Sage Journals: Discover world-class research

Abstract

BACKGROUND:

Radioresistance leads to treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, enhancing the radiosensitivity of NPC cells would likely increase the effectiveness of radiotherapy. Annexin VII (Annexin A7, ANXA7) might be a tumor promoter in NPC but its functions in radiosensitivity remain unclear.

METHODS:

NPC cell lines CNE2-shANXA7 and CNE2-pLKO.1 were generated and CNE2-shANXA7 nude mice xenograft tumor models were established. The main effects and molecular mechanisms of ANXA7 knockdown in NPC radiosensitivity were studied in vitro and in vivo by analyzing cell viability, clonogenicity, apoptosis, cell cycle distribution, tumor radioresponse and immunohistochemistry assay.

RESULTS:

ANXA7 knockdown revealed potentially enhanced NPC cell radiosensitivity via apoptosis and increased the cell number at the G2/M phase. In the xenograft model, NPC cells with ANXA7 knockdown were dramatically sensitive to irradiation and tumor growth was significantly suppressed. Compared to CNE2-pLKO.1 xenografts, CNE2-shANXA7 showed more $\gamma$ -H2AX foci and less phospho-DNA PKcs.

CONCLUSIONS:

ANXA7 knockdown increased the radiosensitivity of NPC by enhancing apoptosis, modulating the cell cycle distribution into more radiosensitive phases, promoting DNA damage, and inhibiting repair. We showed that decreased ANXA7 levels enhanced radiosensitivity and provided insights into the therapeutic targets for NPC radiotherapy.

Keywords

Nasopharyngeal carcinoma radiosensitivity annexin VII

1. Introduction

Nasopharyngeal carcinoma (NPC) is characterized by distinct geographical distributions and is one of the most common malignant tumors in southern China and Southeast Asia, with an incidence ranging from 20 to 50/100,000 [1, 2], and a 5-year survival of 70–80% after appropriate therapy [3]. Radiotherapy (RT) remains the preferred treatment for patients with NPC because of its high sensitivity. However, radioresistance leads to treatment failure in patients receiving radiotherapy for NPC [4, 5]. Hence, preventing radioresistance and predicting and increasing radiosensitivity are critical for further improving patient prognosis and increasing their 5-year survival rates. Annexin VII (Annexin A7, ANXA7), a member of the annexin family, is a calcium and phospholipid-binding protein with diverse properties. The protein was overexpressed in NPC compared to normal tissues [6] and is closely related to apoptosis and metastasis [7]. It might also act as a tumor promoter in NPC [8]. However, the function of ANXA7 in the radiosensitivity of NPC remains poorly understood. Therefore, we investigated the role of ANXA7 and the underlying molecular mechanisms of NPC radiosensitivity.

2. Materials and methods

2.1 Materials

pLKO.1 vector and pLKO.1-ANXA7-shRNA plasmids were obtained from RiboBio Co. Ltd (Guang-zhou, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and Lipofectamine 2000 were bought from Invitrogen (Invitrogen, Carlsbad, CA, USA). Microporous polyvinylidene fluoride (PVDF) transfer membranes were bought from Millipore (Billerica, MA, USA). The anti-ANXA7 monoclonal antibody was obtained from Abcam (ab197586, Cambridge, UK).

2.2 Human NPC tissue samples

Eighty-two human NPC tissue samples (42 radiosensitive NPC tissues and 40 radioresistant NPC tissues) were obtained from the First Affiliated Hospital of the University of South China, China. The patients were pathologically diagnosed as having NPC by experts from the Department of Pathology. Three months after the diagnosis, cancer regression in the neck and nasopharynx (nasal endoscopy and nasopharyngeal computed tomography [CT] examination) was assessed in all patients who completed RT. The short-term therapeutic effect of RT was evaluated according to the international standard, in which progression of disease (PD) was defined as increased tumor volume; stable disease (SD) as a tumor volume decrease by $<$ 50% and most of the nasopharyngeal structures remaining abnormal; partial remission (PR) as a tumor volume decrease by $>$ 50% and most of the nasopharyngeal structures returning to normal; and complete remission (CR) as tumor disappearance and the return of nasopharynx soft tissue to normal. The criteria for clinically radiosensitive and radioresistant patients were patients with CR and PR who were thought to be clinically radiosensitive and those with SD and PD who were considered clinically radioresistant, respectively [9]. No statistically significant differences were noted between the radioresistant and radiosensitive NPC patients in terms of sex, age, histological type, primary tumor (T) stage, lymph node metastasis, and clinical stage. All tissue samples were examined by immunohistochemistry. The study was approved by the local ethical committee and informed consent was obtained from the patients.

2.3 Cell lines

CNE2 and CNE2-IR (a radioresistant subclone cell line derived from CNE2) cells used in our previous study [9] were cultured in DMEM including 10% FBS in a 37 ${}^{\circ}$ C humidified incubator with 5% CO ${}_{2}$ .

2.4 CNE2 cell transfection with ANXA7 shRNA plasmids

To establish a stable knockdown of the ANXA7-expressing NPC cell line, CNE2 cells were transfected with pLKO.1 or pLKO.1-ANXA7-shRNA plasmids by Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. After selection with 1.0 $\mu$ g/ml puromycin (Invitrogen) for 2 weeks, individual puromycin-resistant cell colonies were picked, expanded, and maintained in a medium containing 0.5 $\mu$ g/ml puromycin. Expression levels of ANXA7 in the cloned cells were measured by western blotting. The cell lines stably transfected with pLKO.1 and pLKO.1-ANXA7-shRNA were named CNE2-pLKO.1 and CNE2-shANXA7, respectively.

