EXPLORING THE ANTICANCER POTENTIAL OF GREEN SYNTHESIZED SILVER NANOPARTICLES USING Cymodocea serrulata in MCF-7 CELLS –

AN IN VITRO APPROACH

B N Poojitha 1

M Jayalakshmi 2

Haifa Almukadi 3

Dareen Alyousfi 4

Thoraia Shinawi 5

Khalda K. Nasser 5,6

Ashwaq Hassan Alsabban 7

Babajan Banaganapalli 8

T Meera 9

Noor Ahmad Shaik 8 Phone+966 54 734 9257 Emailnshaik@kau.edu.sa

Amudha Parthasarathy 1✉ Phone+91 7904075676 Emailamudhaa85@gmail.com

Dr.

P Amudha

M.Sc., M.Phil., Ph.D.

1 Emailamudha.sls@vistas.ac.in

1 Department of Biochemistry Vels Institute of Science Technology and Advanced Studies 600117 Pallavaram, Chennai Tamilnadu India

2 Department of Biochemistry Valliammal College for Women 600102 Pallavaram, Chennai Tamilnadu India

3 Department of Pharmacology and Toxicology, Faculty of Pharmacy King Abdulaziz University 21589 Jeddah Saudi Arabia

4 Department of Clinical Biochemistry, Faculty of Medicine King Abdulaziz University 21589 Jeddah Saudi Arabia

5 Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences King Abdulaziz University 21589 Jeddah Saudi Arabia

6 Department of Biological Science, Faculty of Sciences King Abdulaziz University 21589 Jeddah Saudi Arabia

7 Unit of Neurological Disorders, Faculty of Medicine, Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders King Abdulaziz University 21589 Jeddah Saudi Arabia

8 Department of Genetic Medicine, Faculty of Medicine King Abdulaziz University 21589 Jeddah Saudi Arabia

9 School of Agriculture Vels Institute of Science Technology and Advanced Studies 600117 Pallavaram, Chennai Tamilnadu India

Poojitha B N ¹, Jayalakshmi M², Haifa Almukadi³, Dareen Alyousfi⁴, Thoraia Shinawi⁵, Khalda K. Nasser^5,6, Ashwaq Hassan Alsabban⁷, Babajan Banaganapalli⁸,

Meera T ⁹, Noor Ahmad Shaik^8#, Amudha Parthasarathy^1*

¹Department of Biochemistry, Vels Institute of Science Technology and Advanced Studies, Pallavaram, Chennai – 600117, Tamilnadu. India

²Department of Biochemistry, Valliammal College for Women, Pallavaram, Chennai 600102, Tamilnadu. India.

³Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁴Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁵Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁶Department of Biological Science, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁷Unit of Neurological Disorders, Faculty of Medicine, Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁸Department of Genetic Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁹School of Agriculture, Vels Institute of Science Technology and Advanced Studies, Pallavaram, Chennai – 600117, Tamilnadu. India

*Corresponding Author Details

Dr. P Amudha M.Sc., M.Phil., Ph.D.,

Assistant Professor,

Department of Biochemistry,

Vels Institute of Science Technology and Advanced Studies,

Pallavaram, Chennai – 600117.

Mobile Number: +91 7904075676

Mail: amudhaa85@gmail.com, amudha.sls@vistas.ac.in

ORCHID ID: 0000-0001-6828-9558

#Co-corresponding Author Details

Noor Ahmad Shaik

Department of Genetic Medicine,

Faculty of Medicine,

King Abdulaziz University,

Jeddah, Saudi Arabia.

