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A3014
October 13, 2014
1:00 PM - 2:30 PM
Room Room 231-232
Morphine Induces Dose-Dependent Epigenetic Alterations Through Modification of the Pro-Metastatic IDH-R132H Protein Variant
Dean Bunbury, M.B.B.S., F.A.N.Z.C.A., Lawrence Wengle, B.Sc., Manasvi Vanama, B.Sc., Jason T. Maynes, Ph.D., M.D.
Hospital for Sick Children, Toronto, Ontario, Canada
Introduction:

Abnormally methylated DNA is a characteristic feature of many cancers, modifying cellular protein expression and metabolism to the benefit of the cancer cells and controlling metastasis. Alteration of the Krebs Cycle protein IDH1 is a prominent mechanism used by cancer cells to change DNA methylation and mutation of IDH1 (IDH1-R132H) is diagnostic in certain cancers, including glioblastoma. We have previously shown that morphine is a specific inhibitor of IDH1. Here we sought to show that morphine is also an inhibitor of the prometastatic mutant IDH1-R132H and that morphine can alter the epigenetic profile of cancer cells, providing evidence for a cancer-genotype specific opiate pain therapy.

Methods:

Using site-directed mutagenesis, we created the single point mutant IDH1-R132H in the wild-type enzyme. Upon purifying the recombinant protein, we performed colorimetric enzymatic assays to determine if morphine was also an inhibitor of IDH-R132H. The method and potency of inhibition was determined using non-linear, multivariate regression. To determine if morphine altered DNA methylation, HeLa cells were exposed to four concentrations of morphine for a total of six days, total genomic DNA was harvested and the level of methylated DNA determined using a meDNA-fluorescent antibody-based assay. Reversal of DNA methylation was determined by washing the cells after three days morphine exposure, with a further three days for potential reversal.

Results:

Similar to the wild-type enzyme, morphine was shown to be a mixed-mode inhibitor of IDH1-R132H activity. Michaelis-Menten calculations showed the inhibitory constant (Ki) to be 18.1 μM, indicating that morphine more potently inhibits IDH-R132H than IDH-WT. Hydromorphone, despite its very similar chemical structure, did not inhibit IDH-R132H. Consistent with inhibition of IDH1, morphine was shown to affect DNA methylation, increasing the percentage of meDNA in a dose-dependent fashion. The DNA methylation was not reversible, indicating that the epigenetic alterations induced are, at least, semi-permanent.

Conclusion:

We have demonstrated that the prometastatic IDH-R132H is inhibited by morphine, slightly more potently than the wild-type enzyme. Additionally, we showed that morphine is associated with dose-dependent increases in methylated DNA at clinically relevant concentrations, consistent with intracellular IDH1 inhibition. While inhibition of the native enzyme would be beneficial to cancer cells, inhibition of the R312H variant would be detrimental, illustrating the possibility of a cancer-genotype specific pain therapy that can alter cancer growth and metastasis.

Copyright © 2014 American Society of Anesthesiologists