2.5 Western blotting

CNE2-pLKO.1 and CNE2-shANXA7 cell lysates were examined by western blotting as previously described [10]. In brief, the lysates (30 $\mu$ g) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto PVDF membranes purchased from Millipore. The membranes were incubated with the primary anti-ANXA7 antibody (Sigma; 1:500) overnight at 4 ${}^{\circ}$ C, followed by incubation for 1 h with a horseradish peroxidase-linked secondary antibody diluted 1:3000. ECL (Amersham Pharmacia Biotech) was used for detection. Densitometry was performed for quantification using an ImageQuant analysis system.

2.6 Clonogenic assay

After exposure to ionizing radiation (IR), the radiosensitivities of CNE2-shANXA7 and CNE2-pLKO.1 cells were evaluated by the clonogenic assay as previously reported [10]. In brief, the cells were cultured in 6-well plates and exposed to IR at doses ranging from 0 to 8 Gy. After being cultured for 2 weeks, holoclones with $>$ 50 cells were counted. The dose-modifying factors (DMFs), plating efficiency (PE), and survival fraction (SF) were estimated using the following formulas: DMFs = dose to reach the specified survival in resistant cells divided by dose to reach the same survival in control cells, PE $=$ total number of colonies formed/total number of cells seeded $\times$ 100%, SF $=$ colonies counted/(cells seeded $\times$ PE/100) [10].

2.7 Cell viability after irradiation

The cells were seeded into sterile 96-well microtiter plates (2,000 cells/well), cultured for 12 h, and irradiated (5 Gy). Cell viability was assessed by the 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazolium-romide (MTT) assay. After 24 h, 5 g/L MTT (20 $\mu$ l; Sigma) was added to each well and the plates were incubated for 4 h. After removing the medium, dimethylsulphoxide (150 $\mu$ l, Sigma) was added and the plates were incubated at room temperature for 10 min. The absorbance values (A490 value) of each well were examined at 490 nm on a microplate reader (Biotex, USA). The percentage of viable cells was measured by comparison to the A490 readings measured on the first day.

2.8 Apoptosis and cell cycle post-irradiation with flow cytometry

The cells were cultured for 12 h in T-100 flasks and exposed to IR (5 Gy). After culturing for another 24 h, the cells were collected, fixed with chilled 70% ethanol for 1 h at $-$ 20 ${}^{\circ}$ C, and centrifuged for 5 min (1,500 rpm). The cells were then incubated in 0.5% Triton X-100 and 0.05% RNase for 30 min at 37 ${}^{\circ}$ C in PBS and centrifuged for 5 min (1,500 rpm). Next, the cells were treated with propidium iodide (Sigma, 40 $\mu$ g/ml) for 30 min at ambient temperature. Apoptosis and cell cycle distribution were analyzed by flow cytometry (FCM).

2.9 Hoechst 33258 staining of irradiated cells

After culturing for 12 h in 6-well plates, the cells were exposed to 5 Gy of IR. After 72 h, the culture was fixed at 4 ${}^{\circ}$ C for 30 min in 4% paraformaldehyde (PFA) and stained using Hoechst dye 33258 (Sigma, 5 $\mu$ g/ml) for 10 min in the dark. Cells with highly condensed and/or fragmented nuclei were considered apoptotic. A quantitative assessment of apoptosis was made in three different microscopic fields with at least 100 cells counted per field.

Table 1
Expressions of ANXA7 in the radiosensitive and radioresistant NPC tissues

		High (5–6)	Moderate (3–4)	Low (0–2)	$P$
NPC tissues	$n$	Score
Radiosensitivity	42	22	16	4	0.0001 ${}^{*}$
Radioresistance	40	31	7	2

NOTE: * $P<$ 0.05 wtih $\chi^{2}$ test, radiosensitive versus radioresistant NPC tissues. ANXA7, Annexin VII.

2.10 Tumor radioresponse assay in vivo

All experimental protocols involving animals in this study were approved by the local ethical committee. Male athymic Balb/c nude mice (18–24 g, 5–6 weeks of age) were bought from Shanghai Laboratory Animal Center (Shanghai, China) and were adaptively bred in a laminar airflow chamber under specific-pathogen-free conditions. CNE2-shANXA7 and CNE2-pLKO.1 cells were grown at 37 ${}^{\circ}$ C in DMEM with 10% FBS under 5% CO ${}_{2}$ to obtain cells in the logarithmic phase. Then, 50 $\mu$ l of trypsin (0.25%) was used to digest the cell suspension and live cells were counted using the trypan blue exclusion assay. When the cells reached 95% confluency, 5 $\times$ 10 ${}^{7}$ cells/ml (final concentration) were obtained using serum-free DMEM to adjust the cell concentration. For the in vivo tumor radioresponse assay, 0.2 ml of CNE2-shANXA7 or CNE2-pLKO.1 cell suspension was subcutaneously inoculated into the right axilla region of the nude mice (five mice per group, 1 $\times$ 10 ${}^{7}$ cells/mouse). The minimum diameters (width) and maximum diameters (length) of the tumors were monitored daily with a caliper. Tumor size was calculated using the formula: tumor volume $=$ 1/2 length $\times$ width ${}^{2}$ [9]. When the tumor size reached approximately 50 mm ${}^{3}$ , radiation was delivered to the tumor using a linear accelerator once daily with 2 Gy per fraction for five consecutive days for a total dose of 10 Gy. Two weeks post-IR, the mice were sacrificed by cervical dislocation under sodium pentobarbital anesthesia and the xenograft tumors were excised and weighed. Half of the tumor tissues were fixed with formalin and embedded in paraffin for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical staining.

