Hyperforin-mediated anticancer mechanisms in MDA-MB-231 cell line: insights into apoptotic mediator modulation and caspase activation

Abstract

Breast cancer has been recorded as a frequent malignant cancer. The side effects of current chemotherapy drugs are still a severe problem. Hence, naturally occurring phytochemicals can be a possible solution to overcome this issue. Therefore, naturally occurring hyperforin was used to assess its anticancer effect against triple-negative breast cancer cells (MDA-MB-231) and its effectiveness against taxol (clinically used drug). Both compounds’ anticancer activity and mechanism of action were evaluated by analysing various approaches such as  ELISA, flow cytometry and PCR microarray. Our results showed a lower IC50 of hyperforin (7.18 µg/mL) compared to taxol (7.91 µg/mL, P < 0.05) and hyperforin has almost similar anticancer effects as taxol against MDA-MB-231 cells. PCR microarray results showed that hyperforin induced the expression of caspases activator leading to a caspases-dependent cell death pathway whereas taxol uses TNF-mediated cell death. The results suggest that hyperforin may act as a good cell death-inducing agent.

ABBREVIATIONS: TNBC: Triple negative breast cancer; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; ROS: Reactive oxygen species; 5-FU: Fluorouracil; TRAIL: TNF-related apoptosis-inducing ligand; AKT: Protein kinase B; COX-2: Cyclooxygenase-2; PCR: Polymerase chain reaction; Bax: Bcl-2-associated X protein; BCL-2: B-cell lymphoma 2; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; mRNA: Messenger ribonucleic acid; MCL1: Myeloid-cell leukemia 1; LDH: Lactate dehydrogenase; ELISA: Enzyme-linked immunosorbent assay; MMP: Mitochondrial membrane potential; MCP-1: Monocyte chemoattractant protein-1; ATCC: American Type Culture Collection; RPMI: Roswell Park Memorial Institute; XIAP: X-linked inhibitor of apoptosis protein; FBS: Fetal bovine serum; MCL-1: Myeloid-cell leukemia 1; DMEM: Dulbecco’s Modified Eagle Medium; DMSO: Dimethyl sulfoxide; CASP3: Caspase 3; RPMI: Roswell Park Memorial Institute; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal bovine serum; h: Hour; TNF-α: Tumour necrosis factor alpha; P53: Tumor protein P53; min: Minutes; cps: Count presecond; OD: Optical density; rpm: Revolutions per minute; PI: Propidium iodide; SD: Standard deviation; DDR: Death Domoin Receptor; TRADD: Tumor necrosis factor receptor type 1-associated DEATH domain; HRK: Harakiri, BCL2 Interacting Protein; RIPK2: Receptor-interacting serine/threonine-protein kinase 2; BIK: BCL2 interacting killer; DCF: 2’, 7’ –dichlorofluorescein; DCF-DA: Dichlorofluorescin Diacetate; FADD: Fas Associated Via Death Domain; TNFRSF: Tumor necrosis factor receptor superfamily; FITC: Fluorescein isothiocyanate; DMSO: Dimethyl sulfoxide; CFLAR: CASP8 And FADD Like Apoptosis Regulator; BID: BH3-interacting domain death agonist; BIRC: Baculoviral IAP repeat-containing protein; CIDEA: Cell death activator CIDE-A; CIDEB: Cell Death Inducing DFFA Like Effector B; BrdU: Bromodeoxyuridine; RFU: Relative fluorescence unit; NF-kB: Nuclear Factor kappa-light-chain-enhancer of activated B cells; %: Percentage; µL: microliter; µM: Micromolar; °C: degree Celcius; ng/mL: Nanograms per millimeter; mg/mL: Milligrams per milliliter; µg/mL: Micrograms per milliliter; ΔΨm: Mitochondrial membrane potential; TNFα: Tumour necrosis factor α; Bcl-xL: B-cell lymphoma-extra large; MDR1: Multidrug Resistance Mutation; AIB1: Amplified in breast cancer 1; LTBR: Lymphotoxin Beta Receptor

 

Highlights

• Utilization of hyperforin as anticancer agent against triple negative breast cancer.

• Hyperforin exhibited a lower IC50 compared to taxol in inhibiting growth of MDA-MB-231 cells.

• Hyperforin shows similar potential anticancer activity as taxol.

• Hyperforin uses mitochondrial death pathway to induce cell death in MDA-MB-231 cells.

KEYWORDS:

• Apoptosis
• breast cancer
• caspase
• hyperforin
• paclitaxel

1. Introduction

Breast cancer continues to pose a significant paradox within the field of oncology. Despite substantial efforts to combat this disease, it remains the most prevalent cancer worldwide, affecting nearly 2.1 million women annually. GLOBOCAN estimates that the world incidence and mortality rate of breast cancer for 2020 were 47.8 and 13.5 per 100,000 people, correspondingly [1]. Malaysian women are not exempt from this burden, as breast cancer accounts for 32.1% of cancers in females [2] and one in 30 women is expected to succumb to breast cancer in their lifetime [3]. One particularly aggressive subtype of breast cancer is triple negative breast cancer (TNBC), characterized by the absence of three key breast cancer receptors: oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC exhibits a higher likelihood of relapse episodes and is considered more aggressive compared to other breast cancer variants. Alarmingly, the 5-year mortality rate for advanced TNBC has stagnated at 40%, which urges a more sophisticated option of treatment to be devised [1].

Surgery is often the curative option for early-stage breast cancer. At more advanced stages, adjuvant treatment with radiation therapy, chemotherapy, hormonal therapy, stem cell transplant, immunotherapy, and targeted biologic therapy are the mainstay choices [4]. Treatment for TNBC is limited to surgery and non-targeted therapy which majorly comprised of cytotoxic chemotherapy. However, it is plagued with fatal downsides such as rapid development of chemoresistant cells and lethal toxicity [5], which is crucially in need of a novel modality.

In the past decade, the spotlight has been on natural products derived from either plants, animals or microorganisms as anticancer agents as they possess low toxicity, ease of accessibility and minimum risk of causing cancer cells to become resistant to them. There are a few clinically used antineoplastic drugs derived from and inspired by natural products. For example, camptothecin, derived from the plant Camptotheca acuminate, and its derivatives topotecan (Hycamtins), irinotecan (Camptosars) and belotecan (Camptobells). These drugs act on topoisomerase I and deoxyribonucleic acid (DNA) cleavage and inhibit the ligation accounting in DNA strand breakage [6,7]. Other examples include vinca alkaloids and its analogues such as vinblastine (Velbans), vincristine (Oncovins), vindesine (Eldisines) and vinorelbine (Navelbines), are known to disrupt cell division and formation of microtubules [8,9]. The exploration of natural products as potential sources of anticancer agents provides a promising avenue for the development of novel therapeutics with enhanced efficacy and potentially reduced adverse effects.

