Examining Antigenic Similarities: Comparative Analysis of the Light Chain Lectin of Entamoeba histolytica and p53 Protein on the Surface of Colon Adenocarcinoma Cells Biopsy

doi:10.21203/rs.3.rs-3905336/v1

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Examining Antigenic Similarities: Comparative Analysis of the Light Chain Lectin of Entamoeba histolytica and p53 Protein on the Surface of Colon Adenocarcinoma Cells Biopsy

https://doi.org/10.21203/rs.3.rs-3905336/v1

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Background

Entamoeba histolytica ranks as the third leading cause of death among parasitic infections worldwide. Despite its significance, the impact of this protozoan on colorectal cancer incidence and its association with the p53 protein in affected patients remain insufficiently explored. Therefore, this study aims to investigate the presence of the E. histolytica lectin light chain antigen and the p53 protein in colorectal cancer biopsies, with the objective of identifying any antigenic similarity between this protozoan and the surface proteins of tumor cells.

Materials and Methods

A total of 150 colorectal cancer biopsy samples were subjected to examination for the specific E. histolytica antigen and p53 protein through the application of the immunohistochemical technique. To augment diagnostic precision, the western blotting method was employed and its results were compared with those obtained from immunohistochemistry (IHC).

Results

Among the 150 biopsy samples examined, 100 cases tested positive for the p53 protein, while 19 cases exhibited positivity for the E. histolytica lectin antigen. Notably, only 14 cases demonstrated a positive presence for both the p53 protein and the E. histolytica protozoan.

Conclusion

Entamoeba histolytica, a leading cause of parasitic infections, raises questions about its potential link to colorectal cancer, specifically in connection with the p53 protein. This study aims to explore the antigenic similarity between the lectin of E. histolytica and the p53 protein on colorectal adenocarcinoma cells, shedding light on the intricate interactions between this protozoan and cancer cells. The findings presented in this investigation signal the need for further research to comprehensively understand the implications of E. histolytica in colorectal cancer diagnosis and treatment strategies.

Entamoeba histolytica

p53 protein

CRC

immunohistochemistry (IHC)

western blotting

Entamoeba histolytica globally ranks as the third leading cause of death from parasitic infections. Amoebiasis, a disease caused by this protozoan, remains pervasive in underdeveloped countries, claiming approximately 100,000 lives annually [1]. Rosales-Encina and Kars (1987) reported the first isolation of a lectin from E. histolytica, a protein with the ability to agglutinate red blood cells (RBCs). This lectin displays specific recognition and binding to carbohydrates present on colon epithelial cells. [2]. This lectin recognizes and binds to carbohydrates present in colon epithelial cells. Subsequent research highlighted its pivotal role as an adherence factor, enabling E. histolytica to bind to intestinal cells. The lectin, a carbohydrate-binding protein, is a distinguishing indicator of this protozoan from its subgroups, facilitating attachment to intestinal cells. E. histolytica lectin is recognized as an invasive factor, consisting of three subunits: heavy subunit (Hgl), light subunit (Lgl), and medium intermediate subunit (igl) [2]. Moreover, it can break down intestinal mucin polymerase by binding and secreting glycosides. As mucin on the intestinal cell surface decreases, the lectin attachment to intestinal epithelial cells strengthens, triggering the host's inflammatory response [3].

Lectin, a unique protein, specifically recognizes and binds to glycans on the cell surface without causing any change. In contrast, the sticky glycoproteins on the surface of bacteria, viruses, or parasites adhere to the host cells' surface modifications. This property enables E. histolytica to resist the complement system, while also aiding cell lysis through the activation of C5b-9 with the expression of CD59-like protein [4]. Additionally, this protozoan can produce anti-inflammatory pentapeptides, acting as a monocyte inhibiting factor (MIF)[5]. Consequently, Gal/GalNac lectin is considered a multifunctional protein [6]. However, whether this lectin remains on the surface of IECs due to the immune system's effect is still unknown [7]. This persistence contributes to the spread of inflammation, necrosis, and mega colon [8], prompting investigation into the antigenic structure's similarity with proteins on the surface of intestinal tumor cells, such as p53.Meanwhile, p53, frequently mutated in human cancers, plays a significant role in the adenoma-carcinoma transition [9]. E. histolytica gene promoter sequences, exhibiting high homology to oli-p53, form complexes with nuclear proteins that are abrogated by oli-p53. Ehp53 protein levels increase in UV-irradiated trophozoites. However, O-GlcNAcylation changes between different tumor types lack a clear pattern. For instance, lung and colon cancer exhibit an increase in O-GlcNAcylation and OGT compared to normal tissue[10]. O-N-Acetyl glucosamine (O-GlcNAc), associated with serine and threonine residues in cytosolic and nuclear proteins, is an abundant reversible post-translational modification in all higher eukaryotes [10]. GlcNAcylation, a dynamic post-translational modification, involves the addition of N-acetyl glucosamine [11]. Sugars are attached to serine and threonine residues in proteins by the enzyme O-Linked-N-acetyl glucosamine transferase (OGT), while the enzyme O-GlcNAcase (OGA) removes the modifications. However, whether O-GlcNAc alterations in host cells affect E. histolytica-induced cell death and which signal molecules participate in E. histolytica-induced deglycosylation remain unknown. In another study, the binding ability of E. histolytica, based on its High-Mobility-Group Box, to p53 protein and human DNA has been demonstrated (12). As mentioned, E. histolytica possesses Gal/GalNac lectin, a key factor in binding this protozoan to host cells. The pathogenic mechanism of E. histolytica and its cytolytic effect in host cells are not yet well understood. Notably, E. histolytica possesses a protein, Ehp53, similar to human p53, with the ability to bind to the human p53 DNA sequence (oli-p53) [12]. However, the potential impact of this protozoan on colorectal cancer incidence and its relationship with p53 remain largely unexplored. This study, therefore, aims to assess the concurrent presence of E. histolytica lectin and p53 protein on colorectal cancer biopsy cell surfaces, aiming to elucidate any antigenic similarity.

Sample Collection:

A total of 150 paraffin block biopsy samples of colorectal adenocarcinoma, sourced from hospitals in Tehran, were subjected to immunohistochemical (IHC) staining to detect the presence of E. histolytica antigen and p53 protein. Sections measuring 3 to 5 microns were meticulously prepared on Poly-L-lysine slides.

For western blotting protein extraction, 15-micron sections of paraffin tissues were carefully placed in micro tubes, ensuring precision in the collection process.

Immunohistochemical staining

Immunohistochemical staining was employed to assess the antigenic expression of E. histolytica lectin and p53 protein. Following deparaffinization, dehydration, and peroxidase inhibition achieved with 3% hydrogen peroxide and methanol, slides underwent antigen retrieval in citrate buffer (pH 6) at 121°C under 15x pressure for 15 minutes in an autoclave. The slides were subsequently incubated with specific antibodies for E. histolytica (GeneTex, USA) and p53 (Dako, Denmark), following a blocking step to enhance specificity. After the incubation with the secondary antibody (Envision; Dako, Denmark), reactions were visualized using diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin. This meticulous immunohistochemical staining protocol ensured the accurate detection and localization of E. histolytica lectin and p53 protein within the biopsy samples.

Western blotting technique:

To corroborate the findings obtained through immunohistochemistry (IHC), the western blotting technique was employed. Following deparaffinization, tissue lysates were prepared, and subsequent centrifugation facilitated the determination of protein concentrations using the Bradford method. Subsequently, SDS-PAGE electrophoresis separated the proteins, which were then transferred to PVDF membranes and probed with primary antibodies.

The visualization of proteins occurred post-incubation with secondary antibodies. The transfer of samples from the gel to paper was achieved through electric current. In the blocking step, a solution consisting of 2% non-fat dry milk in TBS-T buffer was applied to the paper to prevent non-specific reactions of the primary antibody. The blocking solution, comprising non-fat dry milk, was gently shaken on the PVDF paper for one hour and 15 minutes at room temperature.

Following the blocking period, the paper underwent incubation with a diluted β-actin primary antibody (sc-47778, 1:300) and E. histolytica lectin primary antibody (1:200) for 16 to 18 hours. Subsequent to this incubation, the paper was washed three times for 15 minutes each with TBS-T buffer. Anti-rabbit secondary antibodies at a concentration of 1:1000 for all primary antibodies were then applied to the paper, followed by another one-hour and 15-minute incubation at room temperature.

After this stage, the paper was washed three times for 15 minutes each with TBS-T buffer. Following blotting and the emergence of bands, the concentration of the extracted proteins was measured and normalized with the control sample. This rigorous western blotting protocol ensured the reliability and accuracy of the protein detection process.

Statistical analysis

The processed data were subjected to analysis using Microsoft Excel and the Statistical Package for the Social Sciences (SPSS) software, version 26. To assess both statistical differences and agreement between E. histolytica lectin and p53, the McNemar test and kappa tests were employed. A significance level of P < 0.05 was considered statistically significant, providing a robust framework for evaluating the observed associations and discrepancies between the two variables.

Out of the 150 colorectal adenocarcinoma samples included in the study (73 women, 77 men), immunohistochemical staining (IHC) revealed that 100 samples tested positive for the presence of the p53 protein. Notably, 19 samples (12.67%) exhibited positive staining for E. histolytica lectin, with 14 of these samples co-expressing the p53 protein (Table 1). These findings provide initial insights into the prevalence and co-occurrence of E. histolytica lectin and p53 protein within the colorectal adenocarcinoma samples.

Table 1: The agreement between levels of p53 variants and Eh lectin in colorectal adenocarcinoma samples

Adenocarcinoma (No.=150)
		Eh lectin		Total
		Positive	Negative
p53	Positive	14	86	100
	Negative	5	45	50
Total		19	131	150

Confirmation of Positivity:

All 19 samples that tested positive for E. histolytica lectin through immunohistochemical staining (IHC) were further validated using the western blot method. This meticulous cross-verification revealed a remarkable 100% concordance between the two techniques, highlighting the robustness and reliability of the findings (Figures 1 and 2). This consistency fortifies the confidence in the detected presence of E. histolytica lectin within the colorectal adenocarcinoma samples.

The images in Figure 2 illustrate the distinct staining patterns observed in the immunohistochemical analysis. Panels A and B represent negative staining for E. histolytica lectin, while panels C, D, and E showcase positive staining for E. histolytica lectin. Panels F and G display negative staining for p53, contrasting with panel H, which exhibits positive staining for p53. These visual representations offer a comprehensive view of the immunohistochemical results, emphasizing the variability in staining between E. histolytica lectin and p53.

The combined analysis of immunohistochemical staining (Fig. 1), Western blotting (Fig. 2), and absorbance values (Table 2) collectively signifies the presence of the lectin light chain of the E. histolytica antigen within the colorectal adenocarcinoma samples. To enhance interpretability, a cutoff for absorbance values was determined at 0.68, designating values higher than this threshold as a positive indicator of the E. histolytica antigen.

In-depth insights into the relationship between p53 expression levels and E. histolytica lectin are provided in Table 2. Significantly, a correlation was observed between these variables, reaching statistical significance at P<0.05. These findings contribute to a nuanced understanding of the interplay between p53 and E. histolytica lectin expression in the context of colorectal adenocarcinoma.

Table 2: The amount of absorption obtained in terms of the lectin light chain of E.histolytica antigen in colorectal adenocarcinoma samples based on western blotting results.

Sample		Actin	E.histolytica	Ratio
Negative* (Mean)	1	10128.27	14740.7	0.687095
Positive	2	13172.50	14305.58	0.974051
Positive	3	13987.58	18023.72	0.802065
Positive	4	22104.55	23028.26	1.041788
Positive	5	17931.41	18475.79	1.030359
Positive	6	18084.12	25939.81	1.434397
Positive	7	15710.29	15010.48	1.046621
positive	8	13434.45	14204.58	0.945783
positive	9	16088.99	13988.65	1.150146
Positive	10	13170.45	13382.29	0.98417
positive	11	16012.2	15505.46	1.032681
positive	12	13141.85	12107.46	1.085434
Positive	13	14875.77	13274.51	1.120627
positive	14	15394.87	11231.27	1.370715
Positive	15	8061.865	9985.267	0.807376

* Values higher than 0.68, which is the average result of 131 negative cases, were considered as a cutoff.

Correlation Between E. histolytica (Eh) Lectin and p53 Expression in Colorectal Adenocarcinoma Cells:

P53 positive (P53+) samples:

Among the 100 samples that tested positive for p53, 14 samples also exhibited positive expression for E. histolytica lectin (Eh+). The disparity in E. histolytica lectin expression between these two groups was statistically significant, with a P-value less than 0.005. It is essential to note that this significant difference does not imply concordance between the Eh+ and Eh- samples within the P53+ group.

