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Page 8 of 11 Fonseka et al. J Cancer Metastasis Treat 2020;6:7 I http://dx.doi.org/10.20517/2394-4722.2019.024

A B

Figure 5. Combinatorial therapy increases the survival of mice implanted with neuroblastoma cells. A: graphical representation of
body weight of mice over the period in the presence or absence of curcumin and doxorubicin (n = 3). Treatment with curcumin and
doxorubicin does not impart visible side effects in mice. B: Kaplan-Meier analysis of the survival rate of mice bearing neuroblastoma
tumour undergoing treatment. Combinatorial treatment significantly enhanced the survival of mice (*P < 0.05). DMSO: dimethyl
sulfoxide

DISCUSSION
Neuroblastoma is the most common extracranial solid tumour in children under the age of five. Depending
[50]
on risk factors, neuroblastoma can be divided in to low- and high-risk . Even though the survival rates
of low-risk neuroblastoma have improved significantly, the survival rates of high-risk neuroblastoma have
[51]
remained poor . Among the risk factors, amplification of the oncogene N-Myc is detected in about 20%
[52]
of neuroblastoma patients and considered high-risk . Hence, therapeutic strategies to manage high-risk
N-Myc amplified neuroblastoma cells is needed.

Curcumin and silibinin have shown anticancer properties by modulating several signalling
pathways [34,36,53-55] . More importantly, it is documented that curcumin and silibinin have the ability to
repress proteins that are involved in EMT and metastasis [45,47,56] . We sought to elucidate the role of curcumin
or silibinin in combination with doxorubicin.

First, the results from publicly available RNA-Seq analysis suggest that the mesenchymal-like phenotype
exhibited by neuroblastoma cells could be one of the potential reasons for the aggressiveness. In the current
study, we targeted the utility of curcumin or silibinin in sensitising neuroblastoma cells to doxorubicin.
Combinatorial therapy of curcumin and doxorubicin sensitised the highly aggressive neuroblastoma cells
both in vitro and in vivo. Here, we utilised four neuroblastoma cell lines, among which SK-N-BE2 and
IMR32 possess N-Myc amplification and hence are categorised as high-risk aggressive neuroblastoma.
N-Myc is not amplified in SH-SY-5Y and SK-N-AS cells, which were used to identify the usage of
combinational therapy in a wider range of neuroblastoma cells.

Agreeing with the previous literature, curcumin and silibinin reduced the expression of the mesenchymal
[37]
marker N-Cadherin and induced MET . However, the combinatorial effect with doxorubicin can also be
attributed to other anti-cancer activities of these MET inducers, such as inhibition of p53, pAKt and STAT3
signalling pathway [34,53,57] . Moreover, recent findings also suggest curcumin and silibinin as agents that can
inhibit the cancer stem cells growth [58,59] . Nevertheless, based on these results, curcumin and silibinin are
interesting candidates for combination with standard chemotherapeutic drugs including doxorubicin for