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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Nasopharyngeal carcinoma (NPC), which has the highest incidence in South China, is mainly treated by radiotherapy. However, the survival rate remains low. Angiogenesis is closely correlated with progress of NPC. Thus, the combination of anti-angiogenesis with radiation is an attractive strategy for NPC treatment. A heterogenic xenografted human NPC nude mice model was established to investigate the effect of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic factor, and the combined effect of PEDF and radiotherapy on nasopharyngeal carcinoma. Pigment epithelium-derived factor remarkably suppressed the growth of NPC by 43.52% and decreased the tumor microvessel density (MVD). Pigment epithelium-derived factor had no effects on the proliferation and apoptosis of NPC cell lines by MTT and flow cytometry assay. However, PEDF decreased vascular endothelial growth factor (VEGF) in NPC cell lines by downregulation of hypoxia-inducible factor 1α, a crucial transcriptional factor for VEGF expression, as demonstrated by western blotting and immunofluorescent staining assay. Interestingly, irradiation alone could also effectively downregulate VEGF and MVD of xenografted tumor, which indicates that irradiation suppresses NPC not only by killing tumor cells but also through anti-angiogenesis. Furthermore, combined treatment of PEDF with irradiation enhanced the antitumor efficacy. The MVD and VEGF in the combined therapy were much less than in the treatment with PEDF or radiotherapy alone. Our observation demonstrated that the combination of PEDF with radiotherapy enhances the efficacy of the antitumor effect on NPC by the coordinated inhibition on angiogenesis, which implies the potential role of PEDF as an adjuvant agent for NPC treatment. (Cancer Sci 2011; 102: 1789–1798)

Nasopharyngeal carcinoma (NPC) is rare in most countries, with an age-adjusted incidence less than one per 100 000 population per year. However, the highest incidence is found in Southern China (∼25–30 per 100 000 population per year), especially among people of Cantonese ancestry.(1) Nasopharyngeal carcinoma has the highest incidence of distant metastasis among head and neck cancers.(2,3) Radiotherapy is the main strategy for NPC. With the improvement of modern imaging techniques and radiotherapy planning and delivery, the local–regional control of this neoplasm has been improved.(4,5) Overall survival has also been improved with the addition of concurrent and adjuvant chemotherapy.(6,7) However, the incidence of relapse remains high. Therefore, the development of multidisciplinary therapeutic approaches to improve local–regional control and eradicate micrometastases is required.(8)

Angiogenesis, the formation of new blood vessels from pre-existing vessels, occurs physiologically in growth, development and pathologically in tumors, diabetic retinopathy, rheumatoid arthritis and regeneration.(9) Tumor growth and metastasis are angiogenesis dependent, therefore inhibiting tumor-induced angiogenesis is a promising strategy for the treatment of cancer and potentially leads to tumor regression.(10,11) Several preclinical studies have indicated that anti-angiogenic treatment might enhance the antitumor effect of radiotherapy without increasing damage to normal tissues.(12–14) Angiogenesis inhibitors have been demonstrated promise for cancer therapy.

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is a member of the serpin superfamily encoded by the gene SERPINF1 located on chromosome 17p13.(15,16) Pigment epithelium-derived factor was first identified as a neurotrophic factor purified from the conditioned medium of cultured foetal retinal pigment epithelial cells.(17) Further studies showed that it was a multifunctional protein, including neurotrophic,(18) neuronal differentiation-induction,(19) neural stem cell self-renewal,(20) anti-inflammatory,(21) anti-angiogenic(22,23) and antitumor activities.(24) As an angiogenic inhibitor, PEDF is more potent than any other known endogenous inhibitor of angiogenesis, being more than twice as potent as angiostatin and seven times more potent than endostatin.(22)

Previous studies have shown that PEDF could inhibit the growth of several types of cancers.(24) However, there is no report in NPC. Therefore, the present study focuses on the antitumor effect of PEDF alone, the combined effect of PEDF with radiotherapy on NPC and the underlying mechanism.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell line and culture conditions.  Human well-differentiated and poorly differentiated NPC cell lines CNE-1 and CNE-2 cells, respectively,(25) were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco) and incubated at 37°C, 5% CO2 in a humidified incubator. To induce hypoxia, cells were maintained at an O2 tension of 1% in a Hypoxia Workstation (Ruskinn, Cincinnati, OH, USA).

