Protective Effects of Medicago sativa Extract on Oxidative Stress and Apoptosis in a DMBA-Induced Experimental Breast Carcinogenesis Model

Abstract

Background

Breast cancer is a leading cause of cancer-related morbidity and mortality worldwide, and oxidative stress plays a critical role in early mammary carcinogenesis. Medicago sativa has been reported to possess antioxidant properties; however, its effects on early oxidative stress–apoptosis interplay in breast tissue remain unclear.

Methods

Twenty-eight female Wistar albino rats were randomly allocated into four groups: control, DMBA, Medicago sativa, and DMBA + Medicago sativa (n = 7 each). Breast carcinogenesis was initiated by a single intraperitoneal dose of 7,12-dimethylbenz[a]anthracene (DMBA; 80 mg/kg). Medicago sativa extract was administered orally at 250 mg/kg/day for 10 weeks. Serum malondialdehyde (MDA), total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were measured. Mammary tissues were evaluated histopathologically and immunohistochemically for Bax and Bcl-2 expression.

Results

DMBA administration significantly increased serum MDA, TOS, and OSI levels compared with the control group (p < 0.05). Medicago sativa treatment significantly reduced MDA, TOS, and OSI levels in the DMBA + Medicago sativa group (p < 0.05). No statistically significant difference in TAS levels was observed between the DMBA and DMBA + Medicago sativa groups. Histopathological analyses revealed moderate fibrosis, inflammatory infiltration, degenerative epithelial changes, and ductal hyperplasia in the DMBA group, whereas these alterations were attenuated following Medicago sativa treatment. The immunohistochemical evaluation demonstrated that, compared with the DMBA group, Bax expression with pro-apoptotic function increased, while Bcl-2 expression with anti-apoptotic function decreased in the DMBA + Medicago sativa group.

Conclusions

Medicago sativa mitigates DMBA-induced early mammary tissue damage by reducing oxidative stress and modulating Bax/Bcl-2–mediated apoptotic signaling prior to overt tumor formation, highlighting its mechanistic relevance in targeting early stages of breast carcinogenesis.

Keywords

breast cancer DMBA oxidative stress apoptosis Bax Bcl-2

1. Introduction

In recent years, breast cancer has surpassed lung cancer to become the most common type of cancer worldwide.¹ Due to its high morbidity and mortality rates, it represents a significant health problem for both women and healthcare systems across countries.² According to the World Health Organization (WHO), the incidence of breast cancer in 2022 was estimated at 2.29 million, and with 666,103 deaths in the same year, it was the leading cause of cancer-related mortality among women.³

In recent years, significant progress has been made in the treatment of cancer with respect to surgical and oncological principles. Alongside these advancements, the effects of medicinal plants on cancer treatment have also emerged as a subject of increasing interest. Within this context, the term nutraceutical was first introduced in 1989 by Stephen DeFelice, combining the words nutrition and pharmaceutical. Since its introduction into the literature, numerous plant-based food products have been studied for their preventive and protective roles against breast cancer. These studies have demonstrated that such products are effective not only in cancer prevention but also in reducing recurrence after treatment.^4,5

Lignans, coumarins, ascorbic acid, flavonoids, phytoestrogens, and sterols are important agents that can be utilized as phytotherapeutics. These bioactive phytochemicals have been shown to inhibit oxidative stress, suppress inflammation, and modulate critical signaling pathways in cells through apoptosis and neovascularization. As a result, they can eliminate tumor cells in vitro and prevent cancer development, while in vivo studies have demonstrated their inhibitory effects on tumor growth.⁶ In this regard, alfalfa (Medicago sativa) has been used in the treatment of various diseases, and its pharmacological effects have recently been observed.⁷ In recent years, studies have reported the antioxidant and antineoplastic properties of Medicago sativa.^8,9 Moreover, numerous other studies have shown that Medicago sativa exhibits anticancer properties through its antioxidant activity and has protective effects against coronary artery disease, osteoporosis, diabetes mellitus, and gastric ulcers.^10,11

Polycyclic aromatic hydrocarbons (PAHs) were first identified as molecules capable of inducing malignancy in 1921. Subsequent studies demonstrated their role particularly in the initiation of carcinogenesis.¹² They exert their effects by damaging the immune system, causing immunosuppression and immunotoxicity, and generating free radicals.¹³ Among these, 7,12-dimethylbenz[a]anthracene (DMBA) is a significant carcinogenic compound.¹⁴ It has been widely used to induce cancer in various experimental models of breast and skin cancer. Its effect is indirect and occurs through metabolism by cytochrome P450 enzymes, leading to the formation of diol epoxides and reactive oxygen species that cause cellular damage.¹⁵ These metabolites exert their carcinogenic activity by disrupting the structure of DNA through various oxygen radicals and inducing lipid peroxidation.¹⁶

The aim of this study was to investigate the protective effects of Medicago sativa leaf extract on a DMBA-induced breast cancer model in rats. Oxidative stress and antioxidant status (TOS, TAS, and MDA), histopathological and immunohistochemical changes, and apoptotic processes (Bax and Bcl-2 expression) were evaluated.