2.11 Immunohistochemistry

For immunohistochemistry, 40 radioresistant and 42 radiosensitive NPC formalin-fixed and paraffin-embedded tissue sections were incubated with anti-ANXA7. Tumor tissues from the nude mice with NPC were assayed by immunohistochemistry for phosphorylated H2A histone family member X ( $\gamma$ H2AX) (phospho-S139) and phospho-DNA-dependent protein kinase, catalytic subunit (DNA PKcs [S2056]) as described previously [11]. First, the slides were incubated with anti-ANXA7 (Sigma; 1:100), anti- $\gamma$ H2AX (Abcom, USA; 1:500), or anti-phospho-DNA PKcs (Abcam; 1:200) antibody at 4 ${}^{\circ}$ C overnight and then with a biotinylated secondary antibody (1:2000; Sigma-Aldrich) and avidin-peroxidase complex according to the manufacturer’s protocol. Finally, the slides were stained in 3’,3’-diaminobenzidine (DAB) and counterstained with hematoxylin. For the negative control, the primary antibodies were replaced with PBS. Two blinded researchers evaluated the ANXA7, $\gamma$ H2AX, and phospho-DNA PKcs immunoreactions by light microscopy. Ten high-power visual fields were chosen in each section and at least 1,000 cells were counted. The quantitative scores were estimated by calculating both the scores for staining intensity and staining area for each sample [11]. The samples were categorized based on the combined staining score as follows: $\leqslant$ 2: low staining (no expression); 3–4: moderate staining (expression); 5–6: strong staining (high expression).

2.12 TUNEL assay in situ

Apoptotic cells were detected by TUNEL assay in paraffin-embedded tissue sections obtained from the xenograft tumors after IR according to a standard protocol. The TUNEL-positive cells, indicated by a diffuse distribution of fine brownish-yellow particles or the intracellular distribution of coarse brown particles, were counted under a microscope. To quantitatively evaluate the apoptotic cells, the sections were examined and the number of TUNEL-positive cells per 1,000 cancer cells in 10 random microscopic fields was determined by light microscopy.

2.13 Statistical analysis

The mean $\pm$ standard deviation (SD) was calculated for all numerical data and compared. The significant differences among groups were determined by Student’s $t$ -tests. For all comparisons, $p<$ 0.05 was considered statistically significant. The data were analyzed using PASW Statistics for Windows, version 18.0 (SPSS, Inc., Chicago, IL, USA).

3. Results

3.1 ANXA7 expression in human NPC tissues and cell lines

As shown in Fig. 1A, immunohistochemical analysis showed ANXA7 mainly in the nuclei, with mark-edly lower expression in radiosensitive tissues than that in radioresistant tissues. ANXA7 overexpression was observed in 52.4% (22/42) of radiosensitive human NPC tissue and 77.5% (31/40) of radioresistant human NPC tissue samples (Table 1, $p<$ 0.05). ANXA7 expression levels were detected in the CNE2 and CNE2-IR cells by western blotting. Higher ANXA7 expression was observed in CNE-2-IR cells than in CNE2 cells, indicating that ANXA7 expression was associated with radioresistance in NPC.

Figure 1.

The expressions of ANXA7 in NPC cell lines and human NPC tissues. (A) Immunohistochemistry was performed to examine the expressions of ANXA7 proteins in human NPC tissues. (B) Western blotting was used to analyze the expressions of ANXA7 in CNE2 and CNE2-IR cell lines. (C) The histogram showed the expressions level of ANXA7 in CNE2 and CNE2-IR cells as determined using densitometric analysis. $\beta$ -actin was performed as a loading control. ANXA7, Annexin VII; NPC, nasopharyngeal carcinoma; IS-NPC, irradiation sensitive NPC tissues; IR-NPC, irradiation resistant NPC tissues; * $P<$ 0.05.

Figure 2.

Construction of CNE2-shANXA7 cell line with overexpression of ANXA7. (A) A representative result showed the establishment of CNE2-shANXA7 cell line with knockdown of ANXA7, pLKO.1-ANXA7-shRNA-tansfected CNE2 cells (left), the vector pLKO.1-transfected CNE2 cells (Middle), and non transfected CNE2 cells (right). (B) The histogram showed the transfection rate of CNE2-shANXA7, CNE2-pLKO.1 and CNE2 cells. (C) Immunohistochemistry analysis was used to examine the expressions of ANXA7 in pLKO.1-ANXA7-shRNA-tansfected, pLKO.1-transfected and untransfected CNE2 cells. (D) Western blot analysis was used to examine the expressions of ANXA7 in pLKO.1-ANXA7-shRNA-tansfected, pLKO.1-transfected and untransfected CNE2 cells. (E) The histogram showed the expressions of ANXA7 in CNE2, CNE2-pLKO.1 and CNE2-shANXA7 cells as determined using densitometric analysis ( $\beta$ -actin as a loading control). CNE2-shANXA7 vs. CNE2-pLKO.1 or CNE2. ANXA7, Annexin VII; ${}^{*}P<$ 0.05.

