Green-synthesized silver nanoparticles from peel extract of pumpkin as a potent radiosensitizer against triple-negative breast cancer (TNBC)

Abstract

Background

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Radiation therapy (RT) is a modality for TNBC management. Radiosensitizers can mitigate the adverse effects of RT. Applying green-synthesized silver nanoparticles (Ag-NPs) from biological sources such as plants is a potential strategy to sensitize cancer cells to radiotherapy due to the low toxicity. Therefore, identifying novel natural sources for synthesizing stable and broadly applicable green-Ag-NPs has gained more attention in cancer therapy. In the present study, we synthesized green- Ag-NPs from pumpkin peel extract and elucidated the impact of green-synthesized Ag-NPs as a radiosensitizer in MDA-MB 231 cells (a model of TNBC).

Results

The prepared Ag-NPs had a spherical shape with an average size of 81 nm and a zeta potential of − 9.96 mV. Combination of green-synthesized Ag-NPs with RT exhibited synergistic anticancer effects with an optimum combination index (CI) of 0.49 in MDA-MB-231 cells. Green-synthesized Ag-NPs synergistically potentiated RT-induced apoptosis in MDA-MB-231 cells compared to the corresponding monotherapies. Morphological features of apoptosis were further confirmed by the DAPI–TUNEL staining assay. HIF-1α expression was decreased in cells subjected to combination therapy. Bax and p53 expression increased, whereas Bcl-2 genes decreased. Combination therapy significantly increased the protein level of PERK and CHOP while decreasing cyclin D1 and p-ERK/total ERK levels compared to monotherapies.

Conclusion

These findings indicate the potential effect of green-synthesized Ag-NPs as a radiosensitizer for TNBC treatment.

Graphical Abstract

Background

Breast cancer is the most frequently encountered cancer in women, accounting for 23% of cancer-related deaths. Despite advancements in therapeutic options, it remains the primary cause of cancer-related deaths in females (DeSantis et al. 2019). Breast cancer is classified into subgroups based on the expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor. HER2 (human epidermal growth factor) (ER-/PR-/HER2 +), Luminal-A (ER + /PR + /HER2−), Luminal-B (ER + /PR + /HER2−), and basal-like or triple-negative breast cancer (TNBC) (ER-/PR-/HER2-). TNBC is defined as the absence of ER, PR, and HER2 expression (O’Reilly et al. 2015). TNBC accounts for approximately 10–20% of breast cancer cases and is characterized by a more aggressive biological phenotype, higher recurrence rate, and poorer clinical outcomes (Derakhshan and Reis-Filho 2022). Breast cancer treatment is often based on multimodal approaches, such as surgery, chemotherapy, radiation therapy, hormonal therapy, and immune-based interventions. However, in the case of TNBC, no standard therapeutic options are available owing to the absence of receptors. As TNBC is an aggressive subtype with limited therapeutic options, such as traditional hormonal therapy, radiotherapy (RT) can be considered an effective treatment modality for these patients. RT is a cornerstone modality that utilizes high-energy photon radiation to improve local and regional management, whether after surgery or in patients who are not eligible for surgery (He et al. 2018). RT can directly cause DNA damage, specifically single-strand and double-strand breaks, which further hinder cell division, proliferation, apoptosis, and necrosis in some cases. Additionally, RT can indirectly impact cellular stress and harm cancer cells by generating reactive oxygen species (ROS), which is perhaps the most crucial aspect of RT (Wang 2014). Mounting evidence shows that cells with a high proliferation capacity, such as tumor cells, exhibit greater susceptibility to IR-induced damage. Given the detrimental effects of conventional RT on surrounding normal tissue, the primary objective of this modality is to deliver a carefully regulated radiation dose to a specific target site to minimize adverse effects. In other words, radiosensitizers may control the RT dose to preserve normal surrounding tissue (Song et al. 2017). In this era, nanotechnology-based treatments are promising strategies to enhance the efficacy of traditional cancer treatment approaches (Joseph et al. 2019; Joseph et al. 2017; Nair et al. 2020). Many efforts have been made to enhance radiotherapeutic results, especially using nanoparticles (NPs) that can be delivered to tumor sites to improve radiosensitivity. This can enhance the efficiency of RT absorption by the tumor region while reducing the delivered RT doses. NPs can be used in conjunction with RT to absorb and scatter the high energy of RT selectively. This allows for a more significant localization and enhancement of RT-induced damage. Indeed, delivering a therapeutic dose of radiation to a tumor site remains a great challenge in RT. NPs can intensify the generation of Auger electrons, secondary electrons, and ROS, thereby improving RT effects(Kovács et al. 2022). NPs exhibit favorable properties such as large surface area to mass ratio, small size, and high reactivity, leading to the development of high-precision materials to overcome therapeutic barriers. Silver NPs (Ag-NPs) are widely used owing to their well-known medical properties (Liang et al. 2024; Liu et al. 2024).

Ag-NPs have been found to induce oxidative stress, DNA damage, apoptotic cell death, and cytokine production (Huo et al. 2015; Montazersaheb 2024). Few studies have investigated the effects of green-synthesized Ag-NPs on breast cancer cell lines. Previous studies have shown that PERK/eIF2α/ATF4/CHOP axis plays a critical role in G1/S arrest and induction of apoptosis in MDA-MB-468 cells (Coker-Gurkan et al. 2021; Amiri et al. 2023). ERK1 and ERK2 are involved in the Ras–Raf–MEK–ERK signaling cascade, which regulates cellular events such as proliferation, differentiation, survival, and cell cycle progression (Roskoski 2012). Upon activation, ERK1/2 is translocated to the nucleus to phosphorylate transcription factors and drive cell proliferation via cyclin D1 expression. Cyclin D1 regulates the G1–S transition during the cell cycle (Choi et al. 2019). Indeed, cyclin D1 is downstream of p-ERK, and induction of cyclin D1 requires sustained activation of ERK. Overexpression of cyclin D1 has been confirmed in many cancer cells and is associated with poor outcomes (Pawlonka et al. 2021). HIF-1α is considered an appealing target in cancer therapies, and its expression severely affects the sensitivity to radiotherapy (Jafari et al. 2023a).

Researchers have used various techniques for NP preparation, including physical and chemical procedures. Physical methods require sophisticated equipment to maintain the temperature and high pressure required for the reaction. Chemical techniques are not environmentally friendly. They use toxic chemicals and produce various toxic by-products.

Owing to this limitation, the green synthesis of Ag-NPs has been developed to fabricate NPs that are more eco-friendly than conventional methods(Barabadi et al. 2021). Applying green-synthesized Ag-NPs from biological sources such as plants is a potential strategy to sensitize cancer cells to radiotherapy due to their low toxicity. Therefore, identifying novel natural sources for synthesizing stable and broadly applicable green-synthesized Ag-NPs has gained more attention in cancer therapy.