2. Materials and Methods

2.1. Induction of Experimental Breast Cancer

In experimental breast cancer models, 7,12-dimethylbenz[a]anthracene (DMBA) and N-nitroso-N-methylurea (NMU) are commonly used chemical carcinogens. DMBA has well-documented carcinogenic and immunosuppressive properties and has been widely used to induce mammary tumors in rodents.^17,18 Therefore, DMBA was selected for breast cancer induction in this study.

DMBA (Sigma-Aldrich, Darmstadt, Germany) was administered intraperitoneally at a single dose of 80 mg/kg, following the experimental protocol described by Gülbahçe Mutlu et al. Tumor development has been reported to occur approximately 50–140 days after DMBA administration. Animals were monitored throughout the experimental period for clinical signs and pathological changes.^19-21

2.2. Preparation and Administration of Medicago sativa Extract

The Medicago sativa plant used in this study was collected from a village at an altitude of 1300–1400 m in Elazığ, Turkey, and its taxonomic identification was confirmed by Tonçer, a researcher from the Department of Organic Agriculture. The preparation of the plant extract was based on the method described by Kartal et al²² The removal of the solvent from the crude extract was carried out using a Heidolph rotary evaporator (catalog no: HEID_31011, Schwabach, Germany) at the Pharmacognosy Research Laboratory of Dicle University Faculty of Pharmacy. This process yielded the purified Medicago sativa extract. Prior to administration, the extract was reconstituted in distilled water. The extract was prepared at an appropriate concentration to deliver a dose of 250 mg/kg/day, and the gavage volume was adjusted not to exceed 1 mL per 100 g of body weight. Oral administrations were performed using appropriately sized stainless-steel, rounded-tip gavage cannulas with single-use syringes.^23,24

In a study by Raeeszadeh et al alfalfa (Medicago sativa) ethanol extract was administered to Wistar rats at doses of 250, 500, and 750 mg/kg, and it was observed that the 250 mg/kg dose exhibited significant antioxidant activity. This dose was therefore selected as the starting dose for the experimental design of the present study.²⁵

2.3. Experimental Animals and Study Design

A total of 28 female Wistar albino rats (200–250 g) were included in the study. Animals were housed in standard laboratory cages under controlled environmental conditions (22 ± 3 °C, 12 h light/dark cycle) with free access to standard chow and water. Animals were monitored daily for general health, physical appearance, and behavioral changes. Body weights were recorded regularly, and treatment doses were adjusted accordingly.

The primary aim of this study was to evaluate the potential protective and antioxidant effects of Medicago sativa extract in an experimental breast cancer model induced by DMBA. The study design was planned to compare the biochemical and histopathological outcomes of the plant extract administered alone and in combination with DMBA. A positive control group receiving a known chemotherapeutic or anti-estrogenic agent was not included, as the objective of the study was not to compare with standard treatments but to investigate the potential effects of Medicago sativa within the experimental model.

Animals were randomly allocated into four experimental groups (n = 7 per group):

• Group 1 (Control/Sham): Rats received 1 mL of physiological saline orally via gavage.

• Group 2 (DMBA): Rats received a single intraperitoneal dose of DMBA (80 mg/kg).

• Group 3 (Medicago sativa): Rats received Medicago sativa extract orally via gavage at a daily dose of 250 mg/kg.

• Group 4 (DMBA + Medicago sativa): Rats received a single intraperitoneal dose of DMBA (80 mg/kg), followed 24 h later by oral administration of Medicago sativa extract (250 mg/kg/day).

At the end of the 10-week experimental period, in accordance with ethical principles and approved protocols, all rats were administered general anesthesia prior to the initiation of procedures. General anesthesia was induced by intraperitoneal administration of ketamine hydrochloride at a dose of 90 mg/kg (Ketalar; Pfizer, Istanbul, Turkey) and xylazine at a dose of 10 mg/kg (Rompun; Bayer, Istanbul, Turkey). The depth of sedation was regularly monitored at predefined intervals by assessing responses to skin or toe pinch stimuli.

2.4. Sample Collection

Collected blood samples were centrifuged at 3000 rpm for 10 min, and serum was separated and stored at −80 °C until biochemical analyses. Randomly selected mammary tissue samples were fixed in 10% neutral buffered formalin and transferred to the Histology/Embryology Laboratory for histopathological and immunohistochemical analyses.

2.5. Biochemical Analyses

Serum samples were used to evaluate oxidative stress parameters, including malondialdehyde (MDA), total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI).

2.5.1. Malondialdehyde (MDA) Assay

MDA levels, a major secondary product of lipid peroxidation in cell membranes, were determined using the thiobarbituric acid reactive substances (TBARS) method. The principle of the method is based on the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions (pH 3.4) at high temperature, resulting in the formation of a pink-colored MDA–TBA complex. For this purpose, serum samples were mixed with TBA reagent in an oxygenated acidic environment and incubated at 95 °C. After incubation, the absorbance of the resulting colored complex was measured spectrophotometrically at 532 nm. MDA concentrations were calculated based on the obtained absorbance values.^26,27

Quantitative analysis of serum MDA levels was also performed using a commercial ELISA kit. Rat-specific ELISA kits (Cat. No: E0156Ra) were obtained from Bioassay Technology Laboratory (Shanghai, China) and used according to the manufacturer’s instructions. Measurements were carried out using a microplate reader, and the results were calculated based on the standard curve and expressed as nmol/mL.

2.5.2. Total Antioxidant Status (TAS)

Venous blood samples were collected from rats in all experimental groups into EDTA-containing tubes and centrifuged at 3,000 rpm for 10 minutes at +4 °C. The separated plasma samples were stored at −80 °C until analysis. Prior to analysis, both the kit reagents and plasma samples were allowed to reach room temperature.

Total antioxidant status (TAS) levels were measured using a commercial kit from Rel Assay Diagnostics (Gaziantep, Turkey) according to the manufacturer’s instructions. Absorbance measurements were performed spectrophotometrically using a Beckman Coulter AU5800 biochemical autoanalyzer. Results were expressed as µmol Trolox equivalent per liter (µmol Trolox Equivalent/L). The spectrophotometric measurement wavelength used for TAS analysis was set at 660 nm.²⁸

2.5.3. Total Oxidant Status (TOS)

Serum total oxidant status (TOS) levels were determined using a commercial kit from Rel Assay Diagnostics (Gaziantep, Turkey) according to the manufacturer’s protocol. Measurements were performed spectrophotometrically using a Beckman Coulter AU5800 biochemical autoanalyzer.

The analysis is based on the colorimetric method developed by Erel and Erdemli. In this method, oxidant molecules present in the sample oxidize the ferrous ion–o-dianisidine complex to ferric ion; the resulting ferric ion forms a colored complex with xylenol orange, and the intensity of the color change is directly proportional to the total oxidant level of the sample. Results were expressed as µmol H₂O₂ equivalent per liter (µmol H₂O₂ Equivalent/L). The spectrophotometric measurement wavelength used for TOS analysis was set at 532 nm.^29,30

2.5.4. Oxidative Stress Index (OSI)

To evaluate the overall oxidative stress status, the Oxidative Stress Index (OSI) was calculated. The OSI value was obtained using serum total oxidant status (TOS) and total antioxidant status (TAS) levels according to the following formula:

OSI = (TOS / TAS) \times 100

Prior to calculation, TOS results were standardized in µmol H₂O₂ equivalent/L and TAS results in µmol Trolox equivalent/L. The OSI values were then converted to percentage values and expressed as arbitrary units (AU).³¹

2.6. Histological and Histochemical Analyses

Mammary tissue samples were fixed in 10% formalin for 24 h, washed overnight under running tap water, and embedded in paraffin. Sections of 5 μm thickness were obtained using a microtome (Leica RM2265, Wetzlar, Germany) and mounted on positively charged slides.

Hematoxylin and eosin (H&E) staining was performed to evaluate histopathological changes. Vascularization, inflammatory cell infiltration, apoptotic-degenerative changes, and pleomorphism were semi-quantitatively scored as follows: 0 (none), 1 (very mild, 1–15%), 2 (mild, 16–30%), 3 (moderate-low, 31–45%), 4 (moderate, 46–60%), 5 (severe, 61–75%), and 6 (very severe, 76–100%).³²

2.7. Immunohistochemical Analysis

Apoptosis plays a crucial role in cell proliferation under both physiological and pathological conditions. It is a fundamental process in development and maintenance of homeostasis. Pro-apoptotic factors such as Bax and anti-apoptotic factors such as Bcl-2 balance the apoptotic process. In cancer, apoptosis occurs as a result of the activation of caspases, which belong to the cysteine protease family.^33,34 Caspases eliminate molecules that inhibit apoptosis, thereby promoting cell death through the apoptotic pathway.³⁵ The Bcl-2 family proteins are key regulators of apoptosis, consisting of both pro- and anti-apoptotic members. The likelihood of apoptosis is determined by the Bcl-2/Bax ratio.^36,37

Immunohistochemical staining was performed on randomly selected mammary tissue sections (5 μm) to evaluate apoptotic activity. Sections were stained with primary antibodies against Bax and Bcl-2 to assess pro-apoptotic and anti-apoptotic protein expression. The balance between Bax and Bcl-2 expression was used to evaluate apoptosis-related changes in mammary tissues.

Immunohistochemical evaluation was performed under a light microscope by two independent observers blinded to the experimental groups. The intensity and distribution of immunopositive staining were assessed semi-quantitatively.

The reporting of this study conforms to ARRIVE 2.0 guidelines.³⁸

2.8. Statistical Analysis

Sample size calculation was performed using G*Power software (version 3.1.9.4). Based on Cohen’s criteria, an effect size of 0.70, α = 0.05, and power = 0.80 were used, resulting in a minimum sample size of 28 animals. Statistical analyses were conducted using SPSS for Windows (version 20.0; IBM Corp., Armonk, NY, USA).

