Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Effect of Ubenimex alone or combined with radiation on s.c. HeLa tumor volumes (mm3) over time (days). Mice were treated with PBS, bestatin, 4 Gy+PBS, and 4 Gy+bestatin, and tumor volumes were monitored over time. Mice were sacrificed on 23rd day. (B) Photographs of tumors on the 23rd day. (C) Tumor weight on the 23rd day. Optimal growth inhibition was observed in the 4 Gy+bestatin group compared with the other groups. (D)TUNEL assay of mouse tumor sections. This method allows the direct detection of DNA fragmentation. The bright cells are apoptotic cells. More apoptotic cells were observed in the combined-treatment group than in the other groups. Errors bar represent the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2289833&req=5

Figure 5: (A) Effect of Ubenimex alone or combined with radiation on s.c. HeLa tumor volumes (mm3) over time (days). Mice were treated with PBS, bestatin, 4 Gy+PBS, and 4 Gy+bestatin, and tumor volumes were monitored over time. Mice were sacrificed on 23rd day. (B) Photographs of tumors on the 23rd day. (C) Tumor weight on the 23rd day. Optimal growth inhibition was observed in the 4 Gy+bestatin group compared with the other groups. (D)TUNEL assay of mouse tumor sections. This method allows the direct detection of DNA fragmentation. The bright cells are apoptotic cells. More apoptotic cells were observed in the combined-treatment group than in the other groups. Errors bar represent the standard error of the mean.

Mentions: The effect of combined treatment with Ubenimex and radiation on the growth of HeLa tumors was tested in four separate experiments. As can be seen in Fig. 5A, mice that received combined treatment of 4 Gy radiation and Ubenimex exhibited much smaller tumors compared with mice that received no treatment or were treated with only radiation or Ubenimex. In terms of the tumor volume doubling time, the Ubenimex-alone group showed no difference compared with the no-treatment group (2.4 versus 2.5 days, respectively). In contrast, the combined-treatment group showed a significantly longer doubling time than that of the radiation alone group (17.8 days versus 11.5 days, respectively). As shown in Fig. 5B, to the eye, tumor appearance in the combined-treatment group was different from the tumors of the other groups. Regarding tumor volume, optimal growth inhibition was observed in the combination treatment group compared with the radiation alone group (p < 0.05; combined-treatment group versus ionizing irradiation) (Fig. 5C). Finally, we used the TUNEL method to visualize DNA fragmentation at the single cell level. As shown in Fig. 5D, more apoptotic cells were observed in the tumor xenografts of the combined-treatment group than in the other groups.


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) Effect of Ubenimex alone or combined with radiation on s.c. HeLa tumor volumes (mm3) over time (days). Mice were treated with PBS, bestatin, 4 Gy+PBS, and 4 Gy+bestatin, and tumor volumes were monitored over time. Mice were sacrificed on 23rd day. (B) Photographs of tumors on the 23rd day. (C) Tumor weight on the 23rd day. Optimal growth inhibition was observed in the 4 Gy+bestatin group compared with the other groups. (D)TUNEL assay of mouse tumor sections. This method allows the direct detection of DNA fragmentation. The bright cells are apoptotic cells. More apoptotic cells were observed in the combined-treatment group than in the other groups. 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 5: (A) Effect of Ubenimex alone or combined with radiation on s.c. HeLa tumor volumes (mm3) over time (days). Mice were treated with PBS, bestatin, 4 Gy+PBS, and 4 Gy+bestatin, and tumor volumes were monitored over time. Mice were sacrificed on 23rd day. (B) Photographs of tumors on the 23rd day. (C) Tumor weight on the 23rd day. Optimal growth inhibition was observed in the 4 Gy+bestatin group compared with the other groups. (D)TUNEL assay of mouse tumor sections. This method allows the direct detection of DNA fragmentation. The bright cells are apoptotic cells. More apoptotic cells were observed in the combined-treatment group than in the other groups. Errors bar represent the standard error of the mean.
Mentions: The effect of combined treatment with Ubenimex and radiation on the growth of HeLa tumors was tested in four separate experiments. As can be seen in Fig. 5A, mice that received combined treatment of 4 Gy radiation and Ubenimex exhibited much smaller tumors compared with mice that received no treatment or were treated with only radiation or Ubenimex. In terms of the tumor volume doubling time, the Ubenimex-alone group showed no difference compared with the no-treatment group (2.4 versus 2.5 days, respectively). In contrast, the combined-treatment group showed a significantly longer doubling time than that of the radiation alone group (17.8 days versus 11.5 days, respectively). As shown in Fig. 5B, to the eye, tumor appearance in the combined-treatment group was different from the tumors of the other groups. Regarding tumor volume, optimal growth inhibition was observed in the combination treatment group compared with the radiation alone group (p < 0.05; combined-treatment group versus ionizing irradiation) (Fig. 5C). Finally, we used the TUNEL method to visualize DNA fragmentation at the single cell level. As shown in Fig. 5D, more apoptotic cells were observed in the tumor xenografts of the combined-treatment group than in the other groups.

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