Email: nshaik@kau.edu.sa

Mobile Number: +966 54 734 9257

Abstract

The present study investigated the anticancer potential of silver nanoparticles (AgNPs) sourced from Cymodocea serrulata against the MCF-7 cell line. Flow cytometric assessments, encompassing cell cycle progression, apoptosis induction, and expression of apoptotic and anti-apoptotic proteins, were utilized to elucidate the fundamental mechanisms underlying cytotoxicity. Analysis of the cell cycle indicated that treatment with AgNPs led to a notable accumulation of cells within the G₀/G₁ phase, signifying cell cycle arrest and the inhibition of DNA synthesis. Apoptotic assessment revealed a marked elevation in early apoptotic events, thereby confirming the pro-apoptotic influence of AgNPs. Expression of apoptotic marker proteins further corroborated these observations, specifically the significant upregulation of caspase-3 and caspase-9, alongside a pronounced downregulation of the anti-apoptotic protein Bcl-2. Moreover, DNA fragmentation analysis further reinforced these observations by revealing extensive nuclear degradation in AgNPs-treated cells, signifying the activation of programmed cell death pathways. These collective findings suggest that AgNPs provoke apoptosis via the activation of intrinsic (mitochondrial) apoptotic pathways facilitated by caspase signaling and Bcl-2 inhibition. The documented G₀/G₁ phase arrest and DNA fragmentation further accentuate the role of AgNPs in disrupting cellular proliferation and facilitating programmed cell death. In summary, the investigation underscores the potential utility of Cymodocea serrulata-derived AgNPs as a promising nanotherapeutic agent for the treatment of breast cancer by facilitating the induction of caspase-dependent apoptotic pathways.

Keywords:

Apoptosis

Breast cancer therapy

Cymodocea serrulate

Caspase activation

Flow cytometry

MCF-7 cells

silver nanoparticles

1. Introduction

In contemporary times, inorganic nanoparticles have been the subject of extensive investigation due to their multitude of advantageous applications within the realms of science and technology. The term ‘nanotechnology’ refers to the specialized methodology involved in the creation, manipulation, and utilization of materials possessing dimensions ranging from 1 to 100 nanometers (1). Beyond their diminutive size, nanoparticles exhibit a plethora of attributes, such as a comparatively elevated surface area to volume ratio, pronounced reactivity or stability during chemical interactions, and sustained mechanical strength. Owing to these exceptional characteristics, nanoparticles manifest distinct physical, chemical, and biological properties at the nanoscale that are unparalleled when compared with larger particles. Such properties have rendered nanoparticles unique, facilitating their application across diverse domains. Among the most promising applied sectors for nanoparticles are pharmaceuticals, manufacturing and materials science, environmental applications, electronics, energy harvesting, and mechanical engineering (2). Nanoparticles can be synthesized through chemical, physical, or biological methodologies. Although chemical and physical approaches yield pure and well-defined nanoparticles, they are often associated with high costs, substantial energy consumption, and environmental detriment. Biological synthesis utilizing micro-organisms, enzymes, fungi, and plant materials represents a viable alternative to the aforementioned methods. Notably, plant extracts expedite the formation of stable metal nanoparticles through the rapid reduction of metal ions (3–5). Recently, the applications of silver nanoparticles (AgNPs) have garnered particular attention due to their remarkable and appealing physical, chemical, and biological attributes (6). Among the various categories of biogenically synthesized metal nanoparticles, AgNPs have ascended as the pre-eminent type in recent decades, attributable to their distinctive physicochemical and biological characteristics (7).

Cancer, defined as a pathological condition marked by unregulated cellular proliferation, remains a critical health issue, with more than 10 million new cases recorded globally on an annual basis (8). The most challenging aspect of cancer is metastasis, which has established itself as a primary contributor to mortality worldwide. As the global population expands and lifestyle alterations escalate cancer susceptibility, it is anticipated that the likelihood of developing cancer, alongside the incidence of new cases and cancer-related mortality rates, will rise significantly (2).

Breast carcinoma is classified as the second most widespread variant of neoplastic disease globally, subsequent to pulmonary carcinoma among the 34 acknowledged classifications of cancer (9). In 2020, there were over 2.3 million newly diagnosed breast cancer cases. Analyses forecast that the incidence of breast cancer may escalate to nearly 3 million cases annually by the year 2040 (10). This malignancy is stratified into various subcategories based on histological characteristics and type, with these distinctions corresponding to patterns of cancerous cellular growth. Breast neoplasms are among the most frequently diagnosed malignancies, presenting a high mortality rate and a prognosis that is commonly worse than that of lung cancer in this population (11).

