Introduction

Cancer ranks as the second most common cause of death globally, surpassed only by cardiovascular diseases [1]. In 2020, global cancer rates were estimated at 19.3 million cases, resulting in approximately 10 million deaths [2]. Women worldwide express a significant concern about malignancies, with breast cancer being the most prevalent, constituting about 25% of all gynecological cancers [3, 4]. Various treatment methods such as surgery, chemotherapy, radiation therapy, and monoclonal antibodies are selected based on the patient’s specific circumstances. Nevertheless, the current treatments’ limitations in efficacy and safety necessitate the exploration of novel technologies to develop more effective treatment alternatives [5, 6].

Colorectal cancer (CRC), accounting for 10% of all cancers, holds the third position in the global cancer prevalence hierarchy and is implicated in a substantial portion of cancer-related fatalities [7]. Multiple treatment options for CRC, including surgery, chemotherapy, radiation therapy, and palliative care, are currently available [8]. Although traditional treatments can eliminate cancer cells, they also inadvertently harm healthy cells. Consequently, the quest for innovative treatment strategies has stimulated the invention and progression of novel cancer therapies [6].

In recent years, nanotechnology has made significant advances, especially in the creation of various medications. The production of nanoparticles represents a significant achievement in the effective diagnosis and treatment of diverse diseases. The manufacturing of nanoparticles employs biological materials such as microorganisms, enzymes, cells, and plant extracts [9]. There has been a marked increase in interest in deploying biogenetically engineered metallic nanoparticles (NPs) for cancer theranostics. The biological synthesis of nanoparticles, using organisms such as bacteria, actinomycetes, fungi, and plants, has been explored as a simpler and more cost-effective alternative to other chemical and physical methods [10]. Recently, considerable attention has been given to nanoparticle synthesis through green methods, using plants and bacteria, due to its numerous advantages [11, 12]. One such advantage is that the green synthesis of nanoparticles eliminates the need for cell cultures, which are labor-intensive and expensive, thereby increasing scalability and industrial applicability [13].

The unique characteristics of nanoparticles have recently sparked interest in utilizing microbial strains for nanoparticle synthesis. The distinct physicochemical properties of nanomaterials, compared to their non-nano counterparts, have stimulated extensive research in this field over the past few decades [14, 15]. Organisms have developed sophisticated mechanisms to survive in environments with high concentrations of undesirable ions, including transforming the chemical nature of a toxic ion into a non-toxic form, ultimately leading to the formation of corresponding nanoparticles [16]. Therefore, nanoparticles can be considered a byproduct of an organism’s resistance mechanism to a particular ion [17].

Selenium (Se) is a vital element that has garnered the attention of numerous researchers owing to its unique physicochemical and electrical characteristics [18]. This crucial micronutrient, found in a variety of sources such as organ meats, seafood, plants, cereals, and nuts, is integral to human health [19]. Its distinctive properties and potential uses in the realms of physics, chemistry, and biology have been widely acknowledged [20, 21]. As a trace element fundamental to all forms of life, selenium constitutes a component of selenocysteine in many enzymes, making it indispensable to all living beings [22]. In humans, selenium serves as a coenzyme for glutathione peroxidases and thioredoxine reductases, playing a vital role in the development of the immune system, cancer prevention, and antioxidant and antiviral effects [23]. Selenium nanoparticles (SeNPs), with their unique optical, electronic, electrocatalytic, and biological properties, carry considerable potential for technology applications across diverse fields, including medicine, diagnosis, therapy, electronic devices, catalysis, and chemical sensors [24].

SeNPs exhibit an array of biological attributes, such as antioxidant [25] and antimicrobial effects [26], immune system regulation [27], and tumor prevention [28]. As such, SeNPs have been identified as a key type of nanoparticle in medical research [29, 30]. Studies have shown that SeNPs have less toxicity and display superior biological activity when compared to common forms of selenium, such as se+4 and se+6 ions [31, 32]. Hence, SeNPs have been employed as a new addition to drug nanocarriers for drug delivery systems, particularly as selenium is a necessary dietary micronutrient in the form of SeNPs [33]. When scaled to the nanolevel, selenium functions as a promising chemo-preventive agent with lower toxicity [34, 35]. Several mechanisms of SeNPs’ anticancer effects have been identified, including the production of reactive oxygen species (ROS), activation of apoptotic pathways, mitochondrial dysfunction, DNA fragmentation, cell cycle arrest, and disruption of cellular homeostasis, [36,37,38,39]. In the study at hand, we synthesized SeNPs using Lactobacillus casei (LC-SeNPs) cell-free supernatant to assess their anticancer effects on MCF-7 breast cancer and HT-29 colon cancer cell lines.

