Fig 1.
T2A inhibited HIF-1α expression in MDA-MB-231 and MCF-7 cells.
(A and B) The MDA-MB-231 and MCF-7 cells were treated without or with various concentrations of T2A for 16 hours and then subjected to hypoxia, or remained in normoxia for an additional 8 hours. Whole cell extracts (WCE) and nuclear extracts (NE) were prepared from cells and subjected to Western blot assay using antibodies against HIF-1α, HIF-2α, HIF-1β, and β-actin. (C) Cells were fixed, permeabilized, and processed for immunofluorescence labeling with anti-Tubulin (Red) and anti-HIF-1α (green) antibodies. Nuclei were counterstained with 0.1 μg/ml DAPI (blue). Scale bar represents 10 μm.
Fig 2.
T2A inhibited the transcriptional activity of HIF-1α, but had no significant effects on the HIF-1α mRNA level.
(A) The MDA-MB-231 cells were treated without or with various concentrations of T2A for 24 hour under normoxic or hypoxic conditions. VEGF in the supernatant was evaluated using ELISA. Error bars represent means ± S.D. (n = 3). (B) The MDA-MB-231 cells were transfected with pBI-GL V6L plasmid, pRL-SV-40 plasmid and HIF-1α plasmid (HIF-1α overexpression group) or an empty vector plasmid (control). Cells were treated with T2A or DMSO. The promoter activity of HIF-1α was analyzed as described in the Methods section. **P< 0.01 compared with the control. (C and D) Total cellular RNA was extracted and the HIF-1α, VEGF, Glut1, and EPO mRNA levels were evaluated using real-time PCR. The relative mRNA levels of HIF-1α, VEGF, Glut1, and EPO were normalized according to the β-actin abundance and expressed as percentages of the control. Error bars represent means ± SD (n = 3).
Fig 3.
T2A inhibited HIF-1α expression through the inhibition of protein synthesis rather than the promotion of HIF-1α degradation.
(A) The MDA-MB-231 cells were cultured under normoxic or hypoxic conditions for 8 hours, then pretreated without or with 20 μM T2A for 30 minutes, followed by treatment without or with 20 μM cycloheximide (CHX) for various intervals as indicated. Upper panel: after treatment, whole cell extracts were analyzed by Western blot using antibodies against HIF-1α and β-actin. Lower panel: quantification of the HIF-1α by densitometry following normalization to the β-actin level. The HIF-1α protein levels in untreated or T2A-treated cells were arbitrarily given the value of 100%. (B) Cells were labeled with [35S]methionine as described in the Methods section and chased for the indicated periods (h) in the presence or absence of T2A under normoxic or hypoxic conditions. Upper panel: equal amounts of proteins from each cell lysate were subjected to immunoprecipitation and then separated in SDS-PAGE and examined by autoradiography following normalization to the control. Lower panel: quantification of the autoradiographic HIF-1α signal by densitometry. (C) Cells were pretreated with either vehicle or 20 μM T2A for 16 hours and then subjected to hypoxia, or remained in normoxia for an additional 8 hours. Subsequently, cells were labeled with [35S]methionine in the presence or absence of 20 μM T2A for the indicated time. Upper panel: Equal amounts of proteins were subjected to immunoprecipitation and then separated in SDS-PAGE and examined by autoradiography. Lower panel: quantification of the autoradiographic HIF-1α signal by densitometry. (D) Cells were pretreated with the proteasome inhibitor MG132 (10 μM) for 2 hours, followed by T2A treatment (20 μM) under normoxic or hypoxic conditions as described above. Whole cell extracts were analyzed by Western blot using antibodies against HIF-1α and β-actin.
Fig 4.
The mTOR/p70S6K/ RPS6/4E-BP1 signaling pathway was involved in T2A-induced inhibition of HIF-1α.
(A) The MDA-MB-231 cells were treated without or with various concentrations of T2A for 24 hours under normoxic and hypoxic conditions. Whole cell extracts were prepared from cells and subjected to Western blot assay using antibodies against phospho-mTOR (p-mTOR), mTOR, p70S6 Kinase(p70S6K), p-p70S6K (Thr421/Ser424), p-p70S6K(Thr389), S6 Ribosomal Protein (RPS6), p-RPS6(Ser235/236), p-RPS6(Ser240/244), 4E-BP1, and p-4E-BP1(Thr37/46). (B) Cells were pretreated with rapamycin (20 nM) for 2 hours, followed by T2A treatment (20 μM) under normoxic or hypoxic conditions as described above. Whole cell extracts were analyzed by Western blot using antibodies as indicated. (C) VEGF in the supernatant was evaluated using ELISA. Error bars represent means ± SD (n = 3). Values of cells treated with T2A and rapamycin were significantly reduced compared to values obtained from cells treated with T2A alone based on Student’s t-test; **P <0.01.
Fig 5.
T2A inhibited angiogenesis and tumor growth in vivo.
Xenograft mouse models on the back of nude mice were established using human MDA-MB-231 cells (2 × 106). Tumor mice were treated with T2A or vehicle as described in the Methods section. Tumor growth and body weight of mice were measured once a week. (A) Average tumor volume of vehicle control mice and mice treated with T2A. Values of tumor volume from the T2A-treated group were significantly reducted compared with those from mice of the control group based on Student’s t test; *P < 0.05 or **P < 0.01. (B) Body weight of mice during the eight weeks of T2A treatment. (C) Representative tumors from the control and T2A-treated groups. (D) Average hemoglobin levels in tumor tissues from 4 vehicle control and 4 T2A-treated mice. **P < 0.01 compared with vehicle control. (E) Representative tumor sections were immunostaining by using CD31 antibody. Scale bar represents 20 μm. (F) Microvessel density (MVD) was counted the CD31-positive blood vessels per field (400 × magnification) from four replicate tumor sections. **P < 0.01 compared with vehicle control. (G) Total cellular RNA were extracted from tumors in 4 vehicle control and 4 T2A-treated mice and the VEGF mRNA level was evaluated using real-time PCR. **P < 0.01 compared with vehicle control. (H) Tumors from 1 vehicle control mouse and 1 T2A-treated mice were surgically removed and homogenized. Whole tumor lysates were prepared and subjected to Western blot assay using antibodies as indicated.