Pumpkin belongs to the genus Cucurbita of the Cucurbitaceae family. Pumpkin is traditionally known for several biomedical applications such as anti-bacterial, anti-inflammation, anticancer, and immunomodulatory effects. This edible plant contains many active biological compounds, including unsaturated fatty acids, β-carotene, α- and β-cryptoxanthin, and polysaccharides (Durante et al. 2014; Nkoana et al. 2022). Figure 1 demonstrates the edible fruit of the pumpkin (Cucurbita spp.).

Fig. 1

Image of the edible fruit of the pumpkin (Cucurbita spp.) plant grown in Diyarbakır

In this study, pumpkin peel extract was used to synthesize Ag-NPs. We aimed to elucidate the apoptotic effects of green-synthesized Ag-NPs on MDA-MB 231 cells as a model of human breast cancer through the Bax, Bcl2, P53, ERK, cyclin D, and PERK/CHOP signaling pathways, which have been reported as essential signaling molecules activated in TNBC.

Considering that RT is a common modality in patients with TNBC, using a radiosensitizing agent to minimize the adverse effects of RT may be an ideal option. Due to the ability of Ag-NPs to inhibit cancer cell growth and induce apoptosis(Al-kawmani et al. 2020), we sought to determine the effect of prepared green-synthesized Ag-NPs as a possible radiosensitizer in TNBC treatment through different signaling axes. Therefore, this study aimed to assess the effect of green-synthesized Ag-NPs, alone and in combination with RT, to determine how they influence the sensitivity of TNBC to radiation.

Materials and methods

Preparation of the plant extract

Pumpkin, called sour gourd belonging to the Cucurbitaceae family, grown in Lice of Diyarbakir, is a plant whose fruits are frequently consumed in that region. Cucurbita spp. sample purchased from the local bazaar of lice, Diyarbekir, Türkiye. Our Experimental research on cucurbita, including its collection, complied with the WHO guidelines and legislation of Good Agricultural and Collection Practice (GACP) to ensure the collection of high-quality plant materials. Pumpkins, which can be stored under room conditions in winter, are prepared in many different varieties. The shells of this plant are peeled and removed for use in food preparation. The pumpkins were collected from the Lice Diyabakır region. Peels were removed from the fruit, washed, and dried at room conditions. The dried peels were weighed to 25 g after size reduction and boiled in 75 ml distilled water. The extract was cooled to room temperature and filtered through filter paper. The obtained pumpkin peel extract was stored in a + 4 °C refrigerator for synthesis.

Metal solution preparation and synthesis of green Ag-NPs

In the synthesis of Ag-NPs, a solution with a concentration of 5 mM (mM) was prepared from AgNO₃ solid compound from Sigma-Aldrich (Germany), as the Ag⁺ source.

To produce Ag-NPs, the pumpkin peel extract that had been prepared and the AgNO3 solution, which is a metal source with a concentration of 5 mM, were combined in a 2:1 ratio. During the course of the research that was conducted in a room setting, a shift in color was detected. The creation of Ag-NPs was responsible for the observed color change, which occurred within 30 min and went from yellow to brown. In a span of 90 min, the reaction that took place in the synthesis medium was finished. As a result of the creation of Ag-NPs, samples were collected from the medium used for synthesis. In order to determine the typical absorbances of silver at maximum wavelength absorbances, measurements were taken with a Perkin Elmer One spectrophotometer that covers both ultraviolet and visible wavelengths (UV–vis). The stability of Ag-NPs under physiological conditions was assessed six months after synthesis using Neoflex hypertonic liquid, which is composed of a 3% sodium chloride solution.

Characterization of green-synthesized Ag-NPs

Various analyses were performed to characterize the properties of the Ag-NPs obtained by the green synthesis.

Ultraviolet spectrophotometer

A Perkin Elmer One Ultraviolet spectrophotometer (UV–vis) was used for the maximum wavelength absorbance measurements in the 300–800 nm range, depending on the formation of Ag-NPs.

X-ray diffraction (XRD) for detection of crystalline pattern

For the crystal pattern and size, data obtained through a Rigaku Miniflex 600 model X-ray diffraction (XRD) device measuring 20–80 at 2-Theta were used. The crystal sizes of the Ag-NPs were calculated using the Debye–Scherrer equation (Uzma et al. 2022; Perveen et al. 2021) using the full width at half maximum (FWHM) value of the highest peak of the crystal pattern reflected in the plane obtained from these data.

Morphological analysis

A Jeol Jem 1010 Field Emission Scan Electron Microscopy (FE-SEM), a Park System XE-100 Atomic Power Microscopy (AFM), and Jeol Jem 1010 Transmission Electron Microscopy (TEM) images were evaluated to examine the morphological appearance and structure of the synthesized Ag-NPs.

Particle size and zeta potential determination

Malvern Dynamic Light Scattering (DLS) analysis was used to determine the hydrodynamic size and surface distribution of Ag-NPs(zeta potential).

Fourier transform infrared (FTIR) analysis

The spectra were analyzed using a Perkin Elmer One Fourier transform infrared (FTIR) spectrometer to evaluate the functional groups that may be responsible for the formation, stability, and coating of Ag-NPs in the pumpkin peel extract.

Cell culture and reagents

MDA-MB-231 cells were purchased from the Pasteur Institute (Tehran, Iran) as a model for human triple-negative breast cancer (TNBC). MDA-MB-231 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin–streptomycin (Sigma, USA). The cells were incubated in a 37º incubator with a humidified atmosphere regulated at 5% CO₂. The green-synthesized Ag-NPs were dissolved in distilled water to prepare a stock solution and sonicated for 15 min on the day of treatment. Prior to each treatment, the stock solution was diluted to a predetermined concentration in the final medium. Cells were grown until they reached 70–80% confluency and then subjected to the designed treatment regimens.

Treatment with green-synthesized Ag-NPs and RT of MDA-MB-231 cells

MDA-MB cells were initially cultivated in T25 flasks for 24 h and then subjected to a treatment regimen. MDA-MB-231 cells were left untreated (control group) or treated with green-synthesized Ag-NPs alone at varying concentrations for two h, followed by RT alone or in combination with green-synthesized Ag-NPs. The RT dosage was based on previously published studies (Montazersaheb et al. 2023). The gray irradiations of 4 and 8 were used in this study.

Radiation

The cell culture flasks were radiated using an Elekta linear accelerator machine (Elekta Solutions AB, Sweden). For this purpose, the flasks were sandwiched between water-equivalent slab phantoms at the source to a surface distance of 100 cm and a field size of 20 × 20 cm². The flasks were exposed to a total dose of 4 and 8 Gy from 6 MV photons and a 300 cGy/min dose rate.