3.2 Construction of a stable ANXA7 knockdown CNE2-shANXA7 cell line

To explore the association of ANXA7 expression in NPC cells with radiosensitivity, the pLKO.1 and pLKO.1-ANXA7-shRNA plasmids were transfected into CNE2 cells with puromycin screening. Fourteen days later, the colonies were picked and expanded and the ANXA7 expression was detected by immunohistochemistry and western blotting. As shown in Fig. 2, the ANXA7 protein expression level in CNE2-shANXA7 cells was markedly lower than that in CNE2 and CNE2-pLKO.1 cells ( $p<$ 0.01) but no statistically significant differences were observed between CNE2 and CNE2-pLKO.1 cells. We then developed the stable CNE2-shANXA7 (ANXA7 knockdown) NPC cell line.

Table 2
Cell cycle distribution of CNE2-shANXA7 and CNE2-PLKO.1 cell lines at 24 h after irradiation with 5 gray

Cell line	Number of cells (%)
	G2/M	$P$	S phase	$P$	G0/G1	$P$
CNE2-shANXA7	14.8 $\pm$ 1.7	0.004 ${}^{\ast}$	23.4 $\pm$ 1.3	0.033 ${}^{\ast}$	62.3 $\pm$ 2.7	0.735
CNE2-pLKO.1	7.3 $\pm$ 0.7		33.0 $\pm$ 3.6		59.7 $\pm$ 2.8

Note: ${}^{*}P<$ 0.05 by $\chi$ 2 test, CNE2-shANXA7 vs. Control CNE2-pLKO.1. ANXA7, Annexin VII.

3.3 Effects of ANXA7 knockdown on NPC cell proliferation in vitro after IR

CNE2-pLKO.1 and CNE2-shANXA7 cells were assessed by the clonogenic survival assay after IR with doses from 0 to 8 Gy to investigate the relationship between ANXA7 knockdown and radiosensitivity in NPC cells. As shown in Fig. 3A, the surviving colonies of CNE2-shANXA7 cells were remarkably fewer and smaller than those of CNE2-pLKO.1 cells (Fig. 3B). The DMFs were 0.67 and 0.53 at 1% and 10% of isosurvival levels for CNE2-shANXA7, respectively. The SF2 (SF at 2 Gy) was 0.41 for CNE2-shANXA7 cells and 0.76 for CNE2-pLKO.1 cells. Furthermore, following the exposure of CNE2-shANXA7 and CNE2-pLKO.1 cells to 5 Gy radiation to analyze the effects on cell growth, we observed that the cell viability decreased more slowly in CNE2-pLKO.1 cells than in CNE2-shANXA7 cells (Fig. 3C). Together, these findings showed a markedly higher radiosensitivity of CNE2-shANXA7 cells than of CNE2-pLKO.1 cells, indicating that ANXA7 expression decreased their radiosensitivity.

Figure 3.

Effects of ANXA7 knockdown on radiosensitivity of NPC cells after irradiation in vitro. (A and B) Clonogenic survival assay. CNE2-shANXA7 and CNE2-pLKO.1 cells were plated in six-well plate and were irradiated with radiation doses of 0–8 gray, and the survival fraction was calculated by counting the amount of colonies after 12 days of incubation. (C) Cell viability analysis. CNE2-shANXA7 and CNE2-pLKO.1 cells were plated into 96-well plate, after 12 h of culture, cells were irradiated (5 gray). MTT assays was used to monitor the cell viability at different time intervals. CNE2-shANXA7 vs. CNE2-pLKO.1. ${}^{\ast}P<$ 0.05. ANXA7, Annexin VII.

3.4 Effects of ANXA7 knockdown on apoptosis in NPC cells post-IR in vitro

The differences in apoptosis induced by IR between CNE2-shANXA7 and CNE2-pLKO.1 cells were studied by FCM analysis and Hoechst staining assay. We observed more apoptotic CNE2-shANXA7 cells than control cells.

3.5 Effects of ANXA7 knockdown on cell cycle distributions in NPC cells post-IR

Cell cycle progression at various phases may determine the radiation sensitivity of NPC cells. The cell cycle distributions in CNE2-shANXA7 and CNE2-pLKO.1 cells were examined by FCM 24 h after exposure to 5 Gy IR. The results are shown in Table 2. No differences between the two groups were noted in G0/G1 phases at 24 h post-IR. However, as compared to CNE2-pLKO.1 cells, fewer CNE2-shANXA7 cells were detained in the S and G2/M phases ( $p<$ 0.05). These data suggest that ANXA7 proteins could regulate IR-induced cell cycle progression; thus, ANXA7 protein levels might be closely related to NPC radiosensitivity.

Figure 4.

Effects of ANXA7 knockdown on apoptosis of NPC cells post-irradiation. (A) Hoechst staining assay. CNE2-shANXA7 and CNE2-pLKO.1 cells were irradiated with 5 gray of irradiation, after culturing for 72 h, apoptotic cells were identified by hoechst 33258. Apoptotic nuclei (arrow) being stained with intense fluorescence were indicated. (B) The histogram showed the apoptotic rate of CNE2-shANXA7 and CNE2-pLKO.1 cells. The experiments were repeated three times; mean, points, bars, SD. (C) FCM analysis. CNE2-shANXA7 and CNE2-pLKO.1 cells were irradiated with 5 gray of irradiation, after culturing for 24 h, the apoptosis were assessed with FCM. CNE2-shANXA7 vs. CNE2-pLKO.1, ${}^{\ast}P<$ 0.05. ANXA7, Annexin VII.

