Proteasome inhibitors induce p53-independent apoptosis in human cancer cells - PubMed (original) (raw)

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells

Bulbul Pandit et al. Am J Pathol. 2011 Jan.

Abstract

Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Proteasome inhibitors inhibit the viability of cancer cells in a p53-independent manner. A: Mid-log colon carcinoma HCT 116 (wild type [wt] p53) and p53−/− cells were treated with DMSO (control) or various concentrations of thiostrepton, MG132, and bortezomib for 72 hours and IC50 values were determined by using MTT cell viability assay as described under Materials and Methods. The values shown are mean ± SD for three separate experiments. The IC50 values were 2.1 ± 0.08 and 1.4 ± 0.06 μM/L for thiostrepton, 0.51 ± 0.09 and 0.35 ± 0.1 for MG132, and 18 ± 0.04 and 15 ± 0.07 nM/L for bortezomib against HCT-116 (wt p53 and p53−/−) cells, respectively. B: Mid-log breast carcinoma MCF-7 cells (wt p53 and shp53) were treated with dimethyl sulfoxide or various concentrations of thiostrepton, MG132, and bortezomib for 72 hours; IC50 values were determined by using MTT cell viability assay as described under Materials and Methods. The values shown are mean ± SD for three separate experiments. The IC50 values were 4.5 ± 0.34 and 4.7 ± 0.2 μmol/L for thiostrepton, 0.34 ± 0.08 and 0.28 ± 0.1 for MG132, and 8 ± 0.04 and 11 ± 0.06 nmol/L for bortezomib against MCF-7 wt p53 and shp53 cells, respectively.

Figure 2

Figure 2

p53-independent induction of apoptosis by proteasome inhibitors in colon cancer cells. Colon carcinoma HCT 116 wild type (wt) p53 cells were treated with 3 μmol/L thiostrepton (Thio), 2 μmol/L MG132, and 10 nmol/L bortezomib for 24 hours, then stained with annexin V-PE and 7AAD and analyzed by flow cytometry. Colon carcinoma cells with knockout of p53 (HCT 116 p53−/−) were treated with 3 mmol/L thiostrepton, 2 μmol/L MG132, and 10 nmol/L bortezomib for 24 hours, then stained with annexin V-PE and 7AAD and analyzed by flow cytometry. The values shown are mean ± SD for three separate experiments.

Figure 3

Figure 3

Caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage is p53-independent after treatment with proteasome inhibitor. A: Colon carcinoma HCT 116 wild type (w.t.) p53 and p53−/− cells were treated with 2.5 μmol/L thiostrepton, 2 μmol/L MG132, 25 nmol/L bortezomib, and 500 μmol/L 5-FU for 24 hours, then lysed and subjected to immunoblot analysis with antibodies against PARP, caspase-3 (cleaved), p53, and β-actin. HCT116 cells and HCT-116 cells overexpressing bcl-2 were treated with 2.5 μmol/L and 25 nmol/L bortezomib for 24 hours, then lysed and immunoblotted for PARP, bcl-2, and β-actin. B: Breast carcinoma MCF-7 w.t. p53 and shp53 cells were treated with 10 μmol/L thiostrepton, 2 μmol/L MG132, and 25 nmol/L bortezomib for 48 hours, then lysed and subjected to immunoblot analysis with antibodies against PARP, caspase-3 (cleaved), p53, and β-actin. C: Liver carcinoma HepG2 w.t. p53 and shp53 cells were treated with 2.5 μmol/L thiostrepton, 2 μmol/L MG132, and 25 nmol/L bortezomib for 24 hours, then lysed and subjected to immunoblot analysis with antibodies against PARP, caspase-3 (cleaved), p53, and β-actin.

Figure 4

Figure 4

Effect of PI on p53-regulated pro- and antiapoptotic proteins. A: Colon carcinoma HCT 116 wild-type (wt) p53 and p53−/− cells were treated with 2.5 μmol/L thiostrepton, 2 μmol/L MG132, and 25 nmol/L bortezomib for 24 hours, then lysed and subjected to immunoblot analysis with antibodies against Puma, Noxa, Mcl-1, p21, and β-actin. B: Breast carcinoma MCF-7 w.t. p53 and shp53 cells were treated with 10 μmol/L thiostrepton, 2 μmol/L MG132, and 25 nmol/L bortezomib for 48 hours, then lysed and subjected to immunoblot analysis with antibodies against Puma, Noxa, Mcl-1, p21, and β-actin C: Liver carcinoma HepG2 w.t. p53 and shp53 cells were treated with 2.5 μmol/L thiostrepton, 2 μmol/L MG132, and 25 nmol/L bortezomib for 24 hours, then lysed and subjected to immunoblot analysis with antibodies against Puma, Noxa, Mcl-1, p21, and β-actin.

Similar articles

Cited by

References

    1. Rubin D.M., Finley D. Proteolysis. The proteasome: a protein-degrading organelle. Curr Biol. 1995;5:854–858. - PubMed
    1. Lenz H.J. Clinical update: proteasome inhibitors in solid tumors. Cancer Treat Rev. 2003;29(Suppl 1):41–48. - PubMed
    1. Bhat U.G., Halasi M., Gartel A.L. Thiazole antibiotics target FoxM1 and induce apoptosis in human cancer cells. PLoS One. 2009;4:e5592. - PMC - PubMed
    1. Bhat U.G., Halasi M., Gartel A.L. FoxM1 is a general target for proteasome inhibitors. PLoS One. 2009;4:e6593. - PMC - PubMed
    1. Vousden K.H., Prives C. Blinded by the light: the growing complexity of p53. Cell. 2009;137:413–431. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources