Combination of pigment epithelium‐derived factor with radiotherapy enhances the antitumor effects on nasopharyngeal carcinoma by downregulating vascular endothelial growth factor expression and angiogenesis

Abstract

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 1a, 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 N asopharyngeal 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 localregional 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 preexisting 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...

Metformin inhibits the growth of nasopharyngeal carcinoma cells and sensitizes the cells to radiation via inhibition of the DNA damage repair pathway

“…Cells in the early log phase were trypsinized and plated in a 96-well plate at a density of 1x10 4 cells per well. Twenty-four hours later, the medium was removed and replaced with fresh medium with metformin at the indicated concentrations (0, 4,8,16,25,32, 50 and 64 mM) for 24 or 48 h in the presence of 1% fBS. Cell density was measured using the CCK-8 (Dojindo Molecular Technologies, Japan) assay following the manufacturer's instructions.…”

“…After treatment with metformin and/ or irradiation (6 Gy), sample preparation for immunoblotting was carried out as previously described (16). The membrane was then incubated with the appropriate primary antibody, anti-caspase-9, anti-caspase-3, anti-phospho-histone H2AX(Ser139), anti-ATM, anti-phospho-ATM(Ser1981), anti-ATR, anti-phospho-ATR(Ser428), anti-DNA-PK, anti-Ku70, anti-Ku80, anti-Rad50 and anti-p95/NBS1 (1:1,000; CST, Danvers, MA, uSA), and anti-β-actin (1:1,500; Beyotime Biotech, China).…”

Pigment epithelium-derived factor has a role in the progression of papillary thyroid carcinoma by affecting the HIF1α-VEGF signaling pathway

“…Furthermore, PEDF suppressed tumor growth in a cervical cancer cell line by down regulating the expression of VEGF and HIF1α, which demonstrated the anti-angiogenic activity of PEDF (26). In addition, the HIF1α and VEGF/PEDF signaling pathway is targeted in impacting nasopharyngeal carcinoma cell proliferation and angiogenesis (28). PEDF may not be directly regulated by HIF1α, as indicated by the inverse association between PEDF and HIF1α identified in the current study.…”

“…The anti-angiogenic effect of PEDF is performed primarily through the disruption of microvascular network distribution (25)(26)(27)(28). Vascular endothelial growth factor (VEGF) is an established pro-angiogenic factor and numerous studies have reported an inverse correlation between PEDF and VEGF expression levels in certain tumor models (21,(25)(26)(27)(28).…”

Phosphomimetic Mutants of Pigment Epithelium-Derived Factor with Enhanced Anti-Choroidal Melanoma Cell Activity In Vitro and In Vivo

“…[23][24][25][26] More recent studies show that PEDF may also possess direct antitumor effects that either inhibit proliferation or promote differentiation of tumor cells. [27][28][29] However, the underlying molecular mechanism by In the present study, we investigated whether constitutive expression of PEDF, or its triple phosphomimetic mutant, in choroidal melanoma tumor cells could more efficiently suppress melanoma tumor growth and metastasis, as well as how the triple phosphomimetic mutants act as antitumor agents.…”