Material and Method

Reagents

Sodium selenite (Na2SeO3), RPMI-1640 medium, fetal bovine serum (FBS, courtesy of DENAzist Asia’s Co), and penicillin–streptomycin (PS) 100 X were all procured from Gibco, ThermoFisher Scientific (Waltham, MA, USA). MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] and DMSO (dimethyl sulfoxide) were sourced from Merck (Germany). The Annexin V-FITC kit and 2′-7′-dichlorodihydrofluorescein diacetate (DCFHDA) were obtained from Roche (Munich, Germany). Ultra-pure water was generated using Milli-Q (Millipore, Darmstadt, Germany). DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of Supernatant and Production of LC-SeNPs

The L. casei (Strain 6904) required for the synthesis of selenium nanoparticles was procured from the Iranian Biological Resources Center (IBRC). Initially, the selected strain was grown in 50 mL de Man, Rogosa, and Sharpe (MRS) (Merck Millipore) liquid culture medium in a shaker incubator at 30 °C, with a speed of 150 rpm, over 18 h. Following this, 1 mL of the cultured L. casei was introduced to 100 mL MRS liquid culture medium (1% V/V), and the mixture was incubated at the same conditions for 72 h. The culture supernatant, after 72 h, was collected via centrifugation at 6000 rpm (Hettich, Germany) at room temperature for 20 min. This supernatant was then condensed using a freeze dryer (Alpha 1–2 LDPlus, Christ, Germany) until the volume reduced to 30 mL. This concentrated supernatant was then employed in the synthesis of LC-SeNPs. LC-SeNPs were produced by slightly modifying the protocol established by Xu et al. [40]. To summarize, 4 mM of Na2SeO3 was dissolved in 10 mL of distilled water, followed by the addition of 10 mL of bacterial supernatant, and the mixture was stirred at 40 °C for 3 h and 350 rpm. The emergence of an orange color indicated successful formation of selenium nanoparticles. The precipitate was rinsed with distilled water, then subjected to 10-min centrifugation at 13,000 rpm (Eppendorf 5804R, Germany) three times, and the final product was left to dry in the incubator.

Characterization of the Produced LC-SeNPs

Post-production, the structure, and morphology of the synthesized nanoparticles were confirmed using a variety of methods, such as ultraviolet–visible spectroscopy (UV–vis) (UV 1601, Shimadzu), field emission scanning electron microscopy (SEM) (Zeiss, Sigma VP model), energy-dispersive X-ray spectroscopy (EDX) (Zeiss, Sigma VP model), transmission electron microscopy (TEM) (Leo 906, Zeiss100KV model, Germany), X-ray powder diffraction (XRD) (PANalytical’s X′Pert PRO MRD), and Fourier transform infrared spectroscopy (FT-IR) (Spectrum Two FT-IR Spectrometer (Perkin Elmer)). Dynamic light scattering particle size distribution analysis (DLS) and zeta potential of LC-SeNPs were determined by Malvern Instruments Nano Zetasizer (Worcestershire, UK). The characterization tests were conducted as per our earlier study [41].

Cell Culture

Cell lines of HT-29, MCF-7, and human foreskin fibroblasts (HFF) were purchased from the Pasture Institute Cell Bank in Iran, Tehran. The cells were grown in RPMI 1640 medium (Gibco, USA) that was supplemented with 1% (V/V) penicillin–streptomycin and 10% heat-inactivated FBS, and 2 mM L-glutamine. The cells were kept in a 5% CO2 incubator at 37 °C.

Assessment of LC-SeNP Cytotoxicity via MTT Assay.

The MTT assay was utilized to evaluate the cytotoxic impact of LC-SeNPs on the HT-29 and MCF-7 cell lines. Initially, the cells (1 × 104 cells/well) were seeded. Subsequently, HT-29 and MCF-7 cancer cells were exposed to varying concentrations of the LC-SeNPs (3.125, 6.25, 12.5, 25, 50, and 100 mg/mL) at 24-, 48-, and 72-h intervals at 37 °C and 5% CO2. After incubation, 20 μL of MTT dye solution was added to each well, and the plates were incubated for 24 h. The MTT dye was then removed, and 100 μL of DMSO was added to each well. Ultimately, the absorbance of the microwells was measured at 570 nm using the ELISA reader. This entire process was repeated thrice, along with the control group, and the IC50 value was determined using the obtained OD values as per the formula given in reference [12].

$$\mathrm{Cell}\;\mathrm{viability}(\%)=(\mathrm{Abs}\;\mathrm{test}\;\mathrm{cells}/\mathrm{Abs}\;\mathrm{control}\;\mathrm{cells})\times100$$

Flow Cytometry Assessment of Apoptosis

Apoptotic induction was detected in HT-29 and MCF-7 cells (105 cells/well), treated with IC50 concentration of LC-SeNPs for a duration of 24 h, as per the guidelines provided in the Annexin V-FITC kit (eBioscience, Affymetrix, USA) manual. The stained cells’ analysis was conducted using a flow cytometry apparatus (Biocompare, USA). For comparison, untreated HT-29 and MCF-7 cells were utilized as a baseline control [42].