Viability assay using trypan blue exclusion dye and MTT assay

To elucidate the in vitro cytotoxicity of Ag-NPs synthesized by the green method, an MTT assay was performed using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Aldrich, USA). Briefly, 3 × 10³ MDA-MB-231 cells were seeded in 96-well plates. After 24 h, the cells were treated with varying concentrations of green-synthesized Ag-NPs (0.5–10 µg/mL) and incubated for 48 h. MTT (5 mg/mL) was added to all experimental groups and incubated for 4 h at 37 °C. After removing the supernatants, 200 µL DMSO was added to each well to dissolve the formazan crystals. Finally, optical density was measured at 570 nm using an Elisa Reader (BioTek Instruments, Inc. USA) (Molavi 2020). IC50 was obtained from the dose–response curve, and statistical analysis was performed using GraphPad Prism software. To eliminate the cytotoxic effects of green-synthesized Ag-NPs at higher concentrations, green-synthesized Ag-NPs were used at a concentration around the IC50 for further experiments (Jafari et al. 2022).

Trypan blue exclusion dye was used to determine the cell viability after exposure to RT. Briefly, the MDA-MB-231 cells were cultured in T25 flasks. After 24 h of culture, the cells were exposed to the optimum concentration of green-synthesized Ag-NPs for 2h, followed by 4 and 8 Gy exposure. Cell viability was determined using a microscope at 48 h based on the optimum time obtained by the MTT assay.

Different concentrations of green-synthesized Ag-NPs were added to the culture medium 2 h prior to RT for the combination therapy. After 48 h, the cell viability of all groups was evaluated using a trypan blue exclusion dye assay. Both methods were performed in triplicate, and the results are presented as the mean of three independent experiments. The combination index (CI) of green-synthesized Ag-NPs and RT was determined using the method described by Chou–Talalay. Based on the Chou–Talalay equation, CI < 1 indicates synergism, CI > 1 indicates antagonism, and CI = 1 represents additive. Concentrations close to the IC50 values of green-synthesized Ag-NPs were used for combination therapy. Combination therapy with green-synthesized Ag-NPs and RT showed maximal anticancer effects, as revealed by CI.

Apoptosis determination by flow cytometry analysis using annexin V/PI

The population of apoptotic cells was analyzed by flow cytometry using an apoptosis detection kit (Cat No:88-8005-72; eBioscience). For this purpose, MDA-MB-231 cells were harvested from all experimental groups, including untreated cells and cells treated with green-synthesized Ag-NPs alone, irradiated alone, or a combination of green-synthesized Ag-NPs plus irradiation for 48 h. In the following, the cells were washed with phosphate buffered saline (PBS), and resuspended in a binding buffer at 4 C for 20 min. The cells were exposed to 100 µL of binding buffer containing 5 µL of FITC-conjugated Annexin V and incubated in the dark for 15 min at RT. The cells were washed with binding buffer and exposed to 5 µL PI in 100 µL binding buffer. Finally, the population of apoptotic cells was determined using a FACSCalibur (BD Bioscience). The obtained data were analyzed using the FlowJo software ver. X.0.7. All experiments were performed in triplicate (Valipour et al. 2020).

Evaluation of HIF-1-α expression using flow cytometry

Flow cytometric analysis was conducted to detect HIF-1-α protein. Briefly, the cells were seeded into 6-well plates at a density of 1 × 10⁵ cells/well overnight. The cells were treated with green-synthesized Ag-NPs alone, irradiation alone, or a combination of green-synthesized Ag-NPs and RT for 48 h. After harvesting the cells from all experimental groups, they were fixed with paraformaldehyde in PBS (4% v/v), permeabilized with 0.1% Tween-20 in PBS, and exposed to PE-conjugated anti-HIF-α antibody for 1 h at 4  C. Finally, the stained cells were assessed using a FACSCalibur (BD Bioscience, USA). The obtained data were analyzed using FlowJo software ver. X.0.7 (Farjami et al. 2019).

Real-time PCR for determination of BAX, BCL2, and p53 levels

Real-time PCR was used to evaluate the expression of apoptosis-related genes at the RNA level. MDA-MB-231 cells were harvested from all experimental groups: untreated cells, cells treated with green-synthesized Ag-NPs alone, irradiated alone, and cells treated with a combination of green-synthesized Ag-NPs and Radiation. The harvested cells were washed with PBS and subjected to RNA extraction using a Thermo Scientific kit (K0731) according to the manufacturer’s instructions. RNA concentration in each group was measured using a Nanodrop (ND-1000, Wilmington, DE, USA). Total RNA (500 ng) was reverse-transcribed into cDNA using a previously published method (Montazersaheb et al. 2020). GAPDH was used as the internal control, and all acquired data were normalized to GAPDH expression. Primer sets used in this study are listed in Table 1.

Table 1 Genes, primer sequences, melting temperature, and product size

Western blotting analysis for determination of PERK and CHOP protein levels

Total protein was extracted from all the experimental groups: untreated cells (control), cells treated with green-synthesized Ag-NPs alone, cells treated with RT alone, and cells treated with a combination of RT and green-synthesized Ag-NPs. The harvested cells were washed with cold PBS and lysed with RIPA buffer at 4 °C for 30 min. Following the centrifugation of the homogenates, the protein concentration in each group was measured using a BCA kit (Pierce, Rockford, IL, USA). Fifty micrograms of each sample were loaded in each lane of SDS-PAGE (12%) to separate the proteins. The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. To prevent nonspecific interactions, the membrane was incubated with TBS-T buffer containing 5% skim milk, primary antibodies, C/EBP-homologous protein (CHOP) (Abcam), protein kinase RNA-like endoplasmic reticulum (PERK) and anti-β-actin (Santa Cruz Biotechnology, CA), and incubated overnight at 4 °C. After two washes with TBS-T, the membranes were incubated with a secondary antibody (Santa Cruz Biotechnology, CA) in TBS-T for 60 min at 25 °C. After the final washing with TBS-T, the protein bands were developed by enhanced chemiluminescence (ECL) (Roche, UK) and captured on X-ray film. The intensity of each protein was determined using ImageJ 1.6 software and normalized to that of the corresponding β-actin control (Montazersaheb et al. 2018).

ELISA assay for determining of cyclin D1 and ERK (1/2) levels

ELISA assay was conducted to evaluate the intracellular levels of cyclin D1 protein and the contents of total and phosphorylated ERK1/2 in all experimental groups using PathScan® ELISA kits. Total p44/42 MAPK (Erk1/2) (Cat. # 7050C), Phospho-p44/42 MAPK (Thr202/Tyr204) (Cat. #7177), and Total cyclin D1 Sandwich ELISA Kit (Cat. #7918C) were provided by Cell Signaling Technology (Danvers, MA, USA). ELISA was performed according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells (8 × 10⁵ cells/mL) were seeded in 100 mm petri dishes. After overnight incubation, the cells were treated with 4 µg/ml Ag-NPs for 48 h. The cells were then washed and rinsed with cold PBS. According to the manufacturer’s protocol, a lysis buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich, USA) and PMSF (Sigma-Aldrich, USA) was used to lyse the cells. Then, the cell lysates were centrifuged for 5 min at 10,000 × g at 4 °C. To make equal amounts of total proteins in samples subjected to ELISA, the total protein concentrations of cell lysates were detected using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The optical density was quantified by ELISA Reader (BioTek Instruments, Inc. USA).

