Fisetin Deters Cell Proliferation, Induces Apoptosis, Alleviates Oxidative Stress and Inflammation in Human Cancer Cells, HeLa

Abstract

Background: Fisetin, a flavonol profusely found in vegetables and fruits, exhibited a myriad of properties in preclinical studies to impede cancer growth.

Purpose: This study was proposed to delineate molecular mechanisms through analysing the modulated expression of various molecular targets in HeLa cells involved in proliferation, apoptosis and inflammation.

Methods: MTT assay, flow cytometry, nuclear morphology, DNA fragmentation and Annexin-Pi were performed to evaluate the anti-cancer potential of fisetin. Furthermore, qPCR and proteome profiler were performed to analyse the expression of variety of gene related to cell death, cell proliferation, oxidative stress and inflammation and cancer pathways.

Results: Fisetin demonstrated apoptotic inducing ability in HeLa cells, which was quite evident through nuclear morphology, DNA ladder pattern, decreased TMRE fluorescent intensity, cell cycle arrest at G₂/M and increased early and late apoptosis. Furthermore, fisetin treatment modulated pro-apoptotic genes such as APAF1, Bad, Bax, Bid and BIK at both transcript and protein levels and anti-apoptotic gene Bcl-2, BIRC8, MCL-1, XIAP/BIRC4, Livin/BIRC7, clap-2/BIRC3, etc. at protein levels to mitigate cell proliferation and induce apoptosis. Interestingly, the aforementioned alterations consequently led to an elevated level of Caspase-3, Caspase-8 and Caspase-9, which was found to be consistent with the transcript and protein level expression. Moreover, fisetin downregulated the expression of AKT and MAPK pathways to avert proliferation and enhance apoptosis of cancer cells. Fisetin treatment also improves oxidative stress and alleviates inflammation by regulating JAK-STAT/NF-kB pathways.

Conclusion: Together, these studies established that fisetin deters human cervical cancer cell proliferation, enhances apoptosis and ameliorates inflammation through regulating various signalling pathways that may be used as a therapeutic regime for better cancer management.

Keywords: AKT/mTOR; JAK-STAT/NF-kB; MAPK; cytotoxicity; fisetin; glutathione; phosphorylation.

Figure 1

Cytotoxic effects of fisetin on HeLa cells. (A) Chemical structure of fisetin. (B) Graph represents dose and time-dependent decrease in cell viability of HeLa cells after treatment with fisetin [1–70 µM] for 24 h and 48 h, respectively, whereas fisetin did not demonstrate any significant difference in the cell viability of AC-16 (normal cell line). All the assay-treated cells were compared with DMSO controls. The IC50 of fisetin was found to be 50 µM at 48 h. The data are expressed as the mean ± standard deviation of three independent experiments. Statistically significant differences are marked by asterisks: two-way ANOVA * represents p < 0.05; (C) Microscopic examination of treated cells: Fisetin treated HeLa cells at various concentrations [20, 30 and 50 µM] and time points [24 h and 48 h] show the characteristic feature of rounding off of the cells, signifying apoptosis at 10X magnification. (D) Nuclear morphology of fisetin treated HeLa cells [20, 30 and 50 µM] shows dose-dependent increase in apoptotic index. Orange = prominent intact nuclei, green = membrane blebbing, yellow = nuclear fragmentation, green = apoptotic bodies. (E) HeLa cells treated with different concentrations [20, 30, 50 μM for 48 h] of fisetin were found to produce a DNA laddering pattern consistent with apoptosis. C = DMSO Control, L = DNA ladder.