Hyperforin is an active phytochemical derived from the terpenoids class and mostly accumulated in the leaves and flowers of the Hypericum perforatum, which is also known as St John’s wort [10]. Numerous reports have highlighted its potential as an anticancer agent, as it has demonstrated the ability to suppress the growth of various cancer cell lines through apoptotic mechanisms [11–13]. Intriguingly, hyperforin has exhibited comparable effects to the potent drug taxol in inhibiting the growth of autologous MT-450 breast carcinoma in immunocompetent Wistar rats without significant signs of toxicity [10]. Although the precise mechanisms underlying hyperforin’s action against breast cancer, particularly TNBC, are not yet fully understood, accumulating evidence supports its potential therapeutic relevance. Hence, the aim of the present study is to investigate the capacity of hyperforin to induce cell death in MDA-MB-231 cells, a cell line representing TNBC, and compare its effectiveness to that of taxol, a clinically used drug. This research endeavours to shed light on the significance of hyperforin in the treatment of breast cancer.

2. Materials and methods

2.1. Compounds preparation

Hyperforin is a bioactive ingredient of the St. John’s wort plant. It was reconstituted in tissue culture grade dimethyl sulfoxide (DMSO) to 1 mg/ml, aliquoted into smaller volumes and kept at −20°C till further use. Further dilution of hyperforin was achieved in complete Dulbecco’s modified eagle medium (DMEM) before any assay. The concentration of hyperforin used to determine IC50 (minimum concentration needed to inhibit 50% of growth of cells) ranged between 0 and 10 µg/mL. Meanwhile, taxol has been applied in this study as a positive control isolated from Taxus yannanensis and it was dissolved in tissue culture grade DMSO to a concentration of 50 mg/ml. It was further diluted in complete DMEM medium ranging from 0 to 8 µg/mL.

2.2. Cell culture

MDA-MB-231 and a normal breast cell line (MCF10A) utilized in this study were bought from the American Type Culture Collection (ATCC). MDA-MB-231 cells were extracted from the pleural effusion of a patient with invasive ductal carcinoma. The cell has a diameter of 18.9 ± 0.4 μm and is highly aggressive and invasive. The MDA-MB-231 cells were preserved in DMEM supplemented with 2 mM glutamine and 15% fetal bovine serum (FBS) along with antibiotics such as penicillin (100 IU/mL) and streptomycin (100 ng/mL) in the incubator at 37°C with 5% CO₂ supply. These cells were subcultured every three days once, when they reached confluency, and seeded at a density between 1 and 3 × 10⁴ cells/cm². Meanwhile, the human non-tumourigenic breast epithelial MCF10A cell line was used as a control in the study. The MCF10A cells were grown in complete medium containing Ham’s F-12 and DMEM with 20 ng/mL epidermal growth factor (EGF), 10 μg/mL insulin, 100 ng/mL cholera toxin, and 250 ng/μL hydrocortisone. The effectiveness of the medium was also increased with 10% (v/v) FBS, penicillin (100 IU/mL), and streptomycin (100 ng/mL). The confluent MCF10A cells were detached using 0.05% trypsin and then seeded at a density of 2.5 × 10⁴ cells for further experiment. The cells were maintained in an incubator at 37°C with a CO₂ supply of 5%. All cell culture reagents were procured from Gibco, Carlsbad, California.

2.3. In vitro treatment of cells with hyperforin and taxol

The MDA-MB-231 and MCF10A cells were maintained in complete DMEM and, Ham’s F-12 and DMEM, respectively, for 18 h. The next day, the media was replenished. Both cancer and normal cells were treated with different working concentrations of hyperforin and taxol, respectively, for 24 and 48 h prior to MTT assay.

2.4. Cell viability assay: MTT

The viability of MDA-MB-231 cells upon treatment was analysed using MTT reagent (Sigma-Aldrich, Malaysia) and quantified using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In brief, MDA-MB-231 cells were plated in triplicates on a 96-well plate at a density of 5 × 10⁴ cells/well in 100 μL of culture medium and incubated for 18 h. Various working concentrations of hyperforin and taxol ranging from 8, 7, 6, 5, 4, 3, 2, to 1 μg/mL were added to the cells, respectively, and incubated for 24 and 48 h. The MTT (5 mg/mL) was added to the cells and incubated further for 4 h in a dark condition and lastly, DMSO was added to the formazan followed by quantification of the cell viability. The percentage of cell viability was compared with that of non-treated control cells, which were arbitrarily assigned a viability of 100%. Percentage of cell survival was plotted against the concentration of the compounds (μg/mL). Based on findings derived from the MTT assay, only one incubation period (24 h) of natural compounds was selected. Similar experiments were repeated using MCF10A to confirm the toxicity and safety of these natural compounds against the growth of normal breast cells.

2.5. Morphological changes assay: Giemsa

The morphological changes in MDA-MB-231 cells were evaluated using Giemsa staining whereby the cells were plated in 6-well plates and treated with hyperforin and taxol for 24 and 48 h, respectively. The medium from cell culture was removed, and the cells were fixed with methanol and stained with 4% Giemsa stain for 15 mins. The solution was then removed, and the cells were observed under a Zeiss AX10 inverted phase-contrast microscope with epifluorescence and a digital imaging system (Carl Zeiss Microscopy GmbH, Gottingen, Germany).

2.6. Reactive oxygen species (ROS) assay

The potential formation of cellular ROS after in vitro treatment with the IC50 of hyperforin and taxol in MDA-MB-231 cell culture was measured using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA)-based assay using a commercially available kit (Abcam, Cambridge, UK). The formation of cellular ROS was measured and recorded at 485 nm (excitation) and 535 nm (emission) using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The non-treated MDA-MB-231 cells were included as a negative control.

2.7. Apoptosis assay

The apoptotic effect of hyperforin and taxol on MDA-MB-231 cells was determined with the 7-Aminoactinomycin D (7-AAD) and Bromodeoxyuridine (BrdU) double staining assay performed using BD FACSCanto II (BD Biosciences, San Jose, CA, USA) flow cytometry using a BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. The samples were analyzed using the FACSDiva software (BD Biosciences).

2.8. Cell cycle assay

The PI and BrdU staining were used to examine the MDA-MB-231 cell arrest pattern following in vitro treatment by hyperforin and taxol. The experiments were carried out using the BD BrdU FITC kit (BD Pharmingen, San Diego, CA, USA). Meanwhile, quantification and analyses were carried out using flow cytometry and FlowJo software (BD Pharmingen, San Diego, CA, USA). The non-treated MDA-MB-231 cells were used as negative controls in this experiment.

