Anti-tumor effects of Soyasaponin IV as a natural product in Ehrlich ascites carcinoma bearing mice: modulation of monocyte vascular endothelial growth factor (VEFG) and nuclear factor kappa B (NF-κB) pathways

Abstract

Breast cancer stands as the leading cause of cancer-related fatalities among women globally, presenting a diverse landscape of histological and molecular c characteristics, treatment response, and prognosis. This investigation examined the impact of soyasaponin IV, a natural product, on mice carrying Ehrlich’s ascites carcinoma (EAC), exploring its potential in blostering the hosts’ defense against cancer. The isolated compounds underwent scrutiny for their cytotoxic effects on breast cancer cell line, with Doxorubicin as the reference standard. Notably, Soyasaponin IV exhibited the highest cytotoxicity. Evaluation of its in vivo antitumor efficacy against EAC revealed a substantial reduction in tumor weight and volume, accompanied by improved histological features of tumor cells. EAC-induced abnormalities, including elevated levels of Malondialdehyde (MDA), vascular endothelial growth factor (VEGF), and nuclear factor-kappa B (NF-κB) were all shown to be upregulated by EAC, while glutathione (GSH), were mitigated in a dose-dependent manner by soyasaponin IV treatment. Soyasaponin IV emerged as a promising strategy for suppressing EAC by impeding the NF-κB and VEGF pathways.

Keywords:

• Breast cancer
• Ehrlich’s ascites carcinoma
• soyasaponin IV
• Natural Product—VEGF
• NF-κB
• GSH
• MDA

1. Introduction

Breast cancer ranks among the most prevalent malignancies in women globally (Burstein et al., 2021). Due to the molecular complexity and diversity that contribute to the development of breast cancer, chemotherapy and/or radiotherapy treatment is incredibly challenging and frequently has adverse side effects (Pearson, Caimino, Shabbir, & Baguley, 2021). Breast cancer is no exception to the long-standing use of plants and plant extracts for the treatment of nearly all diseases (Laskar, Lourembam, & Mazumder, 2020). Herbal medicines are safe to employ in the treatment of cancer due to their low toxicity (Saleh et al., 2022). Additionally, the majority of breast cancer patients adopt herbal therapy due to their availability and affordability (Basu et al., 2021, Han, Wang, Tu, Tan, & He, 2020). Over the past decade, various plants and their constituents have demonstrated potential anticancer actions in both in vivo and in vitro models against breast cancer cells. (Abbaszadeh, Keikhaei, & Mottaghi, 2019, Rawat & Vijaya Bhaskar Reddy, 2022). However, the positive benefits of these treatments in the context of breast cancer are still uncertain due to a lack of randomized clinical trials. Cancer is facilitated by inflammation by changing the genetic and epigenetic composition of the injured tissue (Gallagher et al., 2015). Inflammation contributes to genomic instability and genetic alterations, promoting tumor growth and development (Faldoni et al., 2020). Through increased cellular stress, recruitment of inflammatory factors, and accumulation of DNA damage, persistent inflammation can result in tissue damage, autoimmune disorders, and cancer (Souliotis et al., 2019).

In the context of tumor development, inflammation impacts the immune system’s ability to identify tumor cells and enhances cell proliferation and genetic instability, resulting in oncogenic alterations (Wang, Li, Cang, & Guo, 2019). Reactive oxygen species (ROS), which have a tendency to damage DNA, get released by neutrophils and macrophages at the site of inflammation (ElMahdy et al., 2021, Shams, Khairy, Saleh, & Antar, 2017). Immune cells can activate the nuclear factor kappa B (NF-κB) pathway, which, in turn, produces ROS and cytokines, creating a feedback mechanism that increases NF-κB activity in various cells at the inflammatory site (Antar et al., 2022b). Soyasaponin IVs, natural compounds present in soybean seeds, provide notable health benefits and have the potential to influence the flavor of soy-based products (Chitisankul, Murakami, Tsukamoto, & Shimada, 2019). Soybeans contain a verity of phytochemicals, including isoflavones (0.1–0.3%), phytic acids (1.0–2.2%), phytosterols (0.23–0.46%), and soyasaponin IVs (0.17–6.16%). Soyasaponin IVs, characterized as amphilic triterpenoid saponins, interact not only with hydroxyl groups on the aglycone moiety but also with the phospholipid and cholesterol-rich membranes of cancer cell (Xie et al., 2020).

Regarding medicinal uses, soyasaponin intravenous (IV) treatments supplement traditional therapies for a number of ailments. Along with lowering blood cholesterol levels, their known benefits also include hepatoprotective, antioxidant, antimutagenic, and antiviral actions (Colletti, Attrovio, Boffa, Mantegna, & Cravotto, 2020, Singh, Singh, Singh, & Kaur, 2017). Due to the phytochemicals found in soy products, eating soybeans has been linked to a lower risk of diabetes, Alzheimer’s disease, cancer, and cardiovascular disease (Román, Jackson, Gadhia, Román, & Reis, 2019). Additionally, studies show that soyasaponin IVs contribute to their anti-inflammatory actions by blocking the transcription of inflammatory genes mediated by NF-κB (Gu et al., 2021). The most active metabolite was tested for cytotoxic activity in vitro and then its anticancer efficacy in vivo using Ehrlich’s ascites carcinoma-infected mice. Potential pathways were investigated, including a variety of oxidative stress markers and the anti-tumor effects of plasma vascular endothelial growth factor (VEGF).

2. Materials and methods

2.1. Phytochemical investigation

2.1.1. Plant material

The soybean, officially known as Glycine max L., was purchased from a Mansoura-based herbal store. This plant was specifically chosen for acquisition due to its well-established reputation as a noteworthy source of soyasaponin IV. Staff from the faculty of agriculture at Mansoura University attested to the legitimacy of the commercial seeds. In the pharmacognosy department of the pharmacy faculty, a sample of seeds (SB-5-21) was deposited. A sample of seeds (SB-5-21) was then placed at the pharmacy faculty’s pharmacognosy department.

2.1.2. Extraction and isolation

At room temperature, methanol was soaked in 1 kg of powdered soybean. Every 48 h, the alcoholic extract was taken three times. Using a rotary evaporator, the extract was evaporated to dryness at 40 °C. The whole dry extract was produced as a 200 g semisolid. A gravity column chromatography (125 × 5.5 cm) packed with silica gel and the mobile phase made up of CHCl₃:CH₃OH:H₂O were used to separate the whole extract and an equal amount of silica gel (65:35:10, lower layer). The result was 15 fractions (I–XV). To get 10 subfractions, fraction IX (28.5 g) was chromatographed over a silica gel column and eluted with the bottom layer of CHCl₃:CH₃OH:H₂O (65:35:10) (IX1–IX10). After elution with CHCl₃:CH₃OH:H₂O (65:35:10) over a silica gel column, soybeanponin IV (278 mg) was recovered from the subfraction IX3 (300 mg). After column chromatography over RP18 and elution with MeOH–H₂O, soyasponin I (301 mg) was isolated from the subfraction IX6 (450 mg) (50:50).

2.1.3. General experimental procedure

The isolated compounds’ NMR spectra were captured using a Bruker AVANCE III 400 MHz spectrometer. The substance was dissolved in a combination of CD3OD and pyridine-d5. Solvent peaks were used as a benchmark chemical shift to correct compound peaks. The ¹H spectrum was acquired at a temperature of 298.2 K with the following parameters: SF 400.18 MHz, SW 8012.8, LB 0.30 Hz, and NS 16. With SF 100.64 MHz, SW 24038.5, LB 0.30 Hz, NS 6900, and at TE 298.2 K, the ¹³C spectrum was captured. For chromatographic separation, a glass column filled with silica gel was utilized. To monitor the column eluate, thin layer chromatography (TLC) using aluminum foil coated with silica gel 60 F254 was utilized (Merck). Locating and identifying spots on TLC required the use of the spraying reagent vanillin/H₂SO₄. Solvents of analytical grade were applied during the extraction and purification procedures.

