Tocilizumab (TCZ) Decreases Angiogenesis in Rheumatoid Arthritis through its Regulatory Effect on miR-146a-5p and EMMPRIN/CD147

Abstract BackgroundAngiogenesis is an important contributor to the development of Rheumatoid arthritis (RA). Tocilizumab (TCZ), an anti-IL-6 receptor antibody, is used in the treatment of RA patients, and has been shown to exert anti-inflammatory effects. However, its effects on angiogenesis are not fully elucidated, and the molecular mechanisms regulating this effect are unknown. MethodsWe evaluated the concentrations of several pro- and anti-angiogenic factors and the expression levels of several microRNA molecules that are associated with RA and angiogenesis in serum samples obtained from 40 RA patients, before and 4 months after the initiation of TCZ treatment. Additionally, we used an in vitro co-culture system of fibroblasts (the HT1080 cell line) and monocytes (the U937 cell line) to explore the mechanisms of TCZ action. ResultsSerum samples from RA patients treated with TCZ exhibited reduced levels of EMMPRIN/CD147, enhanced expression of miR-146a-5p and miR-150-5p, and reduced angiogenesis as was manifested by the reduced number of tube-like structures formed by EaHy926 endothelial cell line. In vitro, the accumulation of the pro-angiogenic factors EMMRPIN, VEGF and MMP-9 in the supernatants was increased by co-culturing the HT1080 fibroblasts and the U937 monocytes, while the accumulation of the anti-angiogenic factor thrombospondin-1 (Tsp-1) and the expression levels of miR-146a-5p were reduced. Transfection of HT1080 cells with the miR-146a-5p mimic, decreased the accumulation of EMMPRIN, VEGF and MMP-9. When EMMPRIN was neutralized with a blocking antibody, supernatants derived from these co-cultures exhibited reduced migration, proliferation and tube formation in functional assays. ConclusionsOur findings implicate miR-146a-5p in the regulation of EMMPRIN and propose that TCZ affects angiogenesis through its effects on EMMRPIN and miR-146a-5p.

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Tocilizumab (TCZ) Decreases Angiogenesis in Rheumatoid Arthritis Through Its Regulatory Effect on miR-146a-5p and EMMPRIN/CD147

Frontiers in Immunology ◽

10.3389/fimmu.2021.739592 ◽

2021 ◽

Vol 12 ◽

Author(s):

Devy Zisman ◽

Mirna Safieh ◽

Elina Simanovich ◽

Joy Feld ◽

Amalia Kinarty ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Cell Line ◽

Molecular Mechanisms ◽

Tube Formation ◽

Angiogenic Factors ◽

Angiogenic Factor ◽

Serum Samples ◽

Expression Levels ◽

Angiogenic Potential

BackgroundAngiogenesis is a major contributor to the development of inflammation during Rheumatoid arthritis (RA), as the vascularization of the pannus provides nutrients and oxygen for the infiltrating immune cells and proliferating synoviocytes. Tocilizumab (TCZ) is an anti-IL-6 receptor antibody that is used in the treatment of RA patients, and has been shown to exert anti-inflammatory effects. However, its effects on angiogenesis are not fully elucidated, and the molecular mechanisms regulating this effect are unknown.MethodsWe evaluated the concentrations of several pro- and anti-angiogenic factors and the expression levels of several microRNA molecules that are associated with RA and angiogenesis in serum samples obtained from 40 RA patients, before and 4 months after the initiation of TCZ treatment. Additionally, we used an in vitro co-culture system of fibroblasts (the HT1080 cell line) and monocytes (the U937 cell line) to explore the mechanisms of TCZ action.ResultsSerum samples from RA patients treated with TCZ exhibited reduced circulating levels of EMMPRIN/CD147, enhanced expression of circulating miR-146a-5p and miR-150-5p, and reduced the angiogenic potential as was manifested by the lower number of tube-like structures that were formed by EaHy926 endothelial cell line. In vitro, the accumulation in the supernatants of the pro-angiogenic factors EMMPRIN, VEGF and MMP-9 was increased by co-culturing the HT1080 fibroblasts and the U937 monocytes, while the accumulation of the anti-angiogenic factor thrombospondin-1 (Tsp-1) and the expression levels of miR-146a-5p were reduced. Transfection of HT1080 cells with the miR-146a-5p mimic, decreased the accumulation of EMMPRIN, VEGF and MMP-9. When we neutralized EMMPRIN with a blocking antibody, the supernatants derived from these co-cultures displayed reduced migration, proliferation and tube formation in the functional assays.ConclusionsOur findings implicate miR-146a-5p in the regulation of EMMPRIN and propose that TCZ affects angiogenesis through its effects on EMMPRIN and miR-146a-5p.