3.6 ANXA7 knockdown increased NPC cell radiosensitivity in vivo

Xenograft nude mice models were developed using ANXA7 knockdown NPC cells to study the effects of ANXA7 on NPC radiosensitivity in vivo. The radioresponses of the tumors were estimated after irradiation with total doses of 10 Gy. Slower tumor growth was observed in the ANXA7 knockdown mice. The subcutaneous xenografts in the CNE2-shANXA7 group were significantly smaller and lighter than those in the CNE2-pLKO.1 group. The results showed markedly higher radiosensitivity in CNE2-shANXA7 cell tumors with ANXA7 knockdown than that in CNE2-pLKO.1 cells. TUNEL assay revealed that the radiation-induced apoptosis in CNE2-pLKO.1 xenografts was much less than that in CNE2-shANXA7 xenografts, indicating that ANXA7 knockdown markedly increased tumor cell apoptosis in xenografts. In addition, immunohistochemical staining showed that ANXA7 knockdown increased the ratio of $\gamma$ H2AX-positive cells (DNA double-strand breaks [DSBs]) in the xenograft tumors. However, ANXA7 knockdown decreased the number of phospho-DNA-PKcs positive cells; i.e. cells involved in DSB repair, in the xenograft tumors (Fig. 5D). These findings demonstrated that a decreased ANXA7 protein level promoted NPC cell radiosensitivity in vivo.

Figure 5.

CNE2-shANXA7 increases NPC cell radiosensitivity in vivo. (A) The weight and volume of tumors generated by CNE2-shANXA7 cells and CNE2-pLKO.1 cells following 10 gray irradiation. (right) The growth curves of the tumors post-irradiation; (middle) the mean weights of the tumors post-irradiation at the sacrifice; (left) the mice were put to death, and the tumors were photographed 2 weeks post-irradiation. (B) (left) representative image of TUNEL detection of apoptosis in situ in the tumors after irradiation; (right) histogram indicated the percent of apoptotic cells. (C) (left) Representative immunohistochemical staining of $\gamma$ H2AX in tumor tissues generated by CNE2-pLKO.1 cells and CNE2-shANXA7 cells post-irradiation; (right) a histogram indicated percentages of $\gamma$ -H2AX positive cells in the tumors. (D) (left) Representative immunohistochemical results of phospho-DNA PKcs in tumor tissues came into being by CNE2-pLKO.1 cells and CNE2-shANXA7 cells post-irradiation; (right) histogram indicated the percent of phospho-DNA PKcs positive cells in tumor tissues. Mean, SD, and significant different was denoted by ${}^{\ast}P<$ 0.05. Original magnification, $\times$ 200.

4. Discussion

RT is the mainstay of therapy for most patients with NPC because of its high sensitivity. Improving the radiosensitivity of cancer cells is key for strengthening the therapeutic effect of RT. The radiosensitivity of NPC is related to factors such as cell growth and proliferation, apoptosis, cell cycle, and repair of radiation damage. Accumulating evidence indicates that ANXA7 proteins play an important role during tumor development and progression. The deregulation of ANXA7 proteins is associated with differentiation, apoptosis, metastasis, and poor prognosis in various tumors [12, 13, 14]. To elucidate the role of ANXA7 and its underlying mechanism in NPC radioresistance, we constructed stable ANXA7 knockdown (CNE2-shANXA7) and control (CNE2-pLKO.1) cell lines and performed radioresponse tests in vitro. The clonogenic survival assay findings demonstrated remarkably fewer and smaller surviving CNE2-shANXA7 cell colonies than those of CNE2-pLKO.1 cells. The results of the MTT assay showed significantly reduced CNE2-shANXA7 cell viability following IR compared to that in CNE2-pLKO.1 cells. These results suggested that ANXA7 knockdown could promote cell growth and proliferation, as the radiosensitivity of CNE2-shANXA7 cells was higher than that of CNE2-pLKO.1 cells. To elucidate the effects of ANXA7 knockdown on cellular radiosensitivity of NPC in vivo, CNE2-shANXA7 and CNE2-pLKO.1 mouse xenograft models were established. The subcutaneous tumors from the CNE2-shANXA7 group were significantly smaller and lighter than those from the CNE2-pLKO.1 group post-irradiation in vivo. The data showed significantly inhibited xenograft tumor growth post-radiotherapy in the ANXA7 knockdown group and markedly higher radiosensitivity of tumors from CNE2-shANXA7 cells than that for tumors from CNE2-pLKO.1 cells. Hoechst staining and FCM showed higher radiation-induced apoptosis in CNE2-shANXA7 cells than in CNE2-pLKO.1 cells. TUNEL assay in tumor tissue sections showed markedly increased cellular apoptosis in xenografts from ANXA7 knockdown cells. Cell cycle in CNE2-pLKO.1 and CNE2-shANXA7 cells measured by FCM at 24 h post-IR with 5 Gy showed no difference with respect to G0/G1 phases, but more G2/M phase and less S phase arrests were observed in CNE2-shANXA7 cells than in CNE2-pLKO.1 cells. Taken together, our findings demonstrated that ANXA7 knockdown remarkably increased NPC cell radiosensitivity in vivo and in vitro; thus, ANXA7 proteins may participate in the cellular response to irradiation in NPC.