Some traditional therapeutic modalities for oncological treatment encompass chemotherapy, surgical intervention, and radiation therapy (12). Chemotherapy serves as a conventional approach for a multitude of cancer types; however, the preponderance of chemotherapeutic agents demonstrates limited efficacy and unwanted side effects, including the development of drug resistance, challenges in dosage optimization, reduced specificity, and rapid drug metabolism (13, 14). Consequently, the identification of novel anticancer pharmaceuticals with minimal or negligible adverse effects has emerged as a paramount objective for numerous researchers (15). In pursuit of this aim, a plethora of investigators are examining the application of Ag/AgCl nanoparticles (NPs) synthesized via economical, expeditious, and non-toxic green biosynthesis techniques employing plant extracts as a prospective alternative to conventional anticancer therapeutics (16–18). Numerous scholarly articles have documented the efficacy of Ag/AgCl NPs mediated through various plant leaf, root, bark, or fruit extracts against disparate cancer types (16, 18).

In the present study, we synthesized silver nanoparticles (AgNPs) utilizing the hydroalcoholic extract derived from Cymodocea serrulata. C. serrulata is a flowering plant classified within the family Cymodoceaceae (19). This species is exclusively located in tidal zones (20). Within traditional medicine, the seaweed, commonly referred to by various names including Karumbu Pasi, C. serrulata, peria korai pasi, and peria thazai pasi, is utilized by coastal inhabitants and fishermen during maritime excursions, serving both alimentary and medicinal purposes (21, 22). This species constitutes a principal component of seagrass ecosystems and is prevalent in the tropical regions of Indo-Pacific nations (23). C. serrulata has been employed for various therapeutic applications, including relief from muscle fever, pain, gastrointestinal disorders, wounds, and cutaneous ailments (24). The active compound phenylthioketone, characterized by its antibacterial properties, has been isolated from C. serrulata (25). The antimicrobial efficacy of C. serrulata has been evaluated against human pathogens, demonstrating maximal activity against specific pathogens (26). The anti-oxidant potential of this seaweed indicates a definitely abundant source of natural anti-oxidants (27). The anti-oxidant activity of C. serrulata extract has exhibited superior capabilities in seizing free radicals, suggesting that its pronounced anti-oxidant effects may be attributable to the high concentration of phenolic compounds present (28). C. serrulata demonstrates prospective therapeutic efficacy against liver cancer cells contingent upon a certain dosage, exerting an antiproliferative influence on these cells by inhibiting pertinent cell signaling pathways (29). It contains an array of phytochemicals, including phenols, flavonoids, alkaloids, tannins, glycosidesaponins, and anthraquinones (27). These secondary metabolites exhibit significant reducing properties, stabilization and capping potentials, thereby playing a crucial role in the biosynthesis of metal nanoparticles with predetermined characteristics. AgNPs demonstrate robust anticancer and antibacterial properties. The anticancer mechanisms of AgNPs predominantly arise from their capability to disrupt telomerase stability mechanisms, interfere with mitochondrial respiratory processes, induce the generation of ROS beyond a critical threshold, inhibit ATP synthesis, inflict DNA damage, and ultimately impede the proliferation of cancer cells. Moreover, AgNPs offer multiple advantages over alternative silver forms, such as negligible toxicity, highly effective biological actions at minimal concentrations, and an absence of known side effects. The biological activities of C. serrulata, such as antimicrobial, antioxidant, cytotoxicity, and antifouling properties, have been previously documented (7).

However, previous studies highlight the therapeutic relevance of Cymodocea serrulata the anticancer efficacy of AgNPs synthesized from Cymodocea serrulata remains unexplored. Thus, based on literature this study investigates the anticancer efficacy of silver nanoparticles produced from C. serrulata in human breast carcinoma cells (MCF-7 cells) by flow cytometric techniques.