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POS0613 TOCILIZUMAB DECREASES ANGIOGENESIS IN RHEUMATOID ARTHRITIS THROUGH ITS REGULATORY EFFECT ON EMMPRIN/CD147

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1616 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 543.2-543

Author(s):

D. Zisman ◽

M. Safieh ◽

E. Simanovich ◽

J. Feld ◽

A. Kinarty ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Culture System ◽

Molecular Mechanisms ◽

Serum Levels ◽

Angiogenic Factors ◽

Angiogenic Factor ◽

Serum Samples ◽

Blocking Antibody ◽

Endothelial Layer

Background:Angiogenesis is an important contributor to the development of Rheumatoid arthritis (RA). Tocilizumab (TCZ), an anti-IL-6 receptor antibody, is an immunosuppressant used in the treatment of RA patients, but its effects on angiogenesis and the molecular mechanisms regulating new blood vessel formation are not fully elucidated.Objectives:To evaluate the concentrations of pro- and anti-angiogenic factors in serum samples of RA patients, before and after the initiation of TCZ treatment and to explore in an in vitro co-culture system the mechanisms of TCZ action.Methods:We evaluated the concentrations of EMMPRIN, VEGF, MMP-9, IL-6, NGAL, endostatin and thrombospondin-1 (Tsp-1) using commercial ELISA kits from 40 RA patients, before and 4 months after the initiation of TCZ treatment. The levels of secreted EMMPRIN, VEGF MMP-9 and Tsp-1 were measured in an in vitro co-culture system of HT1080 fibroblasts and U937 monocytes with and without addition of anti-EMMPRIN blocking antibody. In the tube formation assay serum samples and supernatnats from the co-cultures were added to endothelial layer. Images were obtained after 6 hours of incubation and the number of closed lumens were counted in two separate fields. In the wound assay, supernatants from the co-cultures, with or without the addition of the anti-EMMPRIN antibody were added to the endothelial layer after scratching. The scratch site area was measured immediately and compared to the area after 24 hours of incubation to assess the distance of cell migration.Results:Study population included 40 RA patients, 33 (82.5%) females, mean age of 57.5±11.1 years, disease duration of 7.7±5.6 years, and 53.9% positive for rheumatoid factor initiating treatment with TCZ. In this patient cohort, 25/40 (62.5%) patients were classified as “responders” according to EULAR criteria.Following 4 mounts of treatment, statistically significant reductions in the levels of EMMPRIN/CD147 (p=0.035), without significant changes in serum levels of MMP-9, VEGF, MMP-3 and MMP-7 and of the anti-angiogenetic factors Tsp-1 and endostatin were found. A statistically significant decrease in the ratio between the pro-angiogenic factor EMMPRIN and the anti-angiogenic factor Tsp-1 that was calculated for each patient 4 months after initiating TCZ was found(p=0.031). The decrease in angiogenesis was manifested by the reduced number of closed lumen tube-like structures formed by EaHy926 endothelial cell line after incubation with serum samples 4 months after initiation of TCZ, relative to the number of closed lumens formed prior to TCZ initiation (p=0.007). The ratio between EMMPRIN and Tsp-1 was significantly reduced in the responding patients versus non-responders (p=0.033), while the levels of VEGF, MMP-9, Tsp-1, and EMMPRIN were unchanged.In vitro, the accumulation of the pro-angiogenic factors EMMRPIN, VEGF and MMP-9 in the supernatants was increased in the co-culture, while the accumulation of the anti-angiogenic factor Tsp-1 was decreased. When EMMPRIN was neutralized with a blocking antibody, supernatants derived from these co-cultures exhibited reduced migration, proliferation, and tube-like structure formation in functional assays.Conclusion:Our findings suggest an important role for EMMPRIN in mediating pro-angiogenic signals in RA patients, with EMMPRIN/Tsp-1 ratio serving as a marker of angiogenesis in RA. When administered to RA patients, TCZ in turn, exerts an anti-angiogenic effect through its regulation of EMMRPIN/CD147 levels.Disclosure of Interests:None declared

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Abstract TP489: Microglia Involved in Soluble Endoglin-Induced Brain Vascular Malformation

Stroke ◽

10.1161/str.51.suppl_1.tp489 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Eun S. Park ◽

P. Roc Chen ◽

Eunhee Kim

Keyword(s):

