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RETRACTED ARTICLE: uPAR and cathepsin B downregulation induces apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma

  • Laboratory Investigation - Human/Animal Tissue
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This article was retracted on 12 March 2021

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Abstract

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ and increased the interaction of BAD with Bcl-xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD, activation of caspase 3 as well as PARP and cytochrome c and SMAC release. These effects were inhibited by FK506 (10 μM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 μg/ml). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.

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Acknowledgment

This research was supported by National Institutes of Health, CA116708 (to JSR). Contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

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Authors and Affiliations

  1. Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL, 61605, USA

    Rama Rao Malla, Sreelatha Gopinath, Christopher S. Gondi, Kiranmai Alapati & Jasti S. Rao

  2. Department of Neurosurgery, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL, 61605, USA

    Dzung H. Dinh, Andrew J. Tsung & Jasti S. Rao

Authors
  1. Rama Rao Malla
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  2. Sreelatha Gopinath
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  3. Christopher S. Gondi
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  4. Kiranmai Alapati
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  5. Dzung H. Dinh
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Corresponding author

Correspondence to Jasti S. Rao.

Electronic supplementary material

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Supplementary material 1 (TIFF 2947 kb)

Localization of calcineurin A and BAD in U251 and 4910 cells. A. Co-localization of calcineurin A and MTCO2 in glioma cells. After overnight growth, cells were fixed and incubated with calcineurin A and MTCO2 antibodies followed by incubation with species-specific Alexa Fluor secondary antibodies. B. Expression of BAD in cytosolic and mitochondrial fractions from U251 and 4910 cell lysates after transfection with plasmids expressing full-length uPAR and cathepsin B or scrambled vector for 48 hrs. After transfection, whole cell lysates (WL) were collected and equal amounts of protein were immunoblotted for uPAR, cathepsin B and BAD. Cytosolic (CF) and mitochondrial (MF) fractions were immunoblotted for BAD.

Supplementary material 2 (TIFF 5879 kb)

Effect of pCU on co-localization of BAD with 14-3-3 or Bcl-xl and mitochondrial membrane potential in U251 and 4910 cells. A. Immunofluorescence assay was used to detect the co-localization of pBAD (S136) and 14-3-3ζ in pSV- and pCUtreated= U251 and 4910 cells. 72 hrs after treatment, cells were fixed and incubated with pBAD (S136) and 14-3-3ζ antibodies followed by incubation with species-specific Alexa Fluor secondary antibodies. B. Co-localization of BAD and Bcl-xl in pSV- and pCU-treated cells. C. MitoLight dye-based quantification of pCU-induced loss of mitochondrial membrane potential in the presence or absence of FK506 in glioma cells. Flow cytometry analysis with MitoLight dye was carried out in pCU-treated cells in the presence or absence of FK506. The percentage of MitoLight-positive cells (emitting red fluorescence) read from the M1 marker and negative cells from the M2 marker. The position of M1 marker was adjusted according to the results of the negative control (cells without dye).

Supplementary material 3 (TIFF 2943 kb)

Interaction of calcineurin A and Bcl-2 in uPAR and cathepsin B-overexpressed glioma cells. A. Expression of Bcl-2 in pCU-treated U251 and 4910 cells. B. Immunoprecipitation of Bcl-2 in uPAR and cathepsin B-overexpressed glioma cells. Total cell lysates from U251 and 4910 cells transfected with plasmids expressing full-length uPAR and cathepsin B were immunoprecipitated with Bcl-2 and then immunoblotted for Bcl-2 and calcineurin A.

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Malla, R.R., Gopinath, S., Gondi, C.S. et al. RETRACTED ARTICLE: uPAR and cathepsin B downregulation induces apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma. J Neurooncol 107, 69–80 (2012). https://doi.org/10.1007/s11060-011-0727-x

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  • DOI: https://doi.org/10.1007/s11060-011-0727-x

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