Moringa oleifera extract inactivates nuclear transcription factor nuclear factor-kappa B p65/50 subunits and inhibits expression of tumor necrosis factor-α, interleukin-6, and interleukin-8 in phorbol 12-myristate 13-acetate-stimulated human breast cancer cells

INTRODUCTION

Breast cancer is a condition characterized by the uncontrolled proliferation of abnormal breast cells, leading to the formation of tumors that can metastasize and pose life-threatening risks if left untreated.^[1] Among the widely used models in breast cancer research are MCF-7 cells, derived from human mammary adenocarcinoma, which closely resemble hormone receptor-positive breast cancer, especially in postmenopausal women.^[2] These cells are instrumental in studying the role of estrogen, androgen, progesterone, and glucocorticoid receptors in breast cancer progression, making them a valuable tool in translational research.^[3] Furthermore, MCF-7 cells undergo apoptosis when exposed to cytokines such as tumor necrosis factor-α (TNF-α) though variant sub- clones have shown resistance to TNF-α-induced apoptosis, providing insights into cancer cell survival mechanisms.^[4] Cytokines such as interleukin-6 (IL-6), TNF-α, and chemokines such as interleukin-8 (IL-8) have gained attention due to their role in cancer pathogenesis.^[5] IL-6, a multifunctional cytokine, plays a critical role in immune regulation and inflammation.^[6] Overexpression of IL-6 has been implicated in promoting cancer cell growth and survival, making it a key target in cancer therapy.^[7] TNF-α is a pro-inflammatory cytokine that was considered a potential cancer therapy.^[8] Studies revealed that chronic exposure to TNF-α in the tumor microenvironment can promote cancer development by facilitating inflammation, tumor growth, and metastasis.^[9] Both IL-6 and TNF-α activate several signaling pathways, including nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), which promote cancer cell survival, proliferation, and angiogenesis.^[10] They also enhance the recruitment of immune cells, such as macrophages and neutrophils, which can further sustain an inflammatory environment conducive to tumor progression.^[11] Both IL-6 and TNF-α have become a target for therapeutic interventions aimed at modulating their activities in various cancers.^[12] Drugs that block IL-6 or TNF-α, such as monoclonal antibodies and receptor antagonists, are currently being explored as adjunct treatments to reduce inflammation and inhibit tumor-promoting effects in cancer patients.^[13] Similarly, IL-8 is known to contribute to tumor progression through its pro-inflammatory and pro-angiogenic effects.^[14]

Moringa oleifera, a tree native to parts of Africa and Asia, has long been recognized for its diverse medicinal properties.^[15,16] Known as the “miracle tree,” it contains a rich array of nutrients and bioactive compounds with potential therapeutic benefits.^[17] Various parts of ME, including its leaves, seeds, and bark, have been shown to possess anti-inflammatory, antioxidant, and anticancer properties.^[18] The single genus Moringa consists of 14 species, including Arabia.^[19,20] In Middle Eastern countries, Moringa seed powder has proven to be an effective and versatile solution for various applications, particularly in water purification. It is especially beneficial in underprivileged regions where access to proper sanitation and clean water remains limited.^[21]

Previous studies have demonstrated that ME extracts can induce apoptosis, inhibit cell proliferation, and reduce viability in a range of cancer cell lines, including breast, prostate, and liver cancers.^[22] This study investigated the effects of ME extracts on phorbol 12-myristate 13-acetate (PMA)-stimulated expression and production of key pro-inflammatory cytokines IL-6, TNF-α, and IL-8 in MCF-7 cells. The novel findings of this study shed light on the potential of ME as an adjunct therapy in breast cancer, targeting cytokine-mediated pathways involved in tumor progression.