DAPI Staining

The DAPI staining protocol is employed to assess apoptosis in HT-29 and MCF-7 cells exposed to LC-SeNPs. The cells are initially seeded in 6-well plates before treating with an IC50 concentration of LC-SeNPs for 24 h and fixation with 0.4% formaldehyde. Following fixation, the cells are stained with 0.5 mg/mL DAPI dye for 5 min at ambient temperature. Afterward, the cells are thoroughly rinsed twice with PBS and inspected under the Olympus IX81 inverted fluorescence microscope (Olympus, Hamburg, Germany). Apoptosis is ultimately deduced from the detected nuclear fragmentation [43].

Apoptotic Genes Expression via qRT-PCR

The relative expression of apoptotic genes, namely, BCL2-associated X (BAX), BCL2 apoptosis regulator (BCL2), Caspase 3 (CASP3), and Caspase 9 (CASP9), is determined through qRT-PCR.

Initially, total RNA from treated and untreated cells is extracted following the instructions of an RNA extraction kit (CinnaGen, Iran). The extracted RNA’s quality and quantity are then verified using a nanodrop (IMPLEN GMBH). This is followed by cDNA synthesis using the Revers AidTM First Strand cDNA Synthesis Kit (Fermentas, USA). The expression of BAX, BCL2, CASP3, and CASP9 genes is then scrutinized using qRT-PCR on a Light Cycler (Bioneer, Daejeon, Korea), with beta-actin (ACTB) employed as an internal control.

The cDNA synthesis involves the addition of 3 μg of total RNA to a blend containing 1 μL of 10 × buffer and 1 μL of DNAse, incubated at 37 °C for 30 min. Afterward, 1 μL EDTA is added and further incubated at 65 °C for 30 min to inhibit DNAse activity. A mixture of 4 μL of 5 × buffer, 1.5 μL of MgCl2, 2 μL of dNTP mix (10 mM), 2 μL of random hexamer primer, 0.5 μL of RNasin (40 Unit/μL), and 1 μL of reverse transcriptase (RT) enzymes is then prepared and incubated at 42 °C for 60 min. Finally, the RT enzyme is deactivated by heating at 70 °C for 10 min.qRT-PCR reactions are set up in a final volume of 20 μL, comprising 10 μL master mix (Bioneer, South Korea), 1 μL of each primer (10 mM) (Table 1), 1 μL of cDNA, and 8 μL of PCR-grade H2O. The thermal cycling parameters consist of initial DNA denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 20 s, 62 °C for 40 s, and 72 °C for 10 s. The relative change in gene expression for all genes, compared to the ACTB control gene, is calculated using the widely accepted 2−△△Ct method [41, 42].

Table 1 Primer characteristics of target genes
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Cell Cycle Assay

HT-29 and MCF-7 cell lines were cultured in 6-well plates (106 cells/well) and treated with a half maximal inhibitory concentration (IC50) of LC-SeNPs for a period of 24 h. Subsequent to the treatment, cells were rinsed twice with PBS and underwent centrifugation at 1500 rpm for 5 min. Thereafter, cells were stained with 250 mL of propidium iodide (PI) dye and incubated for half an hour. A second round of centrifugation was then conducted at 5000 rpm for 5 min at room temperature. The cell cycle stages were identified using a BD FACScan Cell Flow Cytometer (Becton Dickinson, USA). The outcomes were conveyed as the percentage representation of cells in each phase of the cell cycle using the Flow Jo software (Version 7.6.1) [12].

Cellular Uptake of LC-SeNPs.

On 6-well culture plates, 1 × 105 MCF-7 cancer cells were seeded, and the plates were incubated for 24 h at 37 °C in 5% CO2. The cells were treated with IC50 concentration of LC-SeNPs and Cy7 dye (7 μg/mL) for 24 h. Then, the cells were trypsinized, transported to tubes, and centrifuged after being washed three times with PBS to remove the free nanoparticles. For quick examination, the pellets were re-suspended in 0.5 mL of PBS, fixed in 4% paraformaldehyde, and stained with DAPI. At last, the fluorescence amplitude of each well was determined using a microplate reader (Biotek Synergy HT, USA) with an excitation wavelength of 750 nm and an emission wavelength of 775 nm. Becton Dickinson’s Cell Quest software was used to gather and evaluate data from 1 × 104 cells. A confocal laser-scanning microscope (A1, Nikon, Switzerland) was used to detect the cellular uptake of LC-SeNPs [44].