Data normality was assessed using the Shapiro–Wilk test, skewness and kurtosis values, and Q–Q plots. Normally distributed data were analyzed using one-way analysis of variance (ANOVA), whereas non-normally distributed data were analyzed using the Kruskal–Wallis H test. Pairwise comparisons were performed using the Mann–Whitney U test where appropriate. Results were expressed as mean ± standard error (SE), and a p-value < 0.05 was considered statistically significant.

3. Results

3.1. Effects of Medicago sativa on Oxidative Stress Parameters

3.1.1. Serum Malondialdehyde (MDA) Levels

Serum malondialdehyde (MDA) levels differed significantly among the experimental groups. The highest mean MDA level was observed in the DMBA group, whereas significantly lower levels were detected in the DMBA + Medicago sativa group (p < 0.05). No statistically significant difference was found between the Medicago sativa and DMBA + Medicago sativa groups (p > 0.05). These findings indicate that Medicago sativa administration attenuated DMBA-induced lipid peroxidation.

3.1.2. Total Antioxidant Status (TAS)

Analysis of total antioxidant status (TAS) revealed no statistically significant difference between the DMBA and DMBA + Medicago sativa groups (p > 0.05). However, significant differences were observed between the DMBA + Medicago sativa group and both the control and Medicago sativa groups (p < 0.05). The highest mean TAS value was recorded in the DMBA group, whereas the lowest value was observed in the Medicago sativa group.

3.1.3. Total Oxidant Status (TOS)

Total oxidant status (TOS) levels showed significant differences among the groups. The DMBA group exhibited the highest serum TOS levels, while significantly lower values were observed in the DMBA + Medicago sativa group compared with the DMBA group (p < 0.05).

3.1.4. Oxidative Stress Index (OSI)

The oxidative stress index (OSI), calculated as the ratio of TOS to TAS, differed significantly between the groups. The DMBA group demonstrated the highest mean OSI value, whereas the DMBA + Medicago sativa group showed a significantly lower OSI value (p < 0.05). No significant difference was detected between the control and Medicago sativa groups (p > 0.05). The results of MDA, TAS, TOS, and OSI analyses for all experimental groups are summarized in Figure 1. Mean±Standard deviations of serum MDA, TAS, TOS and OSI values averaged between groups are shown in Table 1.

Figure 1.

Statistical analysis of MDA, TAS, TOS, and OSI across all groups

Table 1.

Mean ± Standard Deviation of Serum MDA, TAS, TOS, and OSI Values Calculated for Each Group

Groups	MDA (nmol/mL)	TAS (μmol)	TOS (μmol)	OSI (AU)
Control	1.47±0.30^b	1.21± 0.09^b,d	34.32±1 7.65^b,d	28.29±14.72^b,d
DMBA	2.15±0.32^a,c,d	1.93± 0.02^a,c	171.68±62.31^a,c,d	89.11±33.13^a,c,d
MS	1.73±0.26^b	1.18± 0.03^b,d	26.91±16.23^b,d	22.47±13.06^b,d
DMBA+MS	1.75±0.23^b	1.71± 0.26^a,c	92.05±33.32^a,b,c	52.27±13.12^a,b,c

TAS; Total Antioxidant Capacity (μmol H₂O₂ equivalent/L), TOS; Total Oxidant Capacity (μmol Trolox equivalent/L), MDA; Malondialdehyde (nmol/mL).OSI; Oxidative Stress Index (arbitrary unit). DMBA; 7,12-dimethylbenz[a]anthracene, MS; Medicago sativa extract.

^adifferent from control.

^bdifferent from DMBA.

^cdifferent from MS.

^ddifferent from DMBA+MS.

3.2. Histopathological Findings

At the end of the experiment, the breast tissues obtained were first evaluated macroscopically, and no visible tumor formation was observed in any of the experimental groups. The tissues were then fixed appropriately for histopathological examination and embedded in paraffin blocks after routine tissue processing. The prepared sections were examined microscopically. In the histopathological evaluation, the parameters of vascularization, inflammatory cell infiltration, apoptotic-degenerative changes, and cellular pleomorphism were scored semi-quantitatively. These parameters were graded according to the severity of the lesion.

Mammary tissue sections from the control group exhibited normal histological architecture. In the Medicago sativa group, very mild fibrosis was observed. In contrast, the DMBA group showed moderate fibrosis, inflammatory cell infiltration, degenerative changes in mammary epithelial cells, and ductal hyperplasia. The DMBA + Medicago sativa group displayed mild fibrosis and increased vascularization compared with the DMBA group.

Histopathological parameters were scored semi-quantitatively and statistically compared between groups. The analysis revealed a statistically significant difference in histopathological scores between the DMBA group and the DMBA + Medicago sativa group (p < 0.05).

Representative histological images are shown in Figure 2.

Figure 2.

Histopathological Micrographs of Specimens. (A) Control group: Mammary tissue with normal appearance (H&E, Bar 50 µm). (B) MS group: Mild fibrosis (H&E, Bar 50 µm). (C) DMBA group: Inflammatory cell infiltration, moderate fibrosis, pleomorphic cell structure (H&E, Bar 100 µm). (D) DMBA group: Vascularization and degenerative changes in mammary cells (H&E, Bar 20 µm). (E) DMBA+MS group: Mild proliferation and moderate fibrosis (H&E, Bar 20 µm)

3.3. Immunohistochemical Findings

3.3.1. Bax Expression

Bax immunoreactivity was very weak in the control group and moderately positive in the Medicago sativa group. In the DMBA group, Bax expression was weakly positive to negative, comparable to the control group. In contrast, the DMBA + Medicago sativa group exhibited stronger Bax positivity compared with the DMBA group, indicating enhanced pro-apoptotic signaling. Representative images are presented in Figure 3.