MATERIAL & METHODS

RESULTS

Cell viability against ME, PMA, and ME in combination with PMA

The viability of MCF-7 was assessed after treatment with increasing concentrations of ME (0–40 µg/mL) for 24 h [Figure 1a]. Serum-starved MCF-7 cells were exposed to ME (0–40 µg/mL) for 24 h, and cell viability was quantified using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). The results indicated no significant change in cell viability across the tested ME concentrations for the 24-h treatment period (P > 0.05). Moreover, the effect of treatment duration on cell viability was evaluated using a fixed concentration of ME (10 µg/mL) over increasing time points, as depicted in Figure 1b. The viability of MCF-7 cells remained unchanged even after 72 h of treatment. The study further investigated cell viability following treatment with PMA (40 nM) alone and in combination with varying concentrations of ME (5–10 µg/mL) [Figure 1c]. To further validate these findings, the microscopic images of the main sets of treatment of MCF-7 cells with PMA, ME, and PMA in combination with ME for 24 h were also given in Figure 1dg, respectively. The findings demonstrated that neither PMA alone nor its combination with ME at the tested concentrations significantly affected the viability of MCF-7 cells [Figure 1].

Close

Figure 1:

Cell viability of human breast cancer cells against Moringa oleifera leaf extract (ME). (a) MCF-7 cells were treated with ME (1–40 µg/mL) for 24 h and the percent viability was determined. (b) MCF-7 cells were treated with ME (10 µg/mL) for 0–72 h and percent viability was determined. (c) MCF-7 cells were pretreated with ME (5–10 µg/mL) for 3 h and then stimulated with PMA (40 nM) for 24 h and percent viability was determined. (d) Microscopic picture of MCF-7 cells without treatment. (e) Microscopic picture of MCF-7 cells with PMA (40 nM) treatment for 24 h. (f) Microscopic picture of MCF-7 cells with ME (40 µg/mL) treatment for 24 h. (g) Microscopic picture of MCF-7 cells treated with PMA (40 nM) and ME (40 µg/mL) for 24 h. The cancer cells MCF-7 were 12 h serum starved before treatment with ME or PMA. All microscopic images of cancer cells were taken at 100X magnification and the cells’ viability was determined using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega). PMA: Phorbol 12-myristate 13-acetate.

Export to PPT

ME inhibits TNF-α messenger RNA (mRNA) expression and TNF-α protein production in PMA-stimulated MCF-7 cells

TNF-α, a proinflammatory cytokine implicated in the progression of various cancers, including breast cancer, was evaluated under experimental conditions to determine its mRNA expression levels [Figure 2a]. Untreated MCF-7 cells exhibited negligible TNF-α mRNA expression. However, stimulation with PMA alone significantly upregulated TNF-α mRNA expression (P < 0.01). Notably, this PMA-induced increase in TNF-α mRNA levels was significantly suppressed by ME in a dose-dependent manner (P < 0.05). To investigate whether the changes in TNF-α mRNA expression translated to protein secretion, TNF-α protein levels were measured in the culture supernatants of treated and untreated cells [Figure 2b]. PMA stimulation markedly elevated TNF-α production, which was significantly reduced by ME in a dose-dependent manner (P < 0.05). These findings indicate that ME effectively inhibits both TNF-α mRNA expression and TNF-α protein production in PMA-stimulated MCF-7 cells, further demonstrating its anti-inflammatory potential [Figure 2].

Close

Figure 2:

Effect of Moringa oleifera leaf extract (ME) on TNF-α gene expression and production in PMA-stimulated human breast cells. MCF-7 cells were pretreated with ME (5–10 µg/mL) for 3 h and stimulated by PMA (40 nM) for 24 h. (a) Gene expression of TNF-α was determined by quantitative RT-PCR normalized to GAPDH and then compared with the levels present in untreated cells. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells. (b) Level of TNF-α in the culture medium was quantified by TNF-α-specific sandwich ELISA. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells. Results are representative of mean±standard error of the mean for three independent experiments. PMA: Phorbol 12-myristate 13-acetate, RT-PCR: Real-time polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay, TNF-α: Tumor necrosis factor-α.