2.9. DNA fragmentation assay

The ability of hyperforin and taxol to dissociate total DNA of MDA-MB-231 cells after the in vitro treatment was analyzed via DNA fragmentation assay using 1.4% agarose gel. The gel was later visualized using a UV light source and imaged using a Gel Doc XR gel documentation system (Bio-Rad, Hemel Hempstead, UK). The non-treated MDA-MB-231 cells were used as negative controls in this assay.

2.10. Mitochondrial membrane potential (MMP) assay

The ability of hyperforin and taxol to reduce the activity of mitochondria after the in vitro treatment on MDA-MB-231 cells was determined using the tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial membrane potential detection kit (Biotium, CA, USA). The accumulation of JC-1 in living cells was used to determine the mitochondrial membrane potential which will indirectly represent the condition of mitochondria in the cell. Briefly, MDA-MB-231 cells were seeded into the 96-well plates and grown for 18 h. This was followed by induction of in vitro treatment with hyperforin and taxol accordingly for 24 h. The cell culture medium was replaced with JC-1 reagent at working concentration (1X) for 15 min. The reagent was then removed, cells washed with saline buffer, and fresh saline buffer was added. The plate was read at red fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) using a fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The ratio of red fluorescence divided by green fluorescence was determined whereby the ratio of red to green fluorescence is decreased in dead cells and cells undergoing apoptosis compared to the healthy cells. The non-treated MDA-MB-231 cells served as controls.

2.11. Lactate dehydrogenase (LDH) assay

The ability of hyperforin and taxol to release LDH from MDA-MB-231 cells were examined using a LDH activity assay kit (BioVision, CA, USA). In brief, the cells were treated with IC50 of hyperforin and taxol for 24 h, harvested and washed with ice-cold saline buffer. Homogenization of cells was carried out in a cold assay buffer and the supernatant was stored for further investigation. A nicotinamide adenine dinucleotide (NADH) standard curve was prepared before sample analysis. The reaction mixture containing LDH assay buffer and LDH substrate mix was added into each standard, sample, and positive control sample wells, mixed well and output measured immediately at OD 450 nm on a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The OD measurement was taken in a kinetic mode, every 2–3 mins, for at least 30–60 mins at 37°C protected from light. Untreated cells were also tested to check the actual effectiveness of hyperforin.

2.12. Cyclooxygenase-2 (COX-2) assay

The release of cell-based COX-2/PGHS-2 from hyperforin and taxol treated MDA-MB-231 cells was determined using a commercial sandwich ELISA (R&D Systems, Abingdon, United Kingdom). The cells were subjected to in vitro treatment with hyperforin and taxol for 24 h. The supernatants were collected and added to the plate pre-coated with anti-COX-2 together with standards. The unbound COX-2 was washed, and biotin-conjugated anti-COX-2 was added to the wells. After washing, avidin conjugated horseradish peroxidase (HRP) was added followed by washing of unbound avidin-enzyme reagent. Later, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added to the wells to facilitate colour change in proportion to the amount of bound-COX-2 in the initial step. The enzyme–substrate reaction was terminated by adding sulphuric acid H₂SO₄ and the colour change was measured at 450 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of COX-2 was estimated by comparing the optical density (OD) of samples with the standard curve. The non-treated cells were used as negative control.

2.13. Isolation of ribonucleic acid (RNA) and complementary DNA synthesis

Hyperforin and taxol treated cancer cells were harvested and subjected to RNA isolation following 24 h of treatment. The isolation of total RNA was performed through RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Briefly, the cells were lysed and homogenized with the help of RLT buffer containing 2-mercaptoethanol followed by addition of ethanol to the lysate needed for optimal binding conditions on silica membrane. The lysates were then added into RNeasy mini column whereby only RNA was bound to it and all unneeded components were removed. Finally, the quality of the isolated RNA was assessed using a nanospectrophotometer (Implen, München, Germany). Only good quality RNAs were used for microarray analysis. For the cDNA synthesis, 1 µg of total RNA from each sample was used for first strand cDNA synthesis using RT² First Strand Kit (Qiagen, Valencia, CA, USA) according to manufacturers’ protocols. Concisely, mixture reaction containing buffer GE, 5x buffer BC3, RE3 reverse transcriptase mix, control P2 and nuclease-free water were prepared first and added to the RNA sample to synthesis the cDNA in a CFX96 touch real-time PCR detection system. (Biorad, Hercules, CA, USA). The transcribed cDNA was immediately analysed using PCR microarray.

2.14. PCR microarray

RT² profiler PCR arrays (PAHS-012Z) that contained 84 pro and anti-apoptotic key genes involved in the apoptosis pathway plus 5 housekeeping genes and 7 quality controls (Qiagen, Valencia, CA, USA) were used to determine the pathway-focused gene expression profiling and regulation of apoptosis in hyperforin and taxol treated MDA-MB-231 cells. Three replicates from all the three groups (hyperforin treated MDA-MB-231 cells, taxol treated MDA-MB-231 cells and untreated MDA-MB-231 cells) were used in 9 arrays, respectively. Briefly, 102 µl of cDNA from each sample were mixed with 1350 µl of 2× RT² SYBR Green Master mix (Qiagen, Valencia, CA, USA) and 1248 µl of RNase-free H₂O to the final volume of 2700 µl. A total of 25 µl of sample were used for each well of the RT2 Profiler PCR Array plate. Quantitative Real-Time PCR was performed in a CFX real time PCR thermocycler (Biorad, Hercules, CA, USA). After 10 min of activation at 95°C, 45 cycles were performed with the following cycle parameters: 15 sec at 95°C, 1 min at 60°C (acquisition). After finishing the last cycle, a melting curve analysis was performed. Dissociation curves were performed to verify that only a single product was amplified. Standard −ΔΔCt method was used for determining changes in gene expression during the development. GeneGlobe (Qiagen, Valencia, CA, USA) was used to calculate experimental results including statistical analysis. The data was normalized with beta-actin, house-keeping gene. Isolation and transcription quality controls were evaluated using Gene Globe. Several heatmap with dendrograms indicating co-regulated genes were obtained from the software. Data was represented as the fold-regulation between the experimental groups and the control cells. Fold change values <1 indicate a negative result or downregulation, and the fold -regulation is the negative inverse of the fold change.

2.15. Statistical analysis

GraphPad Prism 6.0 software (GraphPad, La Jolla, CA) were used to analyze the entire data including the comparison of within and/or between variable and significance through one-way ANOVA test. Data was presented as mean ± standard error of the mean (SEM) where n refers to the number of independent experiments. The fitness of the groups with the normal distribution was assessed by the Kolmogorov Smirnov test. The level of significance was considered at P < 0.05, and measures represented by *P < 0.05, **P < 0.01 and ***P < 0.001.

