in tumor progression and metastasis. We found that the loss of STAT3 in ECs did not affect primary Lewis lung carcinoma (LLC) tumor growth, but it reduced in vivo LLC metastasis in experimental and spontaneous metastasis models. Mechanistically, STAT3 activation upregulated cell adhesion molecule expression, including E-selectin and P-selectin, in murine endothelial MS-1 cells treated with tumor cell-conditioned media in vitro and in pre-metastatic lungs of tumor-bearing mice in vivo. We also found that both E-selectin and P-selectin were, at least in part, responsible for STAT3-induced adhesion and invasion of LLC cells through an EC monolayer. However, tumor cell-conditioned media from B16F10 melanoma cells did not activate STAT3 in MS-1 cells. As a result, EC STAT3 knockout did not affect B16F10 melanoma cell metastasis. In addition, various human cancer cells activated STAT3 in human ECs (HUVECs), resulting in increased cell adhesion molecule expression. Collectively, our findings demonstrate that STAT3 activation in ECs promotes tumor metastasis through the induction of cell adhesion molecules, demonstrating a role for ECs in response to tumor cells during tumor metastasis.

Angiopoietin 2 induces astrocyte apoptosis via αvß5-integrin signaling in diabetic retinopathy.

The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in early diabetic mice retina as Ang2 increased. The astrocyte loss and vascular leakage were inhibited by intravitreal injection of Ang2-neutralizing antibody. In vitro, Ang2 aggravated high glucose-induced astrocyte apoptosis via GSK-3ß activation. Ang2 directly bound to αvß5 integrin, which was abundant in astrocyte, and the blockade of αvß5 integrin, in vitro, effectively attenuated Ang2-induced astrocyte apoptosis. In vivo, intravitreal injection of anti-αvß5-integrin antibody inhibited astrocyte loss in early diabetic retinopathy. Taken together, Ang2 induced astrocyte apoptosis under high glucose via αvß5-integrin/GSK-3ß/ß-catenin pathway. Therefore, we suggest that Ang2/integrin signaling could be a potential therapeutic target to prevent the vascular leakage by astrocyte loss in early diabetic retinopathy.

Characterization of JDP genes, an evolutionarily conserved J domain-only protein family, from human and moths.

We characterized evolutionarily conserved J domain containing protein (JDP) genes from human, Bombyx mori, and Manduca sexta. Each of the JDP proteins contains a J domain at its N-terminus and a highly conserved C-terminal domain. Southern blot analysis revealed that the human JDP1 gene is present as a single copy in the human genome. Expression was higher in brain, heart, and testis than in kidney or stomach. Human JDP1 was mapped in silico to chromosome 10q21.1, which exhibits a conserved synteny with the central region of mouse chromosome 10. Drosophila jdp is located at 99F4-99F11 on the right arm of the third chromosome.

Isolation of a new member of DnaJ-like heat shock protein 40 (Hsp40) from human liver.

A new member of Hsp40, HLJ1, consisting of 337 amino acids, was cloned from a human liver cDNA library. The deduced amino acid sequence of HLJ1 has an 84% homology (69% identity) with that of HDJ-1 isolated from human placenta. Northern analysis showed that expression of the HLJ1 gene is heat-inducible and its transcription shows some degree of preference in heart, skeletal muscle, and pancreas.

CXXC5 is a negative-feedback regulator of the Wnt/ß-catenin pathway involved in osteoblast differentiation.

The positive roles of the Wnt/ß-catenin pathway in osteoblast differentiation and bone mineral density (BMD) maintenance have been clearly demonstrated in both animal experiments and clinical investigations. CXXC finger protein 5 (CXXC5), a recently identified negative regulator of the Wnt/ß-catenin pathway, showed altered cellular localization and function, which were dependent on the cell type in previous studies. However, the in vivo function of CXXC5 has not been clearly investigated yet. Here, we characterized CXXC5 as a negative regulator of osteoblast differentiation and bone formation. Deficiency of CXXC5 resulted in elevated BMD in mice without any severe gross developmental abnormalities. CXXC5 exerted a negative-feedback effect on the Wnt/ß-catenin pathway via Wnt-dependent binding to Dishevelled (Dvl) during osteoblast differentiation. Suppression of the Dvl-CXXC5 interaction using a competitor peptide resulted in the activation of the Wnt/ß-catenin pathway and osteoblast differentiation, and accelerated thickness growth of ex vivo-cultured calvariae. Overall, CXXC5 is a negative-feedback regulator induced by Wnt/ß-catenin signaling that inhibits osteoblast differentiation and bone formation via interaction with Dvl.

