1. NMDA RECEPTOR INHIBITION REDUCES SURVIVAL OF MUTANT P53 BREAST
CANCER CELL LINES
INTRODUCTION
• Thirty years ago, p53 was discovered as a cellular partner of simian virus 40
large T-antigen, the oncoprotein of this tumor virus (Levine and Oren, 2009).
p53 is a transcription factor induced by stress that promotes cell cycle arrest,
apoptosis, and senescence, and is undoubtedly among the most extensively
studied genes and proteins (Levine and Oren, 2009).
• The p53 tumor suppressor provides a powerful intrinsic defense against
cancer (Levine and Oren, 2009). Hence, mutations in the TP53 gene are the
most frequent genetic alteration in human cancers. Some frequently
observed p53 mutations also contribute actively to cancer development
through gain-of-function (GOF) activities (Brosh and Rotter, 2009).This may
involve enhancement of invasive properties, attenuation of apoptosis, and
increased genomic instability.
• N-methyl D-aspartate receptors (NMDARs) are glutamate-gated ionotropic
receptors/channels that are central to many physiological processes,
including learning and memory, and are involved in neurotoxicity and
psychiatric disorders (Paoletti, Bellone, and Zhou, 2013). NMDAR expression
has been implicated in multiple tumor cell types, including neuroblastoma
cells, lung cancer cells, and breast cancer cells (North, Gao, Memoli, and Du,
2010). North et al. report that most breast tumor cell lines necessarily
express high levels of NMDAR1 and NMDAR2 (North, Gao, Memoli. Pang,
and Lynch, 2010).
• NMDARs regulate mTOR signaling activity; their inappropriate expression on
several human cancer cell lines represents a potential therapeutic avenue to
control tumor development and progression (Figure 1) (Deutsch, Tang,
Burket, and Benson, 2014). NMDAR antagonists, such as MK-801 (uncomp-
etitive antagonist) were shown to possess anti-proliferative and anti-invasive
effects in human lung adenocarcinoma cells (Lafon-Cazal, Perez, Bockaert,
and Marin, 2002). These properties conflict with prevalent hypotheses that
suggest promoting NMDA receptor activation as a cancer chemotherapeutic
strategy (Zheng and Quirion, 2009).
• Determining the effect of NMDAR on specific tumor cell lines is therefore a
critical step in developing potential NMDAR chemotherapeutics. We studied
these effects in breast cancer by inhibiting NMDAR activity in three mutant
p53 breast cancer cell lines
REFERENCES
METHODS & RESULTS
Donald Gosife Okoye2, & Luis Martinez PhD1
CONCLUSIONS
• These findings indicate that NMDAR activity increases cell survival via an mTOR-dependent
mechanism that can be abrogated by NMDAR antagonists
• They also present the inhibition of the NMDAR pathway as a potentially less risky and less
harmful chemotherapeutic target for mutant p53-based breast cancer treatment.
• Irrespective of whether activation or antagonism is associated with anti-proliferative and
anti-invasive effects for specific types of cancer, emerging data support the exploration of
targeting NMDA receptors expressed on the surface of cancer cells as a therapeutic
strategy.
Future Work:
• Further elucidation of the specific mechanism of activity is required.
• Comparative experiments with other inhibitors and/or activators of NMDAR will be
helpful in confirming results.
1Biochemistry Dept. University of Mississippi Medical Center, 2Sewanee: The University of the South
ACKNOWLEDGEMENTS
My sincerest thanks to Drs. Luis Martinez & Madhu Kollaredy for their guidance and
mentorship. My gratitude as well to Zunamys Carrerro, Krishna Chauhan, and Gopal
Ramakrishnan for all of their help and advice during my stay. Finally, thanks to Mary
Canterbury and the University of Mississippi Medical Center School of Graduate Studies in
the Health Sciences for making my research experience a successful one.
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CELL LINES: We used 3 different cancer cell lines for this experiment: HCC 38 (R273L); BT 549 (R249S); MDA-MB-231
(R280K)
SEEDING: We seeded 5000 cells per well
DRUG TREATMENT: We added the appropriate concentration of the MK-801 drug in DMSO to the experimental
plates (then incubated for 72 hours) normalizing for DMSO toxicity
MTT : We prepared and added 10 microliters of 5mg/ml MTT to each well and incubated for 3 hours noting color
changes; then we added lysis buffer and incubated for another 24 hours
ABSORBANCE AND CONCENTRATION READING (CELL COUNT): We obtained absorbance and thus concentration
readings of the experimental wells and compared them to the absorbance/concentration of the control wells in
order to determine the IC50 of MK-801 for different cell lines.
WESTERN BLOT ANALYSIS
Method: Probing for the presence of phospho-Akt. [Akt has been reported to mediate the anti-apoptotic effects of
NMDAR activity when phosphorylated by kinases upstream of the mTOR pathway (Lafon-Cazal, Perez, Bockhaert, and
Marin, 2002)].
Results: Treatment of the cell lines with MK-801 reduced the levels of phospho-AKT present in all cell lines
Fig. 1. Regulation of mTOR signaling activity through
NMDA receptor activation. The figure depicts NMDA
receptor activation leading to both diminished
transport of arginine via cationic amino acid
transporters (CAT) into the cell, and shortening of the
duration of signaling by the phosphorylated form of
extracellular signal-regulated kinase (ERK) ½
(Originally published by Deutsch, Tang, Burket, and
Benson, 2014).
MK-801 concentration
Fig 2. Colorimetric measurement of
amount of viable mutant p53
breast cancer cells after drug
treatment. Increasing
concentration of MK-801
treatment resulted in fewer
concentration of viable cells in the
wells.
Fig. 3. Determination of IC50
values for MK-801 effect on
mutant p53 breast cancer cell
lines. The calculations, plot and
subsequent extrapolation was
done using Graphpad Prism
Version 5.
24 hrs
treatment
Fig. 4. Western Blot analysis for phosphor-Akt
expressionin MK-801-treated mutant p53 breast
cancer cell lines. *Con refers to control (untreated
cell lines). Native Akt or inactive Akt probed to
ensure that Akt expressionlevels are unaffected.
Actin is used as endogenouscontrol.
Cell MembraneNMDAR
NMDA
AKT-p
mTOR pathway
Cell Proliferation
Cell Growth
Cell Motility Cell survival
Protein synthesis and transcription
Apoptosis
MK-801Synaptic Plasticity, Learning,
and Memory
Fig. 5. Regulation of mTOR signaling activity through NMDA receptor activation. The figure depicts NMDA receptor activation leading to
phosphorylation of Akt and a resulting activation of the mTOR pathway. MK-801 activity prevents the activation of NMDAR.
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