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Doxorubicin-mediated apoptosis in glioma cells requires NFAT3

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Abstract

Nuclear factor of activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. Here, we characterize, for the first time, the role of NFAT3 in doxorubicin (DOX)-mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates that the specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect induced by DOX and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated tumor necrosis factor (TNF)-α induction, its receptor TNFR1, caspase 10, caspase 3, and poly (ADP-ribose) polymerase, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion, as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells.

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Acknowledgments

This research was supported by National Cancer Institute Grants CA75557, CA116708, CA138409 (to JSR). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. We thank Shellee Abraham for helping in manuscript preparation, and Diana Meister and Sushma Jasti for manuscript review.

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Authors and Affiliations

  1. Department of Cancer Biology and Pharmacology, College of Medicine at Peoria, University of Illinois, 1649, Peoria, IL, 61656, USA

    Sreelatha Gopinath, Sravan K. Vanamala & Jasti S. Rao

  2. Department of Pathology, College of Medicine at Peoria, University of Illinois, Peoria, IL, 61656, USA

    Meena Gujrati

  3. Department of Neurosurgery, College of Medicine at Peoria, University of Illinois, Peoria, IL, 61656, USA

    Jeffrey D. Klopfenstein, Dzung H. Dinh & Jasti S. Rao

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  1. Sreelatha Gopinath
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  2. Sravan K. Vanamala
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  6. Jasti S. Rao
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Correspondence to Jasti S. Rao.

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18_2009_157_MOESM1_ESM.tif

Supplementary Fig. 1. Western blot analysis of AIF, p53, and cleaved BID. Immunoblot analysis of total protein from SNB19 and U87 cells after transfection with pSV or shNF3 and/or DOX. Proteins were probed for AIF, p53, and cleaved BID. The expression of these molecules was unaffected with the treatments Supplementary material 1 (TIFF 1509 kb) (TIFF 1509 kb)

18_2009_157_MOESM2_ESM.tif

Supplementary Fig. 2. TNF-α induces apoptosis by upregulating caspase 10 expression and PARP cleavage. Cell lysates were collected from SNB19 and U87 after transfection with SV or shNF3, and treatment with or without TNF-α. Western blot analysis of 50 μg of total cell lysates was performed to check the expression of caspase 10 and PARP. GAPDH was used as a loading control. TNF-α treatment increased the expression of these molecules irrespective of other treatments

18_2009_157_MOESM3_ESM.tif

Supplementary Fig. 3. DOX treatment leads to NFAT3 activation. Cytoplasmic (Cy) and nuclear extracts (Nu) from SNB19 and U87 cells transfected with SV or shNF3 and treated with or without DOX were immunoblotted and probed with anti-phospho NFAT3 antibody and anti-NFAT3 antibody, respectively. When nuclear extracts were probed with anti-NFAT3 antibody, the DOX treatment showed increased expression as compared to the control and no expression with the shNF3 treatment. Similarly, the cytoplasmic extracts, when probed with phospho-NFAT3, showed decreased expression of NFAT3 as compared to the control and less or no expression in the shNF3 treatment

18_2009_157_MOESM4_ESM.tif

Supplementary Fig. 4. DOX induces apoptosis in a dose-dependent manner. SNB 19 and U87 cells were treated with various concentrations of DOX (0.25, 0.5, 0.75, 1.0 μM). DNA content of control and DOX-treated SNB19 and U87 cells were measured by flow cytometric analysis to determine the cell cycle progression and apoptosis

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Gopinath, S., Vanamala, S.K., Gujrati, M. et al. Doxorubicin-mediated apoptosis in glioma cells requires NFAT3. Cell. Mol. Life Sci. 66, 3967–3978 (2009). https://doi.org/10.1007/s00018-009-0157-5

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