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. 2011 Jul-Aug;5(4):332-43.
doi: 10.4161/cam.5.4.17287.

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

Nadejda Vintonenko  1 , Irma Pelaez-Garavito, Hélène Buteau-Lozano, Aurore Toullec, Rosette Lidereau, Gérard Yves Perret, Ivan Bieche, Martine Perrot-Applanat
Affiliations

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

Nadejda Vintonenko et al. Cell Adh Migr. 2011 Jul-Aug.

Abstract

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.

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Figures

Figure 1
Figure 1
Overexpression of VEGF189 in MDA-MB-231 cells induces apoptosis under serum starvation condition, while VEGF 165 improves survival. Cells were incubated for 72 h in 0.5% FBS-containing medium. Adherent cells were harvested and percentage of viable cells, cells in early apoptosis (AP I), late apoptosis (AP II) and necrosis was determined by flow cytometry using Annexin V apoptosis/cell death assay. Each column represents a mean (±SE M) of four independent experiments for viable (Annexin V−, PI−), AP I (Annexin V+, PI−), AP II (Annexin V+, PI+), and necrotic (Annexin V−, PI+) cells. *p ≤ 0.05 vs. corresponding cV clone.
Figure 2
Figure 2
Analysis of neuropilins expression in the different MDA-MB-231 clones used in the study. cV, V189 and V165 clones were stably transfected with the control irrelevant shRNA (sh control) or NRP1 shRNA expression plasmid (shNRP1). Protein extracts (A: 70 µg; B: 100 µg) obtained from these clones were immunoblotted with a NRP1 (A) or NRP2 (B) antibodies; anti-GAPDH or actine antibodies (in A and B, respectively) were used to assess protein loading.
Figure 3
Figure 3
VEGF189 overexpression in shCtl-transfected clones induces apoptosis under serum starvation condition, while VEGF165 improves survival. Cells were incubated for 72 h in 0.5% FBS-containing medium. Adherent cells were harvested and percentage of viable cells, cells in early apoptosis (AP I), late apoptosis (AP II) and necrosis was determined by flow cytometry using Annexin V apoptosis/cell death assay. (A) Each column represents a mean (±SE M) of four independent experiments for viable, API, AP II and necrotic cells. *p ≤ 0.05 vs. corresponding cV shCtl clone. (B) Representative flow cytometry profiles under 0.5% FBS (upper part) and 10% FBS (lower part) conditions.
Figure 4
Figure 4
Overexpression of VEGF189 increases doxorubicin-induced apoptosis. ShCtl transfected clones were treated with 2.5 µM doxorubicin in serum-containing medium for 24 h. Adherent cells were harvested and subjected to Annexin V apoptosis/cell death assay. Each column represents a mean (±SE M) of six independent experiments for viable, AP I, AP II and necrotic cells. *p ≤ 0.05 vs. corresponding cV shCtl clone.
Figure 5
Figure 5
NRP1 knockdown abrogates apoptosis induced by VEGF189 under serum starvation conditions. Annexin V/PI staining was performed on adherent cells harvested after 72 h incubation in 0.5% FBS-containing medium. Histograms of means (±SE M) (left) and representative flow cytometry profiles (right) stand for transfected with shNRP-1 vs. irrelevant shCtl clones overexpressing VEGF189 (A), n = 3; VEGF 165 (B), n = 4; and control cV (C), n = 3. Two clones were tested for each shRNA transfection (see Table 2B) giving similar profiles; shown results stand for cVshNRP1–19, V189shNRP1–10 and V165shNRP1–5. *p ≤ 0.05 vs. corresponding shCtl clone.
Figure 6
Figure 6
NRP1 knockdown reduces apoptosis induced by doxorubicin in VEGF189 overexpressing clones. V189 (A), n = 6, V165 (B), n = 5 and cV clones (C), n = 7 have been transfected with shNRP-1 vs. irrelevant control shRNA clones. Apoptosis assay was performed on cells harvested after 24 h treatment with 2.5 µM doxorubicin in serum-containing medium. Histograms of means (±SE M) stand obtained with the same clones, as shown in Figure 5, are presented. *p ≤ 0.05 vs. corresponding shCtl clone.
Figure 7
Figure 7
Analysis of VEGF expression in the different MDA-MB-231 clones used in the study. V189, V165 and cV clones, transfected with shCtl or shNRP1, were grown in serum free medium supplemented or not with heparin (50 µg/ml) for 24 h. VEGF proteins from the conditioned media were measured using an ELISA kit. Results are expressed as ng VEGF/mg protein extract. Values represent means ± SD of a representative experiment out of two independent experiments performed in triplicate.
Figure 8
Figure 8
Analysis of the expression and activity of uPA in the different clones. (A) NRP1 knockdown decreases uPA expression in VEGF189 overexpressing clones. Cells were maintained in 10% FBS containing medium. Transcripts were analyzed by qRT-PCR. Results are normalized to their corresponding shCtl clones. Values represent means ± SE M of three independent experiments. *p < 0.05 vs. corresponding shCtl clone; (B) NRP1 knockdown results in decreased uPA activity in VEGF189 overexpressing clones. Conditioned medium from cells cultured for 24 h in serum-free DMEM was analyzed using zymography, as described in Materials and Methods. The figure shows a representative from two independent experiments.
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