Knockdown of Annexin A2 Enhances Radiosensitivity by Increasing G2/M-Phase Arrest, Apoptosis and Activating the p38 MAPK-HSP27 Pathway in Nasopharyngeal Carcinoma

Abstract

Annexin A2 (ANXA2) has been found to be involved in cancer proliferation, metastasis and prognosis; however, its exact role in nasopharyngeal carcinoma (NPC) radioresistance remains unknown. We found that ANXA2 expression was correlated with prognosis in NPC patients, and longer overall survival in NPC patients with low ANXA2 expression than those with high ANXA2 expression. ANXA2 knockdown increased the radiosensitivity in radioresistant NPC cells, and ANXA2 overexpression decreased the radiosensitivity in NPC cells. Knocking-down ANXA2 expression increased the irradiation-induced apoptosis of radioresistant NPC cells, and ANXA2 overexpression decreased the irradiation-induced apoptosis of NPC cells. ANXA2 knockdown induced G2/M phase arrest in NPC cells post-irradiation, and ANXA2 overexpression abrogated G2/M phase arrest in NPC cells post-irradiation. ANXA2 overexpression resulted in inhibition of the p38 MAPK-HSP27 pathway, while ANXA2 knockdown resulted in activation of the p38 MAPK-HSP27 pathway. In addition, ANXA2 knockdown increased the radiosensitivity of the xenografted tumors in nude mice. Our data demonstrate that knockdown of Annexin A2 enhanced radiosensitivity in NPC by increasing G2/M-phase arrest, apoptosis and activating the p38 MAPK-HSP27 pathway. ANXA2 may be a promising target used to overcome radioresistance in NPC.

Keywords: annexin A2; apoptosis; cell-cycle arrest; nasopharyngeal carcinoma; radioresistance.

Figure 1

ANXA2 expression in NPC specimens and its association with overall survival and Locoregional recurrence-free survival in NPC patients. (A) immunohistochemical analysis of ANXA2 expression in NPC specimens, scale bar = 50 μm; (B, C) Kaplan-Meier survival curves of NPC patients with high (n = 67) and low (n = 33) ANXA2 expression, and the comparison of overall survival (B) and Locoregional recurrence-free survival (C) were done using a log-rank test.

Figure 2

Generation of ANXA2 knockdown and overexpression in NPC cells. (A) Western blotting determines the ANXA2 expression in shR-ANXA2, shR-C and CNE2(R743) cells, and β-actin serves as a loading control; (B) qPCR assay quantifies the relative ANXA2 expression in shR-ANXA2, shR-C and CNE2(R743) cells; (C) Western blotting determines the ANXA2 expression in pcD-ANXA2, pcD-C and CNE2 cells, and β-actin serves as a loading control; (D) qPCR assay quantifies the relative ANXA2 expression in pcD-ANXA2, pcD-C and CNE2 cells. All values are presented as mean ± SD, n = 3. **P < 0.01.

Figure 3

ANXA2 knockdown increases radiosensitivity of NPC cells and ANXA2 overexpression decreases radiosensitivity of NPC cells. (A) clonogenic survival assay reveals less cell colonies in shR-ANXA2 cells than in shR-C and CNE2(R743) cells following exposure to X-ray irradiation at doses of 0, 2, 4, 6, 8 and 10 Gy; (B) the radiosensitivity is increased in shR-ANXA2 cells than in shR-C and CNE2(R743) cells following exposure to X-ray irradiation at doses of 0, 2, 4, 6, 8 and 10 Gy; (C) clonogenic survival assay shows more cell colonies in pcD-ANXA2 cells than in pcD-C and CNE2 cells following exposure to X-ray irradiation at doses of 0, 2, 4, 6, 8 and 10 Gy; (D) a decrease is seen in the radiosensitivity in pcD-ANXA2 cells relative to pcD-C and CNE2 cells. All results are given as mean ± SD obtained from three independent experiments.

Figure 4

ANXA2 knockdown increases irradiation-induced apoptosis and ANXA2 overexpression decreases irradiation-induced apoptosis in NPC cells. (A) flow cytometric analysis detects the apoptosis of shR-ANXA2, shR-C and CNE2(R743) cells 48 h post-exposure to irradiation at doses of 0 and 4 Gy; (B) a higher apoptotic rate is measured in shR-ANXA2 cells than in shRNA-C and CNE2(R743) cells 48 h post-exposure to irradiation at a dose of 4 Gy; (C) Western blotting determines the expression of apoptosis-related proteins in shR-ANXA2, shR-C and CNE2(R743) cells 48 h post-exposure to irradiation at doses of 0 and 4 Gy, and β-actin serves as a loading control; (D) higher Bax expression and lower Bcl-2 expression is determined in shR-ANXA2 cells than in shR-C and CNE2(R743) cells 48 h after 4 Gy irradiation; (E) flow cytometric analysis detects the apoptosis of pcD-ANXA2, pcD-C and CNE2 cells 48 h post-exposure to irradiation at doses of 0 and 4 Gy; (F) a lower apoptotic rate is detected in pcD-ANXA2 cells than in pcD-C and CNE2 cells; (G) Western blotting determines the expression of apoptosis-related proteins in pcD-ANXA2, pcD-C and CNE2 cells 48 h post-exposure to irradiation at doses of 0 and 4 Gy, and β-actin serves as a loading control; (H) lower Bax and higher Bcl-2 expression is determined in pcD-ANXA2 cells than in pcD-C and CNE2 cells. Data are presented as mean ± SD of three experiments. *P < 0.05, **P < 0.01.

Figure 5

ANXA2 knockdown induces and ANXA2 overexpression abrogates G2/M cell cycle arrest in NPC cells following irradiation. (A) flow cytometric analysis detects the cell cycle distribution of shR-ANXA2, shR-C and CNE2(R743) cells 24 h post-exposure to irradiation at doses of 0 and 4 Gy; (B) the percentage of shR-ANXA2 cells with G2/M cell-cycle arrest is significantly higher than those of shR-C and CNE2(R743) cells 24 h following exposure to irradiation at 4 Gy, no significant difference is seen in the percentage of shR-ANXA2, shR-C and CNE2(R743) cells with G2/M cell-cycle arrest without irradiation; (C) Western blotting determines the expression of cell cycle-related proteins in shR-ANXA2, shR-C and CNE2(R743) cells 24 h post-exposure to irradiation at doses of 0 and 4 Gy, and β-actin serves as a loading control; (D) lower Cyclin B1 and CDK1 expression is determined in shR-ANXA2 cells than in shR-C and CNE2(R743) cells; (E) flow cytometric analysis detects the cell cycle distribution of pcD-ANXA2, pcD-C and CNE2 cells 24 h post-exposure to irradiation at doses of 0 and 4 Gy; (F) a lower proportion of the pcD-ANXA2 cells with G2/M cell-cycle arrest is measured in relative to pcD-C and CNE2 cells 24 h following exposure to irradiation at 4 Gy, and no significant difference was detected in the proportion of pcD-ANXA2, pcD-C and CNE2 cells with G2/M cell-cycle arrest without irradiation; (G) Western blotting determines the expression of cell cycle-related proteins in pcD-ANXA2, pcD-C and CNE2 cells 24 h post-exposure to irradiation at doses of 0 and 4 Gy, and β-actin serves as a loading control; (H) higher Cyclin B1 and CDK1 expression is detected in pcD-ANXA2 cells than in pcD-C and CNE2 cells. Data are presented as mean ± SD of three experiments. *P < 0.05, **P < 0.01.

Figure 6

Downregulation of Vimentin and HSP27 expression has no impact on ANXA2 expression. (A) Western blotting determines the expression of ANXA2 and ANXA2-interacting proteins HSP27 and Vimentin in siR-Vimentin-, siR-NC- (Negative control siRNA) and non-transfected radioresistant CNE2(R743) cells, and β-actin serves as a loading control; (B) densitometric analysis determines lower Vimentin expression in siR-Vimentin-transfected CNE2(R743) cells than in siR-NC- and non-transfected CNE2(R743) cells, and no significant difference was seen in ANXA2 or HSP27 expression among the siR-Vimentin-, siR-NC- and non-transfected CNE2(R743) cells; (C) Western blotting determines the expression of ANXA2 and ANXA2-interacting proteins HSP27 and Vimentin in siR-HSP27-, siR-NC- and non-transfected radioresistant CNE2(R743) cells, and β-actin serves as a loading control; (D) densitometric analysis determines lower Vimentin and HSP27 expression in siR-HSP27-transfected CNE2(R743) cells than in siR-NC- and non-transfected CNE2(R743) cells, and no significant difference was seen in ANXA2 expression among the siR-HSP27-, siR-NC- and non-transfected CNE2(R743) cells. All values are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01.

Figure 7

p38 MAPK-HSP27 pathway is activated by ANXA2 knockdown and inactivated by ANXA2 overexpression. (A) Western blotting determines the expression of the p38 MAPK-HSP27 pathway-associated proteins in NPC cells 48 h post-exposure to X-ray irradiation at a dose of 4 Gy, and β-actin serves as a loading control; (B) lower phospho-HSP27/HSP27 and phospho-p38MAPK/p38MAPK expression are determined in pcD-ANXA2 cells than in pcD-C and CNE2 cells; (C) higher phospho-HSP27/HSP27 and phospho-p38 MAPK/p38 MAPK expression are detected in shR-ANXA2 cells than in shR-C and CNE2(R743) cells. All values are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01. P-Vimentin, phospho-Vimentin; P-HSP27, phospho-HSP27; P-p38MAPK, phospho-p38MAPK.

Figure 8

Knockdown of ANXA2 expression increases the radiosensitivity of xenografted tumors in a nude mice model of NPC. (A) the volumes of the xenografted tumors from shR-ANXA2, shR-C and CNE2(R743) 1, 2 and 3 weeks post-irradiation at doses of 0 and 10 Gy; (B) the size of the xenografted tumors from shR-ANXA2, shR-C and CNE2(R743) 3 weeks post-irradiation at doses of 0 and 10 Gy. Values are presented as mean ± SD. *P < 0.05.