Figure 2

(A) Flow cytometry analysis: Analysis of DNA content of treated HeLa cells with 20, 30 and 50 µM of fisetin for 24 and 48 h was compared with DMSO control cells, after PI staining. It demonstrated G2/M arrest cell cycle arrest with increase in sub-G0 apoptotic population. (B,C) Graph represents % distribution of cells across the different phases of cell cycle in 24 and 48 h, respectively. (D) RQ plot of HeLa cells followed by treatment with fisetin for 48 h resulted in downregulation of various cell cycle regulators, genes involved in PI3K/AKT, MAPK and WNT signalling, while upregulation in TSGs expression compared with the control. The data are expressed as the mean ± standard deviation of three independent experiments. Statistically significant differences are marked by asterisks: two-way ANOVA * represents p < 0.05; ** represents p < 0.01.

Figure 3

Fisetin induces apoptosis in HeLa cells (A) Fisetin-treated HeLa cells with 20 µM, 30 µM and 50 µM for 24 and 48 h in comparison with the DMSO control followed by double staining. Representative picture of dot plots showing different stages of apoptosis. Left lower quadrant (FITC⁻/PI⁻) = viable cells, right lower quadrant (FITC⁺/PI⁻) = early apoptotic cells, right upper quadrant (FITC⁺/PI⁺) = late apoptotic cells. (B) Graph illustrating the percentage distribution of different stages of apoptotic cells in their respective quadrant by flow cytometry. Early and late apoptotic cell proportions was found to be increased both in time and concentration-dependent manner compared with the control. (C) TMRE staining of treated cells showing reduction in fluorescent intensity signifying reduction in mitochondrial membrane potential. Images were captured by fluorescent microscope. (D) Graph representing TMRE fluorescence of treated HeLa cells with fisetin 20, 30 and 50 µM for 48 h, which exhibited reduction in mitochondrial membrane potential from 81% to 64% and 54%, respectively, in comparison with the untreated control. Data are presented as the mean ± standard deviation of three independent experiments. Two-way ANOVA * = p < 0.05; ** = p < 0.01, *** p < 0.001.

Figure 4

Expression analysis. (A). Heat map showing the expression of various genes involved in apoptosis. RQ plot of caspases, extrinsic receptors and ligands, pro-apoptotic gene and anti-apoptotic after fisetin treatment at 20 μM and 50 μM for 48 h. (B) Images of nitrocellulose proteome profiler showing differential expression of the regulatory pathway and apoptotic proteins in the control and fisetin-treated sample (20 and 50 μM of fisetin for 48 h). (C) Graphical representation of protein expression as fold change compared with the control sample. Fisetin treatment increased pro-apoptotic while decreasing the expression of anti-apoptotic proteins [* p ≤ 0.05, *** p < 0.001]. (D) Evaluation of caspase 3, caspase 8 and caspase 9 activity of fisetin-treated HeLa cells at 20, 30 and 50 μM for 48 h. Graph represents an increase in the fold change in caspase 3, 8 and 9 activity compared with the control.

Figure 5

Analysis of inflammatory cytokines (A) Nitrocellulose membrane showing the differential expression of inflammatory cytokines (B) Graph showing the downregulation of pro-inflammatory and chemokines while showing upregulation in anti-inflammatory cytokines expression in fisetin treated (50 µM) compared with the DMSO control sample. (C) Graph showing an increase in total GSH level in fisetin-treated HeLa cells at 20, 30 and 50 μM for 48 h. Data are presented as the mean ± standard deviation of three independent experiments. Two-way ANOVA. ** = p < 0.01, *** p < 0.001.

Figure 6

Differential expression of various phosphorylated proteins associated with different signalling pathways. (A) Images of proteome profiler membranes showing differential expression of the phosphorylated proteins involved in MAPK, AKT, JAK-STAT, NF-ĸB and TGFβ signalling pathway after 50 μM of fisetin treatment for 48 h in comparison with the DMSO control. (B) Graphical presentation of downregulated expression of different proteins in the aforementioned pathways, while the expression of P53 (p-ser241) and P27 (p-Thr198), p38(P-Thr180/Tyr-182) were upregulated. Differential expression is shown as fold change. Data are presented as the mean ± standard deviation of three independent experiments *** p < 0.001.