Export to PPT

ME suppressed IL-6 mRNA expression and IL-6 protein production in PMA-stimulated MCF-7 cells

IL-6, a proinflammatory cytokine implicated in cancer progression, including breast cancer, was evaluated in this study under experimental conditions. As shown in [Figure 3a], untreated MCF-7 cells exhibited negligible IL-6 mRNA expression. However, stimulation with PMA alone significantly upregulated IL-6 mRNA expression (P < 0.01). Notably, this PMA-induced elevation of IL-6 mRNA levels was significantly attenuated when MCF-7 cells were treated with increasing concentrations of ME (P < 0.05). To determine whether changes in IL-6 mRNA expression corresponded to alterations in IL-6 protein secretion, IL-6 protein levels were measured in the culture supernatants of treated and untreated cells [Figure 3b]. PMA stimulation significantly elevated IL-6 protein production, which was dose-dependently reduced by ME treatment (P < 0.05). These results demonstrate that ME effectively suppresses IL-6 mRNA expression and protein production in PMA-stimulated MCF-7 cells, highlighting its potential anti-inflammatory effects [Figure 3].

Close

Figure 3:

Effect of Moringa oleifera leaf extract (ME) on IL-6 gene expression and production in PMA-stimulated human breast cells. MCF-7 cells were pretreated with ME (5–10 µg/mL) for 3 h and stimulated by PMA (40 nM) for 24 h. (a) Gene expression of IL-6 was determined by quantitative RT-PCR normalized to GAPDH and then compared with the levels present in untreated cells. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells (b) Level of IL-6 in the culture medium was quantified by IL-6 specific sandwich ELISA. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells. Results are representative of mean±standard error of the mean for three independent experiments. PMA: Phorbol 12-myristate 13-acetate, IL-6: Interleukin-6, RT-PCR: Real-time polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay.

Export to PPT

ME declined IL-8 mRNA expression and IL-8 protein production in PMA-stimulated MCF-7 cells

IL-8, a proinflammatory cytokine implicated in cancer development, including breast cancer, was evaluated under the experimental conditions. As shown in [Figure 4a], untreated MCF-7 cells exhibited negligible IL-8 mRNA expression. However, stimulation with PMA alone significantly upregulated IL-8 mRNA expression (P < 0.01). Notably, this PMA-induced elevation in IL-8 mRNA levels was significantly reduced when MCF-7 cells were treated with increasing concentrations of ME (P < 0.05). To assess whether changes in IL-8 mRNA expression impacted IL-8 protein secretion, IL-8 protein levels were measured in the culture supernatants of treated and untreated cells [Figure 4b]. PMA stimulation significantly increased IL-8 protein production, which was dose-dependently suppressed by ME treatment (P < 0.05). These findings confirm that ME effectively reduces IL-8 mRNA expression and protein production in PMA-stimulated MCF-7 cells, suggesting its potential anti-inflammatory properties [Figure 4].

Close

Figure 4:

Effect of Moringa oleifera leaf extract (ME) on IL-8 gene expression and production in PMA-stimulated human breast cells. MCF-7 cells were pretreated with ME (5–10 µg/mL) for 3 h and stimulated by PMA (40 nM) for 24 h. (a) Gene expression of IL-8 was determined by quantitative RT-PCR normalized to GAPDH and then compared with the levels present in untreated cells. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells. (b) Level of IL-8 in the culture medium was quantified by IL-8-specific sandwich ELISA. ^#P<0.01 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus **; ^@P>0.05 versus untreated cells. Results are representative of mean±standard error of the mean for three independent experiments. PMA: Phorbol 12-myristate 13-acetate, IL-8: Interleukin-8, RT-PCR: Real-time polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay.

Export to PPT

Role of ME on PMA-induced activation of NF-κB p65 and p50 in MCF-7 cells

To determine the extent of NF-κB pathway involvement, we analyzed nuclear levels of NF-κB subunits p65 and p50 in MCF-7 cells with ME pre-treatment following 30 min of PMA stimulation. Nuclear extracts from PMA-treated cells showed a significant increase in p65 levels compared to untreated controls [Figure 5a], (P < 0.05). The role of p65 in this process was validated using Bay-11-7082, a specific NF-κB inhibitor. Pre-treatment with ME or Bay-11-7082 led to an inhibition of PMA-induced nuclear p65 levels [Figure 5a], (P < 0.01). Similarly, PMA stimulation increased nuclear p50 levels [Figure 5b], (P < 0.05), treatment with ME or Bay-11-7082, significantly reduced PMA-induced nuclear p50 levels [Figure 5b], (P < 0.05). These findings were further validated by estimating the levels of TNF-α, IL-6, and IL-8 at both gene and protein levels in MCF cells pretreated with Bay-11-7082 and then stimulated with PMA [Figure 5c-h]. These findings collectively demonstrate that ME inhibits PMA-induced expression of TNF-α, IL-6, and IL-8 through deactivation NF-κB (p65/p50) pathways in breast cancer cells.

Close

Figure 5:

Moringa oleifera leaf extract (ME) inhibits TNF-α, IL-6, and IL-8 expression and production through NF-κB p65/50 in PMA-stimulated human breast cells. MCF-7 cells were pretreated with ME (5–10 µg/mL) for 3 h and stimulated by PMA (40 nM) for 30 min. (a) Effect of ME on the NF-κB p65 activity in the nuclear extract of PMA-stimulated MCF-7 human breast cells. Bay-11-7082 was used as a positive control. (a) Effect of ME on the NF-κB p65 activity in the nuclear extract of PMA-stimulated MCF-7 human breast cells. Bay-11-7082 was used as a positive control. (b) Effect of ME on the NF-κB p50 activity in the nuclear extract of PMA-stimulated MCF-7 human breast cells. Bay-11-7082 was used as a positive control. (c) Gene expression of TNF-α in MCF-7 cells treated with NF-κB inhibitor (Bay-11-7082) and PMA. (d) TNF-α production in the culture medium of MCF-7 cells treated with Bay-11-7082 and PMA. (e) Gene expression of IL-6 in MCF-7 cells treated with Bay-11-7082 and PMA. (f) IL-6 production in the culture medium of MCF-7 cells treated with Bay-11-7082 and PMA. (g) Gene expression of IL-8 in MCF-7 cells treated with Bay-11-7082 and PMA. (h) IL-8 production in the culture medium of MCF-7 cells treated with Bay-11-7082 and PMA. ^#P<0.05 versus untreated cells; ^#P<0.05 versus *; *P<0.05 versus *; ^#P<0.05 versus @. Results are representative of mean±standard error of the mean for three independent experiments. PMA: Phorbol 12-myristate 13-acetate, NF-κB: Nuclear factor-kappa B, IL-6: Interleukin-6, TNF-α: Tumor necrosis factor-α, IL-8: Interleukin-8.

Export to PPT

DISCUSSION

A study demonstrates how ME reduces inflammation in PMA-stimulated MCF-7 cells. The NF-κB p65/p50 signaling cascade achieves major pro-inflammatory cytokine inhibition through ME, which results in reduced TNF-α, IL-6, and IL-8 expression and secretion. This research extends existing knowledge about the ME leaf extract as a herbal anti-inflammatory agent designed for cancer prevention because it has minimal toxicity.

The therapeutic safety of ME for clinical applications is confirmed through its ability to maintain cell viability in MCF-7 cells at 40 µg/mL concentration for 72 h alongside PMA. The lack of toxicity in ME extracts is consistent with scientific reports that show these extracts affect inflammatory and stress-related situations while sparing non-stimulated cancer cells when used in moderate concentrations. The absence of cell death from ME treatment indicates its effects on cytokine development stem from targeted modulation of inflammatory activities instead of global cellular damage. The treatment option remains a top candidate for specialized therapeutic approaches.^[25] TNF-α major inflammatory protein activates inflammation, which establishes the conditions for tumor development and metastatic spread.^[26]

The results of her research revealed that ME pre-treatment of PMA-stimulated MCF-7 cells led to decreased levels of mRNA TNF-α and less protein production of this cytokine in a manner that depended on the applied dose. Previous research supports the current findings that ME bioactive components use isothiocyanates to reduce TNF-α levels by inhibiting NF-kB signaling cascades.^[27] The research demonstrates a dose-dependent reduction of TNF-α expression levels by ME, which likely occurs through dual control of transcription and translation since NF-κB inactivation causes reduced nuclear p65 and p50 subunit levels in PMA-stimulated cells.

Breast cancer cells under PMA stimulation developed double the normal level of IL-6 expression at both mRNA and protein levels. The results of this study demonstrate that ME pre-treatment caused a dose-dependent suppression of IL-6 gene expression and protein secretion in MCF-7 cells. The biological activity of IL-6 stops neoplastic cells from becoming apoptotic and autophagic through activation of STAT3 and MAPK pathways and MAPK pathways which operate as apoptosis and autophagy suppressors.^[28,29] The ability of ME to suppress IL-6 points to activating these pathways, and so, ME disables them, which explains the reduced inflammatory response and provides a mechanistic basis for breast cancer cell treatment. The development of IL-6 targeting therapies, including monoclonal antibodies for tumor-associated inflammatory response reduction, is an important medical advancement.^[13] IL-8 acts as a pro-inflammatory chemokine in breast cancer, which recruits immune cells to establish a supportive tumor microenvironment that enables tumor growth by blood vessel development and new site spread.^[30] According to the current research, ME90 treatment effectively reduced IL-8 mRNA and protein levels in PMA-treated MCF-7 cells to a degree that most elementary students understand. The current study’s results align with previous research that shows ME bioactive components quercetin and kaempferol, which effectively downregulate IL-8 production through NF-κB and AP-1 inhibition. The evidence of ME’s dosage-dependent effect on IL-8 provides further support that this treatment effectively manages inflammatory reactions. Through the confirmation of ME’s inactivating effect on NF-κB p65 and p50 subunits during nuclear extract analysis with Bay-11-7082 inhibitor, this research makes a significant structural connection to the low inflammation caused by ME. The main mechanism of gene activation through NF-κB in malignant cells leads to damaging cytokine expression, which includes TNF-α, IL-6, and IL-8 that all support tumor growth.^[10] ME functions through earlier NF-κB activation steps to prevent cytokine production, as shown by reduced nuclear p65 and p50 levels in cells that were treated with ME before PMA exposure. The study shows ME has a specific effect on the NF-κB pathway because Bay-11-7082 treatment reduces cytokine expression in cells. The research importance emerges from the exploration of ME leaves in Tabuk, Saudi Arabia as an underused natural resource for future applications. Multiple Middle Eastern regions recognize the Moringa plant properties that allow it to serve for water purification and provide an inexpensive approach to inflammation management in basic medical facilities. The research demonstrates that ME possesses anti-inflammatory properties which suggest it as a viable option for treating postmenopausal breast cancer which predominantly consists of MCF-7 cells because of their hormone receptor-positive subtype.^[2] The therapeutic impact of ME requires thorough study since isothiocyanates, quercetin, and kaempferol, which form its bioactive compounds, demand scientific scrutiny for uniform development of medical treatments.^[27,31] The research indicates that ME leaf extract reduces TNF-α, IL-6, and IL-8 expression in PMA-stimulated MCF-7 breast cancer cells by blocking NF-κB p65/p50 signaling cascades. Research findings demonstrate that ME functions effectively as a breast cancer treatment through inflammation management by showing potential toxicity and tumor development targeting. The addition of ME to medical treatments would provide affordable and culture-specific improvements to standard care. ME shows dose-dependent suppression of pro-inflammatory cytokines TNF-α, IL-6, and IL-8 production along with reduced expression without compromising cell viability. The study determines that the anti-inflammatory effects of ME from its ability to deactivate nuclear factor-kappa B p65 and p50 subunits in MCF-7 cells. Previous studies found MCF-7 cells treated with ME to be highly resistant to cellular damage from the extract at both high doses and extended exposure times of up to 72 h, which indicates its medical safety.^[18] Moreover, the studies also revealed moderate ME extract toxicity on non-stimulated cancer cells at moderate concentrations with demonstrated therapeutic effects during stress conditions.^[15,25] The anti-inflammatory action of ME does not connect to cell death but instead focuses on precise modulation of inflammatory signaling pathways without disturbing normal cellular functions. The molecule TNF-α functions as a key driver of persistent inflammation in tumor environments, which directly promotes aggressive tumor behaviors, including invasion and metastasis.^[9,26] The treatment of MCF-7 cells with PMA led to increased production of TNF-α mRNA and protein, but ME dose-dependently reduced these measurements. The outcomes support previous research, which demonstrates that bioactive ME compounds, isothiocyanates, decrease NF-κB-mediated TNF-α production.