Radiation-Triggered NF-κB Activation is Responsible for the Angiogenic Signaling Pathway and Neovascularization for Breast Cancer Cell Proliferation and Growth

Abstract

Tumors require blood supply to survive, grow, and metastasize. This involves the process of angiogenesis signaling for new blood vessel growth into a growing tumor mass. Understanding the mechanism of the angiogenic signaling pathway and neovascularization for breast cancer cell proliferation and growth would help to develop molecular interventions and achieve disease free survival. Our hypothesis is that the surviving cancer cell(s) after radiotherapy can initiate angiogenic signaling pathway in the neighboring endothelial cells resulting in neovascularization for breast cancer cell growth. The angiogenic signaling pathway is initiated by angiogenic factors, VEGF and FGF-2, through activation of a transcriptional regulator NF-κB, which in turn is triggered by therapeutic doses of radiation exposure Human breast adenocarcinoma cells (MCF-7 cells) were exposed to Cesium-137 ((137)Cs) γ rays to a total dose of 2 Gy at a dose rate of 1.03 Gy/min. The results of mobility shift assay showed that radiation at clinical doses (2 Gy) could induce NF-κB DNA-binding activity. Then, we examined the communication of angiogenic signals from irradiated MCF-7 cells to vascular endothelial cells. At the protein level, the western blot showed induction of angiogenic factors VEGF and FGF-2 in MCF-7 cells irradiated with 2 Gy. Inhibition of NF-κB activation attenuated VEGF and FGF-2 levels. These factors are secreted into the medium. The levels of VEGF and FGF-2 in the extra cellular medium were both increased, after 2 Gy exposures. We also observed corresponding expression of VEGFR2 and FGFR1 in non-irradiated endothelial cells that were co-cultured with irradiated MCF-7 cells. In support of this, in vitro tube formation assays provided evidence that irradiated MCF-7 cells transmit signals to potentiate cultured non-irradiated endothelial cells to form tube networks, which is the hallmark of neovascularization. Inhibition of NF-κB activation attenuated irradiated MCF-7-induced tube network formation. The data provide evidence that the radiation exposure is responsible for tumor growth and maintenance by inducing an angiogenic signaling pathway through activation of NF-κB.

Keywords: NF-κB activation; angiogenic factors; breast cancer; neovascularization.

Figure 1

(A) Representative autoradiogram of the gel demonstrating the induction of NF-κB DNA-binding activity in MCF-7 cells after radiation exposure. Equal amounts of protein (2 μg) from nuclear extracts obtained from cells harvested at 15 minutes and 24 h after the exposure were analyzed by EMSA. NF-κB-specific DNA binding is shown by arrow head. Corresponding increase or decrease in free probe levels indicates the equal loading. (B) Quantitative analysis of NF-κB DNA-binding activity in MCF-7 cells exposed to 2 Gy and 10 Gy. Notes: The increase in stimulation is presented by calculating the ratio of the optical densities of the induced level of NF-κB activity to that of the constitutive level (0 Gy) at each incubation time from three independent experiments (mean ± SD, **P < 0.01).

Figure 2

Radiation-induced soluble VEGF expression in the culture supernatant of MCF-7 cells. (A) Immunoblot showing radiation-induced soluble VEGF expression in the culture supernatant. MCF-7 cells were either mock irradiated (lane 1) or exposed to 2 Gy in the presence (lane 3) or absence (lane 2) of VEGF blocking peptide (1 μg/mL). The expression of VEGF was determined in the culture supernatant. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Note: **P < 0.01.

Figure 3

Radiation-induced intracellular VEGF expression in MCF-7 cells is mediated through NF-κB pathway. (A) Immunoblot showing radiation-induced VEGF expression in the cell lysate extracted from MCF-7 cells 16 h after exposure. MCF-7 cells were either mock irradiated (lane 1) or exposed to 2 Gy in the presence (lane 3) or absence (lane 2) of NF-κB inhibitor isohelenin (10 μM) added one hour before radiation exposure. Immunogen peptide of VEGF antibody (1 μg/mL) was co-incubated with VEGF antibody to ensure the specificity of VEGF detection (lane 4). The expression of VEGF was determined in the cell lysate extracted from MCF-7 cells. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Note: **P < 0.01.

Figure 4

Radiation-induced soluble FGF expression in the culture supernatant of MCF-7 cells. (A) Immunoblot showing radiation-induced soluble FGF expression in the culture supernatant. MCF-7 cells were either mock irradiated (lane 1) or exposed to 2 Gy (lane 2). 17β-estradiol (1 μM) was incubated with MCF-7 cells for 6 hours to induce FGF-2 as a positive control (lane 3). The expression of VEGF was determined in the culture supernatant. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Note: **P < 0.01.

Figure 5

Radiation-induced intracellular FGF expression in MCF-7 cells is mediated through NF-κB pathway. (A) Immunoblot showing radiation-induced FGF expression in the cell lysate extracted from MCF-7 cells 16 h after exposure. MCF-7 cells were either mock irradiated (lane 1) or exposed to 2 Gy in the presence (lane 3) or absence (lane 2) of NF-κB inhibitor isohelenin (10 μM) added one hour before radiation exposure. 17β-estradiol (1 μM) was incubated with MCF-7 cells for 6 hours to induce FGF-2 expression as a positive control (lane 4). The expression of VEGF was determined in the cell lysate extracted from MCF-7 cells. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Note: **P < 0.01.

Figure 6

Irradiated MCF-7 cells trigger VEGFR2 expression in the bystander non-irradiated endothelial cells. (A) Immunoblot showing VEGFR2 expression in the cell lysate extracted from BAEC at 16 h after co-culture with non-irradiated (lane 1) or irradiated MCF-7 cells with 2 Gy exposure (lane 2). Treatment of BAECs for 10 h with VEGF (50 ng/mL) was used as positive control (lane 3). The expression of VEGFR2 was determined in the cell lysate extracted from BAEC cells. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Notes: *P < 0.05; **P < 0.01.

Figure 7

Irradiated MCF-7 cells trigger FGFR1 expression in the bystander non-irradiated endothelial cells. (A) Immunoblot showing FGFR1 expression in the cell lysate extracted from BAEC at 16 h after co-culture with non-irradiated (lane 1) and irradiated MCF-7 cells with 2 Gy exposure (lane 2). PMA (0.1 μM), as a positive control, was directly treated with BAEC for 10 h (lane 3). The expression of FGFR1 was determined in the cell lysate extracted from BAEC cells. β-Actin was used as a loading control. (B) Densitometric quantitation of three independent experiments. Note: **P < 0.01.

Figure 8

Angiogenic tube network formation assay. BAECs co-cultured with irradiated MCF-7 cells in the presence (Panel D) or absence (Panel B) of NF-κB inhibitor or co-cultured with non-irradiated MCF-7 cells (Panel A) in Matrigel for 10 h. BAECs incubated with VEGF as a positive control (Panel C). Densitometric quantitation of three independent experiments (**P < 0.01) (Panel E).