Figure 1.
Isolation of lung cancer xenograft-derived ECs.
LLC xenografts were resected from mice injected subcutaneously at the dorsal flank with LLC cells (3×106 suspended in 50 µL PBS) for 30 days. After removing obvious necrotic tissues or extra fatty compositions, the minced tissues were ground on ice using a glass grinder and were then filtered through cell strainers to eliminate tissue debris. (A) The CD31-expressing lung cancer-derived ECs were isolated from the single-cell suspension by immunomagnetic sorting, as evidenced by scanning microscopy, and cultured in vitro. (B) CD31 was detected in the isolated lung cancer-derived ECs using immunofluorescence. CD31 antibody staining of the lung cancer-derived EC membranes is shown in red, and nuclear DAPI staining is shown in blue. (C) Total protein was isolated from enriched lung cancer-derived ECs, bEnd.3 cells (positive control), and MLE-12 (negative control) cells. Western blot analysis was performed using antibodies against eNOS and E-cadherin, and β-actin was used as the internal control.
Figure 2.
Hypoxia increases SIRT1 expression in lung cancer-derived ECs.
(A) Western blot analysis of SIRT1 in lung cancer-derived ECs exposed to hypoxia (2% O2) for the indicated time using an antibody against SIRT1. (B) SIRT1 mRNA levels, as measured by real-time RT-PCR, of total RNA obtained from lung cancer-derived ECs exposed to hypoxia (2% O2) for the indicated time period. The data represent the average of three independent experiments from each time point performed in triplicate. * P<0.05 as compared to the control.
Figure 3.
SIRT1 negatively regulates N1IC by deacytelation and regulates angiogenic activity in lung cancer-derived ECs.
(A) Lung cancer-derived ECs were transfected with control or SIRT1 siRNAs for 48 h and cultured in the absence or presence of DLL4 for an additional 6 h. Then, N1IC protein expression was detected using western blot analysis. siRNA-mediated blockade of SIRT1 activity in endothelial cells increased the endogenous N1IC protein levels. (B) Lung cancer-derived ECs were transfected with HA-N1IC, pcDNA-SIRT1, pcDNA-SIRT1 H363Y, or pcDNA3 (4 µg/µL each) for 48 h and were then treated with or without 5 nM NAM for an additional 12 h. Afterward, the cell extracts were immunoprecipitated with an anti-HA antibody and subjected to western blot analysis with an anti-acetyl lysine (upper panel) or anti-HA antibody (lower panel). (C) Lung cancer-derived ECs were triple transfected with N1IC, the acetyltransferase p300, and increasing amounts of SIRT1, and the cells were lysed 48 h later. Comparable levels of N1IC were immunoprecipitated and blotted for acetylated lysine residues (IP: HA and IB: anti-acetyl; upper panel). The blot was reprobed for HA, which demonstrated approximately equal levels of HA-N1IC (IP: HA and IB: HA; lower panel). (D) Lung cancer-derived ECs were transfected with N1IC, the acetyltransferase p300, and increasing amounts of SIRT1 together with the Renilla luciferase reporter and the pGL3-CBF plasmid containing a firefly luciferase reporter gene for 24 h. Then, luciferase activity was measured using a dual-luciferase reporter assay. The data were normalized to the Renilla luciferase activity (mean ± SD; n = 3). (E) qChIP analysis of SIRT1 occupancy at the Notch1 proximal promoter region in lung cancer-derived ECs. SIRT1 occupied a specific region at −500 bp of the Notch1 locus. SIRT1 was immunoprecipitated (IP) with anti-SIRT1 sera (SIRT1 IgG) or preimmune sera (normal IgG, used as a control) (mean ± SEM; n = 3). * P<0.05 as compared to the control. (F) Luciferase reporter gene assay results. pGL3, pGL3-Notch1 −500 to +400 (N+400), or pGL3-Notch1+400 to +1750 (N+1750) were transfected into HEK293 cells with or without SIRT1. *P<0.05. The error bars represent the SEM. (G) Expression of the Notch target genes HEY1 and HEY2 were assessed in control siRNA- and SIRT1 siRNA-transfected lung cancer-derived ECs, which were subsequently transfected with empty vector or N1IC for up to 48 h. SIRT1-deficient endothelial cells displayed markedly enhanced HEY1 and HEY2 activity in response to N1IC overexpression. *P<0.05 as compared to the control. (H) Lung cancer-derived ECs were transfected with control or SIRT1 siRNAs and treated with DMSO or 20 mM NAM for 24 h, followed by incubation with Matrigel for an additional 5 h. Next, tube formation was evaluated using an inverted phase microscope. The bar graphs represent the densitometry results. * P<0.05 as compared to the control.
Figure 4.
Effect of SIRT1 on tumor neovascularization in LLC xenografts.
(A) SIRT1 promotes angiogenesis in vivo. Matrigel plugs were subcutaneously injected into the abdomens of control or SIRT1-transgenic mice, and the plugs were extracted 7 days later to determine the extent of vascularization. The amount of hemoglobin present in the plugs was quantified as an indicator of the formation of functional blood vessels (mean ± SD; n = 10 for each group). * P<0.05 as compared to the control. (B) Changes in the median tumor volumes measured in wild-type (control) C57BL/6J, SIRT1, or SIRT1 (H363Y) mice following inoculation with LLC cells for the indicated time period. * P<0.05 as compared to the SIRT1 mice. (C) Photomicrographs of CD31 IHC staining in sections of LLC xenografts (magnification, ×200) from wild-type, SIRT1 or SIRT1(H363Y) mice. When the xenograft tumor volumes reached approximately 100 mm3, the mice were randomly assigned to the control arm (n = 8) or the experimental arm (DLL4 monoclonal antibody; n = 10). The high-magnification fields (×400) were analyzed for microvessel density count using ImageJ software (mean ± SD; n = 10 for each group). *P<0.05 as compared to the SIRT1 mice. The average tumor volumes in the wild-type, SIRT1 and SIRT1 (H363Y) mice were measured at 4 weeks after treatment. * P<0.05 as compared to the control.
Figure 5.
A model for SIRT1-mediated regulation of endothelial DLL4/Notch signaling in lung cancer angiogenesis.
Our study show that the Delta-like ligand 4 (DLL4), which is highly expressed in vascular cells during tumor angiogenesis, binds to the Notch1 receptor and initiates proteolytic cleavages. The final intramembrane cleavage catalyzed by γ-secretase leads to the release of the active Notch1 intracellular domain (N1IC), which translocates into the nucleus and recruits the protein MAML1 and histone acetyltransferases (HATs, such as p300) to the CSL complex. This recruitment leads to the activation of the Notch1 target genes HEY1 and HEY2. Moreover, p300 acetylates N1IC and enhances its transcriptional activity whereas SIRT1 inhibits the acetylated form of N1IC and significantly diminishes N1IC activity induced by p300, thereby limiting the DLL4/Notch signaling response and inhibiting tumor angiogenesis.