^[17,27] The ability of ME to control TNF-α production indicates that it can halt inflammation-driven breast cancer progression for the development of new therapeutic approaches. The cytokine IL-6 functions as a versatile regulator of breast cancer development through its aggressive promotion of tumor cell survival, resistance, and proliferation.^[6,7] The treatment of cells with ME significantly decreased the production of IL-6 mRNA and protein, which indicates the extract interferes with transcription and secretion of the cytokine. The suppression of signaling pathways such as signal transducer and activator of transcription 3 and mitogen-activated protein kinase appears to play a role in controlling IL-6 expression across different types of cancer.^[7,10] The ability of ME to reduce IL-6 production would create a significant impact on inflammation reduction and enhance breast cancer therapeutic approaches. The pro-inflammatory chemokine IL-8 serves as a critical component for breast cancer growth while also enhancing metastasis in most cases.^[14,30] The anti-inflammatory properties of ME are confirmed through its ability to stop the production of IL-8 mRNA and protein when cells undergo PMA stimulation. Previous studies showed that ME phytochemicals, particularly quercetin, are responsible for suppressing IL-8 expression by inhibiting NF-κB and certain transcription factors.^[15,31] Research evidence validates that ME bioactive compounds successfully target multiple inflammatory mediators to reduce tumor progression. ME effectively prevents the activation of NF-κB p65 and p50 subunits in PMA-treated MCF-7 cells. The inflammatory pathway NF-κB controls immune responses centrally and frequently becomes uncontrolled in cancer to produce pro-inflammatory cytokines and various chemokines.^[11,17] The pre-treatment of cells with ME causes a significant decrease in nuclear p65 and p50 levels which Bay-11-7082 confirms through its NF-κB inhibitory effects. The activation of NF-kB is well-known to drive tumor progression and metastasis during breast cancer while creating therapy-resistant tumor cells. ME antagonizes this pathway, which positions it as a beneficial therapy to reduce tumor growth associated with inflammation. The study results present essential information about natural compounds that have become increasingly popular as effective complementary cancer therapies. ME contains a wide range of active compounds such as flavonoids together with phenolic acids and isothiocyanates, which demonstrate strong abilities to fight inflammation while providing antioxidant and anticancer effects.^[15,31] ME extract demonstrates its ability to reduce pro-inflammatory cytokine expression in breast cancer cells. The molecular mechanisms through which ME inhibits NF-κB and cytokine expression require immediate research for a thorough explanation. Scientists can investigate the precise bioactive compounds in ME that produce these effects and their abnormal interactions with upstream MAPK signaling pathways.^[31] Validation of ME effects through animal models with systemic applications requires essential in vivo studies that thoroughly examine its anti-inflammatory and anticancer properties. In this study, ME extract demonstrates strong suppression of TNF-α expression and IL-6 production in MCF-7 breast cancer cells by inactivating NF-κB p65 and p50 pathways. The research indicates that ME functions as a safe anti-inflammatory agent that effectively reduces tumor progression caused by inflammation in specific breast cancer cases. The molecular mechanisms behind bioactive constituents and the in vivo efficacy of ME require detailed investigation for its development as a complementary breast cancer therapeutic approach. In short, this study demonstrates that ME extract effectively inhibits PMA-stimulated expression and production of TNF-α, IL-6, and IL-8 in human breast cancer cells. These findings highlight the therapeutic potential of ME as an anti-inflammatory agent in breast cancer, warranting further investigation into its clinical applications.

CONCLUSION

The present study provides compelling evidence that ME effectively attenuates the inflammatory response in PMA-stimulated MCF-7 human breast cancer cells. Specifically, ME significantly inhibits the expression and secretion of key pro-inflammatory cytokines, including TNF-α, IL-6, and IL-8. This anti-inflammatory effect is closely associated with the suppression of the nuclear transcription factor NF-κB, as ME was found to inactivate both the p65 and p50 subunits, which are critical components of NF-κB signaling. The results suggest that ME modulates inflammatory pathways by interfering with NF-κB activation, thereby downregulating cytokine production. Collectively, these findings highlight the potential therapeutic value of M. oleifera in targeting inflammation-associated pathways in breast cancer and support further investigation into its role as a natural anti-inflammatory and anti-cancer agent.