Apoptosis plays a vital role in the cell death pathway after IR; thus, radiation-induced apoptosis is critical in radiation therapy. Tumor cell radiosensitivity is associated with cell cycle checkpoints, apoptosis, and DNA damage repair [15, 16]. Radiation causes DNA damage, triggering the apoptotic pathway in tumor cells. Cells with repairable damage enter the repair program, while cells with un-repairable damage enter the apoptotic process [17]. Furthermore, apoptosis plays a crucial role in RT as a part of the cell death pathway after irradiation [18].

ANXA7, which co-locates with B-cell lymphoma 2 (Bcl2) in the mitochondria and cytoplasm, was associated with Hca-P cell apoptosis via alteration of the mitochondrial membrane potential and Bcl2 expression [19]. Inhibition of ANXA7 expression enhanced apoptosis of BGC823 cells in vivo and in vitro, and markedly inhibited the growth of tumor xenografts [20]. In the present study, ANXA7 knockdown increased IR-induced apoptosis in NPC cells in vivo and in vitro, indicating that this knockdown heightened NPC radiosensitivity by increasing apoptosis. The cell-cycle distribution is one of the most important determinants for tumor radiosensitivity [21]. The cell’s sensitivity to RT varies at different stages of the cell cycle. Cells arrested at the G2/M phase are more sensitive to radiation, whereas cells arrested at the S phase are more resistant to radiation [22]. Zheng et al. reported that miRNA-200c increased esophageal cancer radiosensitivity through sub-G1 and G2/M phase arrest [23]. Cao and colleagues reported a decreased proportion of A549S1 (radioresistant A549) cells in the G0/G1 phase and a consequent increase in the number of cells in the S phase as compared the proportion of A549 cells in these phases [24]. In this study, we found that the ratio of cells at the G2/M phase was remarkably increased in CNE2-shANXA7 cells in comparison with CNE2-pLKO.1 cells after IR. These phases corresponded to the times in which cells are most sensitive to the effects of IR. An inverse relationship was observed in S-phase fraction analysis. Generally, the cell cycle is arrested at certain phases to repair damaged DNA. The cell cycle progresses when the damaged DNA is successfully repaired; otherwise, apoptosis occurs. This process requires many cytokines to repair the cells blocked at the G2/M phase following IR-induced DNA damage [25]. Our findings indicated that ANXA7 knockdown arrested the cell cycle at the G2/M phase, participated in the regulation of radiosensitivity through cell cycle arrest, and increased CNE2-shANXA7 cell radiosensitivity by changing the distribution of the cell cycle.

IR primarily results in DNA damage of cells and activates the DNA damage repair pathways [26]. The DNA damage is closely associated with cell radiosensitivity and is the main cause of tumor cell death after RT [27, 28]. Phosphorylation of histone H2AX is associated with the accumulation of damage repair proteins and chromatin damage [29] and, thus, is a sensitive reporter for DNA damage [27, 30]. Meanwhile, phosphorylation of histone H2AX is positively related to increased DSBs and increased numbers of $\gamma$ -H2AX foci [31]. Therefore, the phosphorylated form of H2AX is a highly specific and sensitive biomarker for detecting DNA damage and repair induced by IR. The DNA-PKcs DNA-repair gene plays the major role in the non-homologous DNA end-joining pathway, which is the principal pathway for DSB repair [32, 33]. DNA-PKcs also plays an important role in H2AX phosphorylation in response to DNA damage after radiation. It is also a key kinase involved in regulating the level of phosphorylated H2AX [34]. Thus, DNA-PKcs activity is key for DNA DSB repair [35]. To further clarify the mechanism by which ANXA7 knockdown responded to DNA damage and repair, the foci formed by DNA DSBs and phospho-DNA-PKcs were tested in tissues from irradiated xenograft tumors by immunohistochemical analysis. Our results showed a higher $\gamma$ H2AX level and a lower phospho-DNA-PKcs level in the xenograft tumors from the CNE2-shANXA7 group than in those from the control CNE2-pLKO.1 group. We found ANXA7 knockdown increased the number of $\gamma$ -H2AX foci and suppressed the aggregation of DNA-PKcs induced by IR. More $\gamma$ -H2AX foci indicated increased numbers of unrepaired DSBs. Without repair, DSBs cause chromosome break and loss, eventually leading to cell death. Thus, inhibiting DNA DSB repair can increase NPC cell radiosensitivity. Therefore, ANXA7 knockdown promoted DNA damage by increasing $\gamma$ -H2AX foci formation and inhibiting DNA repair by suppressing DNA-PKcs activation, consequently improving NPC cell radiosensitivity.

5. Conclusion

In summary, ANXA7 was crucial for radiation sensitivity of NPC cells both in vivo and in vitro; therefore, downregulated ANXA7 protein expression may improve NPC cell radiosensitivity by reducing colony growth, blocking the cell cycle at the G2/M phase, inducing increased apoptosis, increasing $\gamma$ -H2AX, and decreasing DNA-PKcs activity. The effects and mechanisms of ANXA7 on NPC radiosensitivity are related to the induction of apoptosis and inhibition of radiation-induced DNA damage repair. Our findings provide strong evidence for ANXA7 as a radiosensitizing target in NPC and warrant further study of the exact molecular mechanisms of ANXA7 in NPC radiosensitization.

