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VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

VEGF-A secreted by DJM-1 cells induced tumor angiogenesis and cancer cell proliferation. (A) Frozen sectioned DJM-1 tumors were stained with the endothelial marker CD-31 and hematoxylin. The arrow indicates blood vessels (Scale bar: 100 μm). (B) Quantification of VEGF-A concentrations secreted by DJM-1 cells. After a 72 h treatment with (20 nM, siControl, or siVEGF-A #1–3) or without siRNA (no addition), conditioned media were collected and analyzed by VEGF-A ELISA. (C) HUVEC migration assay. (B,C) Data represent the means±s.d. Percentages from the each mean relative to siControl are indicated below the graph. (D) Endogenous VEGF-A induced colony formation by cancer cells. DJM-1 cells were treated with siControl or siVEGF-A #1 (20 nM each) and seeded in soft agar. The upper panel shows the bright field of MTT staining colonies; the lower panel shows magnified colonies (Red circle: >80 μm diameter, Scale bar: 250 μm). (E) Quantitative analysis of D. The means of colony numbers in 6 fields for each condition are shown with ±s.d. Percentages from each mean relative to the siControl are indicated below the graph. **P<0.005; ***P<0.001.
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BIO010918F1: VEGF-A secreted by DJM-1 cells induced tumor angiogenesis and cancer cell proliferation. (A) Frozen sectioned DJM-1 tumors were stained with the endothelial marker CD-31 and hematoxylin. The arrow indicates blood vessels (Scale bar: 100 μm). (B) Quantification of VEGF-A concentrations secreted by DJM-1 cells. After a 72 h treatment with (20 nM, siControl, or siVEGF-A #1–3) or without siRNA (no addition), conditioned media were collected and analyzed by VEGF-A ELISA. (C) HUVEC migration assay. (B,C) Data represent the means±s.d. Percentages from the each mean relative to siControl are indicated below the graph. (D) Endogenous VEGF-A induced colony formation by cancer cells. DJM-1 cells were treated with siControl or siVEGF-A #1 (20 nM each) and seeded in soft agar. The upper panel shows the bright field of MTT staining colonies; the lower panel shows magnified colonies (Red circle: >80 μm diameter, Scale bar: 250 μm). (E) Quantitative analysis of D. The means of colony numbers in 6 fields for each condition are shown with ±s.d. Percentages from each mean relative to the siControl are indicated below the graph. **P<0.005; ***P<0.001.

Mentions: The DJM-1 cell line was established from a human skin cancer obtained from a patient who died from metastases to the axillary lymph nodes and lungs. DJM-1 cells were orthotopically inoculated into the backs of mice. After 2 weeks, mice were sacrificed and the tumors were isolated. Tumor sections were stained with an anti-CD31 antibody (Arrow: bv) and hematoxylin. The tumors and peritumoral area were highly vascularized (Fig. 1A). The amount of VEGF-A secreted into the DJM-1 cell conditioned media (CM) was 8 ng/ml, as measured by ELISA, while that secreted into the siControl-treated DJM-1 cell CM was 7.5 ng/ml (Fig. 1B). VEGF-A was suppressed by the knockdown using 3 different siRNAs, with siVEGF-A #1 being the most effective (siVEGF-A #1: 90% inhibition, siVEGF-A #2: 88% inhibition, siVEGF-A #3: 65% inhibition, respectively) (Fig. 1B). VEGF-A secreted by DJM-1 cells stimulated the migration of HUVEC. The knockdown of VEGF-A expression suppressed the migration of HUVEC (siVEGF-A #1: 38% and siVEGF-A#2: 48% of siControl, respectively) (Fig. 1C). Colony formation in soft agar indicated cancer proliferation under anchorage-independent conditions. The knockdown of VEGF-A expression suppressed the anchorage-independent proliferation (52% of siControl) of DJM-1 cells themselves (Fig. 1D,E). The addition of exogenous VEGF-A (1 µg/ml) restored the proliferation of siVEGF-A-treated DJM-1 cells to a similar level to that of the siControl-treated cells (siVEGF-A#1+1 µg/ml VEGF-A, 92% of siControl). These results suggested that the endogenous expression of VEGF-A stimulated the proliferation of DJM-1 cells in an autocrine manner.Fig. 1.


VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

VEGF-A secreted by DJM-1 cells induced tumor angiogenesis and cancer cell proliferation. (A) Frozen sectioned DJM-1 tumors were stained with the endothelial marker CD-31 and hematoxylin. The arrow indicates blood vessels (Scale bar: 100 μm). (B) Quantification of VEGF-A concentrations secreted by DJM-1 cells. After a 72 h treatment with (20 nM, siControl, or siVEGF-A #1–3) or without siRNA (no addition), conditioned media were collected and analyzed by VEGF-A ELISA. (C) HUVEC migration assay. (B,C) Data represent the means±s.d. Percentages from the each mean relative to siControl are indicated below the graph. (D) Endogenous VEGF-A induced colony formation by cancer cells. DJM-1 cells were treated with siControl or siVEGF-A #1 (20 nM each) and seeded in soft agar. The upper panel shows the bright field of MTT staining colonies; the lower panel shows magnified colonies (Red circle: >80 μm diameter, Scale bar: 250 μm). (E) Quantitative analysis of D. The means of colony numbers in 6 fields for each condition are shown with ±s.d. Percentages from each mean relative to the siControl are indicated below the graph. **P<0.005; ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582117&req=5

BIO010918F1: VEGF-A secreted by DJM-1 cells induced tumor angiogenesis and cancer cell proliferation. (A) Frozen sectioned DJM-1 tumors were stained with the endothelial marker CD-31 and hematoxylin. The arrow indicates blood vessels (Scale bar: 100 μm). (B) Quantification of VEGF-A concentrations secreted by DJM-1 cells. After a 72 h treatment with (20 nM, siControl, or siVEGF-A #1–3) or without siRNA (no addition), conditioned media were collected and analyzed by VEGF-A ELISA. (C) HUVEC migration assay. (B,C) Data represent the means±s.d. Percentages from the each mean relative to siControl are indicated below the graph. (D) Endogenous VEGF-A induced colony formation by cancer cells. DJM-1 cells were treated with siControl or siVEGF-A #1 (20 nM each) and seeded in soft agar. The upper panel shows the bright field of MTT staining colonies; the lower panel shows magnified colonies (Red circle: >80 μm diameter, Scale bar: 250 μm). (E) Quantitative analysis of D. The means of colony numbers in 6 fields for each condition are shown with ±s.d. Percentages from each mean relative to the siControl are indicated below the graph. **P<0.005; ***P<0.001.
Mentions: The DJM-1 cell line was established from a human skin cancer obtained from a patient who died from metastases to the axillary lymph nodes and lungs. DJM-1 cells were orthotopically inoculated into the backs of mice. After 2 weeks, mice were sacrificed and the tumors were isolated. Tumor sections were stained with an anti-CD31 antibody (Arrow: bv) and hematoxylin. The tumors and peritumoral area were highly vascularized (Fig. 1A). The amount of VEGF-A secreted into the DJM-1 cell conditioned media (CM) was 8 ng/ml, as measured by ELISA, while that secreted into the siControl-treated DJM-1 cell CM was 7.5 ng/ml (Fig. 1B). VEGF-A was suppressed by the knockdown using 3 different siRNAs, with siVEGF-A #1 being the most effective (siVEGF-A #1: 90% inhibition, siVEGF-A #2: 88% inhibition, siVEGF-A #3: 65% inhibition, respectively) (Fig. 1B). VEGF-A secreted by DJM-1 cells stimulated the migration of HUVEC. The knockdown of VEGF-A expression suppressed the migration of HUVEC (siVEGF-A #1: 38% and siVEGF-A#2: 48% of siControl, respectively) (Fig. 1C). Colony formation in soft agar indicated cancer proliferation under anchorage-independent conditions. The knockdown of VEGF-A expression suppressed the anchorage-independent proliferation (52% of siControl) of DJM-1 cells themselves (Fig. 1D,E). The addition of exogenous VEGF-A (1 µg/ml) restored the proliferation of siVEGF-A-treated DJM-1 cells to a similar level to that of the siControl-treated cells (siVEGF-A#1+1 µg/ml VEGF-A, 92% of siControl). These results suggested that the endogenous expression of VEGF-A stimulated the proliferation of DJM-1 cells in an autocrine manner.Fig. 1.

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus