Anticancer Properties of Actinomycetes-Derived Secondary Metabolites on the MCF-7 Breast Cancer Cell Line: Cytological and Molecular Investigations

Introduction

Breast cancer is the most commonly diagnosed type of cancer and a leading cause of cancer-related deaths worldwide [1]. In Egypt, breast cancer is the most prevalent malignancy, accounting for 18.9% of all cancer cases [2]. Common symptoms of breast cancer include nipple discharge, redness or changes in the breast’s appearance, or alterations in its texture. Early detection through regular breast exams is crucial for preventing the disease from spreading [3].

Chemotherapy and surgery remain the primary treatment methods for breast cancer [4]. However, chemotherapy faces challenges due to the toxicity of the drugs and the development of resistance [5]. Tamoxifen, a well-established anti-estrogen drug, has been in use for over 40 years and remains a cornerstone of breast cancer treatment [6].

A significant proportion of approved drugs are derived from natural sources, with over 60% of them originating from nature [7]. Among these, 50% are natural antibiotics derived from actinomycetes, which are also recognized for their antifungal, antibacterial, and anti-inflammatory properties [8]. Despite this, there is a lack of sufficient research on the potential anti-cancer properties of metabolites derived from marine actinomycetes-based drugs. Actinomycetes, often found as symbiotic microbes, are commonly associated with marine sponges [9], making them excellent sources for discovering new bioactive compounds [10]. Various natural substances derived from marine sponge-associated actinomycetes have been evaluated for their anticancer properties against different cancer cell lines [11]. One such promising secondary metabolite is heliomycin, produced by marine bacteria [12].

Breast cancer cell lines, particularly MCF7 and MDA-MB-231, are widely used in basic research due to their significant contributions to understanding breast cancer biology [13]. These cell lines are vital for identifying new therapeutic targets and studying the signaling pathways involved in breast cancer development [14]. MCF7 and MDA-MB-231 cells represent invasive ductal breast cancer, but they exhibit distinct phenotypic and genetic differences: MDA-MB-231 is a triple-negative breast cancer cell line, while MCF7 is hormone-dependent (positive for estrogen and progesterone receptors) [15].

The objective of our study was to assess the anticancer potential of heliomycin in breast cancer treatment.

Materials and Methods

Reagents and chemicals

The following chemicals were sourced from Sigma-Aldrich (USA): RPMI-1640 medium, trypsin (0.25%), fetal bovine serum (FBS), penicillin, streptomycin, and ethanol. Tamoxifen, the chemotherapeutic agent, was obtained from AstraZeneca (UK). The secondary metabolite, heliomycin, derived from actinomycetes, was identified at the Marine Natural Products Unit (MNPRU) of Helwan University, as outlined in Abdelfattah et al. [10]. MCF-7 cells, a human breast cancer cell line, were acquired from VACSERA Holding Company, Egypt. Cultureware was supplied by TPP, Switzerland.

Cell culture conditions

The MCF-7 cell line was cultured in RPMI-1640 medium enriched with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), 20 mM HEPES, and 25 mM sodium bicarbonate. The cells were incubated in a humidified incubator at 37 °C as previously described [16].

Heliomycin extraction and isolation

Actinomycetes strains were isolated and identified according to the methods in Abdelfattah et al. [17].

Preparation of drug solutions

The heliomycin extract was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg/ml stock solution. Tamoxifen tablets were powdered and dissolved in DMSO to achieve a 10 mg/ml concentration.

Cytotoxicity testing

The cytotoxic effects of heliomycin and tamoxifen on MCF-7 cells were assessed using the MTT assay. Cells were seeded in 96-well plates and cultured for 24 hours. Following incubation, various concentrations of the drugs were applied, and cells were further incubated for 24 hours. The untreated cells served as negative controls. Microscopic examination was performed to detect cell degeneration. The degenerated cells were washed with PBS (ADWIA, Egypt). MTT was then added to the viable cells at 0.5 mg/ml and incubated for 4 hours. The cells were analyzed using a BIOTEK ELx-800 Plate ELISA reader at 570 nm, and the IC50 values were calculated using Master Plex-2010 software.

Assessment of anticancer activity

For the anticancer evaluation, MCF-7 cells were treated with the calculated IC50 concentrations of heliomycin and Tamoxifen, with untreated cells acting as controls. After 24 hours of incubation, cells were examined for morphological changes under a microscope. Detached cells were removed by washing with PBS. The adherent cells were harvested by trypsinization, centrifuged at two thousand rpm for ten minutes, and washed with cold PBS. The resulting cell pellets were resuspended in 100 μl of PBS, labeled, and stored at -70 °C for further analysis.

Real-time PCR procedure

Total RNA was isolated from the cells utilizing the RNeasy Plus Mini Kit (Qiagen, Valencia, CA), following the manufacturer’s protocol. The extracted RNA was then used for the synthesis of complementary DNA (cDNA), applying the RevertAid™ H Minus Reverse Transcriptase enzyme (Thermo Fisher Scientific Inc., Fermentas, Canada). For gene expression analysis, the cDNA samples were amplified in triplicate. Quantitative real-time PCR (qRT-PCR) was conducted using Power SYBR® Green Master Mix (Life Technologies, CA) on an Applied Biosystems 7500 Real-Time PCR System, ensuring accurate amplification and detection of target genes. As an internal reference, β-actin was used. The arithmetic formula 2 -ΔΔCT was used to calculate relative mRNA quantification. Primer sequences for the studied genes are as follows HER2  F:5’-GGT CCT GGA AGC CAC AAG G-3’. R: 5’-GGT TTT CCC ACC ACA TCC TCT-3’ p53, F, 5’- CCCCTCCTGGCCCCTGTCATCTTC-3’, R: 5’ -GCAGCGCCTCACAACCTCCGTCAT-3’ TNF-α F: 5’-CTT CAG GGA TAT GTG ATG GAC TC-3’R: 5’- GGA GAC CTC TGG GGA GAT GT -3’ ER-α F, 5’-TCCTGGACAAGATCACAGAC-3’ R: 5’-GGT TTT CCC ACC ACA TCC TCT-3’, TLR-4 F: 5’-TTGAAGACAAGGCATGGCATGC-3’ R: 5’-TCTCCCAAGATCAACCGATG-3’, β-actin, F; 5’-TGACGGGGTCACCCACACTGTGCCCATCTA-3’ R; 5’CTAGAAGCATTGCGGTGGACGATGGAGGG-3’

Flow cytometry and cell cycle analysis

Both adherent and suspended MCF-7 cells were harvested and subjected to two sequential washes with phosphate-buffered saline (PBS) to remove residual media. The evaluation of apoptosis was conducted using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (ab139418, BD Biosciences), following the instructions provided by the manufacturer. Cells were initially incubated with Annexin V conjugated to fluorescein isothiocyanate (FITC) for 5 minutes. Following this step, PI staining was performed by incubating the cells in darkness for an additional 10 minutes. Flow cytometric analysis was then carried out using a fluorescence-activated cell sorter (FAC), and the collected data were processed using Cell Quest software (Becton–Dickinson, USA) to determine the apoptotic cell population and analyze the cell cycle distribution [18].

Cytopathological analysis

To prepare samples for cytological examination, 50 µl of cells treated with heliomycin or tamoxifen were carefully applied onto clean glass slides, with three replicates allocated for each experimental group. The slides underwent a dehydration process through graded ethanol solutions (100%, 90%, 75%, and 50%), air-dried thoroughly, and then fixed with methanol. Slides were subsequently rinsed with distilled water for 5 minutes. Hematoxylin staining was performed for 3 minutes, followed by two washes with distilled water. Thereafter, slides were briefly (5 seconds) immersed in eosin stain and washed again. After complete drying, the slides were cleared using xylene, mounted with Canada balsam, and covered with coverslips. Microscopic examination was conducted using a light microscope connected to a digital camera (Canon, Japan), and images were captured at 400x magnification from ten selected fields per slide. The chosen fields were determined based on the density of apoptotic cells. Photomicrographs were qualitatively analyzed for the presence of morphological characteristics typical of apoptosis.

Immunocytochemistry

For immunocytochemical detection, MCF-7 cell monolayers grown on glass slides were initially stabilized using 10% neutral buffered formalin. Following fixation, the cells were exposed to the primary antibody anti-CK19 (Labvision, Neomarkers, USA) for 90 minutes to target cytokeratin 19 expression. This was succeeded by the application of a secondary antibody using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), with detection performed via the immunoperoxidase staining protocol.

Transmission electron microscopy (TEM)

To prepare MCF-7 cells for ultrastructural analysis, cells were harvested by centrifugation at 2000 rpm for 10 minutes. The resulting cell pellets were immersed in 3% glutaraldehyde prepared in 0.1M sodium cacodylate buffer (pH 7.0) for two hours at ambient temperature. Samples were subsequently washed with the same buffer and post-fixed using 1% osmium tetroxide for an additional 2 hours. Dehydration was executed using ascending ethanol concentrations (10% to 100%), with each step maintained for 15 minutes, then immersed in absolute ethanol for 30 minutes. Resin infiltration was performed gradually with a mixture of epoxy resin and acetone before complete embedding in pure resin. Ultrathin sections were cut, mounted onto copper grids, and contrasted with uranyl acetate followed by lead citrate staining. The prepared sections were visualized using a transmission electron microscope (JEOL-JEM 1010, 80 kV) available at the Regional Center for Mycology and Biotechnology (RCMB) [19].

Statistical analysis

All quantitative data were displayed as means accompanied by standard errors (SE). Statistical interpretation of the results was conducted using SPSS software, version 23. Differences between experimental groups were determined by applying one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. A significance threshold was set at P < 0.05 to determine statistical relevance [20].

Results and Discussion

Cytotoxicity

The cytotoxic effects of heliomycin and tamoxifen were evaluated through the MTT assay, which revealed different IC50 values against MCF-7 cell lines, with varying toxicity observed. Both bleomycin and tamoxifen demonstrated notable toxicity toward the MCF-7 cells (Figures 1a and 1b). Interestingly, heliomycin exhibited greater toxicity compared to tamoxifen (Figure 1c). The IC50 values for heliomycin and tamoxifen were found to be 18.87 µg/ml and 29.6 µg/ml, respectively.

The MTT assay provided insight into the toxicity of the compounds, showing that cell viability significantly decreased, with a dose-dependent response observed. The untreated control cells maintained their typical morphology and behavior. The IC50 values, indicating the concentrations required to reduce cell viability by 50%, revealed a marked difference in the lethal effects between heliomycin and tamoxifen on MCF-7 cells, even at the highest concentrations tested. After treatment, the cell shape altered to become thin and elongated. Compared to the untreated cells, which were more clustered, the tamoxifen-treated cells formed aggregates that were absent in the heliomycin-treated cells after exposure to their respective IC50 concentrations. Several morphological abnormalities were observed, including cell rounding and areas where no cells were present (Figure 1d).

Cytopathology

Analysis using the H&E staining technique revealed notable changes, including a decrease in nuclear size, often associated with varying levels of chromatin condensation in the same culture. Apoptotic bodies displayed smaller cell sizes and the formation of blebs on the cell surface (Figure 2).

Immunocytochemistry

Immunocytochemistry results indicated significant differences in the intensity of color observed between treated and untreated cells due to the CK-19 immuno-antigen-antibody interaction. Both heliomycin- and tamoxifen-treated cells showed reduced CK-19 expression levels compared to the control (Figure 3).

Transmission electron microscopy

• In the control cells, the ultrastructural analysis revealed an abnormally large and irregular nucleus, with chromatin at the periphery and an expanded nucleolus (Figure 3D a).
• For heliomycin-treated cells, the ultrastructure showed signs of degeneration, including reduced nuclear size, with fragmented chromatin (Figure 3D b).
• In tamoxifen-treated cells, ultrastructural changes included cell degeneration, condensed nuclei, fragmented chromatin, and a noticeably enlarged and denser nucleolus (Figure 3D c).

Real-time PCR

Gene expression analysis of pro-apoptotic markers (TNFα, P53, and TLR4) and anti-apoptotic markers (HER-2 and ERα) in cells treated with heliomycin and tamoxifen revealed distinct expression patterns. HER-2 expression was found to be less elevated in tamoxifen-treated cells compared to heliomycin-treated cells and the control. P53 expression was significantly increased in heliomycin-treated cells, in contrast to tamoxifen-treated cells, when compared to the positive control. On the other hand, the expression of ERα, TNFα, and TLR4 was notably downregulated in heliomycin-treated cells compared to tamoxifen-treated cells and the positive control (Figure 4).

Flow cytometry and cell cycle analysis

Flow cytometry analysis indicated a marked increase in both early and late apoptotic cells in the tamoxifen and heliomycin treatment groups compared to the untreated cells. Specifically, tamoxifen-treated cells showed 5.66% early apoptosis and 11.16% late apoptosis, while heliomycin-treated cells had 3.22% and 14.46%, respectively. In contrast, the untreated cells showed only 0.27% early apoptosis and 0.15% late apoptosis. A slight increase in necrotic cells was also observed in both treated groups compared to the control MCF-7 cells (Figure 5).

Regarding the cell cycle, both treated groups displayed a significant increase in the Sub G1 phase, which corresponded with a notable decrease in the G0/G1 phase, compared to untreated cells. The S phase showed a minor decrease in both treated groups. Furthermore, the G2/M phase was significantly higher in the heliomycin-treated group compared to the tamoxifen-treated group when compared to the control. The untreated cells exhibited 1.78%, 52.06%, 39.88%, and 8.06% in the Sub G1, G0/G1, S, and G2/M phases, respectively. In contrast, heliomycin-treated cells showed 25.72%, 36.25%, 31.48%, and 38.27%, while tamoxifen-treated cells exhibited 23.14%, 38.45%, 36.99%, and 24.56% in the same phases (Figures 5 and 6).

Breast cancer remains the leading cancer type among women worldwide and stands as the second most common cause of cancer mortality, just after lung cancer [21]. Natural product extracts are widely regarded as promising sources for new cancer treatment options.

In this study, we examined the effects of heliomycin and tamoxifen on MCF-7 breast cancer cells. The impact of these agents was analyzed through various methods including cell cycle analysis, apoptosis detection by flow cytometry, real-time RT-PCR for ER-α, HER-2, p53, TNFα, and TLR4 expressions, as well as cytopathological and immunocytochemical assessments.

Heliomycin, an aromatic polyketide compound, is recognized for its anticancer [22], antiviral, and antibacterial properties. Previous studies have demonstrated its ability to inhibit RNA polymerase [23], translation, and histone deacetylase [24]. The inhibition of histone deacetylase impairs DNA replication, halts the cell cycle, and stimulates apoptotic mechanisms.

Tamoxifen is a nonsteroidal drug with anti-estrogenic activity, often prescribed in the treatment of estrogen receptor-positive breast cancer [6]. It competes with estrogen for binding to its receptors, forming a complex that prevents DNA synthesis and disrupts estrogen-driven cell actions.

We initially determined the IC50 values as a reference point for subsequent analyses. Our results indicated that heliomycin exhibited an IC50 of 18.87 µg/mL in MCF-7 cells, while tamoxifen showed a higher IC50 of 29.6 µg/mL. These values align with findings from Barba-Ostria et al. [25], who suggested that IC50 values under 30 µg/mL are crucial for identifying effective anticancer agents.

Extracts from actinomycetes, including cell-free extracts, are known to possess anticancer activity due to the presence of quinines and alkaloids in their metabolites [26]. These extracts have been shown to trigger apoptosis, disrupt fusion transcript degradation, encourage cellular differentiation, and reduce angiogenesis and proliferation [27].

CK19, a cytoskeletal protein expressed in epithelial cells, is also found in active tumor cells and metastatic breast cancer [28]. It is widely used as a marker for breast epithelial cell detection [29]. In our study, we observed varying degrees of CK-19 expression in MCF-7 cells: mild expression following heliomycin treatment, moderate expression after tamoxifen treatment, and strong expression in the positive control group. Our findings are consistent with the work of Tanvetthayanont et al. [30], who reported that CK-19 expression increases in stages II and III of breast cancer, and Ghani et al. [31], who found CK-19 expression in MCF-7 and T47D cell lines, which are derived from luminal epithelial cells.

These findings suggest a direct relationship between CK-19 expression and the oncogenic activity of MCF-7 cells. Both heliomycin and tamoxifen showed significant anticancer effects, evidenced by their ability to reduce CK-19 expression compared to the positive control, where CK-19 expression remained high without any anticancer effects.

TNF-α is a pleiotropic cytokine that was originally identified for its role in inducing apoptosis in some cancer types but is also involved in promoting tumor growth. It can stimulate cellular transformation, proliferation, angiogenesis, invasion, and metastasis [32]. High levels of TNF-α are often found in various malignancies, especially in breast cancer, where it is associated with aggressive tumor behavior and a poor prognosis [33].

In our research, we observed a notable reduction in TNF-α levels in MCF-7 cells treated with heliomycin and tamoxifen when compared to untreated control cells. These findings are consistent with the work of Mocellin et al. [34], who reported that TNF-α levels are typically elevated in human epithelial cancers. In MCF-7 cells, TNF-α activates TNFR1, which then stimulates both JNK and PI3K/AKT pathways, leading to the activation of NF-kB. This results in the upregulation of cyclin D1 and enhanced cell proliferation [34, 35]. Similar effects were observed when estradiol was applied [34]. Furthermore, TNF-α contributes to the growth of cancer cells by activating the p42/p44 MAPK pathway, which operates independently of NF-kB and is regulated by TNFR1 and TNFR2, providing an additional mechanism for cancer progression [35]. While the JNK pathway makes MCF-7 cells more susceptible to apoptosis triggered by TNF-α, the p42/p44 MAPK remains minimally engaged [36].

Toll-like receptors (TLRs) are predominantly expressed in epithelial and immune cells and play a key role in triggering inflammatory responses by stimulating the production of cytokines [37, 38]. TLR4 expression in metastatic ductal carcinoma has been closely linked to clinical outcomes [39, 40]. In the current study, we found a reduction in TLR4 expression in MCF-7 cells treated with heliomycin and tamoxifen, in contrast to the untreated control cells. This aligns with the observations of Haricharan and Brown [41], who indicated that TLR4 modulates the balance between growth and apoptotic cytokines in TP53 mutant breast cancer cells, contributing to increased proliferation. Similarly, Yang et al. [42] found that TLR4 was expressed in MCF-7 and MDA-MB-231 cells.

The p53 protein, which regulates numerous cellular functions including the cell cycle, DNA repair, and immune response [42, 43], showed significant upregulation in MCF-7 cells treated with heliomycin and tamoxifen, compared to controls. This finding corroborates the study by Van Slooten et al. [44], who noted that increased mutations and reduced expression of p53 are often associated with higher proliferative potential in cancer cells.

HER2, a receptor in the epidermal growth factor receptor (EGFR) family, encodes a 185-kDa transmembrane protein found on chromosome 17 [44]. Our findings indicated a substantial decrease in HER2 expression in MCF-7 cells treated with heliomycin and tamoxifen, compared to controls. This is in line with Hudis [45], who reported that HER2 is overexpressed in 20-30% of breast cancer cases and is associated with poor prognosis, higher recurrence rates, and aggressive tumor behavior [46].

Estrogen, through its receptor (ER), plays a pivotal role in mammary gland development and has become a crucial target in treating luminal breast cancer [47]. In line with this, antiestrogen therapies such as tamoxifen and fulvestrant directly target ER signaling pathways [48]. The present research revealed a significant reduction in ERα expression in MCF-7 cells treated with heliomycin and tamoxifen, in agreement with [49], who found that elevated estrogen levels contribute to breast cancer progression.

Both tamoxifen and heliomycin were effective in inducing apoptosis and inhibiting the proliferation of MCF-7 cells. In tamoxifen-treated cells, we observed clusters of viable, highly pleomorphic cells with increased nuclear-to-cytoplasmic ratio (N/C ratio), hyperchromatic nuclei, and 1-6 apoptotic cells per high-power field (HPF). Heliomycin-treated cells showed isolated viable cells with hyperchromatic nuclei and eosinophilic cytoplasm, as well as 1 apoptotic cell per HPF. The positive control cells exhibited clusters of viable cells with hyperchromatic nuclei, an elevated N/C ratio, and 1-2 apoptotic cells per HPF. These observations are consistent with those of Ahmad [21], who reported that heliomycin significantly inhibits the growth of various cancer cell lines, including HeLa, HepG2, and HMO2.

Molecular docking studies suggest that heliomycin may target HER2 tyrosine kinase, which is frequently overexpressed in cancer cells [49]. Additionally, heliomycin has been shown to modulate the balance between pro- and anti-apoptotic effects in cancer cells [46].

Apoptosis is characterized by distinct morphological changes, including cell shrinkage, fragmentation into apoptotic bodies, chromatin condensation, and DNA fragmentation [47, 48]. In our research, TEM analysis revealed that control MCF-7 cells exhibited enlarged and irregular nuclei, peripheral chromatin, and enlarged nucleoli. In contrast, heliomycin-treated cells showed degenerated morphology with shrunken nuclei and fragmented chromatin, while tamoxifen-treated cells displayed similar degenerative changes with dense nucleoli. These TEM findings support the conclusions drawn from cytopathological and flow cytometric analyses, indicating that both tamoxifen and heliomycin induce anticancer effects through the promotion of apoptosis [49].

Conclusion

Heliomycin, derived from actinomycetes bacteria, shows promising anticancer properties against MCF-7 breast cancer cells by promoting cell apoptosis and enhancing the expression of tumor-suppressing genes. This suggests its potential as an alternative or complementary therapeutic option alongside traditional chemotherapy treatments such as tamoxifen.

Acknowledgments: None

Conflict of Interest: None

Financial Support: None

Ethics Statement: None