Arf6 as a therapeutic target: Structure, mechanism, and inhibitors

ADP-ribosylation factor 6 (Arf6), a small G-protein of the Ras superfamily, plays pivotal roles in multiple cellular events, including exocytosis, endocytosis, actin remodeling, plasma membrane reorganization and vesicular transport. Arf6 regulates the progression of cancer through the activation of cell motility and invasion. Aberrant Arf6 activation is a potential therapeutic target. This review aims to understand the comprehensive function of Arf6 for future cancer therapy. The Arf6 GEFs, protein structure, and roles in cancer have been summarized. Comprehending the mechanism underlying Arf6-mediated cancer cell growth and survival is essential. The structural features of Arf6 and its efforts are discussed and may be contributed to the discovery of future novel protein-protein interaction inhibitors. In addition, Arf6 inhibitors and mechanism of action are listed in the table. This review further emphasizes the crucial roles in drug resistance and attempts to offer an outlook of Arf6 in cancer therapy.

Effects and mechanism of high ARNO expression on the permeability of human renal glomerular endothelial cells under high glucose condition / 解放军医学杂志

Objective: To explore the effects and mechanism of high expression of ARNO (ARF nucleotide-binding-site opener) on the permeability of human renal glomerular endothelial cells (HRGECs) under high glucose conditions. Methods: HRGECs were cultured in vitro, and ARNO expression and endothelial permeability were detected in high glucose time gradient (15, 30, 45 and 60 min) and concentration gradient (10, 20, 30 and 40 mmol/L) experiments, respectively. Cell lines of HRGECs with ARNO and Arf6 gene silencing were obtained by infecting HRGECs with ARNO siRNA and Arf6 siRNA recombinant lentivirus vectors, respectively. The effects of ARNO and Arf6 gene silencing on endothelial permeability were observed. Results: In the time gradient experiments, compared with control group, ARNO expressions increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (0.670±0.051, 0.960±0.106, 0.716±0.026, 0.531±0.030 vs. 0.242±0.029. P<0.05). Compared with high glucose 15 min group, ARNO expression increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, ARNO expression decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, ARNO expression decreased in high glucose 60 min group (P<0.05). Compared with control group, endothelial permeability increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (1.196±0.004, 1.399±0.012, 1.301±0.052, 1.184±0.030 vs. 1.000, P<0.05). Compared with high glucose 15 min group, endothelial permeability increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, endothelial permeability decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, endothelial permeability decreased in high glucose 60 min group (P<0.05). In the concentration gradient experiments, compared with normal concentration glucose group, ARNO expressions increased significantly in the groups with high glucose of 20, 30 and 40 mmol/L (0.632±0.031, 0.927±0.041, 1.183±0.098 vs. 0.169±0.033, P<0.05), and the ARNO expression level was increased along with the increase of glucose concentration (P<0.05). Compared with normal concentration glucose group, endothelial permeability increased significantly in the groups with high glucose of 10, 20, 30 and 40 mmol/L (1.147±0.015, 1.237±0.023, 1.351±0.015, 1.444±0.019 vs. 1.000, P<0.05), and the endothelial permeability increased along with the increase of glucose concentration (P<0.05). After transfection with lentivirus, compared with normal concentration glucose untransfected group and normal concentration glucose empty vector group, ARNO mRNA transcription reduced significantly (0.255±0.056 vs. 1.000, 1.183±0.297), and ARNO protein expression also reduced significantly (0.088±0.005 vs. 0.246±0.011, 0.237±0.009) in normal concentration glucose ARNO siRNA group (P<0.05). In addition, compared with those groups, Arf6 mRNA transcription was reduced significantly (0.314±0.090 vs. 1.000, 1.140±0.236), and Arf6 protein expression was also reduced significantly (0.690±0.012 vs. 0.917±0.009, 0.919±0.009) in the normal concentration glucose Arf6 siRNA group (P<0.05). After silencing ARNO, compared with high-glucose untransfected group and high-glucose empty vector group, the ARNO protein expression was reduced significantly (0.572±0.021 vs. 0.915±0.005, 0.916±0.012), Arf6 activity was reduced significantly (0.263±0.007 vs. 0.484±0.014, 0.490±0.008), and endothelial permeability was also decreased (0.718±0.017 vs. 1.000, 0.978±0.040) in high-glucose ARNO siRNA group (P<0.05). Besides, after silencing Arf6, compared with high-glucose untransfected group and high-glucose empty vector group, Arf6 protein expression was reduced significantly (0.673±0.015 vs. 0.932±0.020, 0.899±0.022), and endothelial permeability was also decreased (0.768±0.050 vs. 1.000, 0.978±0.040) in high-glucose Arf6 siRNA group (P<0.05). Conclusion: High-glucose induced high permeability of HRGECs is related to the activation of ARNO/Arf6 signal, and inhibition of ARNO expression and Arf6 activity may inhibit the glomerular endothelial hyperpermeability in diabetic nephropathy, and is expected to be a new direction for the treatment of glomerular endothelial dysfunction in diabetic nephropathy.

