Hesperetin effect on MLH1 and MSH2 expression on breast cancer cells BT-549

ABSTRACT

Due to its genetic and phenotypic heterogeneity, breast cancer is very difficult to eliminate. The harmful consequences of conventional therapies like radiation and chemotherapy have prompted the search for organic-based alternatives. Hesperetin (HSP), a flavonoid, has been discovered to possess the ability to hinder the proliferation of cell associated with breast cancer by acting as an epigenetic agent and modifying gene expression. In this investigation, breast cancer cells (BT-549) and normal cells (MCF-10a) were subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test and three different doses (200, 400, and 600 μM/mL) of HSP for real-time polymerase chain reaction and flow cytometry to examine its cytotoxic and anti-malignant potential. HSP was shown to be cytotoxic to both normal and breast cancer cells, but had a more pronounced effect on the cancer cell lines. After 48 h of treatment, the half-maximal inhibitory concentration (IC₅₀) for BT-549 was 279.2 μM/mL, whereas the IC₅₀ for MCF-10a was 855.4 μM/mL. At high HSP concentrations, upregulation of the MLH1 and MSH2 genes was observed in both cell lines. The influence of HSP on MLH1 gene expression was concentration dependent. Moreover, HSP had a concentration-dependent effect on MSH2 gene expression in the BT-549 cell line but not in the MCF-10a cell line. Cell death and early apoptosis were shown to be concentration dependent upon the application of HSP, as determined by flow cytometric analysis. HSP’s capacity to cause apoptosis and its stronger impact on the malignant cell line when analyzed with the normal cell line imply that it might be useful as an effective therapeutic approach for combating breast cancer.

INTRODUCTION

Breast cancer is acknowledged as a complex malignancy characterized by its heterogeneity and propensity to metastasize, posing significant challenges in terms of treatment.[1,2,3] Increased patient survival and decreased health-care spending may be achieved by early diagnosis using tools like mammography and magnetic resonance imaging.[4,5] Hyperproliferation of duct luminal cells causes many metastatic carcinomas, and breast cancer strikes women at a far higher rate than males.[6] In recent years, cancer stem cells (CSCs) have been recognized as critical participants in tumor initiation, immune suppression, and tumor recurrence. Breast cancer treatment is complicated by CSCs since they are resistant to standard therapies like chemotherapy and radiation.[7]

Despite notable progress in comprehending the etiology and therapeutic approaches for breast cancer, it remains the leading cause of cancer-related mortality among women worldwide. In light of the promising results shown in certain patients, interest in the use of targeted medicines and immunotherapies to treat breast cancer has increased in recent years.[8,9] Aberrant gene expression as a possible indicator for survival in malignancies has recently been studied in individuals diagnosed with breast cancer[9] and other carcinomas.[10,11,12] Thanks to genomics and personalized medicine, presently, our comprehensions of the molecular and genetic basis of breast cancer has significantly improved.[13]

Important tumor suppressor proteins, such as those produced by the breast cancer susceptibility genes, namely BRCA1 and BRCA2, are situated on chromosomes 17q21 and 13q12, respectively. BRCA1 is essential for maintaining normal cellular function and preventing abnormalities and apoptosis due to its involvement in controlling cell cycle checkpoints, centromere duplication, and genomic stability and integrity. Retinoblastoma protein and p130/107 have both been demonstrated to suppress BRCA1.[14,15,16,17,18] BRCA2 regulates recombinational repair of DNA double-strand (ds) breaks by interacting with RAD51 and DMC1, and BRCA2 mutations in these genes are correlated with a high susceptibility to invasive ductal carcinomas in breast cancer.[19] Having mutations in both BRCA1 and BRCA2 considerably increases a person’s risk of getting breast cancer.[20] Moreover, risk factors for breast cancer encompasses age, family history, reproductive characteristics, exposure to estrogen (including oral contraceptives and hormone replacement therapy), and lifestyle choices, to name just a few.[21,22]

The flavonoid hesperetin (HSP), which is present in citrus fruits, is discussed in the article as a possible targeted anti-cancer treatment agent for breast cancer, specifically concerning growth factor receptor (HER) 2-positive breast cancer.[23,24] Receptor protein HER2 is essential for cell proliferation, survival, and resistance to apoptosis; however, its overexpression or unchecked activation can promote carcinogenic processes. HSP has been demonstrated to induce apoptosis, which results in cell death and interruption of the cell cycle in HER2-positive cancer cell lines.[25] This is achieved by blocking the signaling pathway that involves the tyrosine kinase HER2. The ability of HSP to form persistent contacts at the HER2 ATP binding site suggests that it might be utilized to treat HER2-positive breast-cancer. The objective of this study was to enhance understanding the effects of HSP on MLH1 and MSH2 gene expression in BT-549 breast cancer cell lines.[26]

MATERIALS AND METHODS

BT-549 and MCF-10a cell lines were gifted by Pasteur Institute, Baghdad, Iraq. Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) powder, Dulbecco’s Modified Eagle Medium (DMEM), penicillin/streptomycin, and dimethyl sulfoxide (DMSO) were purchased from Gibco, USA. RNA extraction kit and CDNA synthesis kit were obtained from Thermo Fisher, USA, and SYBR green from Ampliqon from Denmark. Real-time polymerase chain reaction (PCR) strip was purchased from Gunster Biotech, Taiwan.