Apoptosis evaluation using DAPI-TUNEL assay

Cell apoptosis was also determined by the TUNEL-DAPI double staining assay. Cell slides were placed into the wells of a 6-well plate. The MDA-MB-232 cells were seeded and then treated in the same manner as described in previous experiments and incubated for 48 h at 37°C with 5% CO2. Following harvesting, the cells were washed with PBS before being fixed with 2% paraformaldehyde. Subsequently, the cells were permeabilized in Triton-X 100 (0.1%) for 10 min at room temperature. The TUNEL assay was carried out according to the manufacturer's instructions (E-CK-A334, Elabsciences). Briefly, the cells were incubated in TUNEL reaction solution in a 37 °C humidified chamber for 2 h in dark condition, then rinsed twice with PBS and incubated with DAPI stain (1 mg/mL) for 30 min at 37  C. Finally, the stained cells were examined using a fluorescence microscope (Olympus BX50). TUNEL-positive cells were defined as apoptotic cells.

Statistical analysis

The obtained data were compared using one-way ANOVA followed by Tukey’s post hoc test for comparison between groups. Statistical significance was set at p < 0.05. Data were analyzed using GraphPad Prism version 6.01.

Results

Characterization of Ag-NPs synthesized by the green method

The color of the mixture underwent a quick transformation to a dark brown hue within 30 min of combining the pumpkin peel extract and AgNO₃ solution. Samples were collected from the synthesis medium and subjected to wavelength scans using UV–vis measurements (Fig. 2), depending on the rate and intensity of color change. The rapid change in color from yellow to brown and the highest absorption of light at a wavelength of 443.6 nm (Fig. 2a–c) were specific indicators of the creation of Ag-NPs (Sampaio and Viana 2018; Parvathalu et al. 2023). The stability of Ag-NPs under physiological circumstances (3% hypertonic liquid) was also evaluated six months after synthesis using UV–vis measurements of the maximum wavelength data. As shown in Fig. 2d, the maximum absorbance peak was obtained at 442.8 nm.

Fig. 2

Color change and UV–vis spectra indicating the formation of green-synthesized Ag-NPs with pumpkin extract; a extract; b dark brown color change due to the formation of Ag-NPs in colloidal form; c maximum absorbance data obtained in wavelength scans of Ag-NPs, and d the stability of Ag-NPs under physiological circumstances (3% hypertonic liquid) was evaluated six months after synthesis using UV–vis measurements of the maximum wavelength data

For the purpose of determining the crystal nanosize and evaluating the pattern of crystal structures of the synthesized Ag-NPs, data obtained from XRD measurements taken at 2-Theta were utilized. In the data shown in Fig. 3, the expansions in the Bragg angles at points (111), (200), (220), and (311) show that the crystal pattern reflected on the plane is face-centered cubic (fcc) structure of green Ag-NPs. Bragg's reflection of the crystal structure of Ag-NPs was compatible with JCPDS file no:04–0783(Anjana et al. 2021; Luna et al. 2015; Sharifi-Rad et al. 2024; Rengarajan et al. 2024). The FWHM values of these angles were 38.50, 44.30, 64.14, and 77.07, respectively (Fig. 3). By evaluating the maximum peak at these Bragg angles, it was calculated that the crystal nanosize of Ag-NPs synthesized by the Debye–Scherrer formula was 13.39 nm. A study was undertaken to synthesize Ag-NPs using Delonix regia extract (Abu-Dief et al. 2020). The crystal diameters of the Ag-NPs were determined to be 12 nm using the Debye–Scherrer equation.

Fig. 3

The X-ray diffraction (XRD) data display the crystal structures of silver nanoparticles (Ag-NPs) that were synthesized utilizing the pumpkin extract

FE-SEM, TEM, and AFM data were used to determine the morphological appearance and structures of the Ag-NPs synthesized by green way. The TEM images in Fig. 4a revealed that the synthesized Ag-NPs exhibited a spherical form. The analysis of Figs. 4b and 5 indicated that Ag-NPs exhibited a spherical shape with a size below 100 nm in the FESEM and AFM micrographs, respectively (Rengarajan et al. 2024). Utilizing the data obtained by measurement and collection by DLS, the hydrodynamic size distributions and surface charge (zeta potential) of the synthesized Ag-NPs were determined (Figs. 6 and 7). It was determined that Ag-NPs synthesized using pumpkin peel extract had a spherical appearance, an average hydrodynamic size distribution of 81 nm, and a surface charge of -9.96 mV. A major finding was made that the synthesized Ag-NPs exhibited a negative surface charge. The presence of a negative surface charge is crucial in preventing the occurrence of unfavorable conditions that can disrupt stability, such as agglomeration and fluctuations. The phytochemicals present in the plant contributed to the generation of a negative surface charge of -9.96. Due to the presence of amino, carbonyl, ester, phenolic, and hydroxyl groups, there is a strong tendency for these groups to successfully connect to noble metals through polar and coordinating interactions (Ajaykumar et al. 2023; Singh et al. 2018).

Fig. 4

The morphological appearances of Ag-NPs synthesized using a green method are depicted in a TEM and b FE-SEM micrograph pictures

Fig. 5

AFM micrograph images depict the topographical features of Ag-NPs produced using the green synthesis process

Fig. 6

The provided data present the distribution of surface charges of Ag-NPs as evaluated using DLS analysis

Fig. 7

The hydrodynamic size distributions of the synthesized Ag-NPs were determined using DLS measurements

Analysis spectra were evaluated using a Perkin Elmer FTIR to evaluate the functional groups that may be responsible for the formation, stability, and coating of Ag-NPs in the pumpkin peel extract (Fig. 8). The FTIR spectra revealed frequency shifts resulting from vibrations at 3280 cm⁻¹, 2918 cm⁻¹, 2113 cm⁻¹, 1699 cm⁻¹, 1401 cm⁻¹, 1233 cm⁻¹, and 1013 cm⁻¹ (Fig. 8). The frequency shifts seen at 3280 cm⁻¹ and 2918 cm⁻¹ were identified as hydroxyl groups exhibiting O–H stretching vibrations. The presence of polyphenols can be indicated by peaks at 1013 cm⁻¹, which may be attributed to aliphatic amine or alcohol/phenol C–N stretching vibrations 1314 The frequency change with stretching vibration at 2918 cm^-1 and 2113 cm^-1 belonged to carboxylic acid (–COOH). The frequency changes at 1699 cm^-1 and 1401 cm^-1 were stretching vibrations of primary amines with N–H folds. The frequency changes at 1233 cm^-1 and 1013 cm^-1 were alcohol groups belonging to C–N stretching vibrations 6 15.

