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Page 6 of 11 Fonseka et al. J Cancer Metastasis Treat 2020;6:7 I http://dx.doi.org/10.20517/2394-4722.2019.024

Figure 3. Curcumin and silibinin increases the sensitivity of neuroblastoma cells to doxorubicin. FACS cell death assay on a panel of
neuroblastoma cells treated with 10 μM curcumin or 100 μM silibinin in the presence of 1 μM doxorubicin (n = 3). Data are presented
as mean ± SEM, *P < 0.05; **P < 0.01 as determined by Student’s t-test. Significance of the percentage of cell death in combinational
treatments was calculated in comparison to respective doxorubicin only treatments. DMSO: dimethyl sulfoxide; FACS: fluorescence
activated cell sorting

Combinatorial treatment of curcumin or silibinin with doxorubicin sensitises neuroblastoma
cells
Curcumin and silibinin are natural compounds that have many anti-cancer properties including induction
of MET [34-36] . As neuroblastoma cells, especially high-risk ones, are aggressive and resistant to treatment,
the combinatorial effect of curcumin or silibinin with doxorubicin was examined to understand their
therapeutic potential. A panel of four neuroblastoma cells, namely SH-SY-5Y, SK-N-AS, SK-N-BE2 and
IMR32, was selected for the in vitro analysis. Among these neuroblastoma cells, SH-Y-5Y and SK-N-
[49]
AS cells are low-risk and do not contain N-Myc amplification . On the contrary, SK-N-BE2 and IMR32
[49]
cells are high-risk neuroblastoma cell models with N-Myc amplification (> 0 copies) . Consistent
with the literature, treatment of the low-risk neuroblastoma cells (SH-SY-5Y and SK-N-AS) with the
chemotherapeutic agent doxorubicin (1 µM) induced significant cell death [Figure 3]. Whilst the basal
cell death for SK-N-AS cells was about 1%, incubation with doxorubicin induced nearly 70% cell death,
an increase by 70-fold. Similarly, SH-SY-5Y cells exhibited more than three-fold cell death (30%) upon
doxorubicin treatment. However, the high-risk neuroblastoma cells (SK-N-BE2 and IMR32) showed lesser
percentage of cell death (11.8% and 23%, respectively) when incubated with the chemotherapeutic agent
doxorubicin (1 µM). This relates to a 2.3- and 2.6-fold increase in cell death in SK-N-BE2 and IMR32 cells,
respectively.

Next, the combinatorial effect of curcumin or silibinin with doxorubicin was evaluated in the panel of
neuroblastoma cells. As shown in Figure 3, neither curcumin (10 µM) nor silibinin (100 µM) induced
significant cell death in any of the neuroblastoma cells. The concentration for curcumin and silibinin
was chosen as they reduced the expression of N-Cadherin in neuroblastoma cells. When performing the