Figure 3.

Immunohistochemical Micrographs of Specimens Showing Bax Expression Staining: Bax immunohistochemistry; Counterstain: Hematoxylin; Bar: 50 µm. (A) Control group: Very low level of expression (B) MS group: Moderate level of expression (C) DMBA group: No expression observed (D) DMBA+MS group: Low level of expression

3.3.2. Bcl-2 Expression

Bcl-2 immunoreactivity was negative to very weak in the control and Medicago sativa groups. The DMBA group demonstrated moderately widespread positive Bcl-2 expression in mammary tissues. In the DMBA + Medicago sativa group, Bcl-2 expression was overall weakly positive compared with the DMBA group. Representative immunohistochemical images are shown in Figure 4.

Figure 4.

Immunohistochemical Micrographs of Specimens Showing Bcl-2 Expression Staining: Bcl-2 immunohistochemistry; Counterstain: Hematoxylin; Bar: 50 µm. (A) Control group: Very low level of expression (B) MS group: Very low level of expression (C) DMBA group: Moderate level of expression (D) DMBA+MS group: Low-to-moderate level of expression

4. Discussion

Medicinal and aromatic plants have been used for centuries in the prevention and treatment of various diseases, and scientific interest in their bioactive properties continues to increase. These plants are rich in phenolic compounds, including flavonoids, phenolic acids, and phenolic terpenes, which are particularly abundant in leaves and flowers and are well recognized for their antioxidant and anti-inflammatory activities.³⁹ Such properties have been suggested to play a role in modulating oxidative stress–related diseases, including cancer.

Oxidative stress is a key contributor to breast carcinogenesis, primarily through lipid peroxidation and subsequent cellular damage. Malondialdehyde (MDA), a major end product of lipid peroxidation, forms cross-links with lipids, proteins, and nucleic acids and serves as a reliable biomarker of oxidative damage.^39-43 Elevated MDA levels have been consistently reported in both breast cancer tissues and serum samples, reflecting impaired tissue oxygenation and increased free radical activity during carcinogenesis.^40,41 Moreover, increased lipid peroxidation has been associated with DNA strand breaks and genotoxic damage in experimental breast cancer models.^42,43 In agreement with these findings, the present study demonstrated the highest serum MDA levels in the DMBA group, whereas Medicago sativa co-administration significantly reduced MDA levels. The absence of a significant difference between the MS and DMBA+MS groups suggests that MS effectively counteracted DMBA-induced lipid peroxidation rather than exerting a pro-oxidant effect.⁴⁴

Endogenous antioxidant defense systems play a crucial role in maintaining redox homeostasis and protecting tissues from oxidative injury. Enzymes such as superoxide dismutase and catalase constitute essential components of this defense network, and reduced antioxidant capacity has been reported in various cancer types.^45-47 In the present study, total oxidant status (TOS) was significantly elevated in the DMBA group and markedly reduced following MS treatment, indicating attenuation of systemic oxidative burden. Although total antioxidant status (TAS) did not differ significantly between the DMBA and DMBA+MS groups, this finding should be interpreted with caution. TAS represents a cumulative systemic measure and may not fully reflect early or tissue-specific antioxidant responses during the initial stages of carcinogenesis. Importantly, the concurrent reductions in MDA, TOS, and OSI strongly support a biologically meaningful antioxidant effect of MS despite the lack of a pronounced TAS difference.

The oxidative stress index (OSI), which integrates both oxidant and antioxidant parameters, provides a more comprehensive assessment of redox balance. In this study, OSI values were highest in the DMBA group and significantly reduced in the DMBA+MS group, further confirming the protective effect of MS against DMBA-induced oxidative stress. The lack of difference between the control and MS groups indicates that MS did not disrupt physiological redox homeostasis under non-stressed conditions.

Histopathological alterations are well documented in DMBA-induced breast carcinogenesis and include inflammatory cell infiltration, epithelial hyperplasia, fibrosis, and early neoplastic changes.^48,49 In line with previous reports, breast tissues from DMBA-treated rats in the present study exhibited moderate fibrosis, inflammatory infiltration, degenerative epithelial changes, and ductal hyperplasia. In contrast, MS administration following DMBA exposure resulted in markedly milder histopathological alterations, suggesting preservation of tissue architecture and suppression of early carcinogenic changes. These findings are consistent with previous studies demonstrating the protective effects of phytochemicals against DMBA-induced mammary tissue damage.⁵⁰

Apoptosis plays a central role in eliminating damaged or transformed cells during carcinogenesis. Members of the Bcl-2 protein family regulate mitochondrial apoptotic pathways by balancing pro-apoptotic and anti-apoptotic signals. Bcl-2 overexpression promotes cell survival and has been associated with tumor progression, whereas Bax acts as a pro-apoptotic mediator whose loss enhances neoplastic potential.^51-53 In the present study, weak or absent Bax expression accompanied by moderate Bcl-2 positivity in the DMBA group reflects suppression of apoptotic signaling during early carcinogenic insult. Notably, MS treatment enhanced Bax expression while reducing Bcl-2 immunoreactivity, indicating restoration of pro-apoptotic balance. These findings suggest that MS may facilitate the elimination of damaged mammary cells through modulation of apoptosis-related pathways.

Despite these findings, several limitations of the present study should be acknowledged. The experimental duration was deliberately limited to 10 weeks to focus on early, pre-neoplastic molecular and cellular alterations induced by DMBA rather than macroscopic tumor formation. Accordingly, the effects of Medicago sativa on long-term tumor incidence, growth, and progression could not be assessed. In addition, total antioxidant status represents a cumulative systemic parameter and may not fully reflect dynamic or tissue-specific redox responses during the early stages of carcinogenesis. In this context, the absence of a statistically significant difference in TAS between the DMBA and DMBA + Medicago sativa groups does not negate biologically meaningful antioxidant activity, particularly given the consistent improvements observed in MDA, TOS, OSI, histopathological findings, and apoptosis-related markers. Finally, although Bax and Bcl-2 immunoreactivity provided insight into apoptosis-related signaling, further molecular analyses are warranted to elucidate downstream mechanisms involved in Medicago sativa–mediated protection.

5. Conclusions

The present study demonstrates that Medicago sativa exerts a protective effect against DMBA-induced early breast tissue injury through coordinated modulation of oxidative stress and apoptosis-related pathways. The consistent reduction in lipid peroxidation and systemic oxidant burden, together with improved histopathological architecture and a shift toward a pro-apoptotic Bax/Bcl-2 profile, indicates that Medicago sativa primarily targets early molecular and cellular events preceding overt tumor formation. Importantly, the use of a non-tumor endpoint model allowed the identification of these early mechanistic alterations, highlighting the capacity of Medicago sativa to interfere with carcinogenic signaling before irreversible neoplastic transformation occurs. Collectively, these findings provide mechanistic support for the potential of Medicago sativa as a complementary strategy aimed at the prevention or early intervention of breast carcinogenesis, warranting further studies to explore its long-term and translational relevance.

Supplemental Material

Supplemental Material - Protective Effects of Medicago sativa Extract on Oxidative Stress and Apoptosis in a DMBA-Induced Experimental Breast Carcinogenesis Model

Supplemental Material for Protective Effects of Medicago sativa Extract on Oxidative Stress and Apoptosis in a DMBA-Induced Experimental Breast Carcinogenesis Model by Baran Demir, Mehmet Tolga Kafadar, Eda Yıldızhan, Murat Akkuş in Natural Product Communications

Ethical Considerations

All experimental procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with the ARRIVE guidelines. The study protocol was reviewed and approved by the Local Animal Ethics Committee of Dicle University Prof. Dr. Sabahattin Payzın Health Sciences Research and Application Center (Approval No: [TIP. 22.028], Date: [01.06.2023])

Footnotes

This study was supported by the Dicle University Scientific Research Platform (DUBAP) under Project number TIP.24.007. The authors gratefully acknowledge the contributions of the DUBAP.

ORCID iDs

Baran Demir

Mehmet Tolga Kafadar

Eda Yıldızhan

Murat Akkuş

Author Contributions

Conceptualisation, B.D.; methodology, B.D. and E.Y.; investigation, B.D., M.T.K., E.Y. and M.A.; writing, original draft preparation, B.D. and E.Y.; writing, review and editing, B.D., M.T.K. and E.Y. All authors have read and agreed to the published version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Dicle University Scientific Research Platform (DUBAP) under Project number TIP.24.007.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplemental Material

Supplemental material for this article is available online

References

WHO&International Agency for Reseach on Cancer . Cancer Today, GLOBOCAN; 2020. [Internet] Access Address: https://gco.iarc.fr/today/online-analysis-multi-bars. Access Date: 15/09/2023.

Kashyap

Pal

Sharma

, et al. Global Increase in Breast Cancer Incidence: Risk Factors and Preventive Measures. Biomed Res Int. 2022;2022:9605439. doi:10.1155/2022/9605439

Liao

. Inequality in breast cancer: Global statistics from 2022 to 2050. Breast. 2025;79:103851. doi:10.1016/j.breast.2024.103851

Bozovic-Spasojevic

Azambuja

McCaskill-Stevens

Dinh

Cardoso

. Chemoprevention for breast cancer. Cancer Treat Rev. 2012;38(5):329-339. doi:10.1016/j.ctrv.2011.07.005

Brown

Lippman

. Chemoprevention of breast cancer. Breast Cancer Res Treat. 2000;62(1):1-17. doi:10.1023/a:1006484604454

Mandal

Bishayee

. Mechanism of Breast Cancer Preventive Action of Pomegranate: Disruption of Estrogen Receptor and Wnt/β-Catenin Signaling Pathways. Molecules. 2015;20(12):22315-22328. doi:10.3390/molecules201219853

Bora

Sharma

. Phytochemical and pharmacological potential of Medicago sativa: a review. Pharm Biol. 2011;49(2):211-220. doi:10.3109/13880209.2010.504732

Gatouillat

Magid

Bertin

, et al. Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in sensitive and multidrug-resistant tumor cells. Nutr Cancer. 2014;66(3):483-491. doi:10.1080/01635581.2014.884228

Nigro

Bull

Klopfer

Pak

Campbell

. Effect of dietary fiber on azoxymethane-induced intestinal carcinogenesis in rats. J Natl Cancer Inst. 1979;62(4):1097-1102.