3. Results

3.1. Cytotoxicity of hyperforin by MTT assay

Hyperforin was tested for toxicity properties against MDA-MB-231 following 24 and 48 h of in vitro treatment, in comparison to the treatment with taxol (positive control). Hyperforin was found to significantly inhibit the growth of MDA-MB-231 cells in a dose-proportional manner at both treatment points. It was also found that non-significant and lower IC50 (7.18 µg/mL) was required at 24 h compared to 48 h (7.77 µg/mL), however it was not statistically significant (Figure 1(A)). Besides, taxol also demonstrated similar trend as hyperforin in suppressing the cell proliferation (Figure 1(B)). The IC50 of taxol was also lower for 24 h of treatment (7.91 µg/mL) compared to 48 h of treatment (7.99 µg/mL). Interestingly, the anti-proliferative properties of hyperforin against MDA-MB-231 cells was found to be significant compared to taxol following 24 h of treatment (P < 0.05). However, the effectiveness or strength of hyperforin was found to subdue after 48 h of treatment whereby growth of MDA-MB-231 cells was inhibited at the rate of 40% (8 µg/mL) and 3% (1 µg/mL) while at 24 h of treatment, the cell growth has been suppressed at the rate of 49% (8 µg/mL) and 5% (1 µg/mL). However, the effectiveness of taxol did not subdue even at 48 h of treatment. Hence, 24 h of treatment was applied for both hyperforin and taxol in future experiments. As expected, both hyperforin and taxol have shown little to no toxicity towards breast cell line, MCF-10A at 24 and 48 h of treatment. The effectiveness of hyperforin was seen to be significant (P < 0.05) in comparison to taxol (Figure 1(C)). Hence, MCF10A was not used for further investigation (results were not shown).

Figure 1. Effect of hyperforin and taxol on the viability of MDA-MB-231 cell line. The viability of MDA-MB-231 cells upon exposure to hyperforin and taxol has been performed via MTT assay. (1A) Lower inhibitory concentration of hyperforin was needed to inhibit MDA-MB-231 cells at 24 hours compared to 48 hours. (1B) Lower inhibitory concentration of taxol was needed to inhibit MDA-MB-231 cells at 24 hours compared to 48 hour. (1C) Significant difference in the viability of MDA-MB-231 cells treated with hyperforin and taxol for 24 hours. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, compared with cells using Student's paired t-test.

3.2. Hyperforin reduces MDA-MB-231 cell volume

The untreated MDA-MB-231 cells showed intact cell structures with long as well as thin spindle-shaped cells. Some cells formed clumps or aggregates, which is a common behaviour of cells in culture (Figure 2(A)). Meanwhile, cells treated with hyperforin alone exhibited distinctive morphological signs of apoptosis such as became small, isolated, or detached, showing characteristic signs of blebbing (formation of membrane protrusions), resulting in reduction of cell volume (Figure 2(B)). Similar observation was seen in the cell treated with taxol (Figure 2(C)). These morphological changes are indicative of apoptotic cell death, which is a programmed form of cell death.

Figure 2. Morphological changes in MDA-MB-231 cells. The untreated cells were aggregated and clustered as monolayer forms (Figure 2A). Early morphological changes during apoptosis such as cell shrinkage and membrane blebbing in hyperforin treated MDA-MB-231 cells (Figure 2B). Cells treated with taxol exhibited early apoptosis signs like membrane blebbing and cell shrinkage (Figure 2C). Similar cellular morphology was observed in three independent experiments (magnification × 100).

3.3. Hyperforin increases secretion of ROS in MDA-MB-231 cell culture

Hyperforin was then tested for the ability to induce cell death mediator such as ROS after the in vitro treatment. As a result, hyperforin was found to induce significant level of ROS secretion in MDA-MB-231 cells after the 24 h treatment similar to those of taxol whereby higher (90 cps, count per second) was recorded compared to untreated cells (30 cps). As expected, a higher amount of ROS secretion was found in cell culture treated with taxol (105 cps) compared to hyperforin. Similar to the untreated MDA-MB-231 cell culture, the amount of ROS produced in 0.1% DMSO treated MDA-MB-231 cell culture remained same (Figure 3).

Figure 3. Effect of hyperforin and taxol on the secretion of reactive oxygen species. ROS assay executed by spectrometry-based experiment revealed excessive levels of reactive oxygen species were produced after the treatment of hyperforin, and taxol compared to untreated MDA-MB-231 cell culture. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, ***P < 0.001 compared with cells using Student's paired t-test.

3.4. Hyperforin induces formation of apoptotic cells in MDA-MB-231 cell culture

The activity of hyperforin against MDA-MB-231 cells was later tested by determining occurrence of apoptotic cells after the in vitro treatment. Based on the findings, hyperforin treated cells established the presence of 58% apoptotic cells compared to the 62% apoptotic cells in taxol treated cells, the occurrence of cell death by taxol was statistically significant compared to hyperforin (P < 0.001) (Figures 4 and 5).

Figure 4. Effect of hyperforin and taxol on the formation of apoptotic cells. Apoptotic cell was acquired by Tali® Apoptosis Kit showed elevation of apoptosis event in MDA-MB-231 cells culture after treatment of hyperforin and taxol for 24 h, respectively. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, ***P < 0.001 compared with cells using Student's paired t-test.

3.5. Hyperforin exhibits cell cycle arrest at S phase

The inhibitory act of hyperforin on MDA-MB-231 was further performed to determine the occurrence of cell cycle arrest after the treatment. It was found that about 48% of cells from hyperforin treated MDA-MB-231 cells were arrested at S phase compared to untreated (21%) and taxol treated MDA-MB-231 cells (18%) (P < 0.001). On the other hand, taxol induced cell arrest at G0/G1 phase in which accumulation of 52% of cells was seen compared with untreated (45%) and hyperforin treated MDA-MB-231 cells (39%) (P < 0.001). In addition, the formation of apoptotic cells in the sub-G0/G1 phase of treated MDA-MB-231 cells was evident whereby an increase of 15.8% and 20.1% from hyperforin and taxol treated cells, respectively, was recorded compared to 5% of apoptotic cells from untreated cells (Figures 4 and 5). Meanwhile, the formation of apoptotic cells between taxol and hyperforin treated MDA-MB-231 was highly significant (P < 0.001) (Figure 6).

Figure 5. Effect of hyperforin and taxol on the formation of apoptotic cells. Apoptotic cells were identified by employing 7-AAD and BrdU, which demonstrated an increase in apoptosis activity in the MDA-MB-231 cells culture following treatment with hyperforin and taxol for 24 and 48 hours, respectively. Figure 5A depicts MDA-MB-231 cells that have not been treated, Figure 5B depicts MDA-MB-231 cells treated with 0.1% DMSO, Figure 5C depicts MDA-MB-231 cells treated with hyperforin, and Figure 5D depicts MDA-MB-231 cells treated with taxol. Apoptotic cells are represented by the blue cell population, whereas dividing cells and possibly dead cells are represented by the red cell population.

