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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Tomohisa Horibe, Aya Torisawa, Masayuki Kohno, Koji Kawakami
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-59) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

TH, MK, and KK designed this research work. TH, AT, and MK performed experiments and analyzed data in this manuscript. TH, MK, and KK, interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo.

Results

In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition.

Conclusion

These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer.
Zusatzmaterial
Additional file 1: Analysis of PTEN expression in human breast (BT20 and T47D), lung (H460, A549, H322, and H1299), colon (COLO320HSR), and prostate (DU145) cancer, epithelial carcinoma (A431), and GB (U251) cell lines. Cell extracts from the indicated cell lines were examined for PTEN expression by Western-blot analysis with antibodies against PTEN. β-Actin was used as the loading control. Bands were visualized by chemiluminescence as described in the Materials and Methods section. (TIFF 112 KB)
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Additional file 2: Effect of 17-AAG on Hsp90, Hsp70, Hsp27, and Akt expression in GB cells. A172 cells were treated with different concentrations of 17-AAG for 24 h. After 24 h cells were harvested and examined for Hsp90, Hsp70, Hsp27, and Akt expression by Western-blot analysis with antibodies against these proteins. β-Actin was used as the loading control. (TIFF 111 KB)
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Additional file 3: Time course of cell viability after treatment with Antp-TPR or 17-AAG (A) and effect of Hsp70 inhibitor on the cytotoxic activity of Antp-TPR or 17-AAG to U251 cells (B). (A) U251 cells were treated with Antp-TPR or 17-AAG at the indicated concentrations, and then analyzed for cell viability by WST-8 assay. (B) U251 cells were treated with Antp-TPR or 17-AAG at the indicated concentrations in the presence or absence of PES (2.5 μM), and then cell viability was assessed by WST-8 assay. Data represent the mean ± SD. (TIFF 107 KB)
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Additional file 4: Effect of Antp-TPR peptide on up-regulation of the YME1L1 and GCP60 genes under the mtUPR or Golgi stress condition (A) and Characterization of 17-AAG-induced GB cell killing after induction of the erUPR (B and C). (A) U251 cells were transfected with YME1L1 and GCP promoter-reporter constructs and exposed to mitochondrial or Golgi stress as described in the Materials and Methods section. After induction of mtUPR or Golgi stress, cells were incubated with or without Antp-TPR peptide for 18 h, and then reporter assay was performed by the Dual-Glo Luciferase assay system. Data represent the mean ± SD. (B) Multiparametric flow cytometry. U251 cells were incubated with or without 17-AAG peptide for 18 h in the presence or absence of 0.5 μM Tg, and analyzed for annexin and propidium iodide (PI) staining. (C) Mitochondrial membrane potential after treatment with 17-AAG. After treatment with or without 17-AAG in the presence or absence of Tg, U251 cells were labeled with JC-1 and then images were taken using a confocal laser scanning microscope as described in the Materials and Methods section. All scale bars are 50 μm. (TIFF 195 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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