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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 7 of 11

A B

Figure 4. Combinatorial therapy reduces the tumour burden. A: schematic representation of in vivo work to examine the efficacy of
combinatorial therapy on neuroblastoma tumour burden. B: relative tumour volume across a period in the presence of curcumin and/or
doxorubicin (n = 3). Data are presented as mean ± SEM, *P < 0.05 as determined by Student’s t-test. DMSO: dimethyl sulfoxide

combinational treatments, the cells were treated with curcumin and silibinin 24 h prior to doxorubicin
treatment to allow the induction of MET. Nevertheless, combination of curcumin with doxorubicin induced
significant cell death in both the low- and high-risk neuroblastoma cells. Compared to the doxorubicin
alone treatment, a 2.9-, 2.6-, 1.3- and 1.8-fold increase in cell death was observed in SK-N-BE2, SH-SY-
5Y, SK-N-AS and IMR32 cells, respectively. Moreover, the Combination index (CI) values obtained for the
treatments were < 0.1, indicating a synergism. The CI values were 0.56 and 0.5 for SK-N-BE2 and SH-SY-5Y
cells, respectively. Consistent with these results, combinatorial treatment of silibinin and doxorubicin also
induced significant cell death, compared to doxorubicin alone, in the panel of neuroblastoma cells. Taken
together, these data suggest that curcumin and silibinin can increase cell death induced by doxorubicin in
both low- and high-risk neuroblastoma cells. The data also suggested that curcumin along with doxorubicin
was more effective in inducing cell death in the neuroblastoma cells.

Combinational treatment reduced the tumour burden and increased the survival of mice
implanted with neuroblastoma
Several studies have examined the effect of curcumin or silibinin on inducing cell death in neuroblastoma
[37]
cells . However, the therapeutic potential has not been examined in vivo. Furthermore, it is unclear
whether a combinatorial treatment that increases cell death in neuroblastoma cells also works in vivo.
Hence, to validate the effect of combinatorial treatment in vivo, nude mice were injected with the high-
6
risk SK-N-BE2 neuroblastoma cells (5 × 10 ) subcutaneously. N-Myc amplified SK-N-BE2 cells are more
proliferative and resistant to doxorubicin treatment [Figure 3] than the other neuroblastoma cells used in
this study, hence was chosen as a model cell line. After formation of tumours, the mice were administered
(i.p.) with DMSO (control), doxorubicin, curcumin or a combination of doxorubicin and curcumin
twice per week [Figure 4A]. Here, curcumin was chosen as the curcumin treatment had more effect on
N-cadherin expression level [Figure 2A] and cell death [Figure 3] than silibinin. The tumour volume was
monitored daily in the control and treatment groups. Tumour volume was the highest in mice that received
DMSO or curcumin alone [Figure 4B]. Consistent with the in vitro data and literature, mice that were
treated with doxorubicin alone had a significant decrease in the tumour volume. Encouragingly, the tumour
volume in mice treated with both doxorubicin and curcumin was significantly smaller compared to those
receiving only doxorubicin. There were no visible side effects from any of the treatments and no change
in body weight of the mice was observed [Figure 5A]. Importantly, mice that received combinational
treatment exhibited a higher survival rate [Figure 5B]. Taken together, these results suggest that treatment
of curcumin in combination with a chemotherapeutic drug may be a viable strategy to treat neuroblastoma
patients, most importantly high-risk neuroblastoma patients with N-Myc amplification.