3.6. Hyperforin activates a very slight DNA laddering in MDA-MB-231 cells

The occurrence of DNA fragmentation after treatment of MDA-MB-231 cells with hyperforin was tested since the secretion of excessive ROS could lead to activation of DNA fragmentation whereby DNA degradation in a ladder-like pattern which indicates ultimate cell death can be observed. As a result, a clear DNA degradation has been demonstrated in taxol treated MDA-MB-231 cells which showed late-stage cytotoxic effects of taxol, however, a very slight formation of DNA degradation with a faint DNA ladder pattern was observed in hyperforin treated cells. As expected, no DNA degradation was observed in the untreated and 0.1% DMSO treated MCF-7 cells which were included as controls (Figure 7).

Figure 6. Effect of hyperforin and taxol on the cell cycle of MDA-MB-231 cells. Treated cells stained with PI and BrdU prior to flow cytometry analysis was performed in order to scrutinize their cell cycle pattern. Hyperforin was found to arrest cells at S phase meanwhile taxol arrested cells at G0/G1 phase. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, ***P < 0.001 compared with cells using Student's paired t-test.

3.7. Hyperforin activates the vanishing act of MMP in MDA-MB-231 cells

A vanishing act of MMP leads to the induction of apoptosis in anticancer natural compounds-treated cancer cells. The anticancer effect of hyperforin in MDA-MB-231 cell culture was further proven by investigating the loss of MMP. The findings revealed significant reduction of the ratio of red to green fluorescence emitted by JC-1 on hyperforin (P < 0.001) and taxol (P < 0.001) treated cells in comparison to untreated cells and 0.1% DMSO treated cells (Figure 8), indicating the ability of hyperforin to cause loss of MMP and damage to cell membrane of MDA-MB-231 cells. Nevertheless, the loss of MMP in MDA-MB-231 cell culture caused by hyperforin and taxol, respectively, was not significant.

Figure 7. Effect of hyperforin and taxol on the DNA of MDA-MB-231 cells. DNA fragmentation was observed through the gel electrophoresis. DNA degradation was apparent in taxol treated MDA-MB-231 cells seen in 1.4% agarose gel electrophoresis. A very slight DNA fragmentation pattern hyperforin treated MDA-MB-231 cells compared with untreated and 0.1% DMSO treated cells.

3.8. Hyperforin increases the release of soluble LDH in supernatant culture of MDA-MB-231 cells

Release of a soluble yet stable enzyme, LDH, through damaged cell membrane into the surrounding extracellular space indirectly indicates damage in permeability of a membrane and occurrence of early cell death in the treated cancer cell culture, which can be used to detect cytotoxicity of anticancer extracts. The findings showed that significant amount of LDH was released in supernatants derived from hyperforin and taxol treated MDA-MB-231 cell culture compared with untreated and 0.1% DMSO treated cell culture (P < 0.001) (Figure 9). Although hyperforin promoted a slightly higher release of LDH compared with taxol, it was not significant.

Figure 8. Effect of hyperforin and taxol on mitochondrial membrane potential of MDA-MB- 231 cells. Treated cells undergo staining for MMP analysis using the tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial membrane potential detection kit. Hyperforin and taxol reduced the mitochondrial membrane integrity of MDA-MB-231 cells. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, ***P < 0.001compared with cells using Student's paired t-test.

3.9. Hyperforin decreases the activity of COX-2 in MDA-MB-231 cell culture

It has been said that upregulation of COX-2 in tumour cells will lead to aggressive growth of tumour and inhibition of apoptosis. Similarly, untreated and 0.1% DMSO treated MDA-MB-231 cell culture were seen to express high amount of COX-2 meanwhile treatment with hyperforin and taxol, respectively, were found to reduce a significant amount of COX-2 in MDA-MB-231 cell culture (42 relative fluorescence unit, RFU) compared to taxol (45 RFU) treated MDA-MB-231(P < 0.001) (Figure 10). However, no significant COX-2 inhibitory effect was found in between taxol and hyperforin treated cells culture.

Figure 9. Effect of hyperforin and taxol on the release of LDH the MDA-MB-231 cells. Treated cells were assayed using LDH assay and measured spectrometrically to obtain release of LDH level. Higher cytotoxicity and release of LDH in hyperforin and taxol treated MDA-MB-231 cells was detected. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labeled as *P < 0.05 and **P < 0.01, ***P < 0.001compared with cells using Student's paired t-test.

3.10. Hyperforin activates genes related to mitochondrial and death receptor pathway

Treatment with hyperforin has enhanced the expression of genes related to death receptor pathway, mainly, death domain containing receptor (DDCR) genes, such as CRADD, TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25 and TRADD compared with untreated MDA-MB-231 cells. However, treatment with hyperforin was also found to diminish the expression of other pro-apoptotic genes, such as DAPK1 and FADD. Meanwhile, taxol had greatly induced expression of TNFRSF11B as compared with untreated MDA-MB-231 cell (Figure 11). It also upregulated several genes such as TNFRSF21, TNFRSF25, CRADD, TNFRSF10B and TNFRSF1A. However, certain death domain receptors genes, TNFRSF10A, TRADD, DAPK1, TNFRSF1B and FADD were downregulated in taxol treated cells (Figure 10).

Figure 10. Effect of hyperforin and taxol on the COX-2. Treated cells were subjected to COX-2 analysis via ELISA. The inhibition of COX-2 levels by hyperforin and taxol treated MDA-MB-231 cell culture. Data are mean ± S.E.M. of n = 3 independent experiments carried out in triplicates. Statistically significant differences are labelled as *P < 0.05 and **P < 0.01, ***P < 0.001compared with cells using Student's paired t-test.

Similarly, genes related to extracellular apoptotic signals such as CFLAR, and DAPK1 were also downregulated in hyperforin, and taxol treated MDA-MB-231 cells except for TNFRSF25 (Figure 12). Meanwhile, no changes were observed in expression of CFLAR and DAPK1 in both treated MDA-MB-231 compared with untreated cells (Figure 11). Induction of apoptosis during the treatment with hyperforin could have also been contributed by expression of DNA damage and repair related genes, whereby ABL1 was highly upregulated followed by CIDEA, CIDEB and TP73 compared to untreated cells. Likewise, taxol was found to induce ABL1, TP73 and CIDEA compared to untreated cells but downregulated CIDEB and TP53 (Figure 13).

Figure 11. Fold change of gene expression of death domain receptors in the MDA-MB-231. Gene expression of death domain receptors in treated cells were investigated by PCR microarray analysis. Increase in gene expression of death domain receptors in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

Figure 12. Fold change of gene expression of extracellular apoptotic signal receptors in the MDA-MB-231. Gene expression of extracellular apoptotic signal receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of TNFRSF25 in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

The expression of pro-apoptotic caspases associated with mitochondrial death pathway were evident after the treatment with hyperforin whereby upregulation of several caspases (CASP1, CASP10, CASP2, CASP3, CASP4, CASP5, CASP7, CASP8, CASP9) as well as PYD and CARD domain containing (PYCARD) were seen. Similarly, CASP1 was highly upregulated together with the other caspases, CASP10, CASP14, CASP2, CASP3, CASP5, CASP7, CASP8, CASP9 and PYCARD after the treatment of MDA-MB-231 cell culture with taxol (Figure 14). High expression of caspase activators such as AIFM1, APAF1, BCL2L10, DFFA, and NOD1 in both hyperforin and taxol treated cells could have contributed to the expression of caspases (Figure 15).

Figure 13. Fold change of gene expression of DNA damage and repair receptors in the MDA-MB- 231. Gene expression of DNA damage and repair receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of certain DNA damage and repair receptors in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

Figure 14. Fold change of gene expression of caspases receptors in the MDA-MB-231. Gene expression of caspases receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of caspases in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

Additionally, other pro-apoptotic genes such as BIK followed by TNFSF8, BCL10, BID, GADD45A, TNFRSF9, CYCS, FASLG, LTA, BAK1, TNFSF10, TP53BP2, TRAF3 and DIABLO meanwhile TNF, BAD and BCL2L11 were upregulated and downregulated, respectively, in both treated cells (Figure 16). Additionally, positive regulators of apoptosis including CD70, LTBR, and RIPK2 were activated after the treatment with hyperforin and taxol, respectively. Meanwhile, CD40, TRAF2 and HRK were found to be downregulated (Figure 17). An overall fold changes of gene expression has been described in Figure 1.

Figure 15. Fold change of gene expression of caspase activators receptors in the MDA-MB-231. Gene expression of gene expression of caspases receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of caspases activators receptors in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

Figure 16. Fold change of gene expression of pro-apoptotic receptors in the MDA-MB-231. Gene expression of gene expression of pro-apoptotic receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of pro-apoptotic receptors in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

4. Discussion

Here, we demonstrated the occurrence of cell death in MDA-MB-231 cells that was confirmed by the loss of membrane asymmetry, phosphatidylserine (PS) externalization, cleavage of anti-apoptotic Bcl-2 family proteins, caspase activation, mitochondrial membrane potential, cytochrome C release, alert in cell cycle, nuclear fragmentation, and apoptotic body formation in response to hyperforin and taxol, respectively [14]. Our findings strongly imply that hyperforin exhibits substantial potential, similar to taxol, as an effective chemotherapeutic agent against triple-negative breast cancer Figure 18 and Figure 19.

Figure 17. Fold change of gene expression of positive regulator of apoptosis receptors in the MDA-MB-231. Gene expression of gene expression of positive regulator of apoptosis receptors in treated cells were assayed by PCR microarray analysis. Increase in gene expression of positive regulator of apoptosis receptors in MDA-MB-231 cells after the treatment of hyperforin and taxol. Data are expressed as the hyperforin and taxol induced fold change of regulation relative to untreated cells.

Figure 18. Gene expression of pro-and anti-apoptotic genes in the MDA-MB-231 cells. Gene expression in group of cells were assayed by PCR microarray analysis positive regulator of apoptosis receptors Changes in gene expression of MDA-MB-231 cells after the treatment of hyperforin (group 1) and taxol (group 2).

Figure 19. Predication of apoptosis pathways in MDA-MB-231 cells during treatment of hyperforin and taxol. Based on gene expression and other anticancer assays, the diagram was formed to show occurrence of death receptors cell death pathway (1) and mitochondrial cell death pathway (2) in MDA-MB-231 cells after the treatment of hyperforin and taxol, respectively.

Hyperforin was seen to exhibit a significantly lower IC50 compared to taxol in inhibiting 50% growth of the cells suggesting that hyperforin could have a better drug potency against breast cancer cells compared to taxol. In addition, hyperforin was able to act effectively at 24 h against the cancer cells compared to 48 h of treatment (P < 0.001), demonstrating hyperforin may also potentially induce drug tolerance if used for a longer period of treatment [15]. As expected, all the tested concentration of hyperforin and taxol did not cause any inhibition towards growth of normal breast cells, MCF10A, which shows that these two natural compounds only specifically target breast cancer cells. On the other hand, a previous study on St. John’s Wort ethanol extract that contained both mixtures of hypericin and hyperforin demonstrated inhibition of 50% growth at concentration of 25 µg/mL which was higher than in our study [16]. Although, hyperforin required a lower IC50 to inhibit MDA-MB-231 cells compared to taxol, as expected taxol was found to induce larger volume of early apoptotic cells (62%) compared to hyperforin (60%). This suggest that taxol is more selective against breast cancer cell used in this study. This agrees with a study by Kim et al. [17] which demonstrated that taxol was able to induce higher cytotoxicity and apoptosis in adenocarcinoma (lung, A549 and stomach, MKN45), oral squamous cell carcinoma (OSCC, OECM1) and Hodgkin lymphoma (L428, L540, L591, L1236). Since the occurrence of apoptosis in MDA-MD-231 cells was evident in the study, the morphological changes of MDA-MB-231 cells after the treatment with hyperforin and taxol further confirmed the anticancer properties of these compounds. The observed morphological alterations are consistent with the apoptotic cell death traits, such as cell shrinkage, membrane blebbing, and chromatin condensation [18]. These results imply the combination therapy of hyperforin and taxol may have a good synergistic effects in further boosting apoptotic cell death and support the potential of hyperforin and paclitaxel as medicines for inducing apoptosis in MDA-MB-231 cells. Meanwhile, the release of LDH was studied as well to check whether LDH was upregulated since it is widely known as cell death marker [19–21]. In support of our previous findings, hyperforin was found to induce release of a high percentage of cytotoxicity (66%) in cells by releasing more of LDH compared to taxol treated cells which recorded 63% of cytotoxicity, suggesting that hyperforin may have higher cytotoxic potential in this type of breast cancer.

