In-Vitro Cytotoxicity, Apoptotic Property, and Gene Expression Changes Induced by Naringenin-7-O-Glucoside in Triple-Negative Breast Cancer

Abstract

Introduction: Cancer is one of the most significant health challenges demanding the expansion of effectual therapeutic methods. Triple-negative breast cancer (TNBC) is a form of aggressive cancer with inadequate therapeutic options which lacks the expression of certain hormones.

Materials and methods: The present study investigates the potential of naringenin-7-O-glucoside, a flavanone glycoside extracted from Holarrhena antidysenterica as an anticancer agent against TNBC cell lines. In-vitro analysis to evaluate cytotoxicity, apoptotic-inducing properties and effect on gene expression was conducted.

Results: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay studied the IC-50 of naringenin-7-O-glucoside to be 233.56 µg/µL, revealing the dose-dependent cytotoxicity with minimal effect on Vero cells. Extensive DNA fragmentation confirmed the apoptotic property. Furthermore, a significant downregulation of the epidermal growth factor receptor (EGFR) was noted in treated cells when equated to the control specimen of the sample.

Conclusion: Therefore, naringenin-7-O-glucoside can be a potential targeted therapeutic agent.

Keywords: anticancer potential; egfr downregulation; naringenin-7-o-glucoside; selective cytotoxicity; triple-negative breast cancer.

Figure 1. Dose-dependent inhibition curve of naringenin 7-O-glucoside on MDA-MB-231 and MCF-7 cell lines

The optimum dose-dependent curve illustrates the percentage of inhibition of breast cancer cell lines against naringenin-7-O-glucoside. The dose-dependent curve shows how the percentages of inhibition change as the concentration of naringenin 7-O-glucoside increases from 0 (control) to 1000 µg. The X-axis represents the concentration of the compound and the Y-axis represents the percentage of inhibition on treatment. The control represents the untreated cells, serving as a baseline reference. Series 1 represents the MCF-7 cell line and series 2 represents the MDA-MB-231 cell line.

Figure 2. Naringenin-7-O-glucoside treated (A) MDA-MB-231 cell line and (B) MCF-7 cell line

The image illustrates the effect of the compound against (A) MDA-MB-231 and (B) MCF-7 cell lines. Both cell lines show cell death at the IC-50, but extensive cell death is observed in the MDA-MB-231 cell line compared to the MCF-7 cell line. This observation suggests a greater susceptibility of MDA-MB-231 cells to the cytotoxic effects of naringenin-7-O-glucoside. The image was captured at a magnification of 20X and a scale of 5 microns.

Figure 3. DNA fragmentation analysis using gel electrophoresis showing ladder, control and treated cell lines

The figure shows the effect of naringenin-7-O-glucoside on cellular DNA integrity. The three lanes, namely L, C and N, represent the ladder lane having DNA fragments of known size for reference (100bp to 1000bp); the control lane consisting of untreated MDA-MB-231 cells providing baseline comparisons and cell lines treated with naringenin-7-O-glucoside respectively. The treated lane (N) shows fragmentation of DNA indicating apoptosis of cell lines when treated with the compound. The control lane (C) exhibits minimal fragmentation, consistent with intact DNA in untreated cells. The ladder lane (L) serves as a reference for fragment size determination.

Figure 4. Gene expression analysis demonstrating downregulation of EGFR when treated with naringenin-7-O-glucoside

The figure illustrates gene expression analysis showing the downregulation of EGFR in the MDA-MB-231 cell line following treatment with naringenin-7-O-glucoside. Three lanes are presented: L represents the ladder lane, containing DNA fragments of known sizes for reference; C denotes the control lane serving as a baseline comparison; and NT indicates the treated lane with naringenin-7-O-glucoside. Distinct bands observed at 157 bp in the treated lane signify the downregulation of EGFR expression in response to naringenin-7-O-glucoside treatment. This reduction in EGFR expression suggests a potential inhibitory effect of the compound on EGFR signalling pathways. In contrast, the control lane shows consistent EGFR expression levels, indicating no treatment-induced alterations in gene expression. The ladder lane (L) aids in fragment size determination for accurate analysis. EGFR: epidermal growth factor receptor

Figure 5. Densitometry analysis of EGFR expression levels

The figure illustrates the densitometric analysis of EGFR expression in the MDA-MB-231 cell line when treated with naringenin-7-O-glucoside. Fold change values were calculated to quantify the relative expression levels compared to the control group, which has a fold change of 1. MDA-MB-231 cell lines treated with naringenin-7-O-glucoside show a fold change of 0.3, suggesting a substantial downregulation of EGFR expression in response to the treatment. The p-value for the observed difference in EGFR expression between the control and treated groups is <0.05, indicating statistical significance. EGFR: epidermal growth factor receptor