Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.
Background Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer's disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract. Methodology/Principal Findings Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H2O2 to simulate senescence. After being exposed to 50 µM H2O2 for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50–200 µM H2O2 for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses. Conclusions/Significance NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.
Hyperthermia (HT) has been proven to be able to alter the invasion capacity of cancer cells. However, the detailed mechanisms responsible for the anti-metastasis effects of HT have not been elucidated. N-myc downstream-regulated gene 2 (NDRG2), as a member of the NDRG family, has been suggested to be highly responsive to various stresses and is associated with tumor suppression. The present study aimed to investigate the biological role of NDRG2 in the invasion of human hepatocellular carcinoma (HCC) cells exposed to HT. We found that NDRG2 could be induced by HT at 45°C. In addition, NDRG2 overexpression inhibited the expression of matrix metallo proteinases-2 (MMP-2) and MMP-9 as well as the invasion of HCC cells, whereas knockingdown NDRG2 reversed the anti-invasion effect of HT in vivo. Further investigation revealed that the phosphorylation level of ERK1/2, but not that of JNK and p38MAPK, was reduced in NDRG2 overexpressing cells. Moreover, the knockdown of NDRG2 expression resulted in increased cell invasion, which was rescued by treating the HepG2 cells with the ERK1/2 inhibitor PD98059, but not with the p38MAPK inhibitor SB203580 or the JNK inhibitor SP600125. Finally, the synergistic cooperation of HT at 43°C and NDRG2 expression effectively reduced cytotoxicity and promoted the anti-invasion effect of HT at 45°C. Taken together, these data suggest that NDRG2 can be induced by HT and that it mediates the HT-caused inhibition of invasion in HCC cells by suppressing the ERK1/2 signaling pathway. The combined application of constitutive NDRG2 expression with HT may yield an optimized therapeutic benefit.
Copper oxide nanoparticles (CuO NPs) are widely used as catalysts or semiconductors in material fields. Recent studies have suggested that CuO NPs have adverse genotoxicity and cytotoxicity effects on various cells. However, little is known about the toxicity of CuO NPs following exposure to murine lungs. The purpose of this fundamental research was to investigate whether CuO NPs could induce epithelial cell injury, pulmonary inflammation, and eventually fibrosis in C57BL/6 mice. Our studies showed that CuO NPs aggravated pulmonary inflammation in a dose-dependent manner. CuO NPs induced apoptosis of epithelial cells as indicated by TUNEL staining, flow cytometry and western blot analysis, which was partially caused by increased reactive oxygen species (ROS). In addition, CuO NPs exposure promoted collagen accumulation and expression of the progressive fibrosis marker α-SMA in the lung tissues, indicating that CuO NP inhalation could induce pulmonary fibrosis in C57BL/6 mice. All data provide novel evidence that there is an urgent need to prevent the adverse effects of CuO NPs in the human respiratory system.
The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.
Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)