Irradiation conditions.  All irradiations were delivered using a X-ray machine (Beijing Medical X-ray Company, Beijing, China), operating at 210 kVp and 12 mA with a 0.2-mm Cu filter with a dose rate of 1 Gy/min. Local irradiation of the implanted tumor was administered using customized mouse jig with other parts of the body shielded with lead.

Cell proliferation assay.  Cells were seeded in 24-well plates at a density of 2 × 104 cells/well and grown to 70% confluence. The culture medium was replaced with serum free RPMI-1640 medium administered with various concentrations of PEDF (0, 80, 160, 320, 640 and 1280 nmol/L) for 24 h under normoxia (20% O2, v/v) or hypoxia (1% O2, v/v). Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyl tetrazolium bromide (MTT) (Sigma, St Louis, MO, USA) assay. Data represented absorbance and are expressed as percentages of the respective controls.

Flow cytometry assay.  CNE-2 cells were seeded at 5 × 104 per well in six-well plates. Cells were treated with PEDF at a concentration of 80 and 320 nmol/L for 24 h and then harvested for flow cytometry assay using the AnnexinV-FITC Apoptosis Detection kit (Bender Med Systems, Vienna, Austria). Cells were treated with PBS as negative controls.

Animals.  Care, use and treatment of all animals in the present study were in strict agreement with the institutionally approved protocol according to the United States Public Health Service (USPHS) Guide for the care and use of laboratory animals, as well as the guidelines set forth in the Care and Use of Laboratory Animals by the SUN Yat-sen University. Male 4-week-old athymic nude mice (BALB/c, nu/nu) were obtained from the Laboratory Animal Center of Guangdong, China and the animal license number is SCXK(YUE)2008-0020.

Heterotopic tumor growth assay.  Subcutaneous implants of CNE-2 cells were performed by injecting 5 × 106 cells into the lower right flank of athymic nude mice. When tumor volumes reached approximately 100 mm3, the mice were randomized into four groups, with five mice in each group: PBS; irradiation alone; PEDF alone; and irradiation combined with PEDF. Mice treated with PEDF were injected intraperitoneally (i.p.) at a total dose of 5 mg/kg per day on days 1, 3, 5, 7 and 9. Irradiation treatment was delivered on days 1, 3 and 5 at a dose of 2 Gy/day. As for irradiation combined with the PEDF group, PEDF and irradiation treatment were described above. Tumor volume was monitored by caliper measurement every 3 days and calculated according to the following formula: Volume = length × width2/2. Four weeks after the first treatment, the mice were killed and tumor tissues were dissected, weighed and stored at −80°C. The tumor inhibition ratio was calculated using the formula: Inhibition ratio (%) = ([C−T]/C) × 100%, where C is the average tumor weight of the control group and T is the average tumor weight of the treated group.

Western blot assay.  Cells were seeded in six-well plates at a density of 1 × 105/well. Cells at 70–80% confluence were incubated under normoxic or hypoxic conditions with or without PEDF at the concentration of 0–640 nmol/L for 12 h. For radiation treatment, cells were irradiated at a dose of 6 Gy and then treated with or without PEDF at a concentration of 320 nmol/L for 12 h. Whole cell lysates were collected and the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) expression were determined using a polyclonal rabbit anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a polyclonal rabbit anti-HIF-1α antibody (Santa Cruz) at a dilution of 1:500, respectively. The same membrane was stripped and re-blotted with an anti-β-actin antibody (Sigma) for normalization.

Immunofluorescent assay.  CNE-2 cells were seeded on coverslips and incubated under normoxia or hypoxia conditions with or without 320 nmol/L PEDF for 12 h. The cells were washed, fixed and blocked with 5% bovine serum albumin. Primary antibody against VEGF (rabbit polyclonal antibody, 1:100; Abcam, Cambridge, UK) or HIF-1α (mouse monoclonal antibody, 1:100; BD Pharmingen, San Diego, CA, USA) was then added and incubated at 4°C overnight. FITC-conguated goat anti-rabbit or goat anti-mouse secondary antibody (1:100; Dako, Glostrup, Denmark) was added and incubated for 2 h at room temperature. The cell nuclei was stained with 4,6-diamino-2-phenylindole (DAPI) (Sigma). The images were visualized and representative views of the cells were recorded by fluorescence microscopy with an Olympus IX71 microscope (Olympus, Tokyo, Japan). In negative-control staining, the primary antibodies were omitted.

Microvessel density (MVD) assay.  Tumor tissues were fixed with 4% paraformaldehyde and cut into 5-μm paraffin-embedded sections and immunostained. The slides were incubated with rat anti-mouse CD34 monoclonal antibody (1:50; Abcam, Cambridge, UK) at 4°C overnight after washing for three times, and then treated with HRP-conjugated goat anti-mouse secondary antibody (Envison kit; Dako) for 30 min. The antigen–antibody complex was visualized by incubation with the DAB kit (Envison kit). Finally, all sections were counterstained with hematoxylin. Tumor MVD was quantified using Weidner’s method.(26) Negative controls were incubated without the primary antibody.

Statistical analysis.  All data are expressed as mean ± SD. For two-group comparison, the Student’s t-test method was used. For comparison of more than two groups, one-way anova was used. SPSS 13.0 software (SPSS, Chicago, IL, USA) was used for all statistical analyses. P ≤ 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Pigment epithelium-derived factor suppresses growth of xenografted human NPC.  To evaluate the effect of PEDF on tumor growth, heterotopic tumor xenografts of NPC were established and treated with PEDF or PBS (Fig. 1A). On the 15th day after the first injection, the average tumor volume of the PEDF group was significantly lower than that of the control group (P < 0.05, Fig. 1B). Compared with the PBS group, an average of 43.52% suppression of primary tumor growth was observed in the PEDF-treated group (P < 0.01, n = 5, Fig. 1C,D).

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Figure 1.  Inhibition of tumor growth in a nasopharyngeal xenograft athymic mouse model with pigment epithelium-derived factor (PEDF) treatment. CNE-2 cell heterotopic transplanted tumors were developed as described in the Materials and Methods. Mice received i.p. injection of PEDF (25 mg/kg). Tumor tissues were collected and weighed at 21 days after the first injection of PEDF. (A) Tumor-bearing mice treated with PBS (top) and PEDF (bottom). (B) Tumor growth curves: volumes of PEDF-treated versus PBS-treated groups on the days indicated (**P < 0.01). (C) Heterotopic transplanted tumors treated with PBS (top) and PEDF (bottom). [DOWNWARDS ARROW], PEDF injection point. (D) An average of 43.52% suppression of primary tumor growth was observed (**P < 0.01).

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Pigment epithelium-derived factor inhibits tumor angiogenesis.  Microvessel density was assessed in CNE-2 xenografts by counting the number of CD34-positive vessels present in five random high-power fields from three different nude mice. The MVD of the PEDF-treated group was significantly reduced compared with the PBS-treated group (P < 0.01, Fig. 2). These data demonstrate that PEDF inhibits the neovascularization of NPC.

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Figure 2.  Pigment epithelium-derived factor (PEDF) inhibited tumor xenograft angiogenesis. Tumor xenografts were harvested and 5-μm sections were immunostained with rabbit anti-CD34 antibody. (A) Representative immunohistochemical data for CD34 immunoreactivity. Scale bar, 100 μm. a,c: low power; b,d: high power. (B) Quantification of CD34-positive vessels. Microvessels were counted from five randomly selected fields in tumors from three mice of each group. PEDF significantly reduced microvessel density compared with PBS-treated tumors (PBS, 19.07 ± 5.18; PEDF, 11.13 ± 3.62; n = 5 per group. **P < 0.01 vs control).