2.2. Antitumor activity assessment

2.2.1. In vitro assay

Through VACSERA (Cairo, Egypt), a holding firm for biological products and vaccines, the cell line was obtained from the ATCC. To cultivate the Mammary gland (MCF-7) cell line, Eagle’s minimal essential media was utilized (EMEM, Wako Pure Chemical Industries). The following antibiotics were given to every culture medium: 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 100 U/mL penicillin, and 100 U/mL streptomycin (Wako Pure Chemical Industries, Osaka, Japan). The cytotoxicity of cancer cell lines was evaluated using the MTT colorimetric test. In 96-well plates, cells were cultivated at a density of 0.5*104 cells/well in a humidified atmosphere containing 5% (v/v) CO₂ and 95% (v/v) air at 37 °C. After 24 h, the cells were exposed to various dosages of the tested compound dissolved in DMSO. Doxorubicin was employed as a positive control. By adding media containing MTT reagent (5 mg/mL) to each well, incubation for 4 h at 37 °C, and then adding HCl-isopropanol solution to each well, cell viability was assessed using MTT reagent after 24 h. After 4 h of incubation in the dark, the absorbance was measured at 570 nm with a microplate reader (MTP-310, Corona Electric, Hitachi, Japan). The concentration of the drug under test (M) that caused a 50% decrease in viable cells is known as the IC50 value. The mean value of triplicate data points ± SD is used to express the results.

2.2.2. In vivo assay

2.2.2.1. Preparation of Ehrlich solid tumors

Under aseptic circumstances, ascetic fluid was removed from the peritoneal cavity of mice bearing tumors. By using the Trypan blue dye exclusion technique, EAC cells were tested for viability 7–10 days after being implanted. Only EAC cells with a vitality of at least 90% were applied. Each 0.1 ml of normal saline contains 2.5 × 10⁶ EAC cells that have been suspended in the solution. Using a hemocytometer and a microscope, cells were counted. After shaving this area, 100 μl of EAC suspension was administered subcutaneously on the lower ventral side of each mouse.

2.2.2.2. Animals and experimental design

Swiss albino female mice weighing 25–30 g were purchased from the Egyptian Organization for Biological Products and Vaccines (Vacsera, Egypt). The mice were kept in plastic cages with mesh flooring, hardwood bedding, a constant temperature of 25 °C, a regular light/dark cycle, and unrestricted access to food and water. Prior to the studies, mice were given a week to adjust. They were randomly split into three groups (7 mice each). EAC cells were injected into all other groups. The first group was the EAC control group, and they received saline. The second group was given soyasaponin IV (50 mg/kg, orally) three times each week. The third group was given Soyasaponin IV (100 mg/kg, orally) three times each week (GuangLie, WeiShi, GaiLing, & JianPing, 2013). In saline, all chemicals were dissolved. Day zero was defined as the day of tumor inoculation (0). The treatment for each group began on the seventh day after tumor inoculation and lasted for 2 weeks, until day 21. The in vivo research was carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were given a light ether anesthetic and sacrificed by cervical dislocation one day following the conclusion of the experiment (day 21). In order to estimate the levels of VEGF, NF-κB, malondialdehyde (MDA), and glutathione (GSH), the tumor was divided into two halves. The first half was stored at −20 °C. The second half was divided in two, then fixed for histopathological analysis in 10% phosphate-buffered formalin. Tumor was excised, weighed and tumor volume was calculated by the following equation (V = (W(2) × L)/2 for caliper measurements as described in (Figure 1).

Figure 1. Flowchart illustrating the experimental strategy.

2.2.2.3. Assessment of oxidants/antioxidant stress biomarkers; MDA and GSH, angiogenic marker; VEGF and inflammatory marker; NF-κB

Using ELISA kits for MDA (Cat. No: LS-F28474, LSBio, USA), GSH (Cat. NO: AMS.EA1499Mo, AMSBIO, USA), VEGF (Cat. No: SEA143Mu, Cloud-Clone, USA), and NF-κB (Cat. NO: 85-86081-1, ThermoFisher, USA) in accordance with the manufacturer’s instructions, the levels of MDA, GSH, VEGF and NF-κB were assessed in the tumor homogenates of mice.