Fig. 8

The FTIR spectra of both the extract and post-synthesis liquids contain functional groups of phytochemicals that have a role in the synthesis, stability, and coating of Ag-NPs

Cytotoxic effects of green-synthesized Ag-NPs on MDA-MB-231

The cytotoxic effects of green-synthesized Ag-NPs were determined using the MDA-MB-231 cell line as a TNBC model. The dose–response curves for the inhibitory effects of green-synthesized Ag-NPs are shown in Fig. 9. Based on these findings, the IC50 value for green-synthesized Ag-NPs was 4.3 ± 0.95 μg/ml in MDA-MB-231 cells.

Fig. 9

a Dose–response curve of green-synthesized Ag-NPs in MDA-MB-231 cells. GraphPad Prism software created a dose–response curve and calculated the IC50 value. b Fa–CI plot of combination therapy in MDA-MB-231 cells generated using Compusyn software. c The trypan blue exclusion assay determined the anticancer effect of RT, Ag-NPs, and a combination of RT and Ag-NPs in MDA-MB-231. All experiments were performed in triplicate, and data are reported as the mean ± SD (standard deviation). CI: combination index. Fa: fraction of cells affected by combination therapy. ***P < 0.001

To determine whether green-synthesized Ag-NPs can enhance the anticancer effects of RT, MDA-MB-231 cells were exposed to green-synthesized Ag-NPs alone or in combination with RT. The Fa and CI values were acquired by Compusyn software for monotherapies and their combinations (Table 2). As shown in Fig. 9, treatment of MDA-MB-231 cells with green-synthesized Ag-NPs (5 μg/mL) in combination with RT (8Gray) for 48 h significantly reduced cell viability compared to untreated cells (control) and RT alone. The data revealed that combination therapy had a synergistic anticancer effect on cancer cells. Figure 9b depicts the Fa–CI plot of the combination therapy obtained using Compusyn software. As previously mentioned, a CI < 1 indicates a synergistic effect. In the present study, a combination of 5 µM green-synthesized Ag-NPs and 8 Gy RT caused a strong synergistic effect with a CI value of 0.49.

Table 2 Fraction of affected MDA-MB-231 cells inhibited by combination therapy (Fa) and combination index (CI) for different doses of green-synthesized Ag-NPs and RT

Cell viability was reduced in the irradiated group (64.87 ± 3.2%) and in the green-synthesized Ag-NPs (58.09 ± 3.52) % to 37.2 ± 4.56% in the cells exposed to the combination of green-synthesized Ag-NPs (5 μg/mL), and RT (8 Gy). Combination therapy substantially decreased cell viability compared with the corresponding monotherapies. In this context, 5 µg/mL green-synthesized Ag-NPs, and 8 Gy radiation were used for subsequent experiments.

In an initial screening, the anticancer effect of green-synthesized Ag-NPs was also determined in MCF7 cells as a control set. The IC50 value of green-synthesized Ag-NPs in MCF7 cells was obtained 3.41 ± 0.48. In MCF7 cells, the best CI value was achieved 0.68 when green-synthesized Ag-NPs (4 μg/mL) and RT(8 Gy) were used in combination. It was evident that green-synthesized Ag-NPs also showed a potent anticancer effect against MCF7 cells. However, it seemed that the combination of green-synthesized Ag-NPs with RT resulted in more synergistic anticancer effect in MDA-MB-231 cancer cells compared to MCF7 cells. Data are shown as supplementary Figure.S1 and Table.S1.

Green-synthesized Ag-NPs enhance radiation-induced apoptosis in MDA-MB-231 cells

To determine the apoptotic effects of green-synthesized Ag-NPs alone or in combination with RT on MDA-MB-231 cells, the cells were stained by Annexin V/PI and assessed by Flow Cytometry. Early apoptosis is evidenced by Annexin V-positive and PI-negative cells, and late apoptotic or necrotic cells are marked by Annexin V and PI positivity. Figure 10 shows the contour diagram of Annexin V and PI flow cytometry of MDA-MB-231 cells. The percentage of apoptotic cells was 51.09 ± 4.32% in MDA-MB-231 cells treated with green-synthesized Ag-NPs (p < 0.001). Cells exposed to RT showed 39.42 ± 2.27% apoptosis. Interestingly, the combination of green-synthesized Ag-NPs with RT resulted in a substantial proportion of apoptotic cells (65.2 ± 4.6%) as compared to RT (p < 0.001) and green-synthesized Ag-NPs (p < 0.01) alone. Several types of studies have confirmed that induction of apoptosis in cancer cells is the most appropriate type of cell death for cancers (Jafari, et al. 2023b; Akbarzadeh et al. 2016).

Fig. 10

Apoptosis induced by radiotherapy (RT) (8 Gy), green-synthesized Ag-NPs (5 µg/mL), and a combination of Ag-NPs and RT in MDA-MB-231 cells. a Dot plot flow cytometry analysis and b quantification of apoptosis stained with Annexin-FITC/PI. Each data is representative of the mean of triplicate ± SD. ^###p < 0.001 vs. untreated group. **p < 0.01 and ***p < 0.001 vs. each treatment group

To confirm the apoptotic effects of Ag-NPs and RT in other cells rather than TNBC, we used MCF7 cells. Combinational treatment of MCF7 cells with green-synthesized Ag-NPs and RT significantly enhanced the percentage of apoptotic cell death as compared to each monotherapy group (**p < 0.05 and ***p < 0.001 vs. Ag-NPs and RT, respectively) (data were shown in Figure.S2).

Evaluation of HIF-1α expression in MDA-MB-231 cells exposed to monotherapies and combination therapy

HIF-1-α is strongly induced under hypoxic conditions. It is expressed in cancer cells and contributes to malignancy. RT upregulates HIF-1α expression in some cancer cells (Jafari et al. 2023a). In contrast, Ag-NPs have been shown to suppress the function of HIF-1α in cancer cells (Yang et al. 2016). To determine the possible inhibitory effect of green-synthesized Ag-NPs on radiation-induced HIF-1α expression, we examined HIF-1α expression by flow cytometry. To accomplish this, MDA-MB-231 cells were exposed to green-synthesized Ag-NPs alone, irradiation alone, or a combination of green-synthesized Ag-NPs with RT. Briefly, cells were incubated with a PE-conjugated anti-HIF-1α antibody. As depicted in Fig. 11a, b, green-synthesized Ag-NPs alone (5μg/mL) resulted in a non-significant increase in HIF-1-α expression compared to that in untreated cells (13.1 ± 1.7% vs. 5.40 ± 0.69%) (p > 0.05). Irradiation alone remarkably increased the percentage of HIF-1α-expressing MDA-MB-231 cells from 5.40 ± 0.69 in the untreated group to 61.1 ± 5.7% in the RT-exposed group (p < 0.001). As expected, the combination therapy resulted in a remarkable decrease in the rate of HIF-1α expression in cells (25.29 ± 1.55%) compared to those treated with RT alone (p < 0.001). These results imply that green-synthesized Ag-NPs can mitigate radiation-induced HIF-1α expression.

