The potential of lunasin extract for the prevention of breast cancer progression by upregulating E-Cadherin and inhibiting ICAM-1

Introduction

Breast cancer is the most frequent type of cancer in women across the world.¹ In 2012, according to statistics from the Global Burden of Cancer Study, an estimated 1,671,149 new cases of breast cancer and 521,907 breast cancer deaths occurred in women worldwide, resulting in a prevalence of 25.1% of all cancers.¹ The 2018 Globocan statistics indicated that there was an 11.6% decline in the prevalence of breast cancer compared to the 2012 Globocan data. However, the prevalence of breast cancer remains the greatest in women.²

Breast cancer, in specific a metastatic subtype, is an intricate and multi-staged malignant tumor.³ A study by Müllar et al.⁴ has shown that alterations in the intercellular adhesion molecules can play a vital role in the metastasis of breast cancer. Intercellular Adhesion Molecule 1 (ICAM-1) as a surface glycoprotein, functions as an adhesion molecule on tumor cells that could rearrange actin, promote pro-inflammatory cascade, and mediate multiple signaling pathways to regulate cell metastasis.⁵ Although ICAM-1 expression varies depending on the breast cancer subtype,⁶ its expression is significantly increased, especially in metastatic breast cancer.⁷ Different from ICAM-1, epithelial cadherins (E-Cadherin) is a glycoprotein that functions as an adhesion protein between cells to maintain tissue integrity.⁸ Therefore, low expression of E-Cadherin is associated with the progression of epithelial-mesenchymal transition (EMT) in breast cancer.⁸ Due to the high prevalence of this disease, developing breast cancer treatments have become a priority. Cancer is now treated with surgery, chemotherapy, radiation, and targeted therapy,⁹ which have negative side effects. Chemotherapy as one of the most common cancer treatments has many significant side effects such as mucositis, neurotoxicity, chemotherapy-induced peripheral neuropathy, reduced renal function, and bone marrow depression.¹⁰ Many investigations are now developing cancer treatments using natural substances that might be capable of reducing these negative side effects.¹¹

Over the last several decades, an increasing number of plant-derived products, including soybean [Glycine max (L.) Merr.], have been explored, as it is cheap, easy to grow, and abundant in nature.¹² Soy products are also known to lower the incidence and mortality of breast cancer, prostate cancer, colon cancer, and lung cancer.^13-15 The molecule in soybeans that works as an anti-cancer is considered to be a bioactive peptide called lunasin. This peptide was first discovered from a sample of commercially accessible soybean germplasm, originating in the United States.¹⁶ Lunasin is a lipophilic peptide component made up of 44 amino acids that have anti-cancer, anti-oxidant, and anti-inflammatory properties, as well as the ability to control cholesterol biosynthesis in the body.^17,18

The challenges with synthetic lunasin are that the commercial price is quite high due to its costly and time-consuming synthesis process. Additionally, effective techniques for obtaining a significant amount of pure lunasin from plant sources do not exist.¹⁹ Although Lunasin Extract (LE) from the Grobogan type has been shown to have anti-inflammatory and anti-cancerous effects in the colon, studies into various anti-cancer pathways of LE are required to better understand its molecular effect on breast cancer.¹²

As such, this study aims to investigate the anti-cancer effects of LE from the Grobogan variety in Indonesia. We hypothesized that LE administration could suppress breast cancer progression by decreasing ICAM-1 expression while increasing E-Cadherin expression. By examining the effects of LE on these two molecules, this study is expected to investigate the potential of LE for the prevention of breast cancer progression via ICAM-1 and E-Cadherin expression pathway.

Methods

Results

The representative image of IHC staining for ICAM-1 and E-Cadherin in breast tissue is shown in Figure 1. Each image depicts a different expression based on the group and the biomarker (ICAM-1 and E-Cadherin). Some images, such as Figure 1 B, D, F, G, I, K, and L, show a greater brown color intensity than others, which suggests that the associated biomarker is expressed at a greater level.

Figure 1. Representative image of immunohistochemistry staining for ICAM-1 and E-Cadherin in mouse breast tissue.

Scale bar represents 50 μm. All images were taken at 400× magnification.

However, qualitative evaluation alone is insufficient for reaching a conclusion. As a result, H-score quantification was used to quantify the quantitative expression of ICAM-1 and E-Cadherin. Table 1 shows the mean H-score in every group on ICAM-1 and E-Cadherin. Complete H-score data can be accessed in the extended data.²⁶ Figures 2 and 3 show ICAM-1 and E-Cadherin mean H-score differences between groups, respectively.

Figure 2. Effect of ICAM-1 in all groups.

Normal, positive control (PC), curative, adjuvant, and preventive groups have significantly different ICAM-1 expressions compared to the negative control (NC). Duncan Test was used to determine the difference between groups. * represents significant difference (p < 0.05) of groups versus NC. ○ represents a significant difference (p < 0.05) of groups versus preventive.

Figure 3. Expression of E-Cadherin in all groups.

Duncan Test was used to determine the difference between groups. * represents significant difference (p < 0.05) of groups compared to NC. • represents significant difference (p < 0.05) of groups compare to curative. ○ represents a significant difference (p < 0.05) of groups compare to preventive.

One-Way ANOVA for ICAM-1 showed significant results (p < 0.001). Meanwhile, Duncan’s test results showed that there were significant differences in ICAM-1 expression between adjuvant and NC, normal and NC, PC and NC, adjuvant and preventive, normal and preventive, PC and preventive, adjuvant and curative, normal and curative, PC and curative as seen in Figure 2.

One-Way ANOVA analysis for E-Cadherin indicated significant results (p < 0.001). Meanwhile, Duncan’s test results showed there were significant differences between preventive and NC, adjuvant and NC, PC and NC, normal and NC, adjuvant and curative, PC and curative, normal and curative, normal and preventive as seen in Figure 3.

The results of the Pearson correlation analysis showed r = −0.616 (−0.8165; −0.283) with p = 0.001. The correlation between ICAM-1 and E-Cadherin expressions was negatively correlated with moderate strength. Further regression analysis produces a correlation model between E-Cadherin and ICAM-1 expressions through the equation y = −1.214x + 109.7, where y is E-Cadherin expression and x is ICAM-1 expressions, as seen in Figure 4.

Figure 4. Correlation between ICAM-1 and E-Cadherin expression.

Discussion

In this study, the relationship between LE administration in the DMBA-induced breast cancer model was assessed. ICAM-1 and E-Cadherin, molecules involved in breast cancer adhesion and progression, were evaluated in each group to determine their effect. In the breast cancer model, results revealed an inverse correlation between ICAM-1 and E-Cadherin expressions. Additionally, Tamoxifen, adjuvant LE, and preventative LE all had a significant effect on either lowering ICAM-1 expression or promoting E-Cadherin expression.

DMBA as a breast carcinogen is commonly used to simulate mammary carcinogenesis for tumor research.^27,28 In this study, DMBA had a substantial influence on the expression of ICAM-1 and E-Cadherin. As demonstrated in Figures 2 and 3, there is a substantial difference between NC and normal tissue samples. DMBA-induced breast models showed a significantly increased ICAM-1 expression. This is consistent with the fact that ICAM-1 expression rises with cancer progresses,²⁹ especially during metastasis,³⁰ although it is expressed at lower levels in normal tissues.³¹ Several studies have also investigated DMBA as a tumor model and discovered a significant increase in ICAM-1 expression.^32-34 Although the evidence is inconclusive, the nuclear factor-kappaB (NFkB) pathway is considered to play a role in DMBA-induced ICAM-1 upregulation.^35,36 DMBA is able to potentiate NFkB^37,38 via Ras mutation in the Ras/Raf/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway,^39,40 thereby increasing ICAM-1 expression.^35,36 DMBA, however, decreased E-Cadherin expression. This is consistent with the hypothesis that E-Cadherin is expressed in normal cells, with its absence being a hallmark of EMT and a more migratory, invasive cancer.^41,42 These results are also consistent with other research that demonstrate a reduction in E-Cadherin expression following DMBA induction.^42,43 DMBA's impact on E-Cadherin, like that of ICAM-1, is mediated via NFkB activation^37,38 via the Ras-Raf-MEK-ERK pathway³⁹ thus causing E-Cadherin repression due to transcription factor TWIST expression.⁴⁴ DMBA can also activate aryl hydrocarbon receptors (AhR), promoting transcription of SNAI2 (Slug),⁴⁵ an E-Cadherin repressor protein.⁴⁶ Furthermore, the negative correlation of ICAM-1 and E-Cadherin was consistent with the functions of each molecule. Zhao et al.⁴⁷ has also shown that reduced expression of E-Cadherin was associated with increased expression of ICAM-1 in mice models of breast cancer.

