Differential sensitivity of chemoresistant neuroblastoma subtypes to MAPK-targeted treatment correlates with ERK, p53 expression, and signaling response to U0126.
OBJECTIVE
Neuroblastoma tumors are comprised of neuroblastic (N), substrate-adherent (S), and intermediate (I) cells. Because cell growth and differentiation often involve p44/p42 mitogen-activated protein kinase (MAPK) pathway signaling, we explored MAPK signaling and growth response in three NB cell types after MAPK kinase (MEK) inhibition to evaluate the feasibility of MAPK-targeted treatment strategies.


METHODS
Three human NB cell cultures, SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type), were treated in monolayer cultures with increasing concentrations of the MEK inhibitor U0126. MAPK pathway intermediates MEK and ERK, their activated (phosphorylated) forms p-MEK and p-ERK, and p53 expression were assessed by Western blot at 1 and 24 hours. At 72 hours, cell counts determined growth inhibition and DNA fragmentation ELISA assessed apoptosis.


RESULTS
Among all three lines, total ERK and MEK expression were unaffected by U0126. However, constitutive total ERK and p53 expression were significantly greater in BE(2)-C (I-type) cells than in SH-SY5Y (N-type) and SK-N-AS (S-type). Active ERK (p-ERK) levels decreased in dose response to U0126 at 1 and 24 hours in all lines. Conversely, p-MEK levels increased with increasing U0126 concentrations at 1 hour in SH-SY5Y (N-type) and at 24 hours in all lines. BE(2)-C (I-type) cell counts decreased in concentration-dependent fashion with U0126, whereas SH-SY5Y (N-type) and SK-N-AS (S-type) showed a biphasic response with increased cell counts at 1 micromol/L U0126 and slightly decreased cell counts at 10 mumol/L U0126.


CONCLUSIONS
This study demonstrates that BE(2)-C (I-type) cells exhibit greater constitutive total ERK and p53 expression than SH-SY5Y (N-type) and SK-N-AS (S-type). Although all three lines exhibit p-ERK decreases with MEK inhibition, only BE(2)-C (I-type) cells significantly decrease their proliferation with U0126 treatment. Although MEK inhibition holds promise in targeting I-type NB cells, successfully treating this heterogeneous tumor may require combining agents against N- and S-type cells.
Eppstein
Andrew C
AC
Department of Surgery, Indiana University School of Medicine and Riley Children's Hospital, Indianapolis, IN 46202, USA.
Sandoval
John A
JA
Klein
Patrick J
PJ
Woodruff
Heather A
HA
Grosfeld
Jay L
JL
Hickey
Robert J
RJ
Malkas
Linda H
LH
Schmidt
C Max
CM
D017209:Apoptosis / Q000502:physiology; D002070:Butadienes / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; D016159:Tumor Suppressor Protein p53; C113580:U 0126; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases