Kaempferol Targets Global Epigenetic Modifiers to Impedes Growth and Migratory Ability of HeLa Cells

Abstract

Dietary bioactive agents can curb tumour progression through chromatin alterations. Thus, this study attempts to evaluate the influence of kaempferol on epigenome modification in HeLa cells. Biochemical analysis for global DNA methylation-LINE 1, DNMTs (DNA methyltransferases), HAT (histone acetyl transferase), HDACs (histone deacetylases) and HMTs (histone methyltransferases) were examined with their transcript level expression through qPCR. Also, H3 and H4 histone modification marks were quantitated by an ELISA-based assay. Moreover, qPCR and protein profiler were performed to analyse the expression of migratory genes at both mRNA and protein levels, respectively, that was further substantiated through colony formation, invasion, and scratch wound assays. Finally, DNA methyl-sequencing was performed to analyse the promoter methylation of TSGs (tumour suppressor genes) and corroborated by analysing selected TSGs' expression. Kaempferol treatment did not alter the global DNA methylation-LINE 1 compared to untreated control, however, it reduced the expression and biochemical activities of DNMT and HDAC, which can be linked to their hypermethylation by kaempferol exposure. Concordant with the reduced expression of HMTs, HATs and other epi-enzymes, various histone H3 and H4 marks were also observed to be modulated. Kaempferol exposure led to promoter hypomethylation of various TSGs (such as WIF1, RUNX1, RARβ, SOX1), which subsequently led to enhanced expression at the mRNA level, which corresponds to their reactivation. Molecular studies were consistent with cell-based studies, which demonstrated a strong growth inhibitory and anti-migratory effect of kaempferol. This research helps to understand the probable mechanism used by kaempferol as a potential epigenetic modifier.

Keywords: epigenome; epi‐diet; hypermethylation; hypomethylation; kaempferol; methyl sequencing.

FIGURE 1

(a) Bar graph showing kaempferol impacted % inhibition of DNMT activity at 48 h. (b) Bar graph showing dose dependent inhibition of HDAC activity by kaempferol treatment at 48 h. (c) Bar graph showing kaempferol treatment (48 h) reduced of HAT activity. (d) Bar graph representing kaempferol impacted % inhibition of HMT H3K27 activity. Data are presented as the mean ± standard deviation of three independent experiments. Significance was established at p ≥ 0.05. Two way‐ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Chromatin modifier expression analysis: (a) Kaempferol treatment at 50 μM for 48 h demonstrated to modify various chromatin modification enzyme compared to untreated control. (b) Histone H3 marks modifications: Kaempferol treatment at 50 μM for 48 h exhibited to decrease various Histone H3 modification marks. (c) Histone H4 marks modification: Kaempferol treatment at 50 μM for 48 h exhibited to decrease various Histone H3 modification marks respectively. *p < 0.05, ***p < 0.001.

FIGURE 3

Transcript expression: (a) Bar graph showing qPCR result of kaempferol mediated upregulation of various TSGs expression with a simultaneous downregulation of different oncogenes mainly involved in cell migration with respect to DMSO control HeLa cells. ***p < 0.001. (b) Differential expression of migratory proteins: Images of nitrocellulose membrane and graph representing reduced expression of various MMPs while increased in TIMPs expression followed by kaempferol treatment at 48 h respectively, compared to control. *p < 0.05, **p < 0.01. (c) Images of nitrocellulose membranes showing altered fold change of different phosphorylated proteins involved in TGFB pathways after kaempferol (50 μM of for 48 h) compared to control.

FIGURE 4

Mitigation of migratory capacity of HeLa cells: (a, b) Kaempferol (30, 40 and 50 μM) treated cells illustrated a significantly increased wound width with increasing time and concentration dependent mode while in trans well assay depicted a sharp reduction in number of migrated Hela cells in contrast to control. (c) Clonogenic assay: Colony assay and bar graph showing the control and kaempferol (30, 40, and 50 μM) treated HeLa cells after 2 weeks of growth. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

(a) Venn diagram showing distribution of annotation of differentially methylated regions (DMRs) by gene features. (b) Heat map showing the fold change of difference in methylation percentage of various genes between control and kaempferol treated samples (20 μM at 48 h). (c) Showing PPI network and GO‐biological process showing interaction between different hypermethylated gene of interest and showing PPI network and GO‐biological process of hypomethylated genes as an impact of kaempferol treatment on HeLa cells.