Endothelial Dysfunction ◽

Mouse Brain ◽

Tube Formation ◽

Angiogenic Factors ◽

Angiogenic Factor ◽

Vascular Walls ◽

Soluble Endoglin ◽

Bv2 Cells ◽

Primary Mouse

Introduction: Microglia are closely communicating with endothelial cells and involved in cerebrovascular disease. In brain arteriovenous malformation (bAVM), microglia are highly detected in the vascular walls and brain parenchyma, however, the role of microglia in bAVM are not clear. Meanwhile, soluble endoglin (sENG) has been involved in inflammation and endothelial dysfunction in bAVM. Therefore, we hypothesized that microglia mediate sENG-induced endothelial dysfunction in bAVM. Methods: sENG was subcutaneously injected to mice with VEGF overexpression in the brain. After latex casting to visualize brain vasculature, dysplastic capillaries were measured by Zen software. Microglia was detected by iba-1 staining. In in vitro , sENG or vehicle was treated in BV2 microglial cell line and the cultured media from BV2 (BV2-CM) were transferred to primary mouse brain endothelial cell (EC). We determined the expression of inflammatory/angiogenic factors in the BV2 and EC, and tested the tube formation in the EC. Results: The sENG with VEGF induced dysplastic/enlarged capillaries in the mouse brain (8567 μm 2 ± 2797 vs 2584 μm 2 ± 763, p <0.01) and activated microglia were accumulated around the abnormal vessels. In in vitro study, sENG increased inflammatory cytokines (TNF-α and IL-6) and angiogenic mediators (MMP-9 and VEGF-A) in BV2 cells. The sENG-treated BV2-CM significantly increased angiogenic factors (Notch-1 and TGF-β) but decreased an anti-angiogenic factor (IL-17RD), and it was accompanied with increased pERK1/2 in EC. In addition, we observed that tube formation is significantly increased in EC treated with sENG-treated BV2-CM. Conclusion: The study shows that sENG-stimulated microglia released inflammatory and angiogenic factors and it regulates endothelial functions. It suggests that microglia may contribute to sENG-induced dysplastic capillary via regulating inflammatory and angiogenic factors.

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Acute Mechanical Stress in Primary Porcine RPE Cells Induces Angiogenic Factor Expression and in vitro Angiogenesis

10.21203/rs.2.20909/v2 ◽

2020 ◽

Author(s):

Farhad Farjood ◽

Amir Ahmadpour ◽

Sassan Ostvar ◽

Elizabeth Vargis

Keyword(s):

Mechanical Stress ◽

Tube Formation ◽

Angiogenic Factors ◽

Angiogenic Factor ◽

Rpe Cells ◽

Qrt Pcr ◽

Angiogenic Proteins ◽

The Impact ◽

In Vitro Angiogenesis

Abstract Background Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration. CNV is characterized by new blood vessel growth and subretinal fluid accumulation, which results in mechanical pressure on retinal pigment epithelial (RPE) cells. The overexpression of RPE-derived angiogenic factors plays an important role in inducing CNV. In this work, we investigated the effect of mechanical stress on the expression of angiogenic factors in porcine RPE cells and determined the impact of conditioned medium on in-vitro angiogenesis. Results The goal of this study was to determine whether low levels of acute mechanical stress during early CNV can induce the expression of angiogenic factors in RPE cells and accelerate angiogenesis. Using a novel device, acute mechanical stress was applied to primary porcine RPE cells and the resulting changes in the expression of major angiogenic factors, VEGF, ANG2, HIF-1α, IL6, IL8 and TNF - α, were examined using immunocytochemistry, qRT-PCR, and ELISA. An in vitro tube formation assay was used to determine the effect of secreted angiogenic proteins due to mechanical stress on endothelial tube formation by human umbilical vein endothelial cells (HUVECs). Our results showed an increase in the expression of VEGF, ANG2, IL-6 and IL-8 in response to mechanical stress, resulting in increased in vitro angiogenesis. Abnormal epithelial-mesenchymal transition (EMT) in RPE cells is also associated with CNV and further retinal degeneration. Our qRT-PCR results verified an increase in the expression of EMT genes, CDH2, VIM and FN1, in RPE cells. Conclusions In conclusion, we showed that acute mechanical stress induces the expression of major angiogenic and EMT factors and promotes in vitro angiogenesis, suggesting that mechanical stress plays a role in promoting aberrant angiogenesis in AMD.

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Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-βRI/Smad Signaling

Molecules ◽

10.3390/molecules26061715 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1715

Author(s):

Xin Luo ◽

Qiangqiang Deng ◽

Yaru Xue ◽

Tianwei Zhang ◽

Zhitao Wu ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Cell Line ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Salvia Miltiorrhiza ◽

A549 Cells ◽

Epithelial Cell Line ◽

Human Lung Fibroblast ◽

Magnesium Lithospermate B

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-β)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-β receptor I (TGF-βRI) and regulating the TGF-β/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

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Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

Journal of Clinical Medicine ◽

10.3390/jcm10020183 ◽

2021 ◽

Vol 10 (2) ◽

pp. 183

Author(s):

Nadia Meyer ◽

Lars Brodowski ◽

Katja Richter ◽

Constantin S. von Kaisenberg ◽

Bianca Schröder-Heurich ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Growth Factor ◽

Endothelial Dysfunction ◽

Protein Expression ◽

Tube Formation ◽

In Vitro Assays ◽

Heme Oxygenase 1 ◽

Expression Levels ◽

Functional Capacities

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction.