Hyperforin also prompted excessive levels of ROS in the MDA-MB-231 culture supernatant, indicating the occurrence of oxidative stress after the treatment with hyperforin compared to untreated cells. Nevertheless, the production of ROS was higher in taxol treated cells compared to hyperforin treated cells. These findings therefore suggest that hyperforin and taxol may be using the intrinsic mitochondrial cell death pathway to cause damage to the essential building blocks of cancer cells and lead to apoptosis [22]. A study has mentioned that lower level of ROS is needed by the cancer cells to cause proliferation and metastasis [23]. Hence, a small amount of ROS was seen in the untreated cell culture in this study. Researchers have found that various natural products can activate the excessive level of cellular ROS [24]. In addition, determination of mitochondrial activity of cells treated with hyperforin and taxol through ELISA has revealed a reduction of MMP in hyperforin (26%) and taxol (23%) treated MDA-MB-231 cells. This further suggests the cancer killing mechanism of hyperforin and taxol through reducing the mitochondrial activity of MDA-MB-231 cells, leading to a decrease in ATP production and eventual cell death [25]. Menegazzi et al. conducted a comprehensive review highlighting that hyperforin exhibited the ability to diminish mitochondrial activity in various human myeloid tumour cells, specifically U937, OCI-AML3, NB4, and HL-60. This impairment of mitochondrial function ultimately resulted in cellular death of cancer [26].

Several other apoptosis parameters have proven the antitumor properties of hyperforin towards cells. However, it is also important to study the potential target of hyperforin in the cell cycle phase. The current study has found that hyperforin is able to block cell cycle of MDA-MB-231 cells at the S phase. Meanwhile, taxol arrested MDA-MB-231 at the G0/G1 phase. Contradictory to our finding, cell arrest was also reported to occur at the G2/M phase after the treatment with taxol [27]. This is probably due to the different concentration of IC50 of taxol used in that previous study. Hence, the ability of taxol to arrest cancer cells may change with the different concentrations of taxol used [28]. However, further investigation is needed to confirm this hypothesis. This is the first study to confirm the ability of hyperforin to arrest MDA-MB-231 cells at S phase. In addition, MDA-MB-231 cells following hyperforin treatment has exhibited small and not so well-defined DNA fragmentation compared to taxol treated MDA-MB-231 cell. This may indicate that hyperforin does not trigger late-stage apoptosis in MDA-MB-231 cells compared to its treatment on MCF-7 cells [29,30]. However, more exploration needs to be undertaken to confirm this finding. Along with this, secretion of COX-2, also known as prostaglandin-endoperoxide synthase 2, is activated by oncogenes, which will lead to cancer progression [31]. COX-2 has been studied extensively in recent times. Many naturally occurring phytochemicals such as flavonoids, stilbenes, terpenoids, quinones, and alkaloids are said to selectively inhibit expression of COX-2 [32]. Hence, hyperforin and taxol were found to reduce a significant amount of COX-2 in cell culture. This finding suggests that hyperforin could be a better chemotherapeutic agent suggesting potential use as a selective COX-2 inhibitor in cancer therapy. This finding is in line with a previous study that demonstrated a significant reduction in COX-2 production in the treatment of ovarian cancer in a treatment using taxol in combination with celecoxib (selective COX-2 inhibitor) [33].

This study revealed that taxol has upregulated several DDCRs in MDA-MB-231 cell, especially higher intensities of TNFRSF11B, TNFRSF21 and TNFRSF25, which were probably observed due to the release of TNF-α cytokine [34] that could have been activated the DDCRs and led to activation of CASP8 and CASP10, as well as cell death. This finding indicated that treatment with taxol induced activation of TNF-mediated cell death pathway [35], similar to a report by Kim et al. (2003) which mentioned that taxol can induce death receptor and mitochondrial pathway to induce cell death of MDA-MB-231 cells [17]. However, the activation of death receptor pathway mediated by FADD was not prominent in taxol treated MDA-MB-231 cells. Meanwhile, MDA-MB-231 cells following hyperforin treatment has induced proportionately more genes related to DDCRs compared to taxol. The increased level of CRADD, TNFRSF10B, TNFRSF10A, TNFRSF11B, TNFRSF21, TNFRSF25 and TRADD in MDA-MB-231 cells following hyperforin treatment indicated the possible initiation of genes related to DDCRs and TNF-cell death pathway [36]. As expected, the increased level of TNF-α in culture supernatant of MDA-MB-231 cells following hyperforin treatment (the result was not shown) could have contributed to the overexpression of several DDCRs in the cells, especially TNFRSF25 [37].

Activation of mitochondrial death pathway after the treatment of taxol against MDA-MB-231 cells was seen whereby upregulation of differential gene expression related to apoptosis through mitochondria such as expression of CASP1, CASP3, CASP8 and CASP10 together with increased level of ROS in the culture of taxol treated MDA-MB-231 could lead to apoptosis through mitochondria [38]. The occurrence of mitochondrial death pathway in MDA-MB-231 cells after the treatment with taxol was further confirmed with upregulation of DFFA, suggesting the occurrence of DNA fragmentation which is also known as the end stage to mitochondrial death pathway [39]. Along with this, taxol also induced ABL1, a pro-apoptotic gene that can trigger DNA damage-induced cell death related to mitochondria in certain cellular environments, and inversely it may act as an inducer to inhibit proliferation and invasion of cancer cells [40]. Interestingly, treatment of hyperforin against MDA-MB-231 cells demonstrated upregulation of various genes related to caspases activation such as CASP1, CASP2, CASP3, CASP4, CASP6, CASP7, CASP8, CASP9 and CASP10 compared to taxol treated MDA-MB-231 cells. The increased level of these genes together with ROS confirm the occurrence of apoptosis in MDA-MB-231 that was mostly influenced by the mitochondrial death pathway. Similar to taxol treated MDA-MB-231 cells [41], DFFA expression was also increased following treatment with hyperforin. Activation of CASP8 following hyperforin treatment suggests the inhibition of CFLAR gene, hence this probably contributed to downregulation of CFLAR in both hyperforin and taxol treated MDA-MB-231. This is in agreement with a report by Wang et al. that downregulation of CFLAR was reported resulting in apoptosis of HCC due to activation to CASP8 [42]. In addition, in this study, an increase of CIDEA in hyperforin in comparison to taxol treated cells indicated the DNA damage caused by hyperforin may be able to suppress the growth of MDA-MB-231 cells. A previous study confirmed that Pb-TCMC-trastuzumab through chemotherapy approach was found to suppress the growth of LS 174 T, human colon tumour xenograft model, with the upregulation of several DNA damage genes including CIDEA [43]. Increased level of AIFM1 in hyperforin treated cells in comparison to taxol treated cells further confirmed the activation of CASP7 leading to apoptosis related to mitochondria. AIFM1 is highly upregulated in breast cancer cells undergoing apoptosis [44] (Table 1). Overall, it was noted that hyperforin was able to activate both pathways (mitochondrial cell death and TNF-mediated pathway) to cause cell death in MDA-MB-231, whereas taxol uses TNF-mediated pathway (Table 2). Future work that involves use of different cell lines of TNBCs such as 4T1 and BT-549 for anticancer testing of hyperforin may help to improve understanding of how this specific breast cancer cells respond to treatment of hyperforin. This is essential to provide a comprehensive response of TNBC cells to treatment using hyperforin as each of the TNBC cell lines has its own unique set of characteristics [45]. This eventually can be used to improve the diagnosis, treatment, and prevention of all types of TNBC. In addition, combination treatment of hyperforin and taxol on MDA-MB-231 cells should be continued to determine the reactivity of both natural products, which may play an important role in increasing the effectiveness of the treatment against triple negative breast cancer cells. Besides, further investigation in in vivo animal models needs to be conducted to assess the real efficiency of hyperforin against breast cancer cells. In addition, western blotting should be performed alongside other complementary assays (immunohistochemistry and migration assay) to further strengthen the findings and provide a comprehensive understanding of the anticancer effects of a drug in the treatment. This multi-faceted approach enhances the reliability and significance of the findings in anticancer research which eventually improve the diagnosis, treatment, and prevention of breast cancer.