2. Materials and Methods

2.1 Cell cycle analysis

Cells were inoculated in a 6-well plate (2 x 10^4 cells/2 ml) and subsequently incubated in a CO₂ incubator at a temperature of 37°C for 24 hours. The cells were subjected to treatment with AgNPs at the IC₅₀ concentration for a period of 24 hours. Following the designated time frame, the culture medium was discarded and the cells were rinsed using PBS. A trypsin-EDTA solution (200 µl) was introduced, and the cells were incubated at 37°C for a duration of 4 to 5 minutes. The cells were subsequently harvested into polystyrene tubes by the addition of 2 ml of growth media, followed by centrifugation at 300 g for 5 minutes at 25 C. After meticulously decanting and washing the supernatant with PBS, the cells were fixed with 70% ice-cold ethanol and allowed to incubate for 30 minutes at -20°C in a freezer. Subsequently, the cells were resuspended in PBS containing propidium iodide (PI) at a concentration of 50 µl/ml and RNase at 20 mg/ml, and were kept in the dark for a period of 20 minutes. To assess the cells distribution across various stages of the cell cycle, flow cytometry was utilized, and the resulting data were analyzed using BD Cell Quest Pro Software (version 6.0) (30).

2.2 Apoptosis analysis by flow cytometry

Apoptosis in MCF-7 cells was evaluated utilizing an FITC detection kit in conjunction with PI. In brief, the cultured cells were seeded (0.5 x 10^6 cells/2 ml) in medium supplemented with 10% fetal bovine serum and subsequently treated with AgNPs. Post-treatment, the culture medium in the wells was aspirated and rinsed with PBS. Then cells were incubated for 24 hours at 37°C. Subsequently, the cells were centrifuged, washed three times with PBS, and then resuspended in 1 ml of binding buffer, Annexin V. 5 µl of FITC was supplemented, and the cells were incubated at 25°C for 20 minutes in a dark environment. Following this, 5 µl of PI along with 400 µl of 1X Annexin binding buffer were incorporated and gently vortexed. The analysis was performed using a flow cytometer and the data were interpreted with Cell Quest Pro Software (version 6.0). The processed cells were categorized into four quadrants (Q1-Q4) on a graphical representation to differentiate among the various stages of cellular apoptosis (31).

2.3 DNA Fragmentation study - TUNEL assay

A TUNEL assay was conducted to assess DNA fragmentation in MCF-7 cells at IC₅₀ concentrations by utilizing the APO-DIRECT™ Kit. Cells underwent fixation in 1% paraformaldehyde for a duration of 30 minutes, were subsequently permeabilized with 70% ethanol, and incubated with a DNA labeling solution for one hour at 37°C. Following the washing procedure, cells were subjected to staining with sodium azide and PI/RNase and were incubated in a dark environment for 30 minutes. FITC-dUTP was incorporated at the 3′-OH DNA termini, thereby marking apoptotic cells, which were subsequently analyzed utilizing flow cytometry (32).

2.4 Protein expression analysis by flow cytometry

MCF-7 cells (0.5 x 10^6 cells mL⁻¹) were cultured in DMEM within a 6-well plate at a temperature of 37°C in a CO₂ incubator for a duration of 12 hours, according to the methodology of Silva et al., (33). Following the incubation period, the cells were subjected to a wash with 1 mL of 1X PBS and subsequently treated with AgNPs at their respective IC₅₀ concentrations for a period of 48 hours. Subsequent to the treatment, the trypsinized cells were centrifuged at 1800 rpm for 5 minutes at ambient temperature in 5 mL storage vials, washed with 1X PBS, and subjected to a second centrifugation. The resultant pellets were then resuspended in 1 mL of 70% ice-cold ethanol and incubated at -20°C for 1 hour to facilitate the fixation and permeabilization of the cells. The ethanol was subsequently removed through centrifugation at 1800 rpm for 5 minutes, followed by washing with PBS. The cells were then treated with 20 µL of FITC-conjugated caspase-3 antibody and incubated at room temperature for 30 minutes in a dark environment. Concurrently, the expression levels of caspase-9 and Bcl-2 were evaluated by quantifying the fluorescence emitted by the caspase-9 PE antibody and the Bcl-2 PE antibody. Protein expression analysis was performed utilizing flow cytometry techniques.

2.5 Statistical analysis

The assessments were executed in triplicate, with each data denoting the comprehensive average calculated from a minimum of three independent experimental trials. The outcomes were articulated as mean ± standard deviation (SD). The resultant data were subjected to analysis utilizing GraphPad Prism (Version 7.00).

3. Results and Discussion

3.1 Synthesis and characterization of AgNPs

Although the synthesis of silver NPs derived from C. serrulata adheres to established protocols and is characterized through analytical methodologies, this work has previously been documented by Poojitha B.N et al., (34) (Supplementary Fig. 1). The biosynthesis of silver NPs was facilitated through the utilization of C. serrulata extract. These extracts functioned as both capping and reducing agents, thereby stabilizing the formation of silver NPs. The UV–Vis spectra indicated an absorption peak at 422.30 nm, which confirms the presence of silver NPs. Scanning electron microscopy (SEM) analysis depicted a high density of polydispersed spherical NPs exhibiting a size range between 22 to 65 nm, characterized by their spherical morphology and well-dispersed nature. Transmission electron microscopy (TEM) analysis further elucidated that the particles possess a size distribution from 18 to 65 nm. The EDX elemental analysis of the synthesized AgNPs indicated a predominant weight percentage of silver (80.22%), succeeded by chloride (16.45%) and oxygen (4.43%). The crystalline characteristics of the silver NPs were substantiated through X-ray diffraction (XRD) analysis.

3.2 In vitro cytotoxic activity of AgNPs

In the earlier research, Poojitha et al., (35) highlighted the anticancer properties of silver nanoparticles (AgNPs) by employing the MTT assay, indicating a decline in cellular proliferation in MCF-7 cell. The minimal inhibition recorded was 7.02% at the 12.5 µg/mL concentration, suggesting a limited therapeutic effect at lower dosages. Conversely, the maximal inhibition reached 79.38% at 200 µg/mL, signifying a substantial influence on cellular proliferation at elevated concentrations. In contrast, the standard chemotherapeutic agent Doxorubicin at 5 µg/mL resulted in a growth inhibition of 87.65%, surpassing the efficacy observed with AgNPs. The IC₅₀ value for AgNPs was determined to be 102.03 µg/mL (Supplementary Fig. 2a).

3.3 Cell cycle analysis

In the Sub G₀/G₁ phase, 0% of the cellular population was observed to be arrested in the untreated group, 0% in the group treated with Doxorubicin, and 0% in the group administered with AgNPs. In the G₀/G₁ phase, the proportion of cells exhibiting arrest was recorded at 68.9% in the untreated group, 78.3% in the Doxorubicin-treated group, and 65.9% in the AgNPs-treated group. In the S phase, the percentage of inhibited cells was determined to be 11.3%, 8.55%, and 10.4% in the untreated, standard, and AgNPs-treated groups, respectively. In the G₂/M phase, 19.8% of cells were inhibited in the untreated group, 13.1% in the group exposed to Doxorubicin, and 23.7% in the AgNPs-treated group (Fig.A).

