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Human apurinic/apyrimidinic endonuclease siRNA inhibits the angiogenesis induced by X-ray irradiation in lung cancer cells.

Gu X, Cun Y, Li M, Qing Y, Jin F, Zhong Z, Dai N, Qian C, Sui J, Wang D - Int J Med Sci (2013)

Bottom Line: APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time.Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells.Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China.

ABSTRACT

Objective: Radiotherapy is an important and effective treatment method for non-small cell lung cancer (NSCLC). Nonetheless, radiotherapy can alter the expression of proangiogenic molecules and induce angiogenesis. Human apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein, which has DNA repair and redox function. Our previous studies indicated APE1 is also a crucial angiogenic regulator. Thus, we investigated the effect of APE1 on radiation-induced angiogenesis in lung cancer and its underlying mechanism.

Methods: Tumor specimens of 136 patients with NSCLC were obtained from 2003 to 2008. The APE1 and vascular endothelial growth factor (VEGF) expression, as well as microvessel density (MVD) were observed with immunohistochemistry in tumor samples. Human lung adenocarcinoma A549 cells were treated with Ad5/F35-APE1 siRNA and/or irradiation, and then the cells were used for APE1 analysis by Western blot and VEGF analysis by RT-PCR and ELISA. To elucidate the underline mechanism of APE1 on VEGF expression, HIF-1α protein level was determined by Western blot, and the DNA binding activity of HIF-1α was detected by EMSA. Transwell migration assay and capillary-like structure assay were used to observe the migration and capillary-like structure formation ability of human umbilical veins endothelial cells (HUVECs) that were co-cultured with Ad5/F35-APE1 siRNA and (or) irradiation treated A549 cells culture medium.

Results: The high expression rates of APE1 and VEGF in NSCLC were 77.94% and 66.18%, respectively. The expressions of APE1 was significantly correlated with VEGF and MVD (r=0.369, r=0.387). APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time. The high expressions of APE1 and VEGF on A549 cells were concurrently induced by X-ray irradiation in a dose-dependent manner. Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells.

Conclusion: Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation. Administration of Ad5/F35-APE1 siRNA during radiotherapy could be a potent adjuvant therapeutic approach to enhance the radiotherapy response, effectively eliminate metastasis and improve the efficacy of radiotherapy for NSCLC.

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Effects of Ad5/F35-APE1 siRNA on expression of VEGF. A549 (A) and HUVECs (B) cells were treated with Ad5/F35-APE1 siRNA or Ad5/F35-EGFP and then radiated with 4 Gy X-ray at 48 h after infection. Samples were collected at 24 h post-radiation. RT-PCR analysis of total RNA was done with VEGF mRNA primer and reprobed with β-actin primer as a loading control. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-EGFP+IR; lane 3, Ad5/F35-APE1 siRNA; lane 4, Ad5/F35-APE1 siRNA+IR. *p<0.05 vs. Ad5/F35-EGFP; # p<0.01 vs. Ad5/F35-EGFP+IR. A549 (C) and HUVECs (D) cells were irradiated with different doses of X-ray at 48 h post-infection. Samples were collected at 48 h after irradiation. VEGF protein levels in the culture supernatant were determined by ELISA. *p<0.05 different doses irradiation vs. non-irradiation; #p<0.05 Ad5/F35-APE1 siRNA vs. Ad5/F35-EGFP at same dose of X-ray irradiation.
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Figure 5: Effects of Ad5/F35-APE1 siRNA on expression of VEGF. A549 (A) and HUVECs (B) cells were treated with Ad5/F35-APE1 siRNA or Ad5/F35-EGFP and then radiated with 4 Gy X-ray at 48 h after infection. Samples were collected at 24 h post-radiation. RT-PCR analysis of total RNA was done with VEGF mRNA primer and reprobed with β-actin primer as a loading control. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-EGFP+IR; lane 3, Ad5/F35-APE1 siRNA; lane 4, Ad5/F35-APE1 siRNA+IR. *p<0.05 vs. Ad5/F35-EGFP; # p<0.01 vs. Ad5/F35-EGFP+IR. A549 (C) and HUVECs (D) cells were irradiated with different doses of X-ray at 48 h post-infection. Samples were collected at 48 h after irradiation. VEGF protein levels in the culture supernatant were determined by ELISA. *p<0.05 different doses irradiation vs. non-irradiation; #p<0.05 Ad5/F35-APE1 siRNA vs. Ad5/F35-EGFP at same dose of X-ray irradiation.

Mentions: To investigate the role of APE1 on VEGF mRNA expression level after irradiation, we analyzed the mRNA expression of VEGF in Ad5/F35-APE1 siRNA and irradiation alone group, and combined with Ad5/F35-APE1 siRNA and irradiation group. As shown in Figs.5A and B, the VEGF mRNA expression levels of A549 and HUVECs cells increased after irradiation treatment, and Ad5/F35-APE1 siRNA attenuated the radiation-induced VEGF mRNA expression. There was a dose-dependent increase in VEGF protein in A549 (Fig.5C) and HUVECs (Fig.5D) culture supernatants post-radiation, and the increased VEGF protein expression level was significantly inhibited by Ad5/F35-APE1 siRNA.


Human apurinic/apyrimidinic endonuclease siRNA inhibits the angiogenesis induced by X-ray irradiation in lung cancer cells.