2.3. Histopathological assessment

Following fixation, samples were dehydrated in rising grades of ethyl alcohol, cleaned in xylol, embedded in paraffin wax, and then subjected to standard histological procedures. Hematoxylin and Eosin (H&E) staining was done after sections that were 5–7 m thick had been cut. The Olympus CX 41biocular microscope’s image capture engine software (AMT V600.259) was used to take digital images of representative locations. The percentage of tumor necrotic area in each group was estimated. The following parameters within tumor tissue were calculated in three different high-power fields (HPF) and the average number was calculated: mitotic figures in tumor cells, some apoptotic tumor cells, and intra-tumoral inflammatory cells. The mitotic figures and tumor giant cells were used to perform quantities of histopathological analysis on digital images of solid tumors. The National Institutes of Health’s ImageJ program was used to evaluate ten sections from each experimental group at scale bar 20 μm for counting (Betheda, MD, USA). An unbiased reviewer conducted the histopathological investigations.

2.4. Statistical analysis

Data were collected, tabulated, and presented as the mean ± standard error of the mean (SEM) One-way ANOVA followed by Tukey–Kramer multiple comparison test (P˂ 0.0001) were used to analyze the difference between the experimental groups. A P-value <0.0001 was considered to be statistically significant.

3. Result and discussion

3.1. Identification of isolated compounds

Two compounds were isolated from the soyabean and identified based on the analysis of the NMR spectra. Upon identification it was found that compounds are Soyasaponin I (1) and Soyasaponin IV (2). Figure 2.

Figure 2. Chemical structure of soyasaponin I (1) and IV (2).

3.2. In-vitro cytotoxic activity

In comparison to doxorubcin, a positive control, the cytotoxic activity of the separated compounds was assessed against the mammary gland (MCF-7) cell line. Soyasaponin I showed mild cytotoxic activities with IC50 values of 73.87 ± 3.60 against the MCF-7 cell line, but Soyasaponin IV demonstrated strong cytotoxic activities against this cell line with IC50 values of 32.54 ± 2.40. This outcome led to the selection of compound Soyasaponin IV for the in vivo experiment.

3.3. In vivo antiproliferative evaluation of the total isolated compounds

3.3.1. Effect of therapeutic Soyasaponin IV (50 and 100 mg/kg, orally) on tumor weight and volume

To assess the anticancer activity of Soyasaponin IV in a dose-dependent manner, solid tumor discs of EAC in female mice were separated and weighed. In comparison to Soyasaponin IV (50 mg/kg), treatment with Soyasaponin IV (100 mg/kg) resulted in a significantly substantial decrease in tumor weight and volume (P ˂ 0.001). Treatment with Soyasaponin IV (50 mg/kg) resulted in a remarkable decrease in tumor weight and volume by 78.90% and 73.42%, respectively compared to EAC control group. Furthermore, compared to the EAC group control, Soyasaponin IV (100 mg/kg) reduced tumor weight and volume by 92.18% and 88.28% (Figure 3A, B).

Figure 3. Effect of Soyasaponin IV (50,100 mg/kg) on weight (A) & volume (B) on Ehrlich’s ascites carcinoma growing in female mice. ***P < 0.001 vs. control group, ^##P < 0.01 vs. Soyasaponin IV (100 mg/kg).

These findings are in line with those of in vitro cytotoxicity assay. Previous studies have reported that soyasaponin IV exerts inhibitory function on malignant phenotypes of cancers (Omar et al., 2020). Breast cancer cells’ ability to migrate and actively contribute to tumor tissue invasion was greatly inhibited by soysaponin-IV. In a range of cancer cell lines, saponins have demonstrated anticancer action by inhibiting cell proliferation and inducing apoptosis. There are several uses for certain saponins because they exhibit anti-metastasis, anti-angiogenesis, and anti-inflammatory effects (Shi, Zeng, & Wong, 2019). Additionally, some saponins have been shown to enhance chemotherapy effectiveness and reverse multidrug resistance, suggesting that saponins may be employed as an anticancer agent (Tinoush, Shirdel, & Wink, 2020). Moreover, several families of soyasaponin IVs with a same core structure have been identified as the active anti-cancer components of soybeans (Arulkumar, Jung, Noh, & Chung, 2022).

