Osthole sensitizes with radiotherapy to suppress tumorigenesis of human nasopharyngeal carcinoma in vitro and in vivo

Abstract

Background: Radiotherapy is one of the most comment and useful treatment for nasopharyngeal carcinoma (NPC), but the radioresistance remains a major obstacle. Osthole, a natural coumarin derivative, has been shown to have anti-tumor and anti-inflammatory activity. However, the relationship between osthole and NPC treatment, especially for radiotherapy, is still elusive.

Methods: Osthole with or without X ray radiotherapy treated with CNE2 cells, a human EC cell line. Cell viability, proliferation, migration and apoptosis were measured by MTT, colony formation, Annexin V/PI double staining, Transwell assay, respectively. NPC tumor models were established on BALB/c nude mice by subcutaneously injection of CNE2 cells and the effect of osthole and radiotherapy on tumor growth in vivo was studied.

Results: We found that in a dose-dependent manner, osthole could individually, and synergistically with radiotherapy, reduce NPC cell (CNE2) viability, proliferation, migration, and invasion, and induce apoptosis, respectively. This effect of anti-tumor growth and induction of apoptosis was further confirmed in mice induced by subcutaneously injection with CNE2 cells and following treated with osthole or/and radiation.

Conclusion: Osthole increases the effect of radiotherapy on anti-human nasopharyngeal cancer.

Keywords: apoptosis; human nasopharyngeal carcinoma; osthole; proliferation; radiotherapy; tumorigenesis.

Figure 1

Osthole suppresses growth, migration, and invasion of human NPC in vitro. Notes: (A) MTT assay was performed to measure cell growth at 24, 48, and 72 hours after osthole treatment. (B) Osthole-induced cell apoptosis was quantified using an Annexin V/PI double staining kit and analyzed by flow cytometry. Transwell assay with or without Matrigel for cell invasion (C) and migration (D). Representative transwell assay of cells following staining with crystal violet. The number of migrated cells was measured by counting five randomly chosen fields under a microscope. Bar = 50 µm. Data are mean ± SD from three independent experiments each performed in triplicate. ^***P<0.001 compared with control. Abbreviation: NPC, nasopharyngeal carcinoma.

Figure 2

Osthole with radiotherapy has a synergistic effect on decreasing CNE2 cell proliferation. Notes: MTT assay (A), colony formation assay (B), and its quantification (C) were performed to measure CNE2 cell growth following treatment with osthole or radiotherapy individually or combined. ^*P<0.05; ^**P<0.01; ^***P<0.001 compared with control. Abbreviation: Ctrl, control.

Figure 3

Combined effect of osthole and radiotherapy on CNE2 cell apoptosis. Notes: Representative images (A) and quantification (B) of Annexin V/PI flow cytometry analysis of treatment with osthole and/or radiotherapy on CNE2 cells. Representative Western blot (C) and quantification (D) assay for pro-apoptotic of BAX and anti-apoptotic of BCL-2 expression. GAPDH was the normalized control. ^**P<0.01; ^***P<0.001 compared with control. Abbreviations: Ctrl, control; FITC, fluorescein isothiocyanate.

Figure 4

Osthole and radiotherapy cooperatively suppress NPC growth in vivo. Notes: Representative images of tumor size (A) and tumor growth (B) following treatment with osthole and/or radiotherapy in a CNE2 tumor xenograft model. Representative Western blot (C) and quantification (D) assay for BAX and BCL-2 protein expression in tumors following osthole and/or radiotherapy treatment. GAPDH was the normalized control. ^***P<0.001 compared with control. Abbreviation: Ctrl, control.