Tamoxifen, the most established hormone therapy for breast cancer, is an estrogen receptor antagonist that operates in its active metabolite, hydroxytamoxifen, in breast cancer.⁴⁸ This therapy decreased ICAM-1 expression, and increased E-Cadherin expression in DMBA-induced mouse models compared to NC, as seen in Figures 2 and 3. This finding is consistent with the results of Sun et al.⁴⁸ who discovered that Tamoxifen administration can reduce the Toll-like receptor 4 (TLR4)/NFkB-mediated inflammatory response, resulting in a reduction in ICAM-1 expression. This process might potentially account for the rise in E-Cadherin following Tamoxifen administration. As previously stated, NFkB mediates E-Cadherin repression,⁴⁴ therefore, inhibiting this pathway will increase E-Cadherin expression. Furthermore, Tamoxifen could prevent the activation of Slug and Snail, the E-Cadherin protein inhibitors, by inhibiting Transforming Growth Factor Beta (TGF-β)/Smad, thus enhancing E-Cadherin expression.⁴⁹

In this study, three LE groups: adjuvant, curative, and preventive, had varying effects on ICAM-1 expression. At first impression, the adjuvant group appeared to have excellent outcomes since it was substantially different from NC, preventive, and curative groups, as seen in Figure 2. This demonstrates that combining LE with Tamoxifen can dramatically lower ICAM-1 expression. However, this role may be attributed only to Tamoxifen, implying that the addition of LE had no meaningful effect on decreasing ICAM-1 expression. This was further confirmed as no significant difference between the adjuvant and PC group was found. As a result, an assessment of the effects of LE in the curative and preventive groups is essential. Figure 2 shows that the curative group did not have significant outcomes in terms of ICAM-1 expression. Zhu et al.⁵⁰ reported the same result, indicating that lunasin administered to monocytes was unable to decrease ICAM-1 expression. On the other hand, the preventive group showed some intriguing findings. Figure 2 shows a substantial difference in ICAM-1 expression between the preventive group with the other groups including NC, PC, and normal groups. This indicates that, even though LE at preventive doses considerably reduced ICAM-1 expression relative to NC, it was not as low as in the normal or PC groups. Although there has not been any research yet to show the preventive effect of LE on ICAM-1 expression, these findings may be explained by lunasin's antimitotic activity. A study by Hsieh et al.⁵¹ found that lunasin had an anti-mitotic effect by suppressing mutations in a DMBA-induced mouse model. Jeong et al.⁵² also shown that lunasin can prevent Ras mutations by inhibiting core H3 and H4 histone acetylation. This evidence explains why LE administered preventively (before DMBA induction) was more effective than LE administered curatively (after DMBA induction) in decreasing ICAM-1 expression. This is due to LE's ability to prevent the Ras mutation induced by DMBA, therefore preventing the NFkB pathway from being potentiated, which can enhance ICAM-1 expression.