Wound Healing Assay

Cancer cells under growth were subjected to trypsinization, and 2 × 105 cells were dispensed per well in a 12-well plate, followed by a 24-h incubation period. Once 90% cell confluency was achieved, a scratch was introduced via a 200-μL sterile pipette tip to create a cell-free zone. Cellular debris was then eliminated with a gentle wash using culture medium, and initial images were captured (0 h). The HT-29 and MCF-7 cells were left to invade for 24 h, and the impact of nanoparticles on wound healing was evaluated at the conclusion of the 24-h incubation period via secondary microscopy. The progress of wound closure was analyzed using the Image-J 1.45 software [45].

Analysis of Statistical Data

The GraphPad Prism 8.0.2 software was utilized for statistical analysis. Quantitative data were expressed as mean ± standard deviation (SD), while qualitative information was represented as a percentage. The collected data were then examined using t-test, one-way and two-way analysis of variance (one-way ANOVA), with a P < 0.05 indicating a statistically significant disparity.

Results and Discussion

Physiochemical Characterization of LC-SeNPs Produced Through Eco-friendly Synthesis

The spherical form of LC-SeNPs was affirmed by the visual evidence provided by TEM and FE-SEM images (Fig. 1 A and B) [46]. The EDX spectrum showed a high Se peak at 1.4 keV (64.6%), suggesting the preferred purity of Se in the synthesized LC-SeNPs. In our research, we observed O (6.5%) and C (28.9%) peaks in the EDX graph encircling the LC-SeNPs (Fig. C).

Fig. 1
figure 1

SEM (A) and TEM (B) micrograph of biosynthesized SeNPs; EDX spectrum (C); DLS analysis (D) and ζ-potential of the of biosynthesized SeNPs (E)

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The protein molecules probably involved in capping the synthesized nanoparticles are suggested by the detection of carbon and oxygen peaks. This inference is in line with prior characterization studies of SeNPs synthesized with the use of Streptomyces minutiscleroticus, Pseudomonas aeruginosa ATCC 27853, and Bacillus mycoides [47,48,49].

DLS analysis provided data on the size distribution and the polydispersity index (PDI) of SeNPs (Fig. 1D) [50]. SeNPs had an average size of 90.78 nm and a PDI of 0.28 (Fig. 1D).

Earlier studies have noted a range of sizes for SeNPs produced through bacterial mediation. These sizes were observed using various bacterial strains, including Lactobacillus casei ATCC 393 (spherical shape, 50–80 nm), Escherichia coli (spherical shape, 60 nm), and Streptomyces bikiniensis strainess_ama-1 (spherical shape, 50–100 nm) [40, 51, 52].

The LC-SeNPs displayed a zeta potential of − 17.30 ± 0.56 mV, indicating satisfactory stability (Fig. 1E). The charge distribution on the LC-SeNPs can be assessed by the zeta potential, which can forecast the stability of colloidal SeNPs [53]. This observed event could be due to the inherent capping of the nanoparticles by biomolecules, providing an additional advantage in environmentally friendly biosynthesis [54].

UV–vis spectroscopy verified the existence of SeNPs, formed through the reduction of selenium ions to elemental LC-SeNPs, with a peak observed at approximately 269 nm (Fig. 2A) [55, 56].

Fig. 2
figure 2

UV–visible absorption spectrum (A) and X-ray diffraction spectra (B) of SeNPs synthesized using Lactobacillus casei extract

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Further, XRD analysis was used to determine the crystallinity of SeNPs [57]. The XRD diffractogram demonstrated the creation of SeNPs, courtesy of the bacterial supernatant reducing agents, which led to the reduction of selenium. The XRD pattern of the synthesized selenium SeNPs revealed a broad diffraction peak at lower angles, confirming their amorphous or non-crystalline state. This finding was noted in the 2B dataset. A peak index of 30.06° is associated with the crystalline metal structure of SeNPs (Fig. 2B).

The lack of additional pronounced selenium peaks, coupled with the broadening observed in the existing diffractogram, can be attributed to the binding of bacterial supernatant biomolecules to LC-SeNPs. This correlation brings about a loss of crystallinity, resulting in the formation of an amorphous selenium structure. This outcome concurs with previous scientific endeavors focused on the eco-friendly fabrication of SeNPs [58,59,60]. Moreover, the FT-IR spectrometry of bacterial supernatant was used in the green synthesis of SeNPs to identify the specific biomolecules involved in the reduction and capping of SeNPs [61]. Various biomacromolecules, predominantly proteins, have been found to participate in the reduction of selenite and selenate, leading to the stabilization of these compounds into SeNPs [54].

FT-IR spectrometry displayed a prominent and extensive peak at 3434 cm−1, linked to the tensile absorption of O–H and N–H groups found in polysaccharide compounds and amino groups in bacterial proteins. The broad peak was a result of peak interferences related to the supernatant components, which included the absorption of C-H tensile vibrations connected with the alkene compounds' double bonds above 3000 cm−1. The absorption peak of C = O and C-N bonds in the nucleotides and nitrogenous compounds of supernatant protein prominently appeared at 1618 cm−1 and 1381 cm−1, respectively. The tensile vibration frequencies related to C-O–H and C–O–C bonds of polysaccharide compounds were faintly visible at 1000 cm−1. Off-plane bending vibrations associated with C = C-H bonds were exhibited at 617 and 995 cm−1 (Fig. 3A).