Duplication of genes encoding non-clathrin coat protein gamma-COP in vertebrate, insect and plant evolution.

Coatomer is a major component of COPI vesicles and consists of seven subunits. The gamma-COP subunit of the coatomer is believed to mediate the binding to the cytoplasmic dilysine motifs of membrane proteins. We characterized cDNAs for Copg genes encoding gamma-COP from mouse, zebrafish, Drosophila melanogaster and Bombyx mori. Two copies of Copg genes are present in vertebrates and in B. mori. Phylogenetic analysis revealed that two paralogous genes had been derived from a single ancestral gene by duplication independently in vertebrates and in B. mori. Mouse Copg1 showed ubiquitous expression with the highest level in testis. Zebrafish copg2 was biallelically expressed in hybrid larvae in contrast to its mammalian ortholog expressed in a parent-of-origin-specific manner. A phylogenetic analysis with partial plant cDNA sequences suggested that copg gene was also duplicated in the grass family (Poaceae).

Mit1/Lb9 and Copg2, new members of mouse imprinted genes closely linked to Peg1/Mest(1).

Two mouse genes, Mit1/Lb9 and Copg2, linked to Peg1/Mest on mouse chromosome 6, were identified to be imprinted maternally and paternally, respectively. Mit1/Lb9 encoding untranslated transcripts resides within the intron 20 of Copg2. The gene is maternally imprinted in adult mouse brain, partially imprinted in other tissues. Copg240 kb genomic region, being expressed ubiquitously in mouse tissues with a partial imprinting pattern in embryos, neonates, and adult brain in contrast to maternally imprinted human COPG2. In addition, we identified an antisense transcript of Copg2, Copg2AS, which overlaps 3'-UTRs of Copg2 and Peg1/Mest. The Copg2AS transcript is maternally imprinted in embryos, neonates, and adult tissues.

A disposable, coated wire heparin sensor.

The development of an ion-selective electrode heparin sensor consisting of a specially formulated polymer membrane doped with tridodecylmethylammonium chloride as the heparin complexing agent was recently reported. Because of the simple nature of the membrane technology used, the authors envisioned that the sensor could be configured as a disposable single-use device for rapid clinical or bedside measurement of heparin in a small, discrete sample. To explore this possibility, an inexpensive, disposable heparin sensor was created by dip-coating a copper wire with the specially formulated heparin-sensing polymeric membrane. Coated wire heparin sensors with a broad range of membrane thicknesses, prepared by repeatedly dipping the wire in the membrane solution for various times, were examined. Data show that increasing the membrane thickness of the sensor to a certain degree (more than 10 microns) enhanced the sensor's potentiometric response to heparin, although the time required to achieve 90% of the steady-state potential change was also prolonged. In addition, increasing membrane thickness also magnified the stirring effect on the sensor's response. In undiluted plasma samples, the coated-wire sensor with an optimized membrane thickness yielded a significant (5 to 30 mV) and reproducible response to heparin in a clinically relevant concentration range (0.5 to 12 units/ml, respectively). The clinical utility of the coated wire heparin sensor was shown using the sensor during protamine titration of heparinized plasma to assess the titration end-point. Preliminary results showed that the titration end-points determined by the heparin sensor strongly correlated with those determined by the activated partial thromboplastin time clotting assay. The overall time requirement to complete the titration process using a set of prefabricated coated wire heparin sensors, however, was less than 3 minutes. Further titration studies using undiluted clinical whole blood samples are in progress.

A protamine filter for extracorporeal heparin removal. Development, testing, blood compatibility evaluation, and future direction.

