Zerumbone mediates apoptosis and induces secretion of proinflammatory cytokines in breast carcinoma cell culture

Abstract

Objectives: To investigate the potential anti-breast cancer activity of zerumbone in regulating apoptotic mediators and cytokines in comparison with paclitaxel (positive control).

Materials and methods: In this study, assays such as viability, apoptosis, reactive oxygen species, cell cycle, DNA fragmentation, and cytokines were carried out on MCF-7 cells after treatment with zerumbone and paclitaxel.

Results: The results showed that zerumbone demonstrated a higher (18-fold) IC₅₀ value (126.7 µg/ml) than paclitaxel (7.29 µg/ml) in order to suppress proliferation and induce cell death of MCF-7. The cell cycle arrest at the G0/G1 phase and excessive intracellular ROS production during the in vitro zerumbone treatment indicated occurrence of apoptotic cell death although nuclear DNA fragmentation was not observed. The flow cytometer analysis of treated cells revealed secretion of proinflammatory cytokines suggesting the potential immunomodulatory activity of zerumbone.

Conclusion: Although, zerumbone exhibited a higher IC₅₀ value compared with paclitaxel yet its anticancer activity against MCF-7 cells is still parallel to paclitaxel hence zerumbone has the potential to be an antineoplastic agent in the treatment of breast cancer especially the luminal type A.

Keywords: Apoptosis; Breast; Cytokine; Natural; Zerumbone.

Figure 1

Structure of zerumbone. The figure was derived from Eid et al., 2019 (19)

Figure 2

(A) Dose-dependent survival of MCF-7 cells treated with zerumbone as determined by MTT assay. A higher reduction of MCF-7 cell growth was found at a higher concentration of zerumbone. (B) Dose-dependent survival of MCF-7 cells treated with paclitaxel as determined by MTT assay. Different treatment periods exhibit differential sensitivities to paclitaxel treatment in vitro. Dose-response curves were obtained using a logistic nonlinear regression analysis model. (C) Dose-dependent survival of MCF-10A cells against zerumbone as determined by MTT assay. Data are presented as mean ± standard deviation (SD) from duplicate samples of at least three independent experiments

Figure 3

Zerumbone induces apoptosis in MCF-7 cells after 48 hr. The data represent the means ± standard deviations (SDs) of 3 independent tests. Statistical analysis is defined as significant if *P<0.05, **P<0.01, and ***P<0.001

Figure 4

The quantitative analysis indicated zerumbone arrested cell at the G0/G1 and paclitaxel arrested at the S phases. The data represent the mean ± SD of 3 independent tests. Statistical analysis is defined as significant if *P<0.05, **P<0.01, and ***P<0.001

Figure 5

Reactive oxygen species (ROS) generation in treated MCF-7 cells. Fluorescence intensity after the treatment of IC₅₀ of zerumbone and paclitaxel. The graph shows that zerumbone induces cellular ROS almost similar to paclitaxel. The data represent the mean ± standard deviation (SD) of 3 independent experiments. Statistical analysis is defined as significant if *P<0.05, **P<0.01, and ***P<0.001

Figure 6

A clear DNA fragmentation in MCF-7 treated with paclitaxel. zerumbone did not induce a significant pattern of DNA fragmentation compared with paclitaxel. Faint DNA fragments were visible in the lane of zerumbone treated MCF-7 cells. Lane 1: 1000 bp DNA ladder, Lane 2: DNA from untreated MCF-7 cells, Lane 3: DNA from IC50 of zerumbone treated MCF-7 cells, Lane 4: DNA from IC₅₀ of paclitaxel treated MCF-7 cells

Figure 7

Zerumbone and paclitaxel treatment triggers secretion of proinflammatory cytokines in MCF-7 cells. A significant increase in secretion of IL-6, TNF- α, and IFN-γ upon treatment of naturally occurring phytochemicals upon MCF-7 cells. The data represent the mean ± standard deviation (SDs) of 3 independent experiments. Statistical analysis is defined as significant if *P<0.05, **P<0.01, and ***P<0.001, ns: non-significant

Figure 8

Potential anticancer mechanism behind zerumbone and paclitaxel treated MCF-7 cells such as formation of reactive oxygen species (ROS), apoptotic cells, and pro-inflammatory cytokines along with cell cycle arrest at G0/G1 phase