3.3.2. Effect of therapeutic Soyasaponin IV (50 and 100 mg/kg, orally) on MDA and GSH

Comparing the Soyasaponin IV treatment to the EAC control after the induction of EAC, the GSH level increased significantly. GSH content increased noticeably by 49.17% as a result of soyasaponin IV (50 mg/kg). Additionally, Soyasaponin IV (100 mg/kg) led to a considerable increase in GSH of 132.87%, resulting in even more improvement. The MDA level significantly increased in the EAC group. MDA levels were significantly reduced when Soyasaponin IV was used to treat tumors in mice. MDA levels were significantly reduced by 28.50% when soyasaponin IV (50 mg/kg) was compared to the EAC control. A 39.49% decrease in MDA levels when compared to the EAC control was another benefit of soyasaponin IV (100 mg/kg), which also led to even greater improvement (Figure 4A, B).

Figure 4. Effect of Soyasaponin IV (50 and 100 mg/kg) on (A) tumor MDA and (B) tumor GSH in Ehrlich’s carcinoma solid tumors growing in female mice. ***P < 0.001 vs. control group, ^##P < 0.01 vs. Soyasaponin IV (100 mg/kg).

The synthesis of ROS in the body can be aided by carcinogenic substances (Xian, Lai, Song, Xiong, & Zhong, 2019). This ROS can interact with proteins, lipids, and biomolecules like DNA. The cellular membrane’s lipids may be harmed by a ROS assault reaction, resulting in lipid peroxidation, which produces MDA (Atiba et al., 2022). By attacking polyunsaturated fatty acids with free radicals, MDA with a low molecular weight can be created. The primary sensitive indicator of lipid peroxidation is MDA, which is created when polyunsaturated fatty acids undergo oxidative conversion (Antar et al., 2022a, Tsikas, 2017). MDA can be produced when hydroxyl free radicals like ROS combine with fatty acid components of cell membranes in a process known as fat peroxidation, which is a sign of oxidative stress, especially in a number of clinical illnesses linked to the lipid peroxidation process (Su et al., 2019). Damage to cell membranes will result from the breakdown of the fatty acid chain into hazardous chemicals by the process of fat peroxidation (Antar et al., 2022a). In addition to raising MDA levels, a biomarker of the development of cancer cells, MDA interacts with proteins and DNA to cause gene alterations that lead to the development of cancer cells (Klaunig, 2018). Increased MDA in breast cancer patients is associated with excessive ROS production and deficiency of antioxidant defences (Antar, El-Gammal, Hazem, Moustafa, & Research, 2022c, Jabir & Shaker, 2020). Exposure to chemical, biological, and physical carcinogenic agents leads to excessive ROS generation. Antioxidant defences are less effective and oxidative stress is present at higher levels when there is a considerable increase in MDA in cancer (Valavanidis, Vlachogianni, Fiotakis, & Loridas, 2013). This occurrence is crucial to the etiology of cancers and the gene alterations brought about by elevated MDA levels. The most crucial enzyme in the cell’s antioxidant defense mechanism is GSH. Treatment with soybean oil reduced the high levels of ROS in lipopolysaccharide and boosted superoxide dismutase activity (Jiang et al., 2018).

3.3.3. Effect of therapeutic Soyasaponin IV (50 and 100 mg/kg, orally) on VEGF

Due to its function in the angiogenic pathway, this marker’s presence in tumor tissue was examined. The control EAC group significantly outperformed the treatment groups in terms of VEGF tissue content. When compared to the EAC control group, Soyasaponin IV therapy significantly (P ˂ 0.001) lowered the amount of VEGF. A substantial reduction in VEGF tumor content was observed following soyasaponin IV treatment in a dose-dependent manner. Comparing the soyasaponin IV (50 mg/kg) group to the EAC control group, the soyasaponin IV reduced tumor VEGF content by 39%. With Soyasaponin IV (100 mg/kg), more improvement was seen, with a significant decrease in VEGF tumor content by 67.13% in comparison to the EAC control group (Figure 5).

