Isoalantolactone Enhances the Radiosensitivity of UMSCC-10A Cells via Specific Inhibition of Erk1/2 Phosphorylation

Abstract

Background: Although radiotherapy is one of the mainstream approaches for the treatment of head and neck squamous cell carcinoma (HNSCC), this cancer is always associated with resistance to radiation. In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.

Methods: The radiosensitization of UMSCC-10A cells treated with isoalantolactone was analyzed by colony formation assay. The radiosensitization effects of isoalantolactone on cell proliferation, cell cycle and apoptosis regulation were examined by BrdU incorporation assay, DNA content assay and flow cytometry, respectively. Western blotting was performed to determine the effects of isoalantolactone combined with radiation on the protein expression of Mek, extracellular signal-regulated kinase (Erk1/2) as well as phosphorylated Mek and Erk1/2. Erk1/2 knockdown by siRNA was used to confirm that isoalantolactone specifically inhibited the activation of Erk1/2 signaling pathway in UMSCC-10A cells.

Results: Isoalantolactone enhanced the radiosensitivity of UMSCC-10A cells; the sensitivity enhanced ratios (SERs) were 1.44 and 1.63, respectively, for 2.5 and 5 μM. Moreover, isoalantolactone enhanced radiation-induced cell proliferation and apoptosis and cell cycle arrested at G2/M phase. Furthermore, no marked changes were observed in the expression of total Erk1/2 and Mek protein after radiation treatment. However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent. In addition, the radiosensitivity of UMSCC-10A cells with Erk1/2 knockdown was increased. Isoalantolactone cannot further prevent the proliferation of UMSCC-10A cells with Erk1/2 knockdown which other mechanism regulated cell proliferation.

Conclusion: Our results suggested that isoalantolactone enhanced radiation-induced apoptosis, cell cycle arrested and reduced the cell proliferation of UMSCC-10A cells via specifically inhibited the phosphorylation of Erk1/2. Thus a low concentration of isoalantolactone may be used to overcome the resistance of UMSCC-10A cells to radiation and may be a promising radiosensitizer in cancer therapy.

Fig 1. Isoalantolactone enhances the sensitivity of UMSCC-10A cells to radiation.

(A) Representative images of the clonogenic survival assay. A colony formation assay was performed on UMSCC-10A cells treated with 2.5 or 5 μM isoalantolactone for 4 hours prior to radiation treatment with 0, 2, 4, 6, 8 Gy. After incubation for 14 days, the cells were stained with coomassie blue, and the colonies with more than 50 cells were counted. (B) The chemical structure of isoalantolactone. (C) Cell viability was measured using live/dead cells counted method. UMSCC-10A cells were pretreated with isoalantolactone and then following radiated 4 Gy at 0, 7, 15 days. The data are expressed as the mean±SEM from three independent experiments. *P<0.01compared with the control. (D) Radiosensitization by isoalantolactone on UMSCC-10A cells, other HNSCC cell lines (UMSCC-12 cell (radiation resistant) and Cal-27 cell), other tumor cell line (HepG2 cell) and normal cells (FRT cell). SER was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose after isoalantolactone treated (mean ± SEM, n = 4).

Fig 2. Isoalantolactone combined with radiation inhibits the proliferation of UMSCC-10A cells.

(A) Representative images from a cell proliferation assay illustrate nuclear staining after the cells were treated with isoalantolactone at 5 μM for 72 hours or radiation at 4 Gy for 4 hours alone or with a combination. (B) The proliferative ability of UMSCC-10A cells was tested with BrdU incorporation assays. The results are representative of three independent experiments. Values represent the mean ± SEM, * P<0.05, **P<0.01compared with the control.

Fig 3. Isoalantolactone enhances radiation-induced G2/M cell cycle arrest in UMSCC-10A cells.

(A) The effects of isoalantolactone and radiation alone or in combination on cell cycle distribution. The cells were treated with a combination of isoalantolactone and radiation, which isoalantolactone at a dose of 5 μM was added to the cultured cells 16 hours prior to radiation 4 Gy exposure. Those led to an increase in the proportion of cells in G2/M phase; this also led to a slight increase in the number of cells in S phase and a decrease in the number of cells in G1 phase compared with the control. (B) Representative images of Cyclin B1 protein expression by western blot analysis. β-actin was used as a control. Cells that were treated with a combination of isoalantolactone and radiation showed a significant down-regulation of Cyclin B1 expression compared with cells that were untreated and those that were treated with radiation alone. The results are presented as the mean ± SEM for three independent experiments where similar results were obtained. *P<0.05 and **P<0.01 compared with the control.

Fig 4. Isoalantolactone increases radiation-induced apoptosis in UMSCC-10A cells.

(A) Cells were treated with isoalantolactone or radiation individually or with a combination of isoalantolactone and radiation. Isoalantolactone at a dose of 5μM was added to the cultured cells 16 hours prior to radiation 4 Gy exposure. The effect on apoptosis was analyzed by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining in UMSCC-10A cells. (B) Isoalantolactone enhanced radiation-induced apoptosis in the UMSCC-10A cells. The data are expressed as the means ± SEM of three independent experiments where similar results were obtained. **P<0.01 compared with the control or radiation alone. (C) Bax, Bcl-2, Cleaved-caspase 3 and Pro-caspase 3 protein expression in UMSCC-10A cells was determined by Western blot. (D) The quantification of Bax, Bcl-2, Cleaved-caspase 3 and Pro-caspase 3 protein expression is shown. β-actin was used as a control. Values represent the mean ± SEM, * P<0.01, compared with the control or radiation alone.

Fig 5. Isoalantolactone inhibits radiation-induced Mek and Erk1/2 protein phosphorylation in UMSCC-10A cells.

(A) Whole-cell protein extracts were prepared from UMSCC-10A cells that were treated with radiation and isoalantolactone alone or in combination. Isoalantolactone at a dose of 5 μM was added to the cultured cells 16 hours prior to radiation 4 Gy exposure. The blot was probed with antibodies against Mek, Erk1/2, p-Mek, p-Erk1/2 and β-actin. (B) Mek and Erk1/2 protein expression relative to that of β-actin were assessed by densitometric analysis. p-Mek and p-Erk1/2 were expressed as the radio of p-Mek/total Mek and p-Erk1/2/total Erk1/2. ** P<0.01, compared with the control or radiation alone.

Fig 6. Radiosensitization by siRNA-mediated knockdown of Erk1/2 in UMSCC-10A cells.

(A) Radiosensitization by Erk1/2 knockdown. Cells were transfected using Lipofectamine 2000 and siRNA that targets Erk1/2. Seventy-two hours later, one portion of the cells was reserved for immunoblotting (top) and the other portion was plated for the clonogenic assay (bottom, mean ± SEM, n = 3). (B) The cell survival ratio was measured in UMSCC-10A cells that were treated with isoalantolactone, radiation alone or with a combination of the isoalantolactone and radiation after siRNA-mediated knockdown of Erk1/2. Isoalantolactone at a dose of 5 μM was added to the cultured cells 72 hours prior to radiation 4 Gy exposure. The results are presented as the mean±SEM from three independent experiments.^## P<0.01, compared with the control. * P<0.05, compared with the Erk1/2 siRNA. ** P<0.01, compared with the control siRNA. (C) Representative images for a cell proliferation assay at difference time after cells by Erk1/2 silencing treated with isoalantolactone at 5 μM or combination of isoalantolactone and radiation 4 Gy. The results are presented as the mean±SEM from three independent experiments. ** P<0.01, compared with the control cells. (D) Apoptosis was analyzed by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining in UMSCC-10A cells. Cells were transfected using Erk1/2 siRNA and treated with radiation or isoalantolactone individually. The data are expressed as the means ± SEM of three independent experiments where similar results were obtained. **P<0.01 compared with the control siRNA.