Fig. 3
figure 3

Fourier transform infrared spectroscopy spectra of Lactobacillus casei extract (A) SeNPs synthesized using Lactobacillus casei extract (B)

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In the FT-IR spectrum of reduced SeNPs, the presence of several peaks is associated with the coating of SeNPs by diverse molecules. The absorption peak is related to the tensile vibrations of the O–H and N–H groups of polysaccharides and amino groups, which are broadly due to the peak overlap around 3437 cm−1. The tensile vibrations absorption of the C-H aliphatic bonds of polysaccharide compounds at 2922 cm−1, absorption of the carbonyl group C = O of proteins and nucleotides at 1625 cm−1, the absorption peak of the C-N bond of nitrogenous compounds at 1362 cm−1, C-O bond of polysaccharide derivatives at 1003 cm−1, tensile vibrations of the C-O ester group of fats or phospholipids at 1264 cm−1, and the absorption peak of the tensile vibration of the Se-O bond observed on the surface of SeNPs at 492 cm−1 were also detected. The shift at the tensile absorption peak of the C = O bond from 1618 to 1625 cm−1 compared to the initial spectrum could be connected to the binding of the supernatant organic compounds to the SeNPs’ surface (Fig. 3B) [62]. The results of this study highlight the biosynthesis and stabilization of SeNPs through the involvement of bacterial proteins and metabolites [63].

In Vitro Cytotoxicity of LC-SeNPs

The MTT assay was used to determine the cytotoxicity and cell viability of LC-SeNPs. The study demonstrated a notable decrease in the survival rates of HT-29 and MCF-7 cells when exposed to all concentrations of LC-SeNPs, in contrast to the control group. The most significant drop in cell survival was observed at the 100 μg/mL concentration of LC-SeNPs (P < 0.0001). Additionally, a marked variation in the viability of HT-29 and MCF-7 cells was detected across concentrations of 12.5, 25, 50, and 100 μg/mL (Fig. 4A). Interestingly, MCF-7 cells exhibited substantially lower survival compared to HT-29 cells. The estimated IC50 values of LC-SeNPs for MCF-7 and HT-29 cells were 15.72 and 32.37 μg/mL, respectively (Fig. 4B). A significant difference in the IC50 values of LC-SeNPs was discerned after 24 h (P < 0.01) (Fig. 4C). These findings suggest that as the concentration of LC-SeNPs escalated, the survival rate of cancer cells diminished.

Fig. 4
figure 4

Comparison of the cell viability percentage of MCF-7 and HT-29 cells treated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 mg/mL) of LC-SeNPs compared to the control group (A). Comparison of the IC50 values of cells treated with different concentrations (B). Comparison of the difference between the IC50 values of LC-SeNPs in MCF-7 and HT-29 after 24 h (C). Investigation of the cell viability percentage of normal human foreskin fibroblast (HFF) cells treated with different concentrations of LC-SeNPs compared to the control group (D). (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). The results are expressed as mean SD of three independent experiments (n = 3)

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Green synthesized LC-SeNPs exerted a far lesser impact on normal HFF cells compared to the HT-29 and MCF-7 cancer cells (Fig. 4D). MTT assay demonstrated more than 70% cell viability for HFF cells after treatment with a 100-µg/mL concentration of LC-SeNPs. The IC50 value of LC-SeNPs for HFF cells was calculated as 738 µg/mL. The outcomes indicated a very low toxicity of LC-SeNPs against HFF cells, thus attesting their biocompatibility. The cytotoxic effect of LC-SeNPs on cancer cells aligns with the findings of other studies, reaffirming our results [64, 65].

The uptake and cytotoxicity of metallic nanoparticles (NPs) within cells are influenced by several factors including their size, shape, concentration, the materials coating them, surface charge, and surface functionalization. Furthermore, the method of biosynthesis of metallic NPs, particularly when derived from natural sources, significantly impacts their bioactivity. A variety of biomolecules are involved in microbial synthesis, which act as capping agents during biosynthesis procedures [66]. Numerous research findings have highlighted the exceptional ability of SeNPs to cause cytotoxicity in cancer cells via multiple molecular and cellular mechanisms. These include cell cycle disruption, oxidative stress, mitochondrial damage, DNA destruction, and apoptosis initiation [67].

Recent investigations have revealed that the cellular membrane has a crucial role in the toxicity induced by LC-SeNPs in cancerous cells. SeNPs are found to alter the biomechanical characteristics of cancer cells, notably leading to a marked decrease in adhesion force and Young's modulus [30, 68].

Tabibi et al., through their examination of the anticancer effects of SeNPs on MCF-7 and HT-29 cell lines, revealed that a 100-µM concentration of SeNPs inhibited more than 75% of cancer cell growth [69]. Ranjitha et al. also reported potent cytotoxic activity of synthesized SeNPs against the HT-29 cell line, showing 40.5%, 33%, and 23.7% of cell viability at 2 μg/mL, 4 μg/mL, and 30 μg/mL concentrations, respectively [70].

Wadhwani et al. reported that SeNPs, synthesized using Acinetobacter sp.sW30, resulted in 53.5%, 26.6%, and 77% cell viability at a concentration of 100 μg/mL against 4T1, MCF-7, and HEK293 cells, respectively [71]. Biosynthesized SeNPs, using Idiomarina sp.PR58-8, exhibited a dose-dependent cytotoxicity against HeLa cells, with only 3% viability observed at a concentration of 100 μg/mL. No toxicity was detected against HaCaT normal cells [72].

Biogenic SeNPs, synthesized using Streptomyces minutiscleroticus M10A62, led to 99.5% and 100% cytotoxicity against HepG2 and HeLa cells [47].

The IC50 values of SeNPs, synthesized by Streptomyces bikiniensis strainess_ama-1, were found to be 75.96 μg/mL for HepG2, and 61.86 μg/mL for MCF-7 cancer cells [52].

A significant death rate in MCF-7cancer cells was noted following the treatment with SeNPs synthesized using Bacillus sp. MSh-1 (IC50: 41.5 ± 0.9 μg/mL) [73].

Comparing IC50 values, it was clear that the LC-SeNPs in this study demonstrated potent antiproliferative activity against MCF-7 and HT-29 cancer cells, as compared to other green synthesized SeNPs.

LC-SeNPs Triggered Apoptosis in MCF-7 and HT-29 Cells

In the conducted research, the analysis via flow cytometry revealed the cytotoxic influence of LC-SeNPs resulted in both early and late stages of apoptosis in MCF-7 and HT-29 cells. Upon evaluation of the data, it was observed that 66% of MCF-7 and 73% of HT-29 cells remained viable. Notably, early apoptosis was evident in 10% of HT-29 and 14% of MCF-7 cells, while 13% of HT-29 and 14% of MCF-7 cells were in late apoptosis. Additionally, necrosis was found in 4% of HT-29 and 5% of MCF-7 cells. The results highlighted a notably high toxicity of LC-SeNPs towards the MCF-7 and HT-29 cancer cells (P < 0.0001) (Fig. 5).

Fig. 5
figure 5

Flow cytometry plot to evaluate apoptosis induced by LC-SeNPs on the MCF-7, HT-29, and control cells after 24 h (****P < 0.0001 and **P < 0. 01)

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As depicted in Fig. 6, DAPI staining was performed on MCF-7 and HT-29 cells. Treated MCF-7 and HT-29 cancer cells displayed bright blue fluorescence with higher intensity compared to control-untreated cells. The untreated cells had a round shape. The nuclei stained homogeneously with less bright blue fluorescence. A pronounced blue fluorescence, a result of nuclear chromatin condensation, was noted in treated MCF-7 and HT-29 cells compared to those that were untreated. The condensation patterns of chromatin in the cell nuclei treated with LC-SeNPs provided evidence of apoptosis in MCF-7 and HT-29 cancer cells (Fig. 6). In accordance with previous findings, morphological alterations such as cell shrinkage, membrane blebbing, nuclear fragmentation, and chromatin condensation were observed in Hela, HepG2, and HT-29 cells [70, 74, 75]. Apoptosis is a crucial mechanism in the death of cancer cells [76], and in alignment with prior studies, our results indicate that apoptosis is the primary cause of cancer cell mortality [77, 78].

Fig. 6
figure 6

DAPI staining of MCF7, HT-29, and control cells to show apoptosis induced by LC-SeNPs

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Apoptosis is induced through three primary pathways: (1) the mitochondrial pathway (intrinsic apoptotic pathway), (2) the death receptor pathway (extrinsic apoptotic pathway), and (3) the endoplasmic reticulum (ER) pathway [79]. The mitochondrial-dependent intrinsic apoptosis pathway is triggered in response to intrinsic proapoptotic stimuli like DNA damage, chemotherapeutic agents, and intracellular ROS. The intrinsic mitochondrial pathway is managed by pro and anti-apoptotic structural proteins of the B-cell lymphoma 2 (Bcl-2) family, which regulate the permeability of the mitochondrial outer membrane to proteins such as cytochrome c [80, 81]. The initiation of this pathway eventually results in the release of Bcl-2 pro-apoptotic proteins from the mitochondria, the activation of the caspase cascade, and ultimately, the induction of apoptosis. However, the Bcl-2 anti-apoptotic protein hinders the release of cytochrome c from the mitochondria [80, 82].