Cell culture

Two distinct breast cancer cell lines, BT-549 and MCF-10a, were employed for this study. Cell lines were purchased from the Pasteur Institute and then grown in DMEM with 10% FBS and 1% penicillin-streptomycin. Afterward, 5 mL of fresh culture medium was added to the dish. The cells were then cultured at 37°C with 5% CO₂. Each day of the week, the cell density in the culture flasks was measured, and cell passage was conducted as needed.

Cell passage

Each flask’s cell density was checked at 80% before being passed. The cells were inspected for infection, morphology, and growth rate before passing. After removing the growth medium, the cells were washed with 5 mL of phosphate buffer saline (PBS). After adding a trypsin ethylenediaminetetraacetic acid solution to the culture flask and waiting three to 5 min, the remaining live cells were removed. The cells were centrifuged, washed, and resuspended in fresh culture media before being seeded into new flasks at a predetermined density.[27] Cell density was determined by equation (I):

Cell backup and cryopreservation

The cells were separated from the culture flask bottom using 1 mL of trypsin, and the supernatant was removed for preparation of cryopreservation. The cells were then washed with 5 mL of PBS. Afterward, DMEM was utilized to resuspend the cell plate. Neobar slide was utilized for cell counting, followed by transfer of 2 × 106 cancer cells in cryovial, which involved mixture of 90% FBS, 10% DMSO, and 2 × 10⁶ cells. Following a period of 2 h at −20°C, the cryovials were then subjected to −80°C for 24 h and thereafter transferred to nitrogen tanks at − 196°C.[28]

Cell culture and treatment

HSP was dissolved in DMSO to create a stock solution. A 200 mM solution was prepared for subsequent experiments. Hemocytometers using Trypan blue and Lan dye were used to count the cells. The cells were then treated and incubated with 5% CO₂ at 37°C according to the required incubation time. All experiments were performed using cells from passage 3, and for cell treatment, 2% FBS was utilized for simulation of starvation conditions.[29]

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) cell viability test

MTT colorimetric assay was utilized for determining a cell viability. After seeding 104 cells into each well of a 96-well plate, the cells were grown at 37°C with 5% CO₂ for 48 h in 200 mL of DMEM media containing 10% FBS and different concentrations of HSP (100, 200, 400, 600, 800, and 1000 μM/mL) before being subjected to the MTT test. Following a period of 3 h, the surface culture media was removed and replaced with 150 L of MTT solution (0.5 mg/mL) in each well. The formazan precipitate was dissolved by removing the MTT solution and was replaced with 100 L of DMSO then incubated for 15 min. The resultant solution was analyzed by a spectrophotometer at 570 and 630 nm.[30] The cell viability was determined by equation (II):

Real-time polymerase chain reaction

mRNA levels in tiny samples may be detected using real-time (RT)-PCR approach because of its extreme sensitivity. Two major methods for RT-PCR are the use of fluorescent dyes or probes. This study utilized cyber green fluorescent dye. Cyber green binds to all types of dsDNA, including contaminants like primer dimers and by-products of unintended reactions. The melting curve is a useful tool for detecting impurities, primer dimers, and nonspecific DNA. The melting temperature (Tm) of all PCR products produced by a specific pair of primers is the same. The device gradually increases the Tm between 65°C and 95°C, and the fluorescence at each location is monitored to create a melting curve. The Tm is the peak of the curve.[31]

Flow cytometry and multicolor flow cytometry

Multicolor flow cytometry was employed to investigate the apoptotic effect of different concentration of HSP on BT-549 cells using annexin V and propidium iodide (PI) reagent staining. BT0549 cells were cultured and treated with different concentration (200, 400, and 600 μg/mL) of HSP, alongside a control group. Following a specified treatment period, the cells were harvested and washed. Subsequently, annexin V and PI staining solutions were prepared. The washed BT-549 cells were then incubated in the staining solution, and following the incubation period, the cells were diluted in buffer. The flow cytometer was setup with lasers and detector to detect the fluorescence emitted by annexin V and PI. The data were acquired by analyzing each stained cell suspension using a flow cytometer. The acquired data were processed and analyzed with software and gating strategies were applied to identify and quantify different cell populations.