Fig.  11

a Representative histogram of flow cytometry analysis and b quantification of HIF-1α expression MDA-MB-231 cells. The cells were treated with RT, green-synthesized Ag-NPs, and a combination of RT and green-synthesized Ag-NPs. PE-conjugated anti-HIF-1α was used to stain the cells. Each data is representative of the mean of triplicate ± SD. ^###p < 0.001 vs. untreated group. ^###p < 0.001 vs. control group. * p < 0.05 and ***p < 0.001 vs. each treatment group. ns: not significant

Combined green-synthesized Ag-NPs with RT altered the expression level of pro- and anti-apoptotic genes

Real-time PCR analysis was performed to determine the radiosensitizing effect of green-synthesized Ag-NPs on the expression of pro- and anti-apoptotic proteins. To achieve this, the expression levels of the pro-apoptotic protein (Bax, p53), the anti-apoptotic protein (BCL-2) were measured in all experimental groups. As shown in Fig. 12, treatment with green-synthesized Ag-NPs and RT alone increased the level of Bax (p < 0.001) and decreased BCL-2 levels (p < 0.001). In combination therapy, these effects were intensified. In addition, treatment of MDA-MB-231 cells with the combination of green-synthesized Ag-NPs and RT elevated p-53 levels compared with the untreated control group (p < 0.001) and each monotherapy (p < 0.001 vs. RT and p < 0.01 vs. Ag-NPs).

Fig. 12

Quantification of a Bax and Bcl-2 mRNA expression, b the Bax/Bcl-2 ratio, and c P53 mRNA expression in MDA-MB-231 cells using real-time PCR. The cells were treated with RT (radiotherapy), green-synthesized Ag-NPs, and their combination RT + green-synthesized Ag-NPs for 48 h. All experiments were repeated three times, and the data are presented as the mean ± SD. ^#p < 0.05, and ^###p < 0.001 vs. untreated group *p < 0.05, **p < 0.01, and ***p < 0.001 vs. each treatment group

Combination of green-synthesized Ag-NPs with RT potentiates P-PERK and CHOP protein expression

The PERK–ATF4 axis is crucial for regulating CHOP expression, as elevated expression of this protein leads to cellular apoptosis. CHOP can regulate endoplasmic reticulum (ER) stress, which induces apoptosis by increasing the transcription of pro-apoptotic factors (e.g., Bax) and decreasing the expression of anti-apoptotic factors (e.g., Bcl-2) in the mitochondrial membrane(Coker-Gurkan et al. 2021; Amiri et al. 2023).

To confirm whether green-synthesized Ag-NPs could potentiate the anticancer activity of RT, the expression levels of PERK (total and phosphorylated forms) and CHOP protein were analyzed by immunoblotting. Oxidative stress in the ER leads to the phosphorylation of PERK and increases p-PERK protein levels. After 48 h of treatment with monotherapies (green-synthesized Ag-NPs or RT alone) and combination therapies, the levels of proteins involved in cancer cell survival were evaluated. As depicted in Fig. 13, treatment of MDA-MB-231 cells with green-synthesized Ag-NPs increased the level of p-PERK in MDA-MB-231 cells compared to the control group (untreated). As expected, the level of p-PERK was remarkably elevated in the cells exposed to the combination therapy compared to the individual therapy (p < 0.001). Likewise, there was a notable increase in the level of CHOP protein in combination therapy compared to monotherapy with radiation and green-synthesized Ag-NPs (p < 0.01). These results support the ability of green-synthesized Ag-NPs to potentiate the efficacy of irradiation.

Fig. 13

Western blot analysis of P-PERK and CHOP proteins in MDA-MB-231 cells exposed to green-synthesized Ag-NPs (5 µM), radiation (8 Gy), or combination therapy (5 µM + 8 Gy). a Representative image of western blot analysis and b relative quantification of P-PERK and CHOP. All values are expressed as the mean ± SD. ^##p < 0.01 and ^###p < 0.001 vs. the untreated group. **p < 0.01` and ***p < 0.001 vs. each treatment group

Combination of green-synthesized Ag-NPs with RT reduced the expression of cyclin D1 and phosphorylated-ERK1/2

Cyclin D1 is an essential protein that regulates the cell cycle phases (G1–S). Overexpression of cyclin D1 has been identified in most cancer cells (e.g., MDA-MB-231 cell line) (Choi et al. 2019). The ERK signaling pathway also plays a crucial role in cell proliferation and survival. Furthermore, ERK activation is a crucial signal for the resistance of MDA-MB-231 cells to various therapies, including radiotherapy (Pawlonka et al. 2021). The present study examined the intracellular levels of cyclin D1 and phosphorylated ERK1/2 (p-ERK1/2) in all experimental groups using a specific ELISA kit.

Figure 14 shows that green-synthesized Ag-NPs potentially reduced the level of cyclin D1 in the MDA-MB-231 cell-treated cells compared to the control cells (p < 0.01), implying the role of green-synthesized Ag-NPs in cell arrest. Moreover, the combination of green-synthesized Ag-NPs with RT significantly reduced cyclin D1 levels compared to RT alone (p < 0.001), and green-synthesized Ag-NPs alone (p < 0.001). As expected, combination therapy with green-synthesized Ag-NPs and RT significantly decreased p-ERK/total ERK levels in MDA-MB-231 cells (p < 0.01).

Fig. 14

Percentage of protein expression of a cyclin D1 protein, b phosphorylated and total ERK1 in MDA-MB cells treated with RT (radiotherapy), green-synthesized Ag-NPs, and a combination of RT + Ag-NPs for 48 h. ELISA assay (ELISA) was used to determine protein levels. All experiments were repeated three times, and the data are shown as the mean ± SD. ##p < 0.01 and ###p < 0.001 vs. the untreated group. *p < 0.05 and ***p < 0.001 vs. each treatment group

Green-synthesized Ag-NPs intensified RT-mediated morphological alteration of MDA-MB-231 cells

The morphological changes of apoptotic cells include DNA fragmentation, nucleus condensation, cell membrane blebbing, cell shrinkage, and formation of apoptotic bodies. We assessed the effect of green-synthesized Ag-NPs with RT on the induction of apoptosis by observing morphological structure using a DAPI-TUNEL double staining assay (detecting DNA condensation). The quantified results for the DAPI-TUNEL staining assay showed that green-synthesized Ag-NPs significantly increased the percentage of the cells with nuclear condensation and fragmentation. In addition, there was an increase in the number of TUNEL-positive cells containing fragmented DNA in the cells in response to the treatment regimen. As expected, combination therapy with green-synthesized Ag-NPs and RT significantly intensified the observed changes, as shown in Fig. 15.