The regulation of the cellular cycle holds substantial significance in determining cellular longevity. This complex mechanism is sustained by an array of regulatory factors including tumor suppressor genes, cyclin-dependent kinases, and their respective inhibitors. The manifestation of irregularities during the cell cycle has the potential to induce significant DNA damage and activate the apoptotic signaling pathway (36). The cell cycle adheres to a strict sequential progression through the G₁-S-G₂-M phases, as cellular mechanisms meticulously regulate the synthesis and distribution of chromosomes, nucleic acids, and proteins. However, when the cell cycle becomes dysregulated, irregularities ensue, resulting in aberrations in physiological processes such as cell division and proliferation that are contingent upon the integrity of the normal cycle (37). To clarify the antiproliferative mechanisms involved, we examined the influence of AgNPs on cell cycle. Cell cycle arrest was moderately evident when cells were treated with AgNPs at a concentration of 102.03 µg/ml. The findings of the present study are consistent with the prior observations made by Eldabousy et al. (38), which indicated that AgNPs induce cell cycle arrest at the G₀/G₁ phase, suggesting that cells enter the checkpoint for DNA damage repair and fail to progress past this checkpoint when exposed to apoptotic stimuli, as evidenced by the elevated cell population in this phase, subsequently followed by a reduction in cell populations in the ensuing phases. For instance, Kwon et al. (39) corroborated that Kurarione inhibited human colorectal cancer cells in G0/G1 phase. Similarly, Xia et al. (54) reported the anticancer potential of phenolic extract of wild pink bayberry by inhibiting the MDA-MB-231 cancer cells at G0/G1 phase.

3.4 Apoptosis analysis by flow cytometry

Apoptosis encompasses several distinct mechanisms, including the phosphatidylserine translocation from the inner to the outer layer of the cell membrane, condensation and fragmentation of chromatin, DNA cleavage, and the activation of caspases. Indeed, in the early phase of the apoptotic process, the phosphatidylserine present on the outer layer of the cell membrane serves as a recognition site for phagocytic cells and is frequently bound by the calcium-dependent protein Annexin V. Typically, phosphatidylserine is not accessible to Annexin V, as it resides on the interior surface of the cell membrane. Nonetheless, during the initiation of apoptosis, it translocates to the outer cell membrane and interacts with Annexin V. The translocation of phosphatidylserine is a continuous process (40). The staining procedure employs PI to differentiate between necrotic and late apoptotic cells. The integrity of the plasma membrane is preserved during early apoptosis; however, as late apoptosis or necrosis advances, membrane leakage occurs, leading to an increased number of PI-positive cells (41). In the present investigation, it was observed that in MCF-7 cells subjected to the standard chemotherapeutic agent Doxorubicin, the proportion of early apoptosis was 42.7%; conversely, the percentage of late apoptosis was 3.42%. Similarly, apoptosis induced by AgNPs in MCF-7 cells parallels that of the standard drug (Fig. B); wherein it recorded 48.8% of cells in early apoptosis (An+/PI-) and 4.05% of cells in late apoptosis (An+/PI+). The findings concerning apoptosis correspond with the research conducted by Madaniyah et al. (42), which elucidates the anticancer efficacy of silver NPs synthesized from Acalypha indica by demonstrating apoptosis in the late phase, in addition to the extract of Acalypha indica exhibiting both early and late apoptotic characteristics.

3.5 DNA Fragmentation study - TUNEL assay

The investigation of DNA fragmentation within MCF-7 cells is conventionally conducted to evaluate apoptosis, which is a form of programmed cellular demise marked by the disintegration of DNA into diminutive fragments. The Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) assay elucidates DNA damage (d-UTP expression) utilizing a flow cytometric methodology. In the absence of treatment, 6.9% of the cells exhibited evidence of DNA damage, whereas cells subjected to standard treatment and those treated with AgNPs showed a heightened incidence of DNA damage at 86.1% and 82.7%, respectively. These findings are illustrated in Fig. C.

