Neuroblastoma is the most prevalent extracranial solid
tumor mainly in pediatric patients. We explored the efficacy of the combination of 2[(3-[2,3-dichlorophenoxy]propyl)amino]
ethanol (2,3-
DCPE, a small molecule inhibitor of the
anti-apoptotic protein Bcl-x(L)) and N-(4-hydroxyphenyl)
retinamide (4-HPR, a synthetic
retinoid) in inducing differentiation and apoptosis in human malignant
neuroblastoma cells. Immunofluorescence confocal microscopy and flow cytometry showed that the highest level of Bcl-x(L) expression occurred in SK-N-DZ cells followed by SH-SY5Y and IMR-32 cells. Combination of 20 μM
2,3-DCPE and 1 μM
4-HPR acted synergistically in decreasing viability of SK-N-DZ and SH-SY5Y cells. In situ
methylene blue staining and
protein gel blotting showed the efficacy of this combination of drugs in inducing neuronal differentiation morphologically and also biochemically with upregulation of the neuronal markers such as
neurofilament protein (NFP) and
neuron specific enolase (NSE) and downregulation of the differentiation inhibiting molecules such as N-Myc and Notch-1 in SK-N-DZ and SH-SY5Y cells.
Annexin V-FITC/PI staining showed the synergistic action of this combination
therapy in increasing apoptosis in both cell lines.
Protein gel blotting manifested that combination
therapy increased apoptosis with downregulation of the
anti-apoptotic proteins Bcl-x(L), Bcl-2 and Mcl-1 and upregulation of the
pro-apoptotic proteins Bax, p53, Puma (p53 upregulated modulator of apoptosis), and Noxa, ultimately causing activation of
caspase-3. In conclusion, our results appeared highly encouraging in advocating the use of
2,3-DCPE and
4-HPR as a novel combination
therapy for increasing both differentiation and apoptosis in human malignant
neuroblastoma cells having Bcl-x(L) overexpression.