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Pigment epithelium-derived factor has no direct effect on NPC cells.  To explore whether PEDF inhibits tumor growth by direct effect on tumor cells, CNE-2 cell proliferation was measured using MTT assay and cell apoptosis was measured using flow cytometry assay. The MTT assay showed that PEDF had no effect on the proliferation of CNE-1 (Fig. S1) and CNE-2 (Fig. 3A) cells, even at high concentrations. The flow cytometry assay indicated that PEDF had no effect on the apoptosis of CNE-2 cells, both under normoxia (Fig. 3B) and hypoxia (Fig. 3C).

image

Figure 3.  Pigment epithelium-derived factor (PEDF) had no direct effect on the proliferation and apoptosis of CNE-2 cells. Cell proliferation was measured by MTT assay and cell apoptosis was measured by flow cytometry assay. (A) The MTT assay showed that PEDF had no effect on the proliferation of CNE-2 cells, even at high concentrations. (B,C) The flow cytometry assay indicated that PEDF had no effect on the apoptosis of CNE-2 cells.

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Pigment epithelium-derived factor downregulates VEGF expression of CNE-2 in vitro.  Both western blot analysis (Fig. 4A) and immunofluorescent assay (Fig. 4B) indicated that VEGF expression in CNE-2 cells was upregulated under hypoxia compared with normoxia, while the expression of VEGF in CNE-2 cells was significantly downregulated by PEDF (Fig. 4). The ELISA assay (Data S1) showed that PEDF could significantly inhibit the secretion of VEGF under hypoxic conditions in CNE-2 cells (Fig. S2). These results suggested that PEDF might inhibit tumor angiogenesis through downregulation of VEGF expression and secretion in CNE-2 tumor cells.

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Figure 4.  Pigment epithelium-derived factor (PEDF) downregulated hypoxia-induced vascular endothelial growth factor (VEGF) expression in CNE-2 cells. Western blotting and immunofluorescent staining were used to detect the effect of PEDF on VEGF expression. (A) Representative western blotting of CNE-2 cell lysates using anti-VEGF antibody. Compared with normoxia, levels of VEGF were increased under hypoxia. PEDF decreased VEGF expression in a dose-dependent manner under hypoxia (*P < 0.05). H, hypoxia; N, normoxia. (B) Representative immunofluorescent staining of CNE-2 cells using anti-VEGF antibody (*P < 0.05). Scale bar, 20 μm.

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Pigment epithelium-derived factor inhibits the expression and nuclear translocation of HIF-1α in CNE-2 cells.  To elucidate whether PEDF downregulates the expression of VEGF through HIF-1α, we investigated the amount and subcellular localization of HIF-1α in CNE-2 cells. As shown in Figure 5, HIF-1α protein mainly located in the cytoplasm under normoxia, hypoxia could promote the nuclear translocation of HIF-1α. However, PEDF could reduce HIF-1α translocation into the nucleus under hypoxia (Fig. 5).

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Figure 5.  Pigment epithelium-derived factor (PEDF) inhibited hypoxia-induced hypoxia inducible factor 1α (HIF-1α) expression in CNE-2 cells. Western blotting and immunofluorescent staining were used to detect the effect of PEDF on HIF-1α expression. Expression of HIF-1α was elevated by hypoxia and PEDF could dramatically inhibit HIF-1α elevation and nuclear translocation under hypoxia (*P < 0.05; **P < 0.01). (A) Representative western blotting of CNE-2 cell lysates using anti-HIF-1α antibody. H, hypoxia; N, normoxia. (B) Representative immunofluorescence of CNE-2 cells using anti-HIF-1α antibody. Scale bar, 20 μm.

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Pigment epithelium-derived factor attenuates the upregulation of HIF-1α/VEGF induced by irradiation.  The expression of HIF-1α and VEGF were elevated by irradiation, and PEDF treatment could significantly inhibit irradiation-induced HIF-1α and VEGF elevation in both CNE-2 (Fig. 6) and CNE-1 cells (Fig. S3).

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Figure 6.  Pigment epithelium-derived factor (PEDF) attenuates upregulation of hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) induced by irradiation in CEN-2 cells. (A) Western blotting was used to detect the effect of irradiation and/or PEDF on HIF-1α and VEGF expression in CNE-2 cells. The expression level of HIF-1α and VEGF were elevated by irradiation and treatment by PEDF could significantly inhibit HIF-1α and VEGF elevation. (B) Representative immunofluorescent staining of CNE-2 cells using anti-VEGF antibody. Scale bar, 20 μm. IR, irradiation.

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Pigment epithelium-derived factor plus radiotherapy enhances the antitumor effect in CNE-2 xenografts.  On the 28th day after the first injection, the xenografted tumors were dissected and tumor weights were analysed. It is worth noticing that during the combined treatment with PEDF and radiotherapy, the tumors grew slowly from the third day after treatment (Fig. 7A). The growth of tumors after single or combined treatment was significantly slower than that of the PBS-treated group as assessed by tumor weight (Fig. 7B, P < 0.01). An average of 57.34%, 73.04% and 88.37% suppression of primary tumor growth was observed in the PEDF-treated, irradiation-treated and PEDF plus irradiation groups, respectively (P < 0.01, n = 5, Fig. 7C). Notably, the inhibitory effect of the PEDF plus irradiation group was significantly enhanced (Fig. 7C, P < 0.05 compared with PEDF or irradiation treatment alone).

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Figure 7.  Pigment epithelium-derived factor (PEDF) plus radiotherapy enhanced the inhibitory effect on CNE-2 xenografts. (A) The growth curves of CNE-2 xenografts with PEDF and/or irradiation treatment using PBS as a control treatment. [UPWARDS ARROW], PEDF injection point; inline image, irradiation point. (B) Heterotopic transplanted tumors 28 days after treatments. (C) Tumor weight at 28 days after treatment: *P < 0.05. **P < 0.01. IR, irradiation.

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Pigment epithelium-derived factor plus radiotherapy enhances anti-angiogenesis activities.  To determine the effects of different treatments on tumor angiogenesis, we evaluated the MVD of tumor sections. Our data showed that MVD was significantly decreased by PEDF or irradiation alone compared with the PBS group (Fig. 8A). Pigment epithelium-derived factor plus irradiation exhibited more significant vessel density reduction compared with either the PEDF or irradiation treatment alone (Fig. 8B, P < 0.01). Furthermore, consistent with the results of MVD, the expression of VEGF was significantly inhibited by PEDF and irradiation alone, and PEDF plus irradiation showed more significant VEGF reduction (Fig. 8C, P < 0.05).

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Figure 8.  Pigment epithelium-derived factor (PEDF) plus radiotherapy enhanced the anti-angiogenic effect on CNE-2 xenografts. Tumor vasculature was visualized using anti-CD34 immunohistochemistry. (A) Representative micrographs are sections from CNE-2 xenografts. (a) Control treated with PBS. (b) PEDF-treated group. (c) PBS+IR-treated group; (d) PEDF+IR-treated group. Scale bar, 20 μm. (B) Histograms representing the mean number of vessels per tumor section after each treatment. *P < 0.05. **P < 0.01. HPF, high-power field. (C) Western blotting of VEGF protein expression in nasopharyngeal carcinoma (NPC) tumors treated with PEDF and/or IR at 6 Gy; β-actin was used as the internal control. Histogram representing VEGF protein levels measured using western blotting in NPC tumors. The western blotting was repeated three times. *P < 0.05. **P < 0.01. IR, irradiation.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Preclinical studies have shown that anti-angiogenic threapy could effectively suppress the growth of NPC xenografted tumors.(27,28) Tsuji et al.(29) observed that latent membrane protein 1 enhanced lymph node metastasis via the induction of angiogenesis in NPC. In addition, previous studies have indicated that the metastatic potency of NPC and the prognosis of NPC patients could be estimated by measuring MVD.(30,31) These findings suggest that angiogenesis is closely correlated with NPC growth and metastasis, therefore angiogenic inhibitors could be useful in the treatment of NPC.