Figure 5. Effect of Soyasaponin IV (50 and 100 mg/kg) on VEGF in Ehrlich’s carcinoma solid tumors growing in female mice. ***P < 0.001 vs. control group, ^##P < 0.01 vs. Soyasaponin IV (100 mg/kg).

Through the circulatory system of capillaries, breast cancer cells require continuous hydration and oxygenation (Nagaraju, Truong, Mouneimne, & Nikkhah, 2018). As a result, angiogenesis, or the rapid expansion of blood vessels, is crucial for providing enough oxygen and nutrients for the growth of breast tumor (Madu, Wang, Madu, & Lu, 2020). VEGF and fibroblast growth factors (FGF) are two angiogenic growth factors that are primarily responsible for the beginning and development of tumor angiogenesis (Al-Ostoot, Salah, Khamees, Khanum, & Communications, 2021). According to reports, the amount of angiogenic elements present and the number of vascular networks that result are both indicators of breast cancer survival (Madu et al., 2020). As a result, substances that block the angiogenesis pathway have become increasingly popular in studies on the treatment of breast cancer (Varghese, Liskova, Kubatka, Samuel, & Büsselberg, 2020, Würth et al., 2016). The development, invasiveness, and metastasis of cancer depend on the processes of angiogenesis and vasculogenesis. VEGF, one of the most potent angiogenic factors involved in tumor growth and progression, is the major growth factor and cytokine that controls them. By attaching to the endothelial cells’ expressed VEGFR-1 and VEGFR-2 tyrosine kinase (RTK) receptors, VEGF stimulates endothelial cell proliferation, migration, and tube formation (Jiang, Pi, & Cai, 2020). Additionally, VEGF-B plasma levels were a sensitive diagnostic for breast cancer (Zajkowska, Lubowicka, Malinowski, Szmitkowski, & Ławicki, 2018). Angiogenesis, vasculogenesis, and lymphangiogenesis are the results of VEGF signalling, which is mediated by the binding of VEGF ligand to the receptor tyrosine kinases VEGFR1, VEGFR2, and VEGFR3 (Shaik et al., 2020). The main signalling cascade that is mediated by VEGFR2 is activated by VEGF. The metastasis of liver, bladder, and lung cancers was said to be accelerated by the overexpression of VEGF-B (Ceci, Atzori, Lacal, & Graziani, 2020). In order to treat cancer, targeted inhibition of VEGFR kinase has been researched as a very effective clinical method. When VEGF signalling is blocked, angiogenesis is prevented and cancer growth is minimized. In this study, VEGF reduction caused by Soyasaponin IV had an antiangiogenic impact via inhibiting VEGF signalling. According to the results of the current investigation, soyasaponin IV can reduce VEGF in a dose-dependent manner, suggesting that it may have potential as an anti-angiogenic medication. This might demonstrate how the separated compounds’ antiproliferative impact works.

3.3.4. Effect of therapeutic Soyasaponin IV (50 and 100 mg/kg, orally) on NF-κB

Soyasaponin IV therapy (50 mg/kg) for 14 days resulted in a dose-dependent reduction in tumor NF-κB level of 70.47% when compared to the EAC control group. Treatment with soyasaponin IV (100 mg/kg) decreased NF-κB content by 82.85% when compared to the EAC control group (Figure 6).

Figure 6. Effect of Soyasaponin IV (50 and 100 mg/kg) on NF-κB in Ehrlich’s carcinoma solid tumors growing in female mice. ***P < 0.001 vs. control group, ^##P < 0.01 vs. Soyasaponin IV (100 mg/kg).

