Arsenic induced ROS production in BEAS-2B cells, which was required for angiogenesis.

<p>(A) BEAS-2B cells were seeded into 6-well plates. Cells were treated with different doses of arsenic as indicated in serum-free medium. DCFH-DA at 5 µM was added to the cells for 15 min. Then the cells were washed and fixed, and the fluorescent images were captured using a fluorescent microscope (upper panel). The corresponding phase micrographs were shown in the bottom panel. (B) BEAS-2B cells were seeded into the 6-well plate. The cells were then cultured in serum-free medium with arsenic at 5 µM for different time points as indicated. DCFH-DA staining was performed as above. (C) BEAS-2B cells were infected with adenovirus carrying GFP (Ad-GFP) and catalase (Ad-catalase), respectively at 20 MOI. After 24 h, cells were treated with 5 µM arsenic for 5 h to perform angiogenesis assay. *, indicates that the relative angiogenesis index was significantly decreased when compared with Ad-GFP control group, <i>P</i><0.05.</p

AKT and ERK1/2 pathways are required for arsenic-inducing HIF-1α expression and angiogenesis.

<p>(A) BEAS-2B cells were cultured in serum-free medium for 24 h, then cells were pre-treated with LY294002 or U0126 at 20 µM for 30 min. Arsenic at 5 µM was added to the cells for 2 h (for p-AKT, AKT, p-ERK1/2, and ERK2 expression) and 6 h (for HIF-1α and HIF-1β expression), respectively. Total proteins were analyzed by Western blotting to detect the expression of proteins as indicated (left panel). Relative densities of p-AKT, p-ERK1/2 and HIF-1α were analyzed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020858#pone-0020858-g002" target="_blank">Fig. 2A</a> (right panel). *, indicates significant increase when compared with the control cells, <i>P</i><0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment, <i>P</i><0.05. (B) BEAS-2B cells were treated with 5 µM of arsenic. Cells were trypsinized, mixed with equal volume of Matrigel with or without 15 µM of LY294002 or U0126. Equal volume of DMSO was added as a negative control. Angiogenesis assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020858#pone-0020858-g001" target="_blank">Fig. 1</a>. *, indicates that the relative angiogenesis index was significantly decreased when compared with DMSO control group, <i>P</i><0.05. (C) BEAS-2B cells were infected with adenovirus carrying GFP (Ad-GFP) or AKT dominant negative (Ad-AKT-DN) at 20 MOI (upper panel), or transfected with scrambled control of siRNA (siScramble) or siMAPK at 50 nM (bottom panel). After 24 h, cells were treated with arsenic and angiogenesis assay was performed as above. *, indicates that the relative angiogenesis index was significantly decreased when compared with Ad-GFP or siScramble control group, <i>P</i><0.05.</p

Arsenic induced angiogenesis.

<p>(A) A549 cells were treated without or with 5 µM of arsenic (As) for 5 h, then trypsinized, resuspended in serum-free medium (3×10⁷cells/ml, 0.1 ml), and mixed in 1∶1 ratio with Matrigel (Collaborative Biomedical Products, Bedford, MA). Aliquots of the mixture were then implanted onto the CAM of 9-day-old embryos. After 96 h incubation, the area around the implanted Matrigel was photographed with a Nikon digital camera. Bar: 2 mm (upper panel). The number of blood vessels was obtained by counting the branching of blood vessels, and the relative angiogenesis was obtained by normalizing to that of the control without arsenic treatment. The data represent the mean ± SD of the relative angiogenesis from eight different embryos (bottom panel). *, indicates that the relative angiogenesis index significantly increased in arsenic treatment group when compared with control group, <i>P</i><0.05. (B) BEAS-2B cells were treated with or without arsenic to perform tumor angiogenesis assay as above. Bar: 2 mm. *, indicates that the relative angiogenesis index significantly increased in arsenic treatment group when compared with control group, <i>P</i><0.05.</p

Arsenic treatment induced phospho-AKT and phospho-ERK1/2 activation, and increased HIF-1α and VEGF expression.