The authors previously developed a filter device containing immobilized protamine (termed "protamine filter") that could be used to remove heparin during extracorporeal perfusion. In vivo studies involving dogs showed that the protamine filter removed more than 50% of heparin from the animals' blood circuit in less than 20 min. In addition, the use of the protamine filter did not elicit statistically significant protamine induced hemodynamic and thrombocytopenic responses. Biocompatibility of the protamine filter was also evaluated, with the focus on its effect on the coagulation cascade, the complement system, and the blood antithrombin III levels. Results showed that heparin adsorbed to the protamine coated surface retained 20% of its original activated partial thromboplastin time activity, rendering the coated surface antithrombotic. Activation of the coagulation system by the protamine coated membrane and the untreated cellulose membrane, as measured by the elevation of prothrombin fragment F1 + 2 levels, was statistically identical. The CH50 hemolytic assay showed that the protamine coated membrane produced a reduction of 1.2 +/- 0.8% of the total complement levels, as compared to 9.4 +/- 1.6% by the untreated membrane. In addition, the change in C3a des Arginine levels after 30 min of circulation was 1.5 +/- 0.2 mg/ml by the protamine filter, as compared to 2.1 +/- 0.1 mg/ml by the untreated membrane. Unlike native heparin that would bind with antithrombin, heparin adsorbed on the protamine coated surface was devoid of such activity, and produced no depletion of circulating antithrombin. Because of the limited capacity of the protamine filter, the future system is envisioned to consist of two filters; while one filter is removing heparin the other will be regenerated. With a recently developed heparin sensor, it should be possible to design a sensor directed, biofeedback, two filter heparin removal system.

Clinical application of disposable heparin sensors. Blood heparin measurements during open heart surgery.

The authors previously reported the development of an ion selective electrode type heparin sensor consisting of a specially formulated polymer membrane doped with tridodecylmethylammonium chloride as the heparin complexing agent. They also demonstrated the feasibility of measuring blood heparin levels by protamine titration, using a disposable copper wire sensor coated with the heparin sensing membrane to probe the titration end point. In this article, the results of further titration studies conducted on 44 clinical whole blood specimens obtained from 8 patients undergoing open heart surgery were reviewed. Samples were taken from patients at four different stages during the bypass surgery: 1) before heparin administration; 2) immediately after heparin administration; 3) within 30 min to 3 hr after heparin administration; and 4) within 30 min after protamine administration. Heparin anticoagulant activity in these samples was monitored by the activated clotting time assay, whereas heparin concentrations were measured by protamine titration using either the Hepcon HMS Titrator (Medtronic HemoTec Inc., Englewood, CO) or the coated wire heparin sensor to determine titration end points. Results indicate that heparin levels determined by the sensor method were in good agreement with those determined by the Hepcon HMS Titrator. When the heparin concentrations estimated by the two methods show significant discrepancy (> 1.0 unit/ml), the sensor method seems to provide more precise values, as verified by an additional chromogenic heparin assay. The overall time required to complete the titration process and heparin measurement with a pre made heparin sensor was less than 3 min. Clinically, the heparin sensor could be used as a safeguard to precisely monitor heparin levels during surgical procedures. Alternatively, the sensor could be used to assess the accurate protamine dose required for full heparin reversal.

Cryopreservation of Taxus chinensis suspension cell cultures.

A simple cryopreservation method for suspension cells of Taxus chinensis was established. In this procedure 7 days old suspension cells were used without any pre-culture treatment. At first, cells were incubated in cryoprotectant solution (0.5M DMSO and 0.5M glycerol) on ice for 30 min and then frozen at a cooling rate of 1 degree C/min to -40 degrees C prior to immersion in liquid nitrogen. The average viability of frozen-thawed cells was between 30 to 40%. The recovery of cryopreserved cells in liquid nitrogen for 1 month was accomplished. After rapid thawing, cells were transferred to solid medium and cultivated for 4-6 weeks. The treatment of trehalose as a cryoprotectant enhanced re-growth of frozen-thawed cells. The stable maintenance of paclitaxel biosynthetic ability in cryopreserved cells was confirmed by comparing with that of regularly sub-cultured suspension cells.

Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment.