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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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Modulation of apoptotic molecules by Ubenimex and radiation. HeLa cells were pretreated with 100 μg/ml Ubenimex for 24 h, followed by 16 Gy radiation. At 72 h after radiation treatment, whole-cell protein extracts were prepare and subjected to Western blot analysis of Bcl-xL, Bcl-2, caspase-3, cleaved caspase-3, PARP, and cleaved PARP. The β-actin protein level served as a protein loading control. The combination of Ubenimex and radiation cleaved caspase-3 and PARP, leading to apoptosis (A). Anti-apoptotic molecules, Bcl-xlL and Bcl-2, were down-regulated by Ubenimex and radiation (B).
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Figure 4: Modulation of apoptotic molecules by Ubenimex and radiation. HeLa cells were pretreated with 100 μg/ml Ubenimex for 24 h, followed by 16 Gy radiation. At 72 h after radiation treatment, whole-cell protein extracts were prepare and subjected to Western blot analysis of Bcl-xL, Bcl-2, caspase-3, cleaved caspase-3, PARP, and cleaved PARP. The β-actin protein level served as a protein loading control. The combination of Ubenimex and radiation cleaved caspase-3 and PARP, leading to apoptosis (A). Anti-apoptotic molecules, Bcl-xlL and Bcl-2, were down-regulated by Ubenimex and radiation (B).

Mentions: We investigated the effect of Ubenimex on the modulation of molecules involved in the apoptotic pathway. The expression of molecules related to apoptosis was determined by Western blot analysis. As shown in Fig. 4A, the combination of Ubenimex and radiation enhanced the expression of cleaved caspase-3 and PARP, leading to apoptosis. Also, anti-apoptotic molecules, Bcl-xL and Bcl-2, were down-regulated by Ubenimex and radiation (Fig. 4B). These results showed that the combination of Ubenimex and radiation activated the apoptotic pathway more strongly than the other treatments.


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)

Modulation of apoptotic molecules by Ubenimex and radiation. HeLa cells were pretreated with 100 μg/ml Ubenimex for 24 h, followed by 16 Gy radiation. At 72 h after radiation treatment, whole-cell protein extracts were prepare and subjected to Western blot analysis of Bcl-xL, Bcl-2, caspase-3, cleaved caspase-3, PARP, and cleaved PARP. The β-actin protein level served as a protein loading control. The combination of Ubenimex and radiation cleaved caspase-3 and PARP, leading to apoptosis (A). Anti-apoptotic molecules, Bcl-xlL and Bcl-2, were down-regulated by Ubenimex and radiation (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Modulation of apoptotic molecules by Ubenimex and radiation. HeLa cells were pretreated with 100 μg/ml Ubenimex for 24 h, followed by 16 Gy radiation. At 72 h after radiation treatment, whole-cell protein extracts were prepare and subjected to Western blot analysis of Bcl-xL, Bcl-2, caspase-3, cleaved caspase-3, PARP, and cleaved PARP. The β-actin protein level served as a protein loading control. The combination of Ubenimex and radiation cleaved caspase-3 and PARP, leading to apoptosis (A). Anti-apoptotic molecules, Bcl-xlL and Bcl-2, were down-regulated by Ubenimex and radiation (B).
Mentions: We investigated the effect of Ubenimex on the modulation of molecules involved in the apoptotic pathway. The expression of molecules related to apoptosis was determined by Western blot analysis. As shown in Fig. 4A, the combination of Ubenimex and radiation enhanced the expression of cleaved caspase-3 and PARP, leading to apoptosis. Also, anti-apoptotic molecules, Bcl-xL and Bcl-2, were down-regulated by Ubenimex and radiation (Fig. 4B). These results showed that the combination of Ubenimex and radiation activated the apoptotic pathway more strongly than the other treatments.

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