In vitro evaluation of the anti‑breast cancer properties and gene expression profiles of Thai traditional formulary medicine extracts

Abstract

Breast cancer is a leading cause of cancer-related deaths worldwide. Moreover, standard treatments are limited, so new alternative treatments are required. Thai traditional formulary medicine (TTFM) utilizes certain herbs to treat different diseases due to their dominant properties including anti-fungal, anti-bacterial, antigenotoxic, anti-inflammatory and anti-cancer actions. However, very little is known about the anti-cancer properties of TTFM against breast cancer cells and the underlying molecular mechanism has not been elucidated. Therefore, the present study, evaluated the metabolite profiles of TTFM extracts, the anti-cancer activities of TTFM extracts, their effects on the apoptosis pathway and associated gene expression profiles. Liquid chromatography with tandem mass spectroscopy analysis identified a total of 226 compounds within the TTFM extracts. Several of these compounds have been previously shown to have an anti-cancer effect in certain cancer types. The MTT results demonstrated that the TTFM extracts significantly reduced the cell viability of the breast cancer 4T1 and MDA-MB-231 cell lines. Moreover, an apoptosis assay, demonstrated that the TTFM extracts significantly increased the proportion of apoptotic cells. Furthermore, the RNA-sequencing results demonstrated that 25 known genes were affected by TTFM treatment in 4T1 cells. TTFM treatment significantly up-regulated Slc5a8 and Arhgap9 expression compared with untreated cells. Moreover, Cybb, and Bach2os were significantly downregulated after TTFM treatment compared with untreated cells. Reverse transcription-quantitative PCR demonstrated that TTFM extract treatment significantly increased Slc5a8 and Arhgap9 mRNA expression levels and significantly decreased Cybb mRNA expression levels. Moreover, the mRNA expression levels of Bax and Casp9 were significantly increased after TTFM treatment in 4T1 cells compared with EpH4-Ev cells. These findings indicated anti-breast cancer activity via induction of the apoptotic process. However, further experiments are required to elucidate how TTFM specifically regulates genes and proteins. This study supports the potential usage of TTFM extracts for the development of anti-cancer drugs.

Keywords: anti-cancer; apoptosis; breast cancer; gene expression; traditional medicine; transcriptome.

Figure 1

Inhibitory effect of TTFM on the proliferation of 4T1, MDA-MB-231 and EpH4-Ev cells. (A-C) Cell viability assay and (D) IC₅₀ proliferation of 4T1, MDA-MB-231 and EpH4-Ev cells after 5 days of treatment with TTFM. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05, ^**P<0.01 and ^***P<0.001. TTFM, Thai traditional formulary medicine.

Figure 2

Effect of TTFM on the apoptosis of 4T1 cells. Cell apoptosis data were analyzed for each group after 72 h treatment with TTFM at (A) 0 µg/ml, (C) 25 µg/ml, (D) 50 µg/ml, (E) 100 µg/ml, (F) 200 µg/ml and (G) 400 µg/ml. (B) Positive control cells were induced using 1 mM H₂O₂. (H) Quantitative analysis of the apoptosis rates among groups (n=3). ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. mock.

Figure 3

Impact of TTFM on RNA expression in TTFM-treated cells. Venn diagram of the impact of TTFM on RNA expression in TTFM-treated EpH4-Ev and 4T1 cells compared with their respective, untreated cells. (A) Up-regulated genes and (B) down-regulated genes.

Figure 4

Significantly up- and down-regulated genes in TTFM-treated cells. Volcano plot of the significantly up- and down-regulated genes in TTFM-treated 4T1 cells compared with untreated 4T1 cells. DEGs, differentially expressed genes.

Figure 5

mRNA expression levels TTFM-treated cells. Relative mRNA expression levels of (A) Slc5a8, (B) Arhgap9, (C) Cybb and (D-G) apoptosis-related genes in treated cells compared with untreated cells. ^*P<0.05 and ^**P<0.01. EpH4-Ev, treated EpH4-Ev cells-untreated EpH4-Ev cells; 4T1, treated 4T1cells-untreated 4T1 cells.