Footnotes

Acknowledgments

We would like to thank Editage (www.editage.cn) for English language editing.

References

M.C.

and Yuan

J.M.

, Epidemiology of nasopharyngeal carcinoma, Semin Cancer Biol 12 (2002), 421–429.

M.F.

Sheng

Cheng

W.M.

M.H.

B.H.

Wei

K.R.

F.G.

Lian

S.F.

Wang

P.P.

Quan

Deng

X.H.

Liu

X.D.

Xie

Y.L.

Huang

S.J.

S.X.

Huang

S.L.

Liang

X.J.

S.M.

Huang

H.W.

Xia

S.L.

P.S.

Chen

H.L.

Xie

S.H.

Liu

Hong

M.H.

Yuan

Xia

N.S.

Zhang

and Cao

S.M.

, Incidence and mortality of nasopharyngeal carcinoma: interim analysis of a cluster randomized controlled screening trial (PRO-NPC-001) in southern China, Ann Oncol (2019), pii: mdz231.

Chua

M.L.K.

Wee

J.T.S.

Hui

E.P.

and Chan

A.T.C.

, Nasopharyngeal carcinoma, Lancet 387 (2016), 1012–1024.

Kristensen

C.A.

Kjaer-Kristoffersen

Sapru

Berthelsen

A.K.

Loft

and Specht

, Nasopharyngeal carcinoma. Treatment planning with IMRT and 3D conformal radiotherapy, Acta Oncologica 46 (2007), 214–220.

Lee

A.W.

Poon

Y.F.

Foo

Law

S.C.

Cheung

F.K.

Chan

D.K.

Tung

S.Y.

Thaw

and Ho

J.H.

, Retrospective analysis of 5037 patients with nasopharyngeal carcinoma treated during 1976-1985: overall survival and patterns of failure, Int J Radiat Oncol Biol Phys 23 (1992), 261–270.

Yang

and Liang

, Study the relationship between the expression of Annexin A7 and CT of nasopharyngeal carcinoma, J Chin Clin Med Imaging 22 (2011), 6–9.

Leighton

Eidelman

Jozwik

Pollard

H.B.

and Srivastava

, ANXA7-GTPase as Tumor Suppressor: Mechanisms and Therapeutic Opportunities, Methods Mol Biol 1513 (2017), 23–35.

Guo

Liu

Greenaway

and Sun

M.Z.

, Potential role of annexin A7 in cancers, Clin Chim Acta 423 (2013), 83–89.

Liao

Yan

W.J.

Tian

C.M.

M.Y.

Tian

Y.Q.

and Zeng

G.Q.

, Knockdown of Annexin A1 Enhances Radioresistance and Inhibits Apoptosis in Nasopharyngeal Carcinoma, Technol Cancer Res Treat 17 (2018), 1533034617750309.

10.

Huang

Liao

Wan

Cheng

Chen

Tan

and Zeng

, Downregulation of Annexin A1 is correlated with radioresistance in nasopharyngeal carcinoma, Oncol Lett 12 (2016): 5229–5234.

11.

Tan

Liao

Wan

Y.P.

M.X.

Chen

S.H.

W.J.

Zhao

Q.L.

Huang

L.F.

and Zeng

G.Q.

, Downregulation of selenium-binding protein 1 is associated with poorprognosis in lung squamous cell carcinoma, World J Surg Oncol 14 (2016), 70.

12.

Tan

Liao

Wan

Y.P.

M.X.

Chen

S.H.

W.J.

Zhao

Q.L.

Huang

L.F.

Zeng

G.Q.

Fan

Zhao

Yuan

Tan

and Zhang

, Annexin A7 expression is downregulated in late-stage gastric cancer and is negatively correlated with the differentiation grade and apoptosis rate, Oncol Lett 15 (2018), 9836–9844.

13.

Zhao

Yang

Wang

Tian

Liang

and Li

, AnnexinA7 down-regulation might suppress the proliferation and metastasis of human hepatocellular carcinoma cells via MAPK/ERK pathway, Cancer Biomark 23 (2018), 527–s537.

14.

Leighton

Bera

Eidelman

Bubendorf

Zellweger

Banerjee

Gelmann

E.P.

Pollard

H.B.

and Srivastava

, Tissue microarray analysis delineate potential prognostic role of Annexin A7 in prostate cancer progression, PLoS One 13 (2018), e0205837.

15.

Pan

Mou

Liu

Sun

Meng

Zhou

and Peng

, SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis, Oncol Rep 33 (2015), 2999–3005.

16.

Chang

Huang

Wang

Yan

Zhu

Zhuang

and Zhang

, Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy, BMC Cancer 16 (2016), 190.

17.

Willers

Dahm-Daphi

and Powell

S.N.

, Repair of radiation damage to DNA, Br J Cancer 90 (2004), 1297–1301.

18.

Özyurt

çevik

Ö.

Özgen

Özden

A.S.

Çadırcı

Elmas

M.A.

Ercan

Gören

M.Z.

and Şener

, Quercetin protects radiation-induced DNA damage and apoptosis in kidney and bladder tissues of rats, Free Radic Res 48 (2014), 1247–1255.

19.

