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The impact of arsenic trioxide and all-trans retinoic acid on p53 R273H-codon mutant glioblastoma

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Tumor Biology

Abstract

Glioblastoma (GBM) is the most common primary brain tumor in adults and demonstrates a 1-year median survival time. Codon-specific hotspot mutations of p53 result in constitutively active mutant p53, which promotes aberrant proliferation, anti-apoptosis, and cell cycle checkpoint failure in GBM. Recently identified CD133+ cancer stem cell populations (CSC) within GBM also confer therapeutic resistance. We studied targeted therapy in a codon-specific p53 mutant (R273H) created by site-directed mutagenesis in U87MG. The effects of arsenic trioxide (ATO, 1 μM) and all-trans retinoic acid (ATRA, 10 μM), possible targeted treatments of CSCs, were investigated in U87MG neurospheres. The results showed that U87-p53R273H cells generated more rapid neurosphere growth than U87-p53wt but inhibition of neurosphere proliferation was seen with both ATO and ATRA. U87-p53R273H neurospheres showed resistance to differentiation into glial cells and neuronal cells with ATO and ATRA exposure. ATO was able to generate apoptosis at high doses and proliferation of U87-p53wt and U87-p53R273H cells was reduced with ATO and ATRA in a dose-dependent manner. Elevated pERK1/2 and p53 expression was seen in U87-p53R273H neurospheres, which could be reduced with ATO and ATRA treatment. Additionally, differential responses in pERK1/2 were seen with ATO treatment in neurospheres and non-neurosphere cells. In conclusion, codon-specific mutant p53 conferred a more aggressive phenotype to our CSC model. However, ATO and ATRA could potently suppress CSC properties in vitro and may support further clinical investigation of these agents.

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Acknowledgments

This work was supported by an NYMC intramural grant, the Children’s Hospital Foundation Grant, the NYMC Departments of Pathology and Neurosurgery, and the NYMC Graduate School of Basic Medical Sciences. We would like to thank Dr. Wei Dai for his generous donation of a wild-type p53/pcDNA3.1 construct, Dr. Zeling Chau and Mr. Nicolaus Gulati for their assistance of generation and verification of the U87-p53R273H cell line, Dr. David Frick for support on amplification of the vector, and Dr. Fred Wu for assistance on U87 transfection and clone selection.

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  1. Department of Neurosurgery, University of Utah, 175 North Medical Dr. East, Salt Lake City, UT, 84132, USA

    Michael Karsy

  2. Department of Neurosurgery, New York Medical College, Valhalla, NY, 10595, USA

    Ladislau Albert, Raj Murali & Meena Jhanwar-Uniyal

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Supplemental Figure 1

Differentiation of neurospheres into neuronal lineages and the synergistic impact of combined ATO/ATRA treatment. Immunofluorescence analysis of nestin and TUJ1 was evaluated in U87-p53wt neurospheres following ATO or ATRA treatment. Evaluation of differentiation with combined ATO and ATRA was also performed in order to evaluate synergistic/additive effects. A) Immunofluorescence images indicate predominant nestin expression in controls and lower TUJ1 expression. U87-p53wt cells staining for nestin decreased while cells cell staining for TUJ1 increased after treatment with ATO or ATRA. B) Quantitation of cells expressing nestin or TUJ1 indicate that ATO or ATRA treatment resulted in a significant decrease of nestin-expressing cells and a significant increase in TUJ1 expressing cells. C) Quantitation of the number of cells co-expressing TUJ1 and nestin showed a significant decrease after ATO or ATRA treatment. D) Combined treatment with ATO and ATRA demonstrated a significant reduction in nestin-expressing cells with a significant increase in GFAP or TUJ1 expressing cells. E) Co-expression of GFAP and nestin or TUJ1 and nestin was significantly reduced following combined ATO and ATRA treatment compared to control. *p < 0.05, t test (JPEG 166 kb)

Supplemental Figure 2

Impact of high dose ATO and ATRA on GBM neurospheres. The role of high dose ATO and ATRA was evaluated on GBM integrity. Phase contrast photomicroscopy was used to evaluate various doses of ATO (0.5, 1, 2, 4, and 16 μM) and ATRA (2, 5, 10, 20, and 40 μM). High doses of ATO (4 and 16 μM) demonstrated loss of cell–cell contact, increased cellular debris, and neurosphere detachment suggestive of apoptosis. However, high doses of ATRA (20, 40 μM) did not yield similar features. (JPEG 589 kb)

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Karsy, M., Albert, L., Murali, R. et al. The impact of arsenic trioxide and all-trans retinoic acid on p53 R273H-codon mutant glioblastoma. Tumor Biol. 35, 4567–4580 (2014). https://doi.org/10.1007/s13277-013-1601-6

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