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

Fan Y, Weng Z, Gao H, Hu J, Wang H, Li L, Liu H - PLoS ONE (2015)

Bottom Line: In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Liaoning Medical University, Jinzhou, Liaoning, China.

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.

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Related in: MedlinePlus

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.
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pone.0145790.g006: 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.

Mentions: In addition, we also analyzed the effect of siRNA on Erk1/2 in UMSCC-10A cells by western blot. As shown in Fig 6A left, after the knockdown of Erk1/2 to approximately 40–50% of the original level, we found that the UMSCC-10A cells with Erk1/2 knockdown demonstrated increased radiosensitivity with an SER of 1.58 (Fig 6A, right). Thus, the Erk1/2 levels correlated with radioresistance. Similarly, isoalantolactone radiosensitization was also significantly increased with an SER of 1.65 (P<0.05, Fig 6A, right). We also tested the survival ratio of cells after Erk1/2 knockdown and radiation treatment. A similar survival ratio was found between the Erk1/2 knockdown cells with radiation and cells treated with a combination of isoalantolactone and radiation (P>0.05, Fig 6B). Furthermore, we performed cell proliferation assay on UMSCC-10A cells with Erk1/2 knockdown and combined with isoalantolactone following radiated. As shown in Fig 6C, cells proliferation were inhibited by Erk1/2 knockdown relative to control cells (P<0.01). Although cells with Erk1/2 knockdown would slightly proliferation after radiation 14 days, isoalantolactone cannot further inhibit the proliferation of UMSCC-10A cells (P>0.05). Cells apoptosis also were analyzed on cells with Erk1/2 knockdown induced by radiation or isoalantolactone as Fig 6D shown. Cells with Erk1/2 siRNA induce slightly apoptosis and enhance the level of apoptosis induced by radiation compared with cells treated with radiation only (P<0.05). However, isoalantolactone 5 μM cannot enhance apoptosis induced by cells with Erk1/2 siRNA. This indicated that isoalantolactone combined with radiation can specifically inhibit the cell proliferation by activation of the Erk1/2 signaling pathway. Thus, isoalantolactone may be a potent inhibitor of Erk1/2-mediated radiation resistance of UMSCC-10A cells.


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

Fan Y, Weng Z, Gao H, Hu J, Wang H, Li L, Liu H - PLoS ONE (2015)

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.
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pone.0145790.g006: 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.
Mentions: In addition, we also analyzed the effect of siRNA on Erk1/2 in UMSCC-10A cells by western blot. As shown in Fig 6A left, after the knockdown of Erk1/2 to approximately 40–50% of the original level, we found that the UMSCC-10A cells with Erk1/2 knockdown demonstrated increased radiosensitivity with an SER of 1.58 (Fig 6A, right). Thus, the Erk1/2 levels correlated with radioresistance. Similarly, isoalantolactone radiosensitization was also significantly increased with an SER of 1.65 (P<0.05, Fig 6A, right). We also tested the survival ratio of cells after Erk1/2 knockdown and radiation treatment. A similar survival ratio was found between the Erk1/2 knockdown cells with radiation and cells treated with a combination of isoalantolactone and radiation (P>0.05, Fig 6B). Furthermore, we performed cell proliferation assay on UMSCC-10A cells with Erk1/2 knockdown and combined with isoalantolactone following radiated. As shown in Fig 6C, cells proliferation were inhibited by Erk1/2 knockdown relative to control cells (P<0.01). Although cells with Erk1/2 knockdown would slightly proliferation after radiation 14 days, isoalantolactone cannot further inhibit the proliferation of UMSCC-10A cells (P>0.05). Cells apoptosis also were analyzed on cells with Erk1/2 knockdown induced by radiation or isoalantolactone as Fig 6D shown. Cells with Erk1/2 siRNA induce slightly apoptosis and enhance the level of apoptosis induced by radiation compared with cells treated with radiation only (P<0.05). However, isoalantolactone 5 μM cannot enhance apoptosis induced by cells with Erk1/2 siRNA. This indicated that isoalantolactone combined with radiation can specifically inhibit the cell proliferation by activation of the Erk1/2 signaling pathway. Thus, isoalantolactone may be a potent inhibitor of Erk1/2-mediated radiation resistance of UMSCC-10A cells.

Bottom Line: In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Liaoning Medical University, Jinzhou, Liaoning, China.

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.

Show MeSH
Related in: MedlinePlus