The three LE groups also exhibited distinct effects on E-Cadherin expression. The adjuvant group was able to significantly increase E-Cadherin expression as represented by the significant difference it has with both NC and curative (Figure 3). However, similar to ICAM-1, this result might be attributed to the impact of Tamoxifen alone. Contrastingly, the curative group did not demonstrate a significant increase in E-Cadherin expression. A study by Pabona et al.⁵³ reported that the lunasin administration promoted E-Cadherin expression. However, their study is an in vitro study using synthetic lunasin, which is different from this study. The preventive group, unlike the curative group, showed a significant difference in E-Cadherin expression compared with the normal and NC group. However, this was not seen with the PC group. This suggests that administering a preventive dosage of LE in the breast cancer model was able to significantly enhance E-Cadherin expression relative to NC until it was as high as PC, but not as high as the normal group. Interestingly, unlike ICAM-1, E-Cadherin expression in the preventive group did not differ from that of the Tamoxifen group. This is possible because, as seen in Figure 5, more pathways influence E-Cadherin expression than ICAM-1. This creates increased heterogeneity in the expression of E-Cadherin, which is difficult to identify in this study. Aside from these differences, the preventive group outperformed the curative group in terms of enhancing E-Cadherin expression, compared to the findings on ICAM-1 expression. This finding could be explained by the same mechanism that occurs in ICAM-1, namely mutation-inhibiting action by LE.^51,52

Figure 5. Schematic molecular pathway of LE regarding ICAM-1 dan E-Cadherin expression.

7,12-Dimethylbenz [a] anthracene (DMBA) can cause breast cancer by inducing Ras mutations, which cause nuclear factor-kappaB (NFkB) potentiation via the Ras-RAF-MEK-ERK pathway. ICAM-1 and TWIST expression will be enhanced as a result of NFkB activation. TWIST is a transcription factor that inhibits the production of E-Cadherin. DMBA can also bind to aryl hydrocarbon receptor (AhR) and promote the transcription of Slug, an inhibitor of E-Cadherin production. Lunasin extract suppresses Ras mutations, which contribute to the prevention of breast cancer progression. As a result, NFkB potentiation does not occur, resulting in a decrease in ICAM-1 expression, and an increase in E-Cadherin expression. Tamoxifen, on the other hand, can suppress Toll-like receptors (TLR)-4 and Transforming Growth Factor Beta (TGF-β). TLR-4 suppression may also prevent NFkB potentiation. Furthermore, TGF-β inhibition will disrupt the smad/Slug and smad/Snail pathways, resulting in inhibition of E-Cadherin production.

This study has limitations, such as administrating one dose (500mg/kg BW) of the LE, therefore, the effects could not be compared in a dose-dependent manner. As a result, research with a more diversified dose is recommended. Furthermore, more study on other biomarkers, both in the same and distinct molecular pathways, is required to explore the preventive potential of LE in breast cancer.

Conclusion

A preventive dose of LE was able to reduce ICAM-1 expression while increasing E-Cadherin expression. Our findings show that LE has cancer-preventive effects in a well-characterized animal model of breast cancer. Further study, incorporating more biomarkers, is required to explore the mechanism of action of this peptide on mammary carcinogenesis as well as other forms of cancer.

Data availability

Reporting guidelines

(Harvard Dataverse): ARRIVE checklist for “The potential of lunasin extract for the prevention of breast cancer progression by upregulating E-Cadherin and inhibiting ICAM-1”.

DOI: https://doi.org/10.7910/DVN/EZTWXY.²³

This project contains the following extended data:

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Author contributions

KK, NKR, AMM designed the concept of this research article. KK, EW, NKR, AMM performed the methodology. KK carried out the overall reproducibility of experiments and other research outputs. Application of statistical, mathematical, computational, or other formal techniques to analyze or synthesize study data was done by EW. Conducting the research and investigation process, specifically performing the experiments, or data/evidence collection was done by EW, NKR, AMM. The resources were provided by KK, AE, HS. The Organization and management of the datasets collection, as well as writing the original draft of this article was by EW. Review & Editing was done by KK, NKR, AMM, AE, HS. Verification, of the overall reproducibility of experiments and other research outputs was performed by KK, AMM, AE, HS.