Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696678&req=5

pone.0145790.g005: 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.

Mentions: The Mek/Erk1/2 signaling pathway plays a key role in the regulation of tumor cell proliferation, cell cycle progression and survival [24]. Moreover, radiation at low doses can induce the activation of the Mek/Erk1/2 survival pathway in tumor cells, which results in instant cellular proliferation to compensate for cell loss [16]. This has been thought to be one of the major reasons why tumors are resistant to radiation. With this aim, we investigated the possibility that isoalantolactone might enhance the sensitivity of UMSCC-10A cells to radiation via blockage of Mek/Erk1/2 signaling pathway activation. We first examined the total Erk1/2 protein expression level and its phosphorylation levels after the cells were treated with radiation alone or in combination with isoalantolactone for 24 hours (Fig 5A). No marked changes in the total protein expression of Erk1/2 were found following radiation or combination treatment with radiation and isoalantolactone (P>0.05, Fig 5B). Compared with control cells, the phosphorylation of Erk1/2 was significantly increased in cells treated with radiation alone (P<0.01). However, the combination treatment of radiation and isoalantolactone greatly reduced the phosphorylation of Erk1/2 protein compared with radiation alone (P<0.01, Fig 5C). Moreover, we also tested the cells treated with isoalantolactone alone and observed fewer changes in the phosphorylation level of Erk1/2 (P>0.05). Those data suggested that isoalantolactone inhibits radiation-induced Erk1/2 phosphorylation in 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 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696678&req=5

pone.0145790.g005: 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.
Mentions: The Mek/Erk1/2 signaling pathway plays a key role in the regulation of tumor cell proliferation, cell cycle progression and survival [24]. Moreover, radiation at low doses can induce the activation of the Mek/Erk1/2 survival pathway in tumor cells, which results in instant cellular proliferation to compensate for cell loss [16]. This has been thought to be one of the major reasons why tumors are resistant to radiation. With this aim, we investigated the possibility that isoalantolactone might enhance the sensitivity of UMSCC-10A cells to radiation via blockage of Mek/Erk1/2 signaling pathway activation. We first examined the total Erk1/2 protein expression level and its phosphorylation levels after the cells were treated with radiation alone or in combination with isoalantolactone for 24 hours (Fig 5A). No marked changes in the total protein expression of Erk1/2 were found following radiation or combination treatment with radiation and isoalantolactone (P>0.05, Fig 5B). Compared with control cells, the phosphorylation of Erk1/2 was significantly increased in cells treated with radiation alone (P<0.01). However, the combination treatment of radiation and isoalantolactone greatly reduced the phosphorylation of Erk1/2 protein compared with radiation alone (P<0.01, Fig 5C). Moreover, we also tested the cells treated with isoalantolactone alone and observed fewer changes in the phosphorylation level of Erk1/2 (P>0.05). Those data suggested that isoalantolactone inhibits radiation-induced Erk1/2 phosphorylation in 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