Fig. 15

DAPI-TUNEL double staining assay for detection of apoptosis-induced morphological changes in the MDA-MB-231 cells treated with RT (radiotherapy), green-synthesized Ag-NPs, and their combination for 48 h

Discussion

RT is a cancer treatment approach that often uses high-energy radiation to ionize atoms/molecules to damage dsDNA in cancer cells. To decrease injury to the normal tissue surrounding the tumor site, reducing the RT dosage can be an appropriate option for eradicating cancer cells (Song et al. 2017). To address this issue, various radiosensitizers have been used in irradiation procedures.

Nanotechnology-based approaches use metal NPs with high atomic numbers in combination with RT. NPs with high atomic numbers (e.g., gold, bismuth, platinum, silver) augment the effective dose of ionizing radiation in the tumor site, enhancing the therapeutic effectiveness (Goulis 2021). The safety, low toxicity, and accessibility of plants make them an ideal choice for green-synthesized Ag-NPs. The green synthesis of NPs is a better alternative to conventional methods (e.g., physical and chemical techniques) due to its environmentally friendly characteristics (Barabadi et al. 2021). It has also been reported that plant-mediated Ag-NPs exhibit anticancer properties in various cancer cell lines (Telrandhe 2019).

In the present research, Ag-NPs were synthesized quickly, easily, and cost-effectively in an environmentally friendly manner using the extract obtained from the fruit peels of Cucurbita spp. The dark brown color change (characterized by the formation of Ag-NPs) after mixing the extract and AgNO3 solution in Fig. 2 was due to vibrations occurring on the surface of the surface plasma resonance (SPR) plasma. This was confirmed by the 443.6 nm maximum absorbances measured in UV–vis spectroscopy, which characterized silver NPs (Parvathalu et al. 2023; Kumar et al. 2022; Khanal et al. 2023). In the data of the crystal structure pattern of the synthesized Ag-NPs in Fig. 2, the expansions in the Bragg angles occurring at points (111), (200), (220), and (311) show that the crystal pattern reflected on the plane is cubic with a central face (JCPDS No. 04– 0783). With the FWHM values of the highest peak of these angles, the crystal nano-size was determined as 13.39 nm. Different green synthesis studies support these findings (Khan et al. 2023; Abishad et al. 2022).

FE-SEM, TEM, AFM, and DLS data were used to determine the morphological appearance and structure of the Ag-NPs. It was determined that the Ag-NPs synthesized using the pumpkin extract had a spherical appearance, an average size distribution of 81 nm, and a surface charge of − 9.96 mV. In addition, the polydispersibility index (PDI) of the DLS findings was 0.38. This value showed no aggregation in the synthesized Ag-NPs, and the surface charge of -9.96 mV also supported this finding (Figs. 3, 4 and 5). The size distribution of NPs below 100 nm, having only a negative surface charge, and spherical morphological appearance are important biocompatibility features. In this respect, Ag-NPs synthesized using pumpkin peel extract are suitable in terms of their biocompatible structure (Keskin et al. 2023; Rolim et al. 2019). Their small size facilitates their entry into the cell. Only a negative surface charge prevents negative conditions such as clumping or destabilization. In addition, the fact that the PDI value of the Ag-NPs synthesized in Fig. 4 is 0.38 than one also supports the absence of aggregation. Phytochemicals provide stability and coating of NPs synthesized from plant sources (Sarkar et al. 2018).

The functional groups responsible for forming, stable structure, and coating the Ag-NPs were identified using the FTIR data. Figure 6 shows the FTIR spectra of the extract and the Ag-NPs. The FTIR spectrum of the extract showed an absorption peak at 3280 cm^-1, known as –OH stretching in alcohols and phenolic compounds with strong hydrogen bonds, or may be due to the stretching of the –NH band of amino groups (Jagtap and Bapat 2013). The absorption peaks at approximately 2847 cm1 and 2919 cm^-1 can be associated with aldehyde (-CH) stretch vibrations, and the peak at 1736 cm^-1 indicates the presence of a carbonyl group of an aldehyde. Furthermore, the FTIR spectra of the extract showed peaks at 1599 and 1401 cm^-1, and amide I and amide II were recognized as carbonyl (C = O) groups (Dada et al. 2018). In addition, the absorption peaks in the 1401–1013 cm^-1 range are associated with the C–O, C–N, and C–C stretching vibrations of phenolic compounds, aliphatic amines, and alkanes, respectively (bakht Dalir et al. 2020).

The FTIR spectrum of the Ag-NPs was in the range of 1010–2911 cm⁻¹. The peaks appearing at 2911, 1688, and 1010 cm^-1 in the FTIR spectrum of Ag-NPs are associated with the O–H stretching and N–H stretching vibrations of carboxylic acids, aldehydes, ketones, and C = O functional groups, respectively (Mittal et al. 2014; Hamano et al. 2016). In the synthesis of Ag-NPs, N–H, carbonyl, and alcohol/phenol (-OH) groups containing amides played a role in the reduction, coating, and stability of silver ions to silver NPs by adding its extract to the silver nitrate solution (Yu et al. 2019).

Breast cancer is the most frequent cancer among women worldwide. TNBC is an aggressive subtype, and its treatment is challenging due to the lack of specific molecular targets. Chemotherapy and radiotherapy are the most common modalities used to treat patients with TNBC (Manjunath and Choudhary 2021); however, they elicit some adverse effects, particularly at a higher dose. Among the growing number of nanomaterials, choosing a potential material is critical in cancer therapy. Understanding the potency and toxicity of different NPs enables us to selectively target cancer cells. The combination of chemotherapy and irradiation has been reported in various cancer therapies (Manjunath and Choudhary 2021); however, no study was available regarding the radiosensitizing effect of green-synthesized Ag-NPs on RT. Previous reports exhibited the antitumor effects of Ag-NPs in breast cancer (Jeyaraj et al. 2013). Several studies reported that lysosomal entrapment and acidic pH facilitate the ionization of the Ag-NPs. Ag-NP ions activate ROS formation and oxidative stress, thereby driving the pathways leading to cell death (Akter et al. 2021). The idea of using Ag-NPs as radiosensitizers stems from the phenomenon that high atomic numbers of metals can augment radiation's effects. Mechanistically, the radiosensitizing ability of Ag-NPs can be achieved through physical, chemical, and biological dose enhancements. In the physical phase of interactions, Ag-NPs raise cellular damage through the high energy absorption and production of photoelectrons and Auger electrons. In the chemical phase of interaction, Ag-NPs accelerate the formation of free radicals and ROS production to enhance the RT-induced cellular damage. Finally, in biological phase, Ag-NPs augment the effect of RT by induction of oxidative stress, cell cycle disruption, and DNA repair inhibition (Kovács et al. 2022).