The master regulator NF-κB mediates interactions between cancer and inflammation on various levels (Carrà, Lingua, Maffeo, Taulli, & Morotti, 2020). NF-κB is widely expressed and mediates a variety of biological activities, including immunity, cell proliferation, apoptosis, inflammation, memory, and learning. The buildup of pro-inflammatory cytokines at the tumor site directly contributes to the pro-tumorigenic microenvironment in tumorous tissues with enhanced NF-κB activation (Ahmad et al., 2021). Some of the primary tumor-promoting activities regulated by NF-κB signalling include the stimulation of cell proliferation and suppression of apoptosis, the epithelial-to-mesenchymal transition (EMT), angiogenesis, invasiveness, and metastasis (Liu et al., 2018). NF-κB was shown to be a driver of such crucial mechanisms initiating and propagating tumor growth in a wide variety of malignancies from various organs (Taniguchi & Karin, 2018). Additionally, NF-κB subunits may become constitutively active in response to a large number of oncogenic mutations or a protracted, chronic inflammatory milieu. The formation of future cancers depends on NF-κB (Kaltschmidt et al., 2019). Numerous forms of carcinoma frequently upregulate the NF-κB signalling pathway, which results in the establishment of a permissive microenvironment that is crucial for tumor initiation, tumor progression, or both (Marozzi et al., 2021). Since phosphorylation and degradation of IkB protein are critical steps for activation of the NF-kB signaling pathway, the effect of soyasaponins on the LPS-initiated phosphorylation and degradation of NF-kB was measured. LPS stimulated the phosphorylation and degradation of IkB, and the stimulation was prevented largely by all soyasaponins (A1, A2, or I). These results support the notion that soyasaponins influence NF-κB activation by modulating phosphorylation and degradation of IkB.

3.3.5. Histopathological examination of solid tumors

The various groups’ histolopathological findings were depicted in Figure 7. The control EAC displayed substantial tumor cell development as well as localized, central foci of necrosis within the mass. The mass had more proliferating tumor cells with vesicular nuclei, high levels of mitosis, lots of tumor giant cells, and few apoptotic cells. Additionally, the tumor tissue showed extensive infiltration by mixed inflammatory cells. Soyasaponin IV (50 mg/kg) therapy decreased proliferative activity inside the mass by decreasing the number of cells undergoing mitosis, increasing the number of cells undergoing apoptosis, and decreasing the quantity of both inflammatory cells and tumor giant cells. Following Soyasaponin IV (100 mg/kg), there was a significant decrease in mitosis, an increase in apoptosis, and a decrease in the number of inflammatory cells. Quantitative scoring of the number of inflammatory cells within the tumor tissues was decreased in the experimental groups treated with Soyasaponin IV 50 compared to tumor control groups (32.3 ± 7.5 vs. 65 ± 5) and markedly decreased in Soyasaponin IV 100 group compared to tumor control groups (13 ± 1.7 vs. 65 ± 5) (P < 0.001). Soyasaponin IV (50 and 100 mg/kg) therapy reduced the number of inflammatory cells by 47.69% and 80%, respectively, in comparison to tumor control (Figure 7D). Treatment with Soyasaponin IV (50 and 100 mg/kg) in a dose dependent manner reduced significantly the number of mitotic figures in the tumor tissues compared to tumor control group (13.6 ± 5.1 and 7.6 ± 2.5 vs. 29 ± 4) (P < 0.001). Mitotic figures were decreased by treatment with Soyasaponin IV (50 and 100 mg/kg) by 53.39% and 73.86%, respectively compared to tumor control (Figure 7E).

Figure 7. Effect of Soyasaponin IV (50 and 100 mg/kg) on histopathological changes in tumor specimen isolated from EAC control group (A), EAC/Soyasaponin IV (50 mg/kg) group (B) and EAC/Soyasaponin IV (100 mg/kg) treated group(C). Quantitative scoring of inflammatory cells (D), mitotic figures in tumor cells (E), and number of apoptotic tumor cells (F) within the Ehrlich’s carcinoma solid tumors. ***P < 0.001 vs. EAC control group, ^##P < 0.001 vs. Soyasaponin IV (100 mg/kg) treated group.