Table 1: Summary of fold change of genes. Expression related to various genes related to death receptor pathway (DRP), mitochondrial death pathway (MDP) and survival after the treatment of hyperforin and paclitaxel against untreated cells.

MDA-MB-231		HYPERFORIN
		DRP: CRADD (345-fold), TNFRSF10A (13.33-fold), TNFRSF10B (354-fold), TNFRSF11B (130.95-fold), TNFRSF1A (54.69-fold), TNFRSF21 (154-fold), TNFRSF9 (65.44-fold), caspase 8 (128-fold), caspase 10 (24.74-fold), CD70 (11.44-fold), LTBR (71.66-fold) and RIPK2 (55.71-fold)
		MDR: caspase 1 (16.17-fold), caspase 2 (150.4-fold), caspase 3 (12.90-fold), caspase 7 (51.86-fold), caspase 9 (144.65-fold), DFFA (55.36-fold), BIK (600.3-fold), CYCS (55.20-fold), DIABLO (3.92-fold), CIDEA (123-fold), CIDEB (21.33-fold), BID (85-fold), IFM1 (307.22-fold), APAF1 (86.21-fold), BCL2L10 (103.72-fold), DFFA (55.36-fold) and NOD1 (14.25-fold)
		Survival: BAG3 (28.65-fold), BIRC5 (86.86-fold), BCL2 (14.33-fold) BIRC6 (10.22-fold), BAG1 (8.36-fold)
		DRP: FADD (20.33-fold), TRAF2 (117-fold), TNFRSF25 (11.33-fold), TNFRSF1B (1.68-fold)
		MDR: caspase 6 (970.6-fold),
		TAXOL
		DRP: TNFRSF11B (541.3-fold), TNFRSF21 (26.54-fold), TNFRSF25 (24.20-fold), caspase 8 (2.76-fold), caspase 10 (8.09-fold), CRADD (13.18-fold), CD70 (2.53-fold), LTBR (19.58-fold) and RIPK2 (3.86-fold)
		MDR: caspase 1 (166.7-fold), caspase 2 (1.37-fold), caspase 3 (2.44-fold), caspase 6 (6.57-fold), caspase 9 (8.67-fold), DFFA (86.02-fold), BIK (205-fold), CYCS (4.38-fold), DIABLO (27.41-fold), CIDEA (48.89-fold), ABL1 (494.5-fold)
		Survival: BIRC5 (7.44-fold), BCL2 (32.95-fold), XIAP (300-fold)
		DRP: TRADD (1.4-fold) TRAF2 (603-fold), CFLAR (375.18-fold) TNFRSF1B (7.16-fold), TNFRSF10A (-1.4-fold), FADD (148.3-fold)
		MDR: CFLAR (375-6-fold), CIDEB (1.4-fold), caspase 6 (350-fold), BAX (-165.6)

Table 2: Comparison of anticancer properties of hyperforin and taxol against MDA-MB-231 cells. Hyperforin showed similar potential anticancer activities againts MDA-MB-231 cells compared to taxol (positive control)

Cell lines	MDA-MB-231
Compounds	Paclitaxel	Hyperforin
Parameter
MTT (IC50) at 24 hours	7.91 µg/mL	7.18 µg/mL
Cellular morphology	Higher reduction in cell volume	Lower reduction in cell volume
Secretion of ROS	105 cps	90 cps
Percentage of apoptotic cells	62%	58%
Cell cycle arrest	G0/G1 phase	S phase
DNA fragmentation	Visible DNA fragmentation	Slight DNA fragmentation
Reduction of the ratio of red to green fluorescence (loss of MMP)	53	50
Release of LDH (percentage of cytotoxicity)	72%	78%
Inhibition of COX-2	48 RFU	50 RFU
Secretion of cytokines	Increased in IL-6, IL-2, TNF-α, IL-17A, IFN-γ	Increased in IL-6, IL-2, TNF-α, IFN-γ
ATP	7.5 pg/mL	7.7 pg/mL
HSP70 & 90	1.68 ng/mL & 1.69 ng/mL	1.51 ng/mL & 1.3 ng/mL
Apoptosis pathway	TNF-mediated pathway	caspase mediated-mitochondrial & TNF-mediated death receptor death pathway

5. Conclusion

The present study concludes that hyperforin carries potent action specifically against triple negative breast cancer cells, MDA-MB-231, and is a suitable candidate for a new anti-breast cancer drug with properties such as minimal toxicity towards normal cells, MCF10A, and lower IC50 compared to taxol. The results also revealed that taxol is one step ahead of hyperforin in exhibiting anticancer properties such as inducer of ROS, apoptotic cells, cell cycle arrest, LDH, DNA fragmentation and inflammatory cytokines, as well as inhibitor of MMP and COX-2 in MDA-MB-231 cells, yet hyperforin exhibited almost the similar properties as taxol. This unique mechanism can qualify hyperforin to become an alternative treatment option for triple negative breast cancer. This study has established one of the novel approaches to breast cancer in vitro treatment that are being investigated whereby this is first study to examine anticancer effect of hyperforin against TNBCs and also compare the effectiveness against clinically used drug, taxol. Future studies are needed to further validate these findings, such as in hypoxic condition whereby it is a common feature of solid tumours and can impact their response to treatments. Investigating the efficacy of natural products in hypoxic environments can provide insights into their potential effectiveness in realistic tumour microenvironments.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used and/or analyzed during the current study is available from the corresponding author on request.

Additional information

Funding

This work was supported by Fundamental Research Grant Scheme (FRGS) (FRGS/1/2019/SKK15/UM/01/2), a grant from Malaysian Ministry of Higher Education (MOHE) for a study entitled “Anticancer activity of hyperforin against breast cancer cells: identification of cell death and autophagy pathways.” The PhD student (Barathan Muttiah) was supported by the University Malaya Student Financial Aid by Institute of Research Management and Monitoring, Universiti Malaya. However, the funders had no role in this study.