Accordingly, the present study determined the combination therapy of green-synthesized Ag-NPs with radiation in MDA-MB-231 cells as a model of TNBC. This study revealed the potential of green-synthesized Ag-NPs in enhancing radiation sensitivity in MDA-MB-231 cells through a series of events.

Ag-NPs can enhance the efficacy of irradiation by accumulating more in MDA-MB-231 cancer cells, subsequently increasing therapeutic effectiveness. These effects may be attributed to the high absorption of ionizing radiation and secondary electron emissions with Compton scattering and photoelectric effect (Goulis 2021). On the other hand, the bioactive compounds such as carotenoids, flavonoids, vitamins, and other phenolics in pumpkin might have a role in the observed anticancer activity (Hussain et al. 2022).

It has been reported that Ag-NPs can prevent cell growth and induce apoptosis in various cancer cells, such as breast cancer (Al-kawmani et al. 2020). Our results showed that green-synthesized Ag-NPs induced apoptosis and altered morphological structure in MDA-MB-231 cells. Expectedly, the combination with RT augmented the apoptotic properties. Administration of Ag-NPs has been found to induce apoptosis through ER stress mediation, altered ubiquitination, and ROS production (Huo et al. 2015). The generation of intracellular ROS is crucial in oxidative stress and inducing apoptosis (Zhang et al. 2012). The literature search showed that Ag-NPs can increase ROS production in cancer cells, subsequently increasing oxidative stress and radiation-induced damage. This is mediated by restricting the cells to a radiation-sensitive state. In addition, Ag-NP-induced mitochondrial damage can cause cells to release apoptotic stimulators, making them more prone to radiation and sensitizing the surviving cancer cells to radiotherapy (Akter et al. 2021).

Interestingly, radiation can also cause DNA damage and oxidative stress and arrest the cell cycle in the S-phase, triggering the apoptotic intrinsic pathway (Wang 2014). An increase in mitochondrial permeability can release pro-apoptotic factors closely associated with a series of proteins belonging to the Bcl-2 family. In addition to causing damage to DNA and cells, irradiation-induced apoptosis is mediated by the activation of Bax and p53 (Pisani et al. 2020).

In the present study, real-time PCR results demonstrated that green-synthesized Ag-NPs increased the protein expression of p53 in MDA-MB 231 cells. p53 upregulates the genes involved in apoptosis and cell cycle arrest. In addition, P53-induced apoptosis upregulates the expression of the pro-apoptotic gene (Bax) and downregulates the anti-apoptotic (Bcl-2) gene. Homodimerization of Bax protein leads to apoptosis induction, while heterodimerization of Bax with Bcl-2 inhibits apoptosis, enhancing the survival of cancer cells. In this context, p53-induced apoptosis enhances Bax/Bcl2 ratio, resulting in apoptosis. Indeed, p53 behaves as a transcriptional activator of Bax, and inactivator of Bcl-2 (Aubrey et al. 2018). The current findings showed that green-synthesized Ag-NPs upregulated Bax and downregulated Bcl-2 expression in MDA-MB-231 cells. Upon combination therapy, these effects were intensified. We also found an increased ratio of Bax/Bcl-2. These findings proposed that upregulated p53 activates downstream target genes involved in cell apoptosis.

It has been reported that radiotherapy upregulates the expression of HIF-1α in various cancer cells, specifically breast cancer. HIF-1α upregulation influences breast cancer cells' progression and metastasis by upregulating angiogenesis genes (Liu et al. 2015). Our findings suggest that the co-treatment with green-synthesized Ag-NPs and RT led to a more significant reduction than either individual treatment. The results implied that green-synthesized Ag-NPs partly act as a radiosensitizer agent by reducing radiation-induced HIF-1α in TNBC cells.

It is well-documented that ERK1/2 causes cell survival and chemoresistance through activating Bcl-2 and degrading pro-apoptotic proteins such as Bcl-2-modifying factor (BMF), Bcl2-interacting mediator of cell death (BIM), and P53-upregulated modulator of apoptosis (PUMA) (Darling et al. 2017). Additionally, numerous studies have shown that targeting the ERK1/2 pathway with anticancer agents can enhance apoptosis and reduce metastasis in various cancers. Therefore, suppressing ERK1/2 activation by green-synthesized Ag-NPs may induce apoptosis and inhibit the cell cycle.

In this study, ELISA assay results showed that green-synthesized Ag-NPs inactivate ERK1/2, as indicated by a significant reduction of p-ERK/total ERK ratio in MDA-MB-231 cells that were strengthened in combination therapy. Moreover, we demonstrated that green-synthesized Ag-NPs significantly reduced Cyclin D1 levels in MDA-MB231 cells compared to the control group. Upon combining with RT, this reduction was further augmented. Cyclin D1 is a well-established oncogene and a critical mediator in the cell cycle in various types of cancer cells such as MDA-MB-231 cells (Choi et al. 2019).

The ER is a critical intracellular organelle that maintains cellular homeostasis. Under normal conditions, a balance exists between ER protein loading and folding capacity. Adverse external factors such as hypoxia and oxidative stress can induce ER stress, leading to dysfunction of ER and the accumulation of misfolded and unfolded proteins. This condition activates the unfolded protein response (UPR) to resist the unfavorable external milieu (Sano and Reed 2013). It has been reported that ER stress can induce various kinds of cell death, such as apoptosis. When cells experience ER stress, the activated UPR can cause two different outcomes. Cells suffering short-term ER stress can enhance ER ability to restore homeostasis and cell survival. In contrast, under severe ER stress, cells undergo cell death through multiple pathways (Oliyapour et al. 2023). In the current study, exposure of MDA-MB-231 cells to green-synthesized Ag-NPs led to apoptosis, associated with oxidative and ER stress. Combined therapy intensified the observed effects. We found that green-synthesized Ag-NPs remarkably upregulated the protein level of the PERK signaling axis, including PERK and CHOP, compared to the control group.

Conclusion

This study is the first report on the combination therapy of green-synthesized Ag-NPs and RT on MDA-MB-231 cells. We observed synergistic effects of green-synthesized Ag-NPs when co-administered with irradiation, which suggests green-synthesized Ag-NPs are a suitable radiosensitizer agent. In other words, combination therapy shows greater inhibitory effects than individual therapy. Upregulation of apoptotic-related genes was observed in MDA-MB-231 cells with monotherapy, and these effects were intensified with combinational therapy. Activating the apoptosis pathway and induction of ER stress may play an essential role in radiosensitizing effects on green-synthesized Ag-NPs. Combination therapy exhibited the most helpful role of green-synthesized Ag-NPs in inhibiting radiation-induced HIF-1 α, and decreasing the cyclin D1and p-ERK expression.

Given the limitations of treatment options for TNBC patients, this study may contribute to developing novel anticancer strategies for clinical use. Further investigation will be needed to clarify the underlying mechanisms of green-synthesized Ag-NPs and RT on TNBC.