The extrinsic or death receptor pathway induces apoptosis through the binding of death ligands (FasL, TRAIL) to their respective death receptors (TNFRSF), forming the death signaling complex (DISC), and subsequently activating caspase [80, 83]. BAX, Caspase 3, and Caspase 9 proteins, encoded by related genes, are classic proteins involved in the intrinsic pathway of apoptosis. It is well established that Caspase 9 activates Caspase 3, which is critical for the execution phase of apoptosis, and an increase in these protein levels is an indication of apoptosis [84].

The authors’ hypothesis concerning the anticancer properties of SeNPs revolves around the interaction with endogenous copper, particularly copper bound to chromatin, which leads to Reactive Oxygen Species (ROS) generation. Notably, certain types of cancer exhibit higher copper levels within cells and in serum. SeNPs have the capacity to form a complex with copper bound to chromatin, consequently reducing Cu (II) to Cu (I). The reoxidation of Cu (I) then triggers substantial ROS production, which serves to effectively eliminate cancer cells [52]. Thus, SeNPs emerge as potential chemopreventive and chemotherapeutic agents against human cancers [85].

This study explored the gene expression changes in HT-29 and MCF-7 cells following treatment with LC-SeNPs, particularly focusing on genes linked to apoptosis induction. The qRT-PCR results revealed an increase in the expression of BAX, CASP3, and CASP9 genes as compared to normal cells, indicating that LC-SeNPs therapy stimulates apoptosis. However, no marked decrease was noted in the expression of the BCL2 gene, an anti-apoptotic agent, post LC-SeNPs treatment in both cell types. Moreover, the expression of BAX, CASP3, and CASP9 genes in MCF-7 cells exposed to LC-SeNPs was significantly higher than in HT-29 cells (Fig. 7). The study by Huang et al. demonstrated that Tf-SeNPs exert a strong cytotoxic effect on cancer cells by inducing apoptosis through both intrinsic and extrinsic pathways [36]. Othman et al. explored the anticancer properties of SeNPs, revealing that SeNPs containing berberine can stimulate apoptosis by down-regulating BCL2 and upregulating the expression of BAX and CASP3 genes [86]. Pi et al. reported that SeNPs notably enhanced apoptosis and necrosis in MCF-7 cells compared to control cells (12.74% versus 4.27%). Additionally, the expression of key apoptosis regulatory proteins like Caspase 9 and Caspase 3 was markedly higher than in the control group [87]. Furthermore, biosynthesized black ginger-selenium nanoparticles were found to induce apoptosis by upregulating BAX, Caspase 3, and downregulating BCL2 [88]. The anticancer properties of biosynthesized SeNPs were also reported against A549 cancer cells and human cervical cancer cells, with findings indicating a significant increase in caspase-3 activity, ROS overproduction, and mitochondrial dysfunction [51].

Fig. 7
figure 7

The comparison of expression levels of genes responsible for apoptosis induction after 24 h of treatment with LC-SeNPs

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Collectively, these findings align with prior research results, reinforcing the concept that SeNPs possess cytotoxic effects and promote apoptosis in cancer cell lines [89, 90].

Impact of LC-SeNP Treatment on Cell Cycle Arrest

This study observed a hindered proliferation of HT-29 and MCF-7 cells when treated with SeNPs, resulting in cell cycle arrest in the sub-G1 phase. Cell cycle analysis revealed that LC-SeNPs obstructed the cell cycle, leading to sub-G1 phase arrest in both MCF-7 and HT-29 cells, which effectively inhibited cell proliferation. Consequently, the percentage of cells in the sub-G1 phase was significantly higher in HT-29 and MCF-7 cells treated with LC-SeNPs compared to the control group (17.69% and 25.29% vs. 0.62%, respectively). In contrast, the number of HT-29 cells in the G1 phase decreased significantly compared to the control. Additionally, the number of MCF-7 cells in the S and G2 phases showed a significant reduction compared to the control (Fig. 8). Several studies have demonstrated the impact of SeNPs on cell cycle arrest and cell death pathways in different cancer cell lines. Our findings align with these studies, as sub-G1 phase cell cycle arrest was observed, as reported in other studies as well [74, 91].