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Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model

International Journal of Molecular Sciences ◽

10.3390/ijms22083873 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3873

Author(s):

Gabriel Luta ◽

Mihail Butura ◽

Adrian Tiron ◽

Crina E. Tiron

Keyword(s):

Cell Line ◽

Ex Vivo ◽

Glioma Cells ◽

In Vitro Study ◽

Immunofluorescent Staining ◽

Expression Levels ◽

Disease Diagnostics ◽

Phosphorylated Akt ◽

Significant Impairment

Background: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. Methods: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. Results: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. Conclusions: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.

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YWHAZ interacts with DAAM1 to promote cell migration in breast cancer

Cell Death Discovery ◽

10.1038/s41420-021-00609-7 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Jie Mei ◽

Yan Liu ◽

Xinqian Yu ◽

Leiyu Hao ◽

Tao Ma ◽

...

Keyword(s):

Breast Cancer ◽

Poor Prognosis ◽

Molecular Mechanisms ◽

Transcriptional Regulator ◽

Expression Levels ◽

Functional Roles ◽

Normal Tissues ◽

Rhoa Activation ◽

Promote Cell Migration

AbstractDishevelled-associated activator of morphogenesis 1 (DAAM1) is a critical driver in facilitating metastasis in breast cancer (BrCa). However, molecular mechanisms for the regulation of DAAM1 activation are only partially elucidated. In this research, the expression levels of YWHAZ and DAAM1 were examined by immunohistochemistry (IHC) staining in BrCa tissues. The functional roles of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ)–DAAM1 axis and their regulator microRNA-613 (miR-613) in BrCa cells and associated molecular mechanisms were demonstrated in vitro. As results, the expression levels of DAAM1 and YWHAZ were significantly upregulated in BrCa tissues compared with normal tissues and remarkably associated with poor prognosis. Besides, DAAM1 and YWHAZ were positively correlated with each other in BrCa tissues. YWHAZ interacted and colocalized with DAAM1 in BrCa cells, which was essential for DAAM1-mediated microfilament remodeling and RhoA activation. Moreover, miR-613 directly targeted both YWHAZ and DAAM1, contributing to inhibiting BrCa cells migration via blocking the complex of YWHAZ–DAAM1. To sum up, these data reveal that YWHAZ regulates DAAM1 activation, and the YWHAZ–DAAM1 complex is directly targeted by the shared post-transcriptional regulator miR-613.

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Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

International Journal of Molecular Sciences ◽

10.3390/ijms22031163 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1163

Author(s):

Gaia Palmini ◽

Cecilia Romagnoli ◽

Simone Donati ◽

Roberto Zonefrati ◽

Gianna Galli ◽

...

Keyword(s):

Cell Line ◽

Molecular Mechanisms ◽

Osteogenic Sarcoma ◽

Cancer Cell Line ◽

Microscopic Features ◽

In Vitro Establishment ◽

Profile Characteristic ◽

First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

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Silencing VEGF enhances the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R by inhibiting autophagy through the activation of mTOR pathway

10.21203/rs.3.rs-26678/v1 ◽

2020 ◽

Author(s):

Li Chen ◽

Guoxiang Lin ◽

Kaihua Chen ◽

Fangzhu Wan ◽

Yongchu Sun ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Western Blotting ◽

Xenograft Model ◽

Mtor Pathway ◽

Angiogenic Factor ◽

Cell Counting

Abstract Background: Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced to the radioresistance of tumors. The purpose of our study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R and the underlying mechanisms.Methods: The radiosensitivity of CNE-2R cells after silencing VEGF was detected by cell counting kit 8 (CCK-8) and clonogenic assay, cell cycle and apoptosis was subjected to flow cytometry. DNA damage and autophagy were observed by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation analysis. In vivo, the effect of VEGF on radiosensitivity of NPC cells was investigated through xenograft model, furthermore, immunohistochemistry and TUNEL assay were used to further verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion.Results: Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of CNE-2R cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in CNE-2R cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through the activation of mTOR pathway.Conclusion: VEGF depletion increased radiosensitivity of NPC radioresistant cell CNE-2R by suppressing autophagy via the activation of mTOR pathway.

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