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Fonseka et al. J Cancer Metastasis Treat 2020;6:7 I http://dx.doi.org/10.20517/2394-4722.2019.024 Page 3 of 11
[30]
induction of EMT with the upregulation of Twist and the down regulation of E-Cadherin . Similarly,
activation of Snail in CRC cell lines led to increased motility and invasiveness with increased resistance to
[31]
5-FU . Collectively, these data suggest that EMT plays a role in chemoresistance. Neuroblastoma cells,
due to their origin, are more mesenchymal and it is highly likely that the inherent resistance to treatment
could be partly attributed to EMT. It is unclear whether the utility of MET inducers along with standard
chemotherapeutic drugs could increase the sensitivity of the neuroblastoma cells.

Curcumin and silibinin are natural components that have exhibited anti-cancer activities and can induce
MET in many adult cancers with less or no toxicity [32,33] . These natural active ingredients have been
implicated in suppressing various growth and pro-invasive signalling pathways as well as inducing cell
death in a variety of cancer cells [34-36] . In non-N-Myc amplified SH-SY-5Y neuroblastoma cells, curcumin
[37]
was shown to induce cell death in vitro . However, in vivo studies that highlight the therapeutic potential
of curcumin and silibinin in treating high-risk neuroblastoma cells are currently lacking. In addition,
the utility of these natural compounds in combinatorial therapy with doxorubicin in vivo has not been
examined. In this study, we examined the combinatorial effect of curcumin or silibinin in sensitising
neuroblastoma cells to the chemotherapeutic drug doxorubicin both in vitro and in vivo.

METHODS
RNA-Seq analysis
[38]
The RNA-Seq data for NBL and CRC cell lines and tissues were downloaded from published literature .
The mRNA expression (log RPKM values) of genes categorised as epithelial and mesenchymal were plotted
2
using MATLAB. Genes (315) classified as epithelial and mesenchymal were downloaded from published
[39]
literature . Statistical analysis to calculate the significance was performed using the Pearson’s chi-square
test. P-values less than 0.05 were considered statistically significant.

Cell culture
2
The neuroblastoma cell lines SK-N-BE2, SH-SY5Y and SK-N-AS were cultured in 150 cm tissue culture
flasks (BD Falcon ) in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, Life Technologies) medium
TM
supplemented with 10% (v/v) fetal calf serum (GIBCO, Life Technologies) and 100 units/mL of penicillin-
streptomycin (GIBCO, Life Technologies). IMR32 neuroblastoma cells were cultured in Minimum Essential
Medium (GIBCO, Life Technologies) medium. The cells were incubated at 37 °C with 5% CO . SK-N-BE2
2
TM
TM
(CRL-2271 ) and IMR32 (CCL-127 ) cells were purchased from ATCC®, while SH-SY5Y and SK-N-AS
cells were kindly gifted by Dr Julie Atkin.
Whole cell lysate preparation
Neuroblastoma cells were treated with 10 µM curcumin and 100 µM silibinin for 24 and 48 h prior to
[40]
preparation of cell lysates. Cells were lysed as described previously using 4 × sodium dodecyl sulfate (SDS)
loading dye [2% (w/v) SDS, 125 mM Tris-HCl pH 7.4, 12.5% (v/v) glycerol and 0.02% (w/v) bromophenol
blue]. Briefly, loading dye (1.5 mL) was added to culture dishes and evenly spread using the cell lifter (Fisher
Biotec). The lysate was then collected in thick wall polyallomer tubes (Beckman Coulter) and centrifuged
at 100,000 g for 1 h (TLA 100.2, Beckman). Supernatant was collected and stored at -80 °C for further
analysis.

SDS-PAGE and Western blotting
Equal amount of protein samples was prepared in 4 × SDS loading buffer with 100 mM DTT (Astral).
Samples were then denatured by heating at 95 °C for 2 min and were run on a NuPAGE® 4%-12% Bis-Tris
precast gel (Life Technologies). The gels were run at 150 V for 1 h in NuPAGE® MES SDS Running Buffer
(Life Technologies). Proteins were transferred on to nitrocellulose membrane using iBlot dry blotting system