10.

Rana

Katbamna

Padhya

, et al. In Vitro Antioxidant And Free Radical Scavenging Studies Of Alcoholic Extract of Medicago Sativa L. Romanian Journal Of Biology-Plant Biology. 2010;55(1):15-22.

11.

Krakowska-Sieprawska

Rafińska

Walczak-Skierska

Kiełbasa

Buszewski

. Promising Green Technology in Obtaining Functional Plant Preparations: Combined Enzyme-Assisted Supercritical Fluid Extraction of Flavonoids Isolation from Medicago Sativa Leaves. Materials (Basel). 2021;14(11):2724. doi:10.3390/ma14112724

12.

Özben

. Oxidative Stres and Apoptosis: Impact on Cancer Therapy. J Pharm Sci. 2006;96(9):2181-2196. doi:10.1002/jps.20874

13.

Ertekin

Türel

Oto

, et al. The Effect of Nettle Herb on The Levels of Lipid Peroxidation, Antioxidant Substances and Nitrite - Nitrate in Rabbits Induced With Dimetylbenzanthracene. Y.Y.U. Journal of the Faculty of Veterinary Medicine. 2008;2:11-15. 978-605-80730-4-3.

14.

Liu

Kundu-Roy

Matsuura

, et al. Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice. Oncotarget. 2016;7(40):64878-64885. doi:10.18632/oncotarget.11713

15.

Lim

Korourian

Todorova

Kaufmann

Klimberg

. Glutamine prevents DMBA-induced squamous cell cancer. Oral Oncol. 2009;45(2):148-155. doi:10.1016/j.oraloncology.2008.04.008

16.

Ohnishi

Hiraku

Hasegawa

, et al. Mechanism of oxidative DNA damage induced by metabolites of carcinogenic naphthalene. Mutat Res Genet Toxicol Environ Mutagen. 2018;827:42-49. doi:10.1016/j.mrgentox.2018.01.005

17.

Khan

Qindeel

Ahmed

Khan

Rehman

. Development of novel pH-sensitive nanoparticle-based transdermal patch for management of rheumatoid arthritis. Nanomedicine (Lond). 2020;15(6):603-624. doi:10.2217/nnm-2019-0385

18.

Zeweil

Sadek

Taha

El-Sayed

Menshawy

. Graviola attenuates DMBA-induced breast cancer possibly through augmenting apoptosis and antioxidant pathway and downregulating estrogen receptors. Environ Sci Pollut Res Int. 2019;26(15):15209-15217. doi:10.1007/s11356-019-04920-w

19.

Russo

. Significance of Rat mammary tumors for human risk assessment. Toxicol Pathol. 2015;43:145-170. doi:10.1177/0192623314532036

20.

Alvarado

Lopes

Faustino-Rocha

, et al. Prognostic factors in MNU and DMBA-induced mammary tumors in female rats. Pathol Res Pract. 2017;213:441-446. doi:10.1016/j.prp.2017.02.014

21.

Gülbahçe

Baltacı

. Zinc and melatonin supplementation ameliorates uterus tissue damage in dmba-induced breast cancer in rats. General Medical J. 2020;30(3):190-196.

22.

İrtem

Şayak

. Determination of Antioxidant Capacity of Medicago sativa. Journal of Natural & Applied Sciences of East. 2020;3(1):9-18.

23.

Al-Dosari

. In vitro and in vivo antioxidant activity of alfalfa (Medicago sativa L.) on carbon tetrachloride intoxicated rats. Chinese Medicine. 2012;40(4):779-793.

24.

Taysi

Gumustekin

Demircan

, et al. Hippo phaerhamnoides attenuates nicotine-induced oxidative stress in rat liver. Pharmaceutical Biology. 2010;48(5):488-493.

25.

Raeeszadeh

Moradi

Ayar

Akbari

. The Antioxidant Effect of Medicago sativa L. (Alfalfa) Ethanolic Extract against Mercury Chloride (HgCl2) Toxicity in Rat Liver and Kidney: An In Vitro and In Vivo Study. Evid Based Complement Alternat Med. 2021;2021:8388002. doi:10.1155/2021/8388002

26.

Satoh

. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta. 1978;90(1):37-43. doi:10.1016/0009-8981(78)90081-5

27.

Yagi

. Assay for blood plasma or serum. Methods Enzymol. 1984;105:328-331. doi:10.1016/s0076-6879(84)05042-4

28.

Erel

. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin Biochem. 2004;37(2):112-119. doi:10.1016/j.clinbiochem.2003.10.014

29.

Erel

. A new auto mated colorimetric method formeasuring total oxidant status. Clin Biochem. 2005;38:1103-1111. doi:10.1016/j.clinbiochem.2005.08.008

30.

Erdemli

Aksungur

Gul

, et al. The effects of acrylamide and vitamin E on kidneys in pregnancy: an experimental study. J Matern Fetal Neonatal Med. 2019;32(22):3747-3756. doi:10.1080/14767058.2018.1471675

31.

Harma

Erel

. Increased oxidative stress in patients with hydatidiform mole. Swiss Med Wkly. 2003;133(41–42):563-566.

32.

Goldschmidt

Peña

Rasotto

Zappulli

. Classification and grading of canine mammary tumors. Vet Pathol. 2011;48(1):117-131. doi:10.1177/0300985810393258

33.

Henson

Hume

. Apoptotic cell removal in development and tissue homeostasis. Trends Immunol. 2006;27:244-250. doi:10.1016/j.it.2006.03.005

34.

Tomatır

. Apoptosıs: Programmed Cell Death. J Med Sci. 2003;23:499-508.

35.

Elmore

. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516. doi:10.1080/01926230701320337

36.

Yerlikaya

Dokudur

. The importance of protein degradation. Uludag University Faculty of Medicine Journal. 2009;35:93-99.

37.

Stajner

. Season of breast cancer diagnosis and probability of death from breast cancer in the United States. Int J Cancer. 2010;126(12):3010-3013. doi:10.1002/ijc.24848

38.

Percie du Sert

Hurst

Ahluwalia

, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMJ Open Sci. 2020;4(1):e100115. doi:10.1136/bmjos-2020-100115

39.

Javanmardi

Stushnoff

Locke

. Antioxidant Activity And Total Phenolic Content Of Iranian Ocimum Accessions. Food Chemistry. 2003;83(4):547-550. doi:10.1016/S0308-8146(03)00151-1

40.

Gungor

Ilhan

Eroksuz

. The effectiveness of cyclooxygenase-2 inhibitors and evaluation of angiogenesis in the model of experimental colorectal cancer. Biomedicine Pharmacotherapy. 2018;102:221-229. doi:10.1016/j.biopha.2018.03.066

41.

Khanzode

Muddeshwar

Khanzode

Dakhale

. Antioxidant enzymes and lipid peroxidation in different stages of breast cancer. Free Radic Res. 2004;38(1):81-85. doi:10.1080/01411590310001637066

42.

Portakal

Ozkaya

Inal

Bozan

Koşan

Sayek

. Coenzyme Q10 concentrations and antioxidant status in tissues of breast cancer patients. Clin Biochem. 2000;33(4):279-284. doi:10.1016/s0009-9120(00)00067-9

43.

Gerber

Astre

Ségala

, et al. Tumor progression and oxidant-antioxidant status. Cancer Lett. 1997;114(1-2):211-214. doi:10.1016/s0304-3835(97)04665-x

44.

Huang

Sheu

Lin

. Association between oxidative stress and changes of trace elements in patients with breast cancer. Clin Biochem. 1999;32(2):131-136. doi:10.1016/s0009-9120(98)00096-4

45.

Shukla

Srivastava

Shankar

, et al. Oxidative Stress and Antioxidant Status in High-Risk Prostate Cancer Subjects. Diagnostics (Basel). 2020;10(3):126. doi:10.3390/diagnostics10030126. Published 2020 Feb 27.

46.

Kennedy

Sandhu

Harper

Cuperlovic-Culf

. Role of Glutathione in Cancer: From Mechanisms to Therapies. Biomolecules. 2020;10(10):1429. doi:10.3390/biom10101429

47.

Griñan-Lison

Blaya-Cánovas

López-Tejada

, et al.

Antioxidants for the Treatment of Breast Cancer: Are We There Yet?

Antioxidants (Basel). 2021;10(2):205. doi:10.3390/antiox10020205

48.

Feng

Spezia

Huang

, et al. Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. Genes Dis. 2018;5(2):77-106. doi:10.1016/j.gendis.2018.05.001

49.

Bonacho

Rodrigues

Liberal

. Immunohistochemistry for diagnosis and prognosis of breast cancer: a review. Biotech Histochem. 2020;95(2):71-91. doi:10.1080/10520295.2019.1651901

50.

Seshadri

Oyouni

AAA

Bawazir

, et al. Zingiberene exerts chemopreventive activity against 7,12-dimethylbenz(a)anthracene-induced breast cancer in Sprague-Dawley rats. J Biochem Mol Toxicol. 2022;36(10):2212. doi:10.1002/jbt.23146

51.

Ola

Nawaz

Ahsan

. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem. 2011;351(1-2):41-58. doi:10.1007/s11010-010-0709-x

52.

Janumyan

Cui

Yan

Sansam

Valentin

Yang

. G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation. J Biol Chem. 2008;283(49):34108-34120. doi:10.1074/jbc.M806294200

53.

Youle

Strasser

. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9(1):47-59. doi:10.1038/nrm2308

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