RESULTS

The effects of the anticancer drug HSP on both normal and cancer cells were examined using the MTT test, and the levels of expression of the MLH1 and MSH2 genes were evaluated using RT-PCR. Subsequently, gene expression in the examined breast cancer cell line was evaluated using flow cytometry to determine the impact of HSP dosing.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

Seven different HSP concentrations (0, 100, 200, 400, 600, 800, and 1000 μg/mL) were tested on two separate occasions at 37°C on the breast cancer cell line BT-549 and the normal cell line MCF-10a for 48 h. Half-maximal inhibitory concentration (IC₅₀) and impact of HSP on normal and cancer cells were measured using MTT test.

HSP was shown to be cytotoxic in both BT-549 and MCF-10a cells, and the toxicity was increased with increase in doses. As the concentration of HSP was increased, reduced viability was observed, that indicated a dose-dependent decrease in cell-viability with increased HSP levels. After 48 h of treatment with HSP, the IC₅₀ was determined to be 279.2 g/mL for the cancer cell line BT-549 and 855.4 g/mL for the normal cell line. In a time and concentration dependent way, HSP suppressed the proliferation of BT-549 cell lines, with the IC₅₀ value shifting over the course of 48 h. The results of the cytotoxic effect for BT-549 cell lines indicated that HSP had a greater effect on the cancerous line than its effect on the normal cell line as shown in Figure 1.

Figure 1.

Line graph (a) and bar chart (b) of cell viability for MCF-10a and BT-549 cell lines treated with hesperetin and show IC₅₀

Real-time polymerase chain reaction test

MLH1 and MSH2 gene expressions were compared in BT-549 and MCF-10a cell lines using RT-PCR. Different concentrations of these genes’ expressions were analyzed (200, 400, and 600 μg/mL) using HSP. The results indicated that in the BT-549 cell line, the highest concentration of HSP (600 μg/mL) significantly increased MLH1 gene expression (P = 0.0427), while the other two concentrations did not show significant differences in gene expression compared to the control group (P = 0.1974 and P = 0.2520). These findings suggest that MLH1 gene is an antitumor gene that functions to correct errors in the cell’s genetic material, and its expression is positively correlated with HSP concentration. In contrast, in the MCF-10a cell line, HSP did not significantly affect MLH1 gene expression at any of the concentrations tested (P = 0.544, P = 0.899, and P = 0.633 for the 200, 400, and 600 μg/mL concentrations, respectively), as shown in Figure 2.

Figure 2.

HSP has a concentration-dependent effect on MLH1 gene expression in the breast cancer (BT-549) cell line. HSP: Hesperetin

Whereas, the expression of the MSH2 gene did not vary significantly across HSP concentrations in the MCF-10a cell line, as seen by the same median values and nonsignificant P values (P > 0.05). These results suggested that HSP modulates MSH2 gene expression in the BT-549 cell line according to concentration, but not in the MCF-10a cell line. Evidence from studies of HSP’s influence on MSH2 gene expression in breast cancer and normal cell lines suggests that the compound may, at high enough concentrations, alter the expression of anti-tumor genes, as demonstrated in Figure 3. Our results demonstrated that HSP content in breast cancer and normal cell lines is positively linked with the expression of MLH1 and MSH2. Furthermore, the findings suggested that the anti-tumor effect of HSP was more pronounced at higher concentrations (600 μM/mL) for MLH1 and (600 μg/mL, 400 μM/mL) for MSH2 in BT-549 cell line. These observations expand the understanding that HSP mat hold therapeutic potential for the treatment of breast cancer.

Figure 3.

HSP has a concentration-dependent effect on MSH2 gene expression in the breast cancer (BT-549) cell line. HSP: Hesperetin

The results revealed that the highest concentrations of HSP were associated with overexpression of the MSH2 gene in both cell lines. However, the concentration-dependent effect of HSP on MSH2 gene expression in the BT-549 cell line was different from that in the MCF-10a cell line. In the BT-549 cell line, HSP showed a concentration-dependent effect on MSH2 gene expression. As the concentration of HSP increased from 0 to 600 μg/mL, there was a significant increase in MSH2 gene expression, as indicated by the increasing median values and the statistically significant P value (P = 0.0087). Therefore, these genes are considered antitumor genes, as their increased expression is associated with a higher efficacy in correcting errors in the cell’s genetic material. Our study showed that the potent effect of HSP was more pronounced at higher concentrations (600 μg/mL) than at lower concentrations (400 μg/mL and 200 μg/mL) and the control.