An advanced assay that employs the detection of DNA strand breaks in situ through the application of fluorochrome labeling has been developed to dentify and quantify apoptotic cells via fluorescence microscopy or cytometry (43). The TUNEL assay is designed to identify DNA segments that display free 3′-OH groups as a result of DNA fragmentation, thereby functioning as a dependable technique for the detection of cells undergoing the apoptotic process (44). The TUNEL assay is crucial for recognizing apoptotic cells that experience substantial DNA damage during the terminal stages of apoptosis (45). In the current study, breast cancer MCF-7 cells treated with AgNPs exhibited significant DNA damage. TUNEL-positive cells were detected through flow cytometry, signifying the presence of apoptotic cells characterized by fragmented DNA. The present research supports the findings established by Njud S. Alharbi et al. (46), which documented the apoptotic efficacy of AgNPs synthesized from Olea europaea through the mechanism of DNA fragmentation in MCF-7 cells. Simultaneously, the results of current study align with the study of Abdullah et al (2010) (55), reported that more than 80% of cells were TUNEL-positive on treating the cells with 7,3′,5′-trihydroxyflavanone (3HFD) a flavonoid derivative isolated from Hydnophytum formicarium.

3.6 Protein expression in MCF-7 cell lines

Caspases belonging to cysteine-aspartic protease family, serves an indispensable function as a mediator within the apoptotic signaling cascade. Caspases are classified based on their roles in apoptosis into initiator and effector caspases. In this context, the initiator caspases are activated in response to apoptotic signals. Conversely, effector caspases function as the primary downstream executor caspases and are integral to the apoptotic process (47). Apoptosis is instigated by caspase-3, a pivotal constituent of the caspase family, in conjunction with caspase-8 and caspase-9. Caspase-3, an executioner or terminal caspase protein, is critically essential for the process of apoptosis and exists in an inactive zymogen form as Pro-Caspase-3 in healthy cells (48). Nevertheless, during the execution of apoptosis, Pro-Caspase-3 is activated through both intrinsic and extrinsic pathways. As a fundamental executor for apoptosis, activated Caspase-3 can target and degrade proteins involved in DNA replication, proteins that inhibit apoptosis, cytoskeletal proteins, and others, thereby facilitating increased cellular apoptosis (49). In the intrinsic apoptotic pathway, caspase-9 acts as an initiator caspase (50). In this study, caspase expression analysis in MCF-7 cells indicated that post-treatment with AgNPs caused a notable upregulation of caspase-3 and caspase-9 activity by 87.5% and 81.6%, respectively, in comparison to the standard Doxorubicin, which exhibited 80.8% and 88.8%, respectively (Fig. D & E).

In relevant to Shimizu et al. (51), Bcl-2 functions as a critical inhibitor of apoptosis in cells, safeguarding mitochondrial integrity during apoptotic events and various forms of necrotic cell death. In the current study, Bcl-2 expression was employed as a parameter to elucidate the mechanisms underlying apoptosis in cancer cells (40). The study analyzed the effects of exposure to AgNPs relative to Doxorubicin. Both the Doxorubicin-treated and AgNPs-treated cells downregulated the Bcl-2 protein activity. In contrast to other previously identified oncogenes that promote cell proliferation, the upregulation of Bcl-2 fosters cell survival and mitigates cell death (52). In this study, flow cytometric analysis of untreated cells demonstrated that 92.4% of cells exhibited upregulated Bcl-2 protein (Fig. F). However, upon exposure to Doxorubicin, Bcl-2 expression was downregulated to 55.7%. Conversely, treatment with AgNPs resulted in 40.9% of cells exhibiting downregulated Bcl-2 expression. The findings of the present study align with those reported by Mobaraki et al. (53), indicating that testicular embryonic cancer cells treated with AgNPs significantly upregulated caspase-3, -9 and Bax while simultaneously downregulating Bcl-2.

4. Conclusion

The current investigation presents robust evidence that AgNPs produced utilizing C. serrulata demonstrate significant anticancer properties against breast cancer MCF-7 cells via a complex mechanism of action. Flow cytometric analysis indicated a significant arrest in the cell cycle at the G₀/G₁ phase, which signifies the inhibition of cellular proliferation. The initiation of early apoptosis, as evidenced by annexin V/PI staining, implies that AgNPs trigger programmed cell death as opposed to necrosis. Moreover, the upregulation of caspase-3 and caspase-9, together with the downregulation of Bcl-2, emphasizes the activation of the intrinsic mitochondrial apoptotic pathway. Additionally, DNA fragmentation assessments validated the occurrence of nuclear degradation consistent with apoptotic cell death. In aggregate, these in vitro findings underscore the potential of biologically synthesized AgNPs as a promising subject for further investigation within the realm of breast cancer research. However, in light of the limitations associated with cell-line studies, these outcomes should be regarded as foundational rather than conclusive. Future research should focus the validation of these findings through extensive in vivo studies, accompanied with detailed pharmacokinetic, toxicity and biodistribution assessments to enhance the understanding of the therapeutic implications and safety profile of these nanoparticles.