The present study reported for the first time that PEDF could effectively inhibit the growth of xenografted NPC. Compared with the PBS group, an average of 43.52% suppression of primary tumor growth was observed in the PEDF-treated group (P < 0.01, Fig. 1). The present study showed that MVD in CNE-2 xenografted tumor tissues treated with PEDF was markedly decreased (Fig. 2), suggesting that PEDF inhibited tumor angiogenesis. Pigment epithelium-derived factor did not affect proliferation or apoptosis of CNE-2 and CNE-1 cells even at a high dose, indicating that PEDF had no direct anticancer properties by targeting cancer cells (Figs 3,S1). These data demonstrate that PEDF was a potent angiogenic inhibitor and suppressed tumor growth by anti-angiogenesis in NPC.

Solid tumors contain low oxygen level areas, known as hypoxia. Hypoxia results in stabilization and activation of HIF-1α, which in turn triggers the production of VEGF. HIF-1α is a crucial transcriptional factor of VEGF expression under hypoxia; VEGF is the most potent and specific known proangiogenic factor and is secreted by almost all solid tumor cells.(32) Considerable clinical and experimental evidence suggests that VEGF plays a pivotal role in tumor angiogenesis and metastasis, thus VEGF has become an important target for cancer therapy.(33) Several studies have observed that overexpression of HIF-1α and VEGF in NPC or squamous cell head and neck cancer (SCHNC) patients was not only associated with poor prognosis, but also had prognostic significance in patients receiving chemoradiotherapy.(34,35) A meta-analysis of 12 studies demonstrated that expression of VEGF was evaluated in 1002 patients with head and neck squamous cell carcinoma (HNSCC) and positive VEGF staining was associated with an almost two fold higher risk of death at 2 years. In a series of 103 patients with NPC, 67% of tumors were found to overexpress VEGF and this feature was correlated with higher local recurrence.(36) Therefore, blocking the HIF-1α/VEGF pathway might be an effective approach for NPC treatment.

In the present study we demonstrated that PEDF reduced production of VEGF in CNE-2 cells. Western blot analysis showed that the expression of VEGF in cultured CNE-2 cells treated with PEDF was significantly decreased under hypoxia. Similar results were observed by immunofluorescent assay (Fig. 4). Consequently, to explore the mechanism by which PEDF downregulates VEGF, we detected the effect of PEDF on HIF-1α expression in CNE-2 cells. Both western blotting and immunofluorescent assay showed that PEDF treatment remarkably decreased the expression of HIF-1α and nuclear translocation in CNE-2 cells under hypoxia (Fig. 5). ELISA assay showed that PEDF could significantly inhibit the secretion of VEGF under hypoxic conditions (Fig. S2). The inhibitory effect of PEDF on hypoxia-induced HIF-1α/VEGF upregulation was also consistently observed in CNE-1 cells (Fig. S3). These data suggest that PEDF notably inhibited the growth and angiogenesis of NPC by, at least in part, blocking the HIF-1α/VEGF pathway.

Radiotherapy is extensively used in cancer therapy. Although rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence implies that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity.(37) It has been suggested that the sensitivity of tumor vasculature to radiation is a major determinant of overall response to radiotherapy.(38) Irradiation was reported to induce tumor angiogenesis.(39,40) However, some studies have shown that irradiation could prevent tumor angiogenesis by inducing apoptosis of endothelial cells, inhibiting endothelial cell survival, proliferation, tube formation and invasion, thus leading to tumor cell death by destroying the tumor-feeding vasculature.(38,41,42) In the present study we also observed that irradiation could induce the apoptosis of HUVEC in vitro (data not shown). Furthermore, our results demonstrated that irradiation alone could effectively downregulate the level of VEGF protein and the MVD of xenografts in vivo (Fig. 8), which indicates that irradiation suppresses the growth of tumor not only by killing tumor cells but also through anti-angiogenesis.