Flow cytometry

Flow cytometry was utilized to explore the apoptotic effect of HSP on BT-549 cells using annexin V and PI reagent staining. This approach allows for the differentiation between early, late, and dead cells. Flow cytometric analysis revealed that HSP treatment at concentrations of 200 and 400 μg/mL caused a slight increase in early apoptotic effects in BT-549 cells, with a respective increase in population to 15.6% and 16.3%, compared to cells that were not treated. Moreover, the population count of apoptotic cells increased from 7.0% to 11.9% and dead cells from 3.3% to 8.1% after HSP treatment at concentrations of 200 and 400 μg/mL, respectively. Interestingly, the treatment with 600 μg/mL of HSP resulted in a significant increase in apoptotic cell population by 61.1% and dead cell population by 17.5%, with a remarkable reduction in cell viability down to 20.3% as shown in Figure 4.

Figure 4.

Flow cytometric analysis of apoptosis in BT549 cells treated with increasing concentrations (200, 400, and 600 μg/mL) of hesperetin. SCC: Side scatter, FSC: Forward scatter

DISCUSSION

The objective of the study was to examine and analyze the possible anticancer effects of HSP on the breast cancer cell line BT-549 and the normal cell line MCF-10a. The IC₅₀ of HSP and its influence on gene expression of the BT-549 cell line were determined using the MTT test and flow cytometry, respectively. HSP was shown to be cytotoxic to BT-549 and MCF-10a cells in a concentration-dependent manner, with increasing effects at higher doses. The IC₅₀ was determined to be 279.2 μM/mL for BT-549 after 48 h of exposure, whereas it was 855.4 μM/mL for MCF-10a. The research also showed that HSP suppressed BT-549 cell line growth in a concentration-dependent manner, with the IC₅₀ concentration changing after 48 h.

Consistent with earlier research, our findings support HSP’s anticancer potential. HSP was shown to limit breast cancer cell growth via activating the AMP-activated protein kinase pathway,[32] which in turn induced apoptosis and stopped cell proliferation. Another research shown that HSP exhibited the ability to suppress the proliferation of triple-negative breast cancer cells by triggering cell cycle arrest and death through modulation of the PI3K/Akt/mTOR signaling pathway.[33] Evidence from this and previous research suggests that HSP could have potential therapeutic applications in breast cancer applications.

Flow cytometry results demonstrated that HSP induced early apoptotic effects in BT-549 cells at concentrations of 200 and 400 μg/mL, with a respective increase in population to 15.6% and 16.3%, compared with untreated cells. In addition, the population count of apoptotic cells increased from 7.0% to 11.9% and dead cells from 3.3% to 8.1% after HSP treatment at concentrations of 200 and 400 μM/mL, respectively. A significant increase in apoptotic cell population by 61.1% and dead cell population by 17.5% was observed after treatment with 600 μM/mL of HSP, with a remarkable reduction in cell viability down to 20.3%.

Consistent with previous research, this study confirms HSP’s apoptotic effect on breast cancer cells. Researchers found that HSP activated the p53 pathway, leading to the death of breast cancer cells.[34] The Bcl-2/Bax signaling pathway was also modulated by HSP, as shown in a separate study,[35] demonstrating that it caused apoptosis in breast cancer cells. Together with the current study, these findings suggest that HSP-induced apoptosis could be a promising therapeutic approach for treating breast cancer.

Moreover, flow cytometry analysis confirmed that a substantial rise in the apoptotic cell population and the dead cell population occurred after treating BT-549 cells with 600 M/mL HSP. These results indicate that HSP, possibly as a therapeutic strategy for treating breast cancer, induces apoptotic effects in a concentration-dependent manner. Further investigation is necessary to elucidate the mechanism underlying the action of HSP in breast cancer cells, as well as to determine its impact on other types of cancer. HSP’s anticancer activities have been documented on a number of cancer cell lines before.[35,36,37,38,39] HSP’s capacity to cause apoptosis, cell cycle arrest, and suppression of cell proliferation, migration, and invasion have all been implicated as modes of action in cancer cells. The signaling pathways PI3K/Akt, nuclear factor-B, mitogen-activated protein kinase, and Wnt/-catenin, among others, have all been documented to be modulated by HSP, which has anti-cancer effects.[35,36,38,39]

CONCLUSION

HSP, a flavonoid found in citrus fruits, has been extensively studied for its potential anti-cancer properties, particularly on the BT-549 human breast cancer cell line. The results from in vivo experiments demonstrated that HSP could induce programmed cell death and inhibit cancer cell proliferation. In addition, HSP has been found to induce cellular differentiation, which can further help suppress cancer cell growth. However, the optimal concentration of HSP needed to achieve these effects remains unclear, with some studies suggesting higher concentrations are required, while others demonstrate lower concentrations, such as 279.2 μg/mL, to be effective in reaching the IC₅₀ of the BT-549 cell line. The mechanisms through which HSP exerts its anticancer properties are still unclear, and further study is required to establish the ideal concentration and dose for therapeutic applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.