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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

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).
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pone.0145790.g001: 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).

Mentions: In order to explore the interaction of isoalantolactone and radiation, we first needed to know whether UMSCC-10A cells were sensitive to radiation. The radiosensitivity of UMSCC-10A cells was examined by clonogenic assay after exposure to various doses of radiation (0 to 8 Gy). As shown in Fig 1A, the survival cells were reduced accompany with radiation dose increased. This suggested that UMSCC-10A cells were relatively sensitive to radiation. To determine whether a low dose of isoalantolactone can enhance the radiosensitivity of UMSCC-10A cells, a concentration much lower than the IC50 value was used, which generated an inhibition rate lower than 10% when given alone. Based on data from our previous study [20], doses of 2.5 and 5 μM isoalantolactone (chemical structure shown in Fig 1B) and radiation at a dose of 4 Gy were chosen to treat the cells. After radiation treated (0, 7, 15 days), cells were counted to determine the cell viability in UMSCC-10A cells. As shown Fig 1C, combination with low doses of isoalantolactone led to more cells death than treated with radiation alone (P<0.01). Moreover, the clonogenic assay was used to determine the extent of reproductive death caused by radiation alone or combination of radiation and isoalantolactone in UMSCC-10A cells (Fig 1A). Isoalantolactone at 2.5 and 5 μM led to a reduced number of radiation-induced colonies compared with cells treated with radiation alone. We then analyzed the SF (survival fraction) values used UMSCC-10A cell, other HNSCC cell lines (UMSCC-12 cell (radiation resistant) and Cal-27 cell), other tumor cell line (HepG2 cell) and normal cell (FRT cell), which are shown in Fig 1D. A dose-dependent radiosensitization by isoalantolactone was observed in UMSCC-10A cells with SERs (sensitivity enhancement ratio) of 1.44 and 1.63 (P<0.01). However, isoalantolactone could not enhance the radiosensitivity of UMSCC-12, Cal-27, HepG2 and FRT cells (P>0.05). These results strongly indicated that isoalantolactone could enhance the sensitivity of UMSCC-10A cells to radiation and that a relatively low dose of isoalantolactone was needed for 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)

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).
© Copyright Policy
Related In: Results  -  Collection

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pone.0145790.g001: 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).
Mentions: In order to explore the interaction of isoalantolactone and radiation, we first needed to know whether UMSCC-10A cells were sensitive to radiation. The radiosensitivity of UMSCC-10A cells was examined by clonogenic assay after exposure to various doses of radiation (0 to 8 Gy). As shown in Fig 1A, the survival cells were reduced accompany with radiation dose increased. This suggested that UMSCC-10A cells were relatively sensitive to radiation. To determine whether a low dose of isoalantolactone can enhance the radiosensitivity of UMSCC-10A cells, a concentration much lower than the IC50 value was used, which generated an inhibition rate lower than 10% when given alone. Based on data from our previous study [20], doses of 2.5 and 5 μM isoalantolactone (chemical structure shown in Fig 1B) and radiation at a dose of 4 Gy were chosen to treat the cells. After radiation treated (0, 7, 15 days), cells were counted to determine the cell viability in UMSCC-10A cells. As shown Fig 1C, combination with low doses of isoalantolactone led to more cells death than treated with radiation alone (P<0.01). Moreover, the clonogenic assay was used to determine the extent of reproductive death caused by radiation alone or combination of radiation and isoalantolactone in UMSCC-10A cells (Fig 1A). Isoalantolactone at 2.5 and 5 μM led to a reduced number of radiation-induced colonies compared with cells treated with radiation alone. We then analyzed the SF (survival fraction) values used UMSCC-10A cell, other HNSCC cell lines (UMSCC-12 cell (radiation resistant) and Cal-27 cell), other tumor cell line (HepG2 cell) and normal cell (FRT cell), which are shown in Fig 1D. A dose-dependent radiosensitization by isoalantolactone was observed in UMSCC-10A cells with SERs (sensitivity enhancement ratio) of 1.44 and 1.63 (P<0.01). However, isoalantolactone could not enhance the radiosensitivity of UMSCC-12, Cal-27, HepG2 and FRT cells (P>0.05). These results strongly indicated that isoalantolactone could enhance the sensitivity of UMSCC-10A cells to radiation and that a relatively low dose of isoalantolactone was needed for 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