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. 2023 Jul;149(7):3313-3323.
doi: 10.1007/s00432-022-04237-1. Epub 2022 Aug 5.

Anticancer bioactivity of zerumbone on pediatric rhabdomyosarcoma cells

Cristian Urla  1 , Matias Julian Stagno  1 , Jörg Fuchs  1 , Steven W Warmann  1 , Evi Schmid  2
Affiliations

Anticancer bioactivity of zerumbone on pediatric rhabdomyosarcoma cells

Cristian Urla et al. J Cancer Res Clin Oncol. 2023 Jul.

Abstract

Purpose: Natural products are generally regarded as safe and have been shown to mediate anticancer activities against a variety of cell types. Zerumbone is a natural cyclic sesquiterpene derived from the rhizome of Zingiber zerumbet, which has attracted extensive attention in the recent decade for anticancer activities. The present study investigates the in vitro effect of zerumbone on rhabdomyosarcoma cells.

Methods: Two rhabdomyosarcoma cell lines (RD and RH30) were used as the model system. The growth inhibition of zerumbone was measured by MTT-assay, apoptosis via flow cytometry, gene expression by real-time PCR, the migration by transwell assay, and intracellular signaling by Western blotting.

Results: Zerumbone shows anticancer effects on RD and RH30 cells in a dose-dependent manner via cell growth inhibition and induction of apoptosis. Exposure of RD and RH30 cells on zerumbone also resulted in a decrease of migration and downregulation of the hedgehog pathway.

Conclusions: Taken together, our study provided the first evidence that zerumbone imparted strong inhibitory and apoptotic effects on pediatric rhabdomyosarcoma cell lines and merit further investigation as a promising candidate for the anticancer therapy.

Keywords: Apoptosis; Cell proliferation; Pediatric; Rhabdomyosarcoma; Zerumbone.

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Conflict of interest statement

The authors of this manuscript declare that they have no conflicts of interests or financial ties to disclose.

Figures

Fig. 1
Fig. 1
The effect of zerumbone on the viability of RD and RH30 cells as well as SkMCs. Arithmetic means ± SEM (n = 4) of the relative numbers of viable RD (a), RH30 (b), and SkMCs (c) after 72 h incubation in the presence of zerumbone (black bars) relative to the untreated control (white bar). *(p < 0.05), ****(p < 0.0001), indicate statistical significance to untreated control (ANOVA, Dunnett correction)
Fig. 2
Fig. 2
The influence of zerumbone on viability of spheroids. Arithmetic means ± SEM (n = 4) of the relative numbers of viable RD (a) and RH30 spheroids (b) following 72 h incubation in the presence of zerumbone (black bar) relative to the absence of zerumbone (white bar). **(p < 0.01); ***(p < 0.001) indicates statistical significance to untreated control (ANOVA, Dunnett correction)
Fig. 3
Fig. 3
The effect of zerumbone on migration of RMS cells. Arithmetic means ± SEM (n = 5) of the percentage of migrated RD (a) and RH30 cells (b) in the absence (white bar) and presence of 10 µM and 25 µM zerumbone (black bars). ****(p < 0.0001) indicates statistically significant difference to untreated control (ANOVA, Dunnett correction)
Fig. 4
Fig. 4
The effect of zerumbone on colony-forming capacity of RMS cells. Arithmetic means ± SEM (n = 5) of the relative numbers of evolving clones of RD (a) and RH30 (b) cells following incubation for 72 h in the absence (white bars) and presence of various concentrations of zerumbone (black bars). ***(p < 0.001), ****(p < 0.0001) indicate statistically significant difference to untreated control (ANOVA, Dunnett correction)
Fig. 5
Fig. 5
The effect of zerumbone production of reactive oxygen species (ROS), Caspase 3/7 activity, and on induction of apoptosis on RMS cells. ROS level measurement after treatment with two different concentrations of zerumbone in RD (a) and RH30 cells (b) for 24 h. Cells were treated with DCFDA and measured via flow cytometry. Caspase 3/7 activity was measured in RD (c) and RH30 cells (d) after 24 h treatment with zerumbone; Data are shown as arithmetic means ± SEM (n = 5). *(p < 0.05), ***(p < 0.001), ****(p < 0.0001) indicate statistical significance (ANOVA, Dunnett correction). Arithmetic means ± SEM (n = 4) of the relative numbers of viable RD (e) and RH30 (f) cells after incubation with zerumbone for 72 h incubation with 10 µM and 25 µM zerumbone (black bars) relative to the absence of the treatment (white bar). **(p < 0.01), ***(p < 0.001), ****(p < 0.0001) indicates the statistical significance (ANOVA, Dunnett correction)
Fig. 6
Fig. 6
The influence of zerumbone on c-Myc expression in RMS cells. Transcriptional expression of NF-κB, cyclin D1, C-myc, and CXCR4 in RD (a, c, e, g) and RH30 cells (b, d, f, h). For quantification of mRNA levels, quantitative RT-PCR was performed using TBP as internal control. The expressions are given as relative values against to untreated control. *(p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001) indicate statistical significance (ANOVA, Dunnett correction); (n = 3)
Fig. 7
Fig. 7
The influence of zerumbone on expression and abundance levels of NF-κB and cyclin D1. Arithmetic means (n = 3) of protein levels of NF-κB and cyclin D1 in RD (a, c) and RH30 cells (b, d) before (white bars) and after treatment with Zerumbone (black bars); *(p < 0.05) and **(p < 0.01) indicate statistically significant difference compared to control (ANOVA, Dunnett correction); β-actin was used as loading control
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