<p>(A) A549 and BEAS-2B cells were treated with different doses of arsenic (As) for 6 h, total proteins are subjected to Western blotting for HIF-1α and HIF-1β expression. A549 and BEAS-2B cells were cultured in serum-free medium for 24 h, then treated with different doses of arsenic for 2 h. Total proteins were subjected to Western blotting analysis for the levels of phospho-AKT, total AKT, phospho-ERK1/2, and ERK2 expression (upper panel). Relative densities of p-AKT, p-ERK1/2 and HIF-1α were analyzed by the ratio of p-AKT/AKT, p-ERK1/2/ERK2 and HIF-1α/HIF-1β using ImageJ software and normalized to those of control cells. The data represents the mean± SD from duplicate experiments (bottom panel). *, indicates significant increase when compared with the control cells, <i>P</i><0.05. (B) BEAS-2B cells were seeded in 12-well plate. Cells were co-transfected with VEGF reporter and β-galactosidase (β-gal) plasmids and cultured for 15 h. Arsenic at 0, 2.5, and 5 µM was added for 24 h. Luciferase assay was performed by using luciferase assay system. The activity of β-gal was used as internal control of transfection efficiency. The relative luciferase activity was calculated as the ratio of luciferase/β-gal activity, and normalized to the control group. *, indicates that the relative luc activity significantly increased in arsenic treatment group when compared with the control group, <i>P</i><0.05. <i>C,</i> BEAS-2B cells were treated without or with 5 µM of arsenic for 24 h. Total RNAs were extracted by Trizol and subjected to RT-PCR analysis of VEGF and GAPDH expression.</p

ROS are required for AKT and ERK1/2 activation, HIF-1α expression, and angiogenesis.

<p>(A) BEAS-2B cells were cultured in serum-free medium for 24 h, then cells were pre-treated with DPI or catalase for 30 min. Arsenic at 5 µM was added to the cells for 2 h or 6 h as above. The specific proteins are analyzed by Western blotting (left panel). The relative densities of p-AKT, p-ERK1/2 and HIF-1α were determined as above. *, indicates significant increase when compared with the control cells, <i>P</i><0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment alone, <i>P</i><0.05. (B) BEAS-2B cells were infected with adenovirus carrying GFP and HIF-1α siRNA (Ad-GFP and Ad-siHIF-1α, respectively) at 20 MOI for 24 h, then the cells were treated with 5 µM arsenic for 5 h and angiogenesis assay was performed as above. *, indicates that the relative angiogenesis index was significantly decreased when compared with the control group, <i>P</i><0.05.</p

VEGF is required for arsenic-inducing angiogenesis.

<p>(A) BEAS-2B cells were treated with DPI or catalase for 30 min, then with 5 µM arsenic for 24 h. Total RNAs were extracted by Trizol, and analyzed by RT-PCR for VEGF and GAPDH expression (upper panel). Relative density of VEGF was analyzed by the ratio of VEGF/GAPDH using ImageJ software and normalized to control cells. The data represents the mean± SD from duplicate experiments (bottom panel). *, indicates significant increase when compared with the control cells, <i>P</i><0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment alone, <i>P</i><0.05. (B) BEAS-2B cells were transfected with VEGF siRNA and scrambled siRNA (siVEGF and siScramble, respectively). After the transfection for 24 h, cells were treated with 5 µM arsenic for 5 h to perform angiogenesis assay. *, indicates that the relative angiogenesis index was significantly decreased in siVEGF treatment group when compared with siScramble group, <i>P</i><0.05.</p

Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy

<div><p>Cisplatin is commonly used in ovarian cancer treatment by inducing apoptosis in cancer cells as a result of lethal DNA damage. However, the intrinsic and acquired resistance to cisplatin in cancer cells remains a big challenge for improving overall survival. The cyto-protective functions of autophagy in cancer cells have been suggested as a potential mechanism for chemoresistance. Here, we reported <i>MIR152</i> as a new autophagy-regulating miRNA that plays a role in cisplatin-resistance. We showed that <i>MIR152</i> expression was dramatically downregulated in the cisplatin-resistant cell lines A2780/CP70, SKOV3/DDP compared with their respective parental cells, and in ovarian cancer tissues associated with cisplatin-resistance. Overexpression of <i>MIR152</i> sensitized cisplatin-resistant ovarian cancer cells by reducing cisplatin-induced autophagy, enhancing cisplatin-induced apoptosis and inhibition of cell proliferation. A mouse subcutaneous xenograft tumor model using A2780/CP70 cells with overexpressing <i>MIR152</i> was established and displayed decreased tumor growth in response to cisplatin. We also identified that ATG14 is a functional target of <i>MIR152</i> in regulating autophagy inhibition. Furthermore, we found that EGR1 (early growth response 1) regulated the <i>MIR152</i> gene at the transcriptional level. Ectopic expression of EGR1 enhanced efficacy of chemotherapy in A2780/CP70 cells. More importantly, these findings were relevant to clinical cases. Both EGR1 and <i>MIR152</i> expression levels were significantly lower in ovarian cancer tissues with high levels of ERCC1 (excision repair cross-complementation group 1), a marker for cisplatin-resistance. Collectively, these data provide insights into novel mechanisms for acquired cisplatin-resistance. Activation of EGR1 and <i>MIR152</i> may be a useful therapeutic strategy to overcome cisplatin-resistance by preventing cyto-protective autophagy in ovarian cancer.</p></div