In addition, some endogenous angiogenic inhibitors have shown an additive effect in combination with irradiation in preclinical studies, such as angiostatin,(43) plasminogen kringle 5 (K5)(44) and endostatin.(45) However, the combined effect of PEDF and irradiation remains unknown. Our data showed that PEDF and irradiation therapy alone modestly inhibited tumor growth compared with the PBS-treated group; however, the PEDF plus radiotherapy group exhibited a significant additive growth inhibitory effect, which is more significant than either treatment alone (P < 0.05, Fig. 7). Moreover, the tumor vasculature numbers in the group receiving the combined therapy were much less than that in the groups receiving either PEDF or radiotherapy alone (Fig. 8).

It was considered that the effects of irradiation combined with angiogenic inhibitors are by concomitant targeting to tumor vasculature.(13,43) The study by Jin et al.(44) indicated that the significant antitumor effect of rK5 plus irradiation was associated with a direct suppression effect on early angiogenesis and tumor cell apoptosis. This was further verified by another study, which found that prior irradiation significantly sensitized brain microvessel endothelium cells to rK5-induced apoptosis by 500-fold and prior irradiation would have a dose-sparing effect on rK5 anti-angiogenic therapy for brain tumor.(46) Endostatin consistently showed significantly enhanced effects on tumor growth inhibition, endothelial cell and tumor cell apoptosis induction of radiation therapy in human NPC and human lung adenocarcinoma xenografts.(45) However, the molecular basis for the enhanced inhibition on angiogenesis is not clear. Our results indicate that combined treatment with irradiation and PEDF efficiently decreases the expression of VEGF more than that in the groups receiving either PEDF or radiotherapy alone (Fig. 8). The additive effect might come from the coordinated downregulation on VEGF expression.

Preclinical and clinical studies have shown that anti-angiogenic agents could normalize tumor vasculature, leading to improved blood supply and oxygenation, and thus enhancing the antitumor effect of chemoradiotherapy.(47,48) Therefore, as a potent endogenous angiogenic inhibitor, PEDF probably enhances the antitumor effect of radiation via tumor vascular normalization. It is worth exploring more with further study.

In conclusion, our data demonstrate that PEDF can significantly inhibit angiogenesis of xenografted NPC, thus suppressing its growth. Downregulation of VEGF by inhibiting HIF-1α, thus attenuating the paracrine effect of VEGF in tumor cells on endothelial cells, might represent a mechanism for the activity of PEDF. The combination of PEDF with radiotherapy enhances the efficacy on NPC. The additive effect might partially come from the coordinated inhibition on angiogenesis. These findings imply the potential role of PEDF as an adjuvant agent for NPC treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This study was supported by the National Nature Science Foundation of China, grant numbers: 30600724, 30700120, 30872980, 30971208, 30973449, 81070746, 81001014; National Key Sci-Tech Special Project of China, grant number: 2009ZX09103-642; Program for Doctoral Station in University, China, grant number: 20100171110049; Key Project of Nature Science Foundation of Guangdong Province, China, grant number: 10251008901000009; Program for Changjiang Scholars and Innovative Research Team in University, China, grant number: PCSIRT0947; Guangdong Natural Science Fund, China, grant number: 10151008901000007; Key Sci-tech Research Project of Guangdong Province, China, grant number: 2008B080703027; Key Sci-tech Research Project of Guangzhou Municipality, China, grant number: 2008Z1-E231; and Program for Young Teacher in University, China, grant numbers: 09YKPY73, 10YKPY28.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1. Pigment epithelium-derived factor had no direct effect on the proliferation of CNE-1 cells.

Fig. S2. Pigment epithelium-derived factor downregulates vascular endothelial growth factor secretion in CNE-2 cells.

Fig. S3. Pigment epithelium-derived factor inhibits hypoxia- or irradiation-induced hypoxia inducible factor 1α and vascular endothelial growth factor upregulation in CNE-1 cells.

Data S1. Vascular endothelial growth factor ELISA assay.

FilenameFormatSizeDescription
CAS_2013_sm_DataS1.doc22KSupporting info item
CAS_2013_sm_fS1-3.doc133KSupporting info item

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