The number of apoptotic tumor cells was increased in the experimental groups treated with Soyasaponin IV 50 compared to tumor control groups (15 ± 2 vs. 9.3 ± 1.1), Soyasaponin IV 100 compared to tumor control groups (17.6 ± 2.5 vs. 9.3 ± 1.1) (P < 0.05). Apoptosis was increased after treatment with Soyasaponin IV (50 and 100 mg/kg) by 60.77% and 89.38%, respectively compared to control EAC group (Figure 7F).

3.3.6. Effect of therapeutic Soyasaponin IV (50 and 100 mg/kg, orally) on the percentage of tumor necrosis

In comparison to tumor control groups, Soyasaponin IV (50 and 100 mg/kg) treatment increased necrosis in the tumor tissues in the experimental groups(Figure 7G). Within the mass of the EAC group, the tumor cell had pronounced diffuse nuclear expression of Ki-67 expression. While the Soyasaponin IV-treated groups displayed a dose-dependent reduction in Ki67 expression inside the bulk (Figure 7G(Figure 8).

A comprehensive review of the literature has revealed saponins’ great capacity to cause the death of cancer cells through apoptosis, oncotic necrosis, necroptosis, and autophagy (Yu et al., 2020). There have been several types of saponin-induced cell death noted, including cell lysis, necrosis, apoptosis, and autophagy (Figure 8). Increased cellular volume, the development of benign cysts, the disintegration of the plasma membrane, and the release of the cytoplasm into the environment are all signs of necrosis. Enzymes that depend on Ca²⁺ and have the ability to lyse the cytoskeleton are activated in response to these alterations. Membrane rupture is the result of saponin membrane lysis and saponin-induced pore creation, which also cause osmotic swelling. Because saponin-induced gap creation may increase intracytosolic Ca²⁺, activate Ca²⁺-dependent enzymes, and cause necrosis, the two cell deaths are related (Bafundo, Duerr, McNaughton, & Johnson, 2021).

Figure 8. Ki-67 immunoexpression within Ehrlich’s carcinoma solid tumor. EAC group showing marked nuclear expression within the entire mass (A), treated groups with Soyasaponin IV (50 and 100 mg/kg) (B and C respectively) revealing dose-dependent decrease of Ki-67 expression within the mass. Quantitative scoring of Ki-67 within the mass (D). ***P < 0.001 vs. EAC control group, ^##P < 0.001 vs. Soyasaponin IV (100 mg/kg) treated group.

4. Conclusion

In Conclusion, our research revealed a unique molecular mechanism through which soysapnin IV alleviates certain conditions. This improvement was achieved through various techniques, including: (1) mitigating oxidative stress, (2) inhibiting antioxidant suppression, (3) hindering the elevation of pro-inflammatory cytokines NF-κB, (4) impeding the elevation of VEGF. It is important to note that the anti-angiogenic, antioxidant, and anti-inflammatory effects of soyasaponin IV exhibit dose-dependent. The main mechanism believed to underline the observed therapeutic potential revolves around Soyasponin IV's modulatory action on the VEGF/NF-κB pathway.

Statistical analysis

All data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer multiple comparison test

Ethical approval

Kafrelsheikh University’s Animal Ethics Committee (KFS-04/2021).

Author contributions

S.A.A., A.A., A.E.K., L.Q. and A.A.Z performed the project work, conceived and designed the experiments; S.A.A., W.A, A.A. and A.A.Z carried out the experiments; S.A.A., W.A, A.A., A.E.K., L.Q., and A.A.Z., A.E.S. analyzed the data; S.A.A., W.A, A.A. and A.A.Z. wrote the manuscript; all the authors revised the manuscript; L.Q.: obtained funding.

Disclosure statement

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

Availability of data and materials

On request, the datasets used in and/or analyzed during the current research are available. This published article contains all of the data generated or analyzed during this research. This manuscript has never been published before, and it is not currently being considered by another journal. The paper has been approved and agreed to be submitted to this journal by all authors.

Additional information

Funding

This work was sponsored by the special projects for local science and technology development guided by China central government (grant no. 2019L3011).