Fig. 8
figure 8

Cell cycle analysis of MCF-7 and HT-29 cells treated with LC-SeNPs compared to the control group (****P < 0.0001, ***P < 0.001, and *P < 0.05)

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Fig. 9
figure 9

Fluorescence images of MCF-7 cells after 24 h incubation with LC-SeNPs and Cy7 dye, (A) MCF-7 untreated cells (control) (B) MCF-7 cells treated with IC50 concentration of LC-SeNPs and Cy7 dye. (C) Quantitative analysis of cellular uptake of LC-SeNPs and Cy7 dye by MCF-7. Data represent means ± standard deviations (n=3). Significant difference specified as *P < 0.05

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The mechanisms of cell cycle arrest and cell death can vary depending on the specific selenium compounds used and the cell phenotype [92]. SeNPs can selectively enter cancer cells through endocytosis and induce cancer cell apoptosis via signal transduction pathways [87, 93]. Conversely, various studies have reported selenite’s ability to arrest the cell cycle at the S or G2/M phases [94,95,96]. For instance, Bidkar et al. explored SeNPs as a delivery system and demonstrated that paclitaxel-loaded SeNPs arrested cancer cells in the G2/M phase in a dose-dependent manner, resulting in a significant antiproliferative effect on A549, MCF-7, HeLa, and HT-29 cells [97]. Lopez-Heras et al. proposed that SeNPs inhibit the growth of HepG2 cells by inducing cell cycle arrest in the S phase. This effect is mediated by the re-regulation of the eIF3 protein complex, which impacts the protein synthesis machinery and inhibits cell cycle progression [98].

Uptake of LC-SeNPs

Figure 9A and B show fluorescence microscopy photographs of MCF-7 cells after 24 h of treatment with LC-SeNPs at 37 °C. It appears that the cells’ fluorescence was boosted following treatment with the IC50 concentration of LC-SeNPs. The intensity of the fluorescence of the cells was monitored using a microplate reader in order to further quantify the cellular uptake efficiency of the LC-SeNPs (Fig. 9C). Figures also depict the cellular uptake of LC-SeNPs by malignant cells after 24-h incubation at the IC50 concentration of LC-SeNPs at 37 °C. When cells were treated with LC-SeNPs, the efficiency of cellular uptake had improved (Fig. 9). Previous studies have suggested that SeNPs have the capacity to penetrate various cell types through unique endocytosis pathways, thus eliciting toxic effects [99]. Moreover, SeNPs have exhibited greater selectivity between normal and cancer cells compared to Se+IV at similar concentrations, in addition to their distinct anticancer effectiveness. Specifically, cancer cells selectively internalize SeNPs via endocytosis, which then induce cell apoptosis by activating apoptotic signal transduction pathways [87, 93]. To date, numerous studies on intracellular mechanisms have suggested that SeNPs enter cancer cells via endocytosis and trigger apoptosis of cancer cells through signal transduction pathways [36, 100, 101]

Suppression of Migration in MCF-7 and HT-29 Cell Lines by LC-SeNPs

In addition to studying the impact of metal nanoparticles on cell proliferation, this research also investigated their effect on cell motility. Notably, metastasis is a critical characteristic of cancer, and approximately 90% of cancer-related deaths are attributed to metastasis [102]. Consistent with prior studies, SeNPs were found to inhibit cell migration and invasion [89]. Our data also indicate that apart from their antiproliferative effect, SeNPs have inhibitory effects on the migration of cancer cells. The scratch test was employed to examine the migration capacity of HT-29 and MCF-7 cells in the presence of selenium nanoparticles. As illustrated in Fig. 10A, 24 h after SeNP treatment, the migration rate of MCF-7 cells significantly decreased compared to the control group (P < 0.01). Furthermore, the scratch test revealed a significantly lower migration rate in HT-29 cells treated with LC-SeNPs within 24 h compared to the control group (P < 0.05) (Fig. 10B).

Fig. 10
figure 10

The scratch test to investigate the capacity of MCF-7 (A) and HT-29 (B) cells to migrate under the presence of LC-SeNPs. The migration rate of MCF-7 (**P < 0.01) and HT-29 (*P < 0.05) cells during 24-hour treatment with LC-SeNPs compared to the control group

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After 24 h, the wounds of the MCF-7 control, HT-29 control, and LC-SeNPs in MCF-7 and HT-29 groups showed approximately 80%, 70%, 35%, and 20% healing, respectively (Fig. 10C).

Similarly, Xia et al. reported a wound healing rate of approximately 21.9% after 24 h with SeNPs@Am treatment. The use of SeNPs@Am inhibited the aggressiveness of cancer cells [75].

Li et al. demonstrated that biologically synthesized SeNPs with Galangin significantly suppressed the migration of HepG2 cells [103].

Conclusion

In summary, this study reveals the anticancer activity of LC-SeNPs synthesized by L. casei, which inhibits the proliferation and cell cycle of HT-29 and MCF-7 cells, and induces apoptosis through the up-regulation of apoptosis-related genes. Furthermore, LC-SeNPs exhibit inhibitory effects on the migration of HT-29 and MCF-7 cells, providing a potential strategy to prevent the invasion of cancerous cells. These findings offer in vitro evidence supporting the anticancer and anti-metastatic effects of LC-SeNPs, forming a basis for further pre-clinical and clinical investigations.