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Aminopeptidase N (APN)/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer.

Tsukamoto H, Shibata K, Kajiyama H, Terauchi M, Nawa A, Kikkawa F - BMC Cancer (2008)

Bottom Line: Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells.Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan. shiba@med.nagoya-u.ac.jp. tukamoto@med.nagoya-u.a

ABSTRACT

Background: Radiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN)/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies.

Methods: We investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both in vitro and in vivo. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis) and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.

Results: We demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both in vitro and in vivo. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis in vitro and in vivo.

Conclusion: Although further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine cervical cancer.

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(A)Annexin V and PI (propidium iodide) staining was performed, followed by flow cytometry. (B) The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubenimex groups. In the radiation and combined-treatment groups there was a high percentage of apoptosis. Combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the radiation alone group (p < 0.05). Errors bar represent the standard error of the mean.
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Figure 3: (A)Annexin V and PI (propidium iodide) staining was performed, followed by flow cytometry. (B) The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubenimex groups. In the radiation and combined-treatment groups there was a high percentage of apoptosis. Combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the radiation alone group (p < 0.05). Errors bar represent the standard error of the mean.

Mentions: To investigate whether the induction of apoptosis plays a role in Ubenimex-induced radiosensitization, annexin V and PI (propidium iodide) staining by flow cytometry was performed (Fig. 3A and 3B). The level of apoptosis was analyzed at 72 h after 16 Gy radiation, because, at this time point, the highest level of apoptosis was noted. The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubeninex-alone groups. However, in the 16 Gy radiation-alone and combined-treatment groups, there was a high percentage of apoptosis. Further, combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the 16 Gy radiation-alone group. A significant difference was observed between only radiation and combined treatment with Ubenimex and radiation (p < 0.05).


Aminopeptidase N (APN)/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer.

Tsukamoto H, Shibata K, Kajiyama H, Terauchi M, Nawa A, Kikkawa F - BMC Cancer (2008)

(A)Annexin V and PI (propidium iodide) staining was performed, followed by flow cytometry. (B) The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubenimex groups. In the radiation and combined-treatment groups there was a high percentage of apoptosis. Combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the radiation alone group (p < 0.05). Errors bar represent the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289833&req=5

Figure 3: (A)Annexin V and PI (propidium iodide) staining was performed, followed by flow cytometry. (B) The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubenimex groups. In the radiation and combined-treatment groups there was a high percentage of apoptosis. Combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the radiation alone group (p < 0.05). Errors bar represent the standard error of the mean.
Mentions: To investigate whether the induction of apoptosis plays a role in Ubenimex-induced radiosensitization, annexin V and PI (propidium iodide) staining by flow cytometry was performed (Fig. 3A and 3B). The level of apoptosis was analyzed at 72 h after 16 Gy radiation, because, at this time point, the highest level of apoptosis was noted. The percentage of apoptotic cells was counted (Fig. 3A, areas 2 and 3). Almost 3–4% apoptosis was found in the control and Ubeninex-alone groups. However, in the 16 Gy radiation-alone and combined-treatment groups, there was a high percentage of apoptosis. Further, combined treatment with Ubenimex and radiation resulted in a higher percentage of apoptosis than in the 16 Gy radiation-alone group. A significant difference was observed between only radiation and combined treatment with Ubenimex and radiation (p < 0.05).

Bottom Line: Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells.Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan. shiba@med.nagoya-u.ac.jp. tukamoto@med.nagoya-u.a

ABSTRACT

Background: Radiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN)/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies.

Methods: We investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both in vitro and in vivo. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis) and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.

Results: We demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both in vitro and in vivo. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis in vitro and in vivo.

Conclusion: Although further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine cervical cancer.

Show MeSH
Related in: MedlinePlus