Gu X, Cun Y, Li M, Qing Y, Jin F, Zhong Z, Dai N, Qian C, Sui J, Wang D - Int J Med Sci (2013)

Effects of Ad5/F35-APE1 siRNA on expression of VEGF. A549 (A) and HUVECs (B) cells were treated with Ad5/F35-APE1 siRNA or Ad5/F35-EGFP and then radiated with 4 Gy X-ray at 48 h after infection. Samples were collected at 24 h post-radiation. RT-PCR analysis of total RNA was done with VEGF mRNA primer and reprobed with β-actin primer as a loading control. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-EGFP+IR; lane 3, Ad5/F35-APE1 siRNA; lane 4, Ad5/F35-APE1 siRNA+IR. *p<0.05 vs. Ad5/F35-EGFP; # p<0.01 vs. Ad5/F35-EGFP+IR. A549 (C) and HUVECs (D) cells were irradiated with different doses of X-ray at 48 h post-infection. Samples were collected at 48 h after irradiation. VEGF protein levels in the culture supernatant were determined by ELISA. *p<0.05 different doses irradiation vs. non-irradiation; #p<0.05 Ad5/F35-APE1 siRNA vs. Ad5/F35-EGFP at same dose of X-ray irradiation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675501&req=5

Figure 5: Effects of Ad5/F35-APE1 siRNA on expression of VEGF. A549 (A) and HUVECs (B) cells were treated with Ad5/F35-APE1 siRNA or Ad5/F35-EGFP and then radiated with 4 Gy X-ray at 48 h after infection. Samples were collected at 24 h post-radiation. RT-PCR analysis of total RNA was done with VEGF mRNA primer and reprobed with β-actin primer as a loading control. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-EGFP+IR; lane 3, Ad5/F35-APE1 siRNA; lane 4, Ad5/F35-APE1 siRNA+IR. *p<0.05 vs. Ad5/F35-EGFP; # p<0.01 vs. Ad5/F35-EGFP+IR. A549 (C) and HUVECs (D) cells were irradiated with different doses of X-ray at 48 h post-infection. Samples were collected at 48 h after irradiation. VEGF protein levels in the culture supernatant were determined by ELISA. *p<0.05 different doses irradiation vs. non-irradiation; #p<0.05 Ad5/F35-APE1 siRNA vs. Ad5/F35-EGFP at same dose of X-ray irradiation.
Mentions: To investigate the role of APE1 on VEGF mRNA expression level after irradiation, we analyzed the mRNA expression of VEGF in Ad5/F35-APE1 siRNA and irradiation alone group, and combined with Ad5/F35-APE1 siRNA and irradiation group. As shown in Figs.5A and B, the VEGF mRNA expression levels of A549 and HUVECs cells increased after irradiation treatment, and Ad5/F35-APE1 siRNA attenuated the radiation-induced VEGF mRNA expression. There was a dose-dependent increase in VEGF protein in A549 (Fig.5C) and HUVECs (Fig.5D) culture supernatants post-radiation, and the increased VEGF protein expression level was significantly inhibited by Ad5/F35-APE1 siRNA.

Bottom Line: APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time.Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells.Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China.

ABSTRACT

Objective: Radiotherapy is an important and effective treatment method for non-small cell lung cancer (NSCLC). Nonetheless, radiotherapy can alter the expression of proangiogenic molecules and induce angiogenesis. Human apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein, which has DNA repair and redox function. Our previous studies indicated APE1 is also a crucial angiogenic regulator. Thus, we investigated the effect of APE1 on radiation-induced angiogenesis in lung cancer and its underlying mechanism.

Methods: Tumor specimens of 136 patients with NSCLC were obtained from 2003 to 2008. The APE1 and vascular endothelial growth factor (VEGF) expression, as well as microvessel density (MVD) were observed with immunohistochemistry in tumor samples. Human lung adenocarcinoma A549 cells were treated with Ad5/F35-APE1 siRNA and/or irradiation, and then the cells were used for APE1 analysis by Western blot and VEGF analysis by RT-PCR and ELISA. To elucidate the underline mechanism of APE1 on VEGF expression, HIF-1α protein level was determined by Western blot, and the DNA binding activity of HIF-1α was detected by EMSA. Transwell migration assay and capillary-like structure assay were used to observe the migration and capillary-like structure formation ability of human umbilical veins endothelial cells (HUVECs) that were co-cultured with Ad5/F35-APE1 siRNA and (or) irradiation treated A549 cells culture medium.

Results: The high expression rates of APE1 and VEGF in NSCLC were 77.94% and 66.18%, respectively. The expressions of APE1 was significantly correlated with VEGF and MVD (r=0.369, r=0.387). APE1 and VEGF high expression were significantly associated with reduced disease free survival (DFS) time. The high expressions of APE1 and VEGF on A549 cells were concurrently induced by X-ray irradiation in a dose-dependent manner. Silencing of APE1 by Ad5/F35-APE1 siRNA significantly decreased DNA binding activity of HIF-1α and suppressed the expression of VEGF in A549 cells, moreover, significantly inhibited the endothelial cells immigration and capillary-like structure formation induced by irradiated A549 cells.

Conclusion: Our results indicate that APE1 may play a crucial role in angiogenesis induced by irradiation. Administration of Ad5/F35-APE1 siRNA during radiotherapy could be a potent adjuvant therapeutic approach to enhance the radiotherapy response, effectively eliminate metastasis and improve the efficacy of radiotherapy for NSCLC.

Show MeSH
Related in: MedlinePlus