Bai

Guo

Wang

Zhao

Huang

and Tang

, 47kDa isoform of Annexin A7 affecting the apoptosis of mouse hepatocarcinoma cells line, Biomed Pharmacother 83 (2016), 1127–1131.

20.

Fan

Zhao

Yuan

Tan

and Zhang

, Effect of annexin A7 suppression on the apoptosis of gastric cancer cells, Mol Cell Biochem 429 (2017), 33–43.

21.

Geldof

A.A.

Plaizier

M.A.

Duivenvoorden

Ringelberg

Versteegh

R.T.

Newling

D.W.

and Teule

G.J.

, Cell cycle perturbations and radiosensitization effects in a human prostate cancer cell line, J Cancer Res Clin Oncol 129 (2003), 175–182.

22.

Dillon

M.T.

Good

J.S.

and Harrington

K.J.

, Selective targeting of the G2/M cell cycle checkpoint to improve the therapeutic index of radiotherapy, Clin Oncol (R Coll Radiol) 26 (2014), 257–265.

23.

Zheng

Liu

Zhang

Zhao

and Deng

, miRNA-200c enhances radiosensitivity of esophageal cancer by cell cycle arrest and targeting P21, Biomed Pharmacother 90 (2017), 517–523.

24.

Cao

Ding

Xue

Zou

Huang

and Peng

, SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer, Radiat Oncol 8 (2013), 178.

25.

Liang

Zhu

Wen

and Wu

, Extracellular matrix metalloproteinase inducer (CD147/BSG/ EMMPRIN)-induced radioresistance in cervical cancer by regulating the percentage of the cells in the G2/m phase of the cell cycle and the repair of DNA Double-strand Breaks (DSBs), Am J Transl Res 8 (2016), 2498–2511.

26.

Ashwell

and Zabludoff

, DNA damage detection and repair pathways – recent advances with inhibitors of checkpoint kinases in cancer therapy, Clin Cancer Res 14 (2008), 4032–4037.

27.

Zheng

Chen

Z.W.

Wang

S.Y.

Yan

Y.Q.

Q.Z.

Zhang

S.M.

and Zhou

P.K.

, Radioprotection of 4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells isassociated with minimizing DNA damage and activating Akt, Eur J Pharm Sci 33 (2008), 52–59.

28.

Mah

L.J.

Vasireddy

R.S.

Tang

M.M.

Georgiadis

G.T.

El-Osta

and Karagiannis

T.C.

, Quantification of gammaH2AX foci in response to ionising radiation, J Vis Exp 38 (2010), 174–192.

29.

Celeste

Fernandez-Capetillo

Kruhlak

M.J.

Pilch

D.R.

Staudt

D.W.

Lee

Bonner

R.F.

Bonner

W.M.

and Nussenzweig

, Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks, Nat Cell Biol 5 (2003), 675–679.

30.

Rogakou

E.P.

Pilch

D.R.

Orr

A.H.

Ivanova

V.S.

and Bonner

W.M.

, DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139, J Biol Chem 273 (1998), 5858–5868.

31.

Sone

Piao

Nakakido

Ueda

Jenuwein

Nakamura

and Hamamoto

, Critical role of lysine 134 methylation on histone H2AX for γ-H2AX productionand DNA repair, Nat Commun 5 (2014), 5691.

32.

Zhu

Fisher

L.A.

Bessho

and Peng

, Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining, Nucleic Acids Res 45 (2017), 10583–10594.

33.

Dong

Zhang

Ren

Wang

Ling

C.C.

G.C.

Wang

and Wen

, Inhibiting DNA-PKcs in a non-homologous end-joining pathway in response to DNA double-strand breaks, Oncotarget 8 (2017), 22662–22673.

34.

Huang

Y.C.

Q.Z.

Zhou

L.J.

Shang

Z.F.

Huang

Wang

Liu

X.D.

D.C.

and Zhou

P.K.

, DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression, BMC Mol Biol 11 (2010), 18.

35.

Burdine

L.J.

Burdine

M.S.

Moreland

Fogel

Orr

L.M.

James

Turnage

R.H.

and Tackett

A.J.

, Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway, PLoS One 10 (2015), e0127943.

Knockdown of annexin VII enhances nasopharyngeal carcinoma cell radiosensitivity in vivo and in vitro

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Materials

2.2 Human NPC tissue samples

2.3 Cell lines

2.4 CNE2 cell transfection with ANXA7 shRNA plasmids

2.5 Western blotting

2.6 Clonogenic assay

2.7 Cell viability after irradiation

2.8 Apoptosis and cell cycle post-irradiation with flow cytometry

2.9 Hoechst 33258 staining of irradiated cells

Table 1 Expressions of ANXA7 in the radiosensitive and radioresistant NPC tissues

2.11 Immunohistochemistry

2.12 TUNEL assay in situ

2.13 Statistical analysis

3. Results

3.1 ANXA7 expression in human NPC tissues and cell lines

Table 2 Cell cycle distribution of CNE2-shANXA7 and CNE2-PLKO.1 cell lines at 24 h after irradiation with 5 gray

3.5 Effects of ANXA7 knockdown on cell cycle distributions in NPC cells post-IR

5. Conclusion

Footnotes

Acknowledgments

References

Table 1
Expressions of ANXA7 in the radiosensitive and radioresistant NPC tissues

Table 2
Cell cycle distribution of CNE2-shANXA7 and CNE2-PLKO.1 cell lines at 24 h after irradiation with 5 gray