Induction of Apoptosis and Regulation of MicroRNA Expression by (2 E,6 E)-2,6- bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) Treatment on MCF-7 Breast Cancer Cells

Abstract

(2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) is a synthetic curcumin analogue, which has been reported to possess anti-tumor, anti-metastatic, and anti-invasion properties on estrogen receptor (ER) negative breast cancer cells in vitro and in vivo. However, the cytotoxic effects of BHMC on ER positive breast cancer cells were not widely reported. This study was aimed to investigate the cytotoxic potential of BHMC on MCF-7 cells using cell viability, cell cycle, and apoptotic assays. Besides, microarray and quantitative polymerase chain reaction (qPCR) were performed to identify the list of miRNAs and genes, which could be dysregulated following BHMC treatment. The current study discovered that BHMC exhibits selective cytotoxic effects on ER positive MCF-7 cells as compared to ER negative MDA-MB-231 cells and normal breast cells, MCF-10A. BHMC was shown to promote G2/M cell cycle arrest and apoptosis in MCF-7 cells. Microarray and qPCR analysis demonstrated that BHMC treatment would upregulate several miRNAs like miR-3195 and miR-30a-3p and downregulate miRNAs such as miR-6813-5p and miR-6132 in MCF-7 cells. Besides, BHMC administration was also found to downregulate few tumor-promoting genes like VEGF and SNAIL in MCF-7. In conclusion, BHMC induced apoptosis in the MCF-7 cells by altering the expressions of apoptotic-regulating miRNAs and associated genes.

Keywords: 2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC); MCF-7; apoptosis; breast cancer; miRNA.

Figure 1

Morphological changes of MCF 7 cells viewed under light microscope after exposure to: (A) Untreated, (B) 8.2 µM of BHMC for 48 h. Fluorescent microscopy of acridine orange and propidium iodide dual staining of human breast cancer cells lines (MCF-7) (C) untreated and (D) 8.2 µM of BHMC for 48 h. Note: Figures shown were representative of one of at least three independent replicates with similar parameter (magnification 100×).

Figure 2

DK2 induces apoptosis in MCF 7 cells via G1/S cell cycle arrest. (A) Histogram analysis of the cell cycle machinery in MCF-7 after BHMC treatment for 24, 48, and 72 h. (B) Detection of phosphatidylserine (PS) exposure through the detection of Annexin V-PE and 7ADD fluorescence uptake in BHMC treated MCF-7 cells. Note: Values are mean ± SD of three replicates and significantly different from the untreated group (* p < 0.05) by ANOVA and followed by Duncan’s multiple range test. Figures shown are representative of one of at least three independent replicates with similar parameter.

Figure 3

miRNA microarray data revealed differential gene expression between control and BHMC treated MCF-7 cell; (A) heatmap cluster analysis depicting differential miRNA (>2-fold change, p < 0.05) for BHMC treated cells and control MCF-7 cells. Up-regulated genes are depicted in red, down-regulated genes are in blue (see color bar); (B) principal component analysis plot; Tthe closer the dots, the more similar the gene expression profiles are; the farther apart the dots are, the greater the differences are; (C) volcano plot.

Figure 4

Validation of known miRNA and targeted genes using qPCR. Values are mean ± SD of three replicates and significantly different from the untreated group (* p < 0.05) by ANOVA and followed by Duncan’s multiple range test. Figures shown were representative of one of at least three independent replicates with similar parameter.

Figure 5

Chemical structure of BHMC [8].