Overexpression of HER2 and HER3 reverses miR-199a- and miR-125b-inhibited tumor angiogenesis.

<p>(A) Immunoblotting to confirm establishment of an A2780 cell line stably overexpressing HRE2 and OVCAR-3 cell line stably overexpressing HER3 using pBABEpuro and pReceiver-Lv105 vector, respectively. (B) Tube formation assay using HUVEC cells was described as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. (C) Total tube lengths for each treatment were analyzed and presented as mean ± SE (millimeter) from six replicates for each treatment. *Significantly different vs vector+miR-control (P<0.05). (D, E) A2780 and OVCAR cells were transfected pre-miR-control, pre-miR-199a or pre-miR-125b, respectively; and implanted onto the CAMs to perform angiogenesis assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. The representative images from each group were shown here. The total number of blood vessels in each group was quantified. *,**Significantly different compared with that of the same cell line transfected with pre-miR-control with *P<0.05 and **P<0.01.</p

PTEN was the downstream target of miR-21 in inducing angiogenesis.

<p>(A) DU145 cells were infected with adenovirus carrying GFP (Ad-GFP) or PTEN (Ad-PTEN). After 24 h, cells were transfected with pre-miR-21 or scrambled control. The next day, cells were trypsinized and angiogenesis assay was performed as above. The representative plugs from treatments of Ad-GFP plus scrambled control, Ad-GFP plus pre-miR-21, and Ad-PTEN plus pre-miR-21 were shown in the picture. <i>Scale</i>, 2 mm. (B) The relative angiogenesis was showed in the bar graph. Results from each group are presented as mean ±SD of 6 samples. * indicates the significant increase when compared to the Ad-GFP plus scrambled control group (<i>p</i><0.05). ^#, indicates the significant decrease when compared to Ad-GFP plus miR-21 group (<i>p</i><0.05).</p

MiR-199a and miR-125b suppress tumor angiogenesis associated with reduction of HIF-1α and VEGF expression.

<p>(A) HUVEC cells were cultured in serum free medium overnight and re-suspended in basic EBM-2 medium. To perform the tube formation assay, HUVEC cells were incubated in basic EBM-2 medium; conditioned medium prepared from OVCAR-3 or A2780 transfected with pre-miR-control, pre-miR-125b and pre-miR-199a, respectively. Tube formation was determined under light microscope in 12 h. Total tube length (mm) was presented as mean ± SE from six replicates for each treatment. **Significantly different compared with OVCAR-3 control (P<0.01). ^##Significantly different compared with A2780 control (P<0.01). (B) A2780 cells were transfected with 30 nM pre-miR-125b, pre-miR-199a and pre-miR-control precursor, respectively. After transfection (24 h), 2×10⁶ cells were trypsinized, suspended, and mixed with equal volume of Matrigel, and implanted onto the chicken CAMs of 10-day-old chicken embryos. The branches of blood vessels were counted as the index of angiogenesis was obtained from the CAMs of 8–10 embryos per treatment 96 h after implantation. The data represent as mean ± SE of blood vessel numbers, which were normalized to that of the control. **Significantly different compared with that of the control (P<0.05). (C) A2780 cells and OVCAR-3 cells were cultured under normoxic condition, and transiently transfected as above to analyze HIF-1α and β-actin protein expression by immunoblotting and VEGF mRNA levels (VEGF-165 and VEGF-121 isoforms) by RT-PCR. Quantification was performed by scanning densitometry. The results are obtained from triplicate experiments and presented as mean ± SE. *Significantly difference compared with the same cell lines transfected with miR-cont. (D) VEGF mRNA levels in human ovarian cancer tissues (n = 33) were analyzed by SYBR Green qRT-PCR. The Pearson correlations were analyzed between miR-125b or miR-199a expression and its corresponding VEGF expression in cancer tissues.</p