Figures

Figure A: Effect of AgNPs and Doxorubicin on cell cycle distribution in MCF-7 cells on comparison with untreated cells, (a) Untreated MCF-7 cells, (b) MCF-7 cells treated with standard-Doxorubicin, (c) MCF-7 cells treated with AgNPs at IC ₅₀ concentration. Cells were then labelled with PI and analysed by flow cytometry. The values presented indicate the distribution of cells across various phases of the cell cycle. The data depicted are indicative of three separate experimental trials yielding similar outcomes.

Figure B: Apoptosis inducing effect of AgNPs on MCF-7 cells are detected by Annexin V-FITC (AV)/PI method ((a)Untreated MCF-7 cells, (b) MCF-7 cells treated with standard-Doxorubicin, (c) MCF-7 cells treated with AgNPs at IC ₅₀ concentration).

Figure C: DNA damage (d-UTP expression) study in MCF-7 cells after treatment with the IC ₅₀ concentration of AgNPs and doxorubicin by using BD FACS Calibur, (a) Untreated MCF-7 cells, (b) MCF-7 cells treated with standard-Doxorubicin, (c) MCF-7 cells treated with AgNPs. Histograms show number of cell channels vs. PI fluorescence.

Figure D: Representative flow cytometry histograms illustrating caspase-3 protein expression in MCF-7 cells across three experimental conditions: (a) untreated control cells, (b) cells treated with silver nanoparticles (AgNPs) at the IC₅₀ concentration, and (c) cells treated with doxorubicin. The increased caspase-3 signal in treated groups reflects activation of downstream executioner caspase activity, indicative of apoptosis induction. Histogram shifts and changes in fluorescence intensity demonstrate treatment-dependent modulation of apoptotic signaling.

Figure E: Flow cytometry histograms showing caspase-9 expression profiles in (a) untreated MCF-7 cells, (b) cells exposed to AgNPs at the IC₅₀ concentration, and (c) cells treated with doxorubicin. The enhanced fluorescence intensity observed in treated groups signifies activation of the initiator caspase-9, supporting the involvement of the mitochondrial (intrinsic) apoptotic pathway. The comparative expression patterns highlight the pro-apoptotic impact of AgNPs relative to the standard chemotherapeutic agent.

Figure F: Representative histograms depicting Bcl-2 expression in (a) untreated control cells, (b) cells treated with AgNPs at the IC₅₀ concentration, and (c) cells treated with doxorubicin. A reduction in fluorescence intensity in the treated groups reflects downregulation of the anti-apoptotic protein Bcl-2, indicative of suppression of survival pathways during treatment. The observed decrease in Bcl-2 expression complements the caspase activation findings, collectively validating apoptotic pathway engagement.

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Supplementary Material 1

Supplementary Material 2

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Declarations

Funding:

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript

Competing Interests:

The authors have no relevant financial or non-financial interests to disclose

Author Contributions:

All authors contributed to the study conception and design. Study designed by Amudha Parthasarathy and Noor Ahmad Shaik. Material preparation, data collection and analysis were performed by Poojitha B N, Jayalakshmi M, Haifa Almukadi, Dareen Alyousfi, Thoraia Shinawi, Khalda K. Nasser. The first draft of the manuscript was written by Ashwaq Hassan Alsabban, Babajan Banaganapalli, T. Meera and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript

Conflict of Interest:

The authors declare no conflicts of interest

Clinical trial number

Not applicable

Data Availability Statement:

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.