Over-expression of ROBO4 reduces permeability of human renal glomerular endothelial cells in high glucose medium / 解放军医学杂志

Objective To investigate the effect of over-expression of ROBO4 on permeability of human renal glomerular endothelial cells (HRGECs) in high glucose medium.Methods HRGECs infected with recombinant lentiviral vector ROBO4 were cultured in high glucose or low glucose medium in vitro.The protein levels of ROBO4 and ARF6 in each group were detected by Western blotting.The endothelial permeability was measured by the effiux of fluorescein isothiocyanate-dextran (FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell model system.The cell viability after lentivirus transfection was measured by CCK8 assay.Results The transfection rate of lentiviruses in HRGECs reached 80％ 72h after,and obvious overexpression of ROBO4 protein was in transformed cells compared with the empty vector group (P＜0.05).The lentivirus-mediated ROBO4 transfection did not affect cell viability of HRGECs.Compared with the low glucose group,the expression of ROBO4 increased obviously after 12h,but declined after 24h (P＜0.05),and reached to minimun after 72h (P＜0.05).On the contrary,the expression of ARF6 increased after 12h,and the increase reached to the maximum after 72h (P＜0.05).Furthermore,the vascular permeability increased gradually after 24h,and reached to the maximum after 72h (P＜0.05) in high glucose group.Compared with the empty vector group,the over-expression of ROBO4 inhibited the expression of ARF6 significantly,and the FITC-Dextran permeability reduced obviously.Conclusion Over-expression of ROBO4 may significantly enhance the barrier functions of HRGEC in high glucose medium,and ROBO4 activation may be a potential therapeutic approach in diabetic nephropathy.

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

Expression and clinical significance of ROBO4/ARF6 signal pathway in renal glomerulus tissue of patients with diabetic kidney disease / 解放军医学杂志

Objective To investigate the expression of ROBO4/ARF6 and its correlation to glomerular endothelial cells (MGECs) permeability at each pathological stage of diabetic kidney disease (DKD). Methods Thirty patients with DKD were enrolled (patient group), and their renal tissues obtained by biopsy were divided into incipient, manifest and advanced stages (10 each) with Tervaert pathological staging, and confirmed normal renal tissues adjacent to the renal angiomyolipoma were assigned as control group. Immunohistochemistry method of MaxVision was used to detect the expression of ROBO4/ARF6 in the specimens, and then the correlation of the expression with the clinicopathological parameters (patients' HbA1c, albuminuria, serum creatinine, eGFR) were determined using statistical software SPSS 21.0. Results ROBO4 was abundantly but ARF6 rarely expressed in normal MGECs. In DKD patients, ROBO4 was mainly expressed in MGECs, while ARF6 was expressed in both MGECs and renal tubular epithelial cells. Compared with the control group, the positive intensity of ROBO4 in patient group decreased significantly (P0.05), but was positively correlated with 24h-urinary albumin (r=0.603, P<0.01) and HbA1c (r=0.582, P<0.01). Conclusion The low expression of ROBO4 and high expression of ARF6 in MGECs of DKD patients suggest that ROBO4/ARF6 signaling pathway may participate in the DKD glomerular endothelial and vascular pathological injury and urinary protein increase.

Expression and prognostic role of ADP-ribosylation factor 6 in human endometrial carcinoma / 中华内分泌外科杂志

Objective To investigate the expression and prognostic role of ADP-ribosylation factor 6 (ARF6) in human endometrial carcinoma.Methods 51 endometrial carcinoma tissues and matched tumor-adjacent tissues were collected from Jan.2010 to Dec.2011 in People's Hospital of Linzi District of Zibo City and Penglai People's Hospital.mRNA expression of ARF6 was detected by qRT-PCR,and the protein expression of ARF6 was detected by immunohistochemistry.The correlation between ARF6 and clinicopathological features was analyzed by chi-square test,and the Kaplan-Meier survival curves were drawn to describe the prognostic effects of ARF6 expression.Results Both mRNA and protein expression of ARF6 were down-regulated in endometrial carcinoma tissues than those in matched tumor-adjacent tissues (P＜0.05).Positive expression of ARF6 was associated with lynphatic metastasis (P＜0.05) and advanced TNM stage (Ⅲ+Ⅴ,P＜0.05).Both the 3-year overall survival rate and disease-free survival rate were lower in ARF6 positive expression group than in ARF6 negative expression group(P＜0.05).Conclusion Positive expression of ARF6 in human endometrial carcinoma is related to the malignant clinicopathological features and poor prognosis.