P53 negative (P53-) samples:

Within the subset of samples testing negative for p53 (50 samples), 5 were identified as positive for E. histolytica lectin (Eh+). Conversely, 45 samples in this category did not express E. histolytica lectin (Eh-). The variation in E. histolytica lectin expression in this cohort was also statistically significant, with a P-value less than 0.005. This variation contradicts the expectation of a uniform antigenic structure in Eh- and Eh+ samples within the p53-negative group.

Amoebiasis and Diagnostic Challenges:

Amoebiasis, caused by Entamoeba histolytica and Entamoeba dispar, remains a considerable global health concern [13]. Despite recent advancements in immunohistochemistry facilitating the detection of E. histolytica in fresh stool samples, challenges persist in diagnosing amebiasis, often requiring invasive procedures like colonoscopy and pathology-based assessments[14]. Notably, the efficacy of certain diagnostic methods on paraffin-embedded samples remains a subject of investigation[15].

Antigenic Mimicry and Previous Studies:

Earlier studies have hinted at potential antigenic mimicry between E. histolytica and tumoral cells in the colon. Notably, the immunohistochemical detection of E. histolytica lectin antigen in colon adenocarcinoma samples has been reported[16]. However, the exploration of antigenic similarity between E. histolytica and the tumor cell surface, particularly in comparison with the p53 protein, has not been thoroughly examined[17].

p53 and Its Significance in Cancer:

The discussion delves into the vital role of the p53 protein in growth, genome stability, aging, and tumor suppression. Mutations in the p53 gene are identified in a significant proportion of colorectal tumors. Activated in response to DNA damage and oncogenic stress, p53 regulates various cellular responses crucial for tumor suppression. The discussion emphasizes the importance of p53 as a guardian of genomic stability, underlining its involvement in apoptosis, cell cycle arrest, and angiogenesis inhibition.It is widely recognized that p53 plays a central role in growth, genome stability, aging, and tumor suppression. Mutations in the p53 gene have been identified in 34% of proximal colon tumors and 45% of distal colorectal tumors. Activated by DNA damage and oncogenic stress, p53 orchestrates a myriad of cellular responses essential for tumor suppression. The transcription factor p53 induces the activation of MDM2, a primary E3 ubiquitin ligase targeting p53 for proteasomal degradation, forming a negative feedback loop. DNA repair defects can induce genome instability, leading to cancer[18]. The p53 gene, mutated in over 50% of human cancers, has earned monikers like "Guardian of the Genome" and "Guardian Angel Gene" due to its paramount role in maintaining genomic stability and tumor suppression, primarily by triggering apoptosis, cell cycle arrest, and angiogenesis inhibition[19].

Evolutionary Perspective and Ehp53:

The study introduces Ehp53, identified as the first p53 analog in protozoans and speculated to be the evolutionary precursor of mammalian p53. The investigation aims to explore potential antigenic similarity between E. histolytica lectin and the p53 protein in tumoral cells, presenting preliminary findings suggesting a tentative likeness.[20].

Results and Lack of Antigenic Agreement:

Contrary to expectations, the study's results indicate no discernible antigenic agreement between E. histolytica lectin and the p53 protein in colorectal adenocarcinoma cells. The weak agreement observed is critically examined, and the potential reasons for the positive outcomes with western blotting and immunohistochemistry are highlighted.

Implications and Future Directions:

There is a requirement for further in-depth analysis, considering the positive outcomes in western blotting and immunohistochemistry related to the E. histolytica lectin light chain's antigenic index. The potential role of a different monoclonal antibody, such as the Macrophage Inhibitory Factor (MIF), is suggested for exploration. Additionally, the clinical implications of this antigen as a diagnostic biomarker, potentially influencing prognosis and treatment strategies, are proposed as avenues for future research.

Conclusion and Call for Further Research:

In summary, the study findings indicate that only 14 colorectal adenocarcinoma samples exhibited positivity for both the p53 protein and the E. histolytica antigen. This observation suggests a modest likelihood of antigenic agreement between the lectin of E. histolytica and the p53 antigen. The implications of these results prompt a broader consideration of the challenges associated with amoebiasis diagnosis.

Examining the significance of p53 in cancer, particularly its role in maintaining genomic stability and acting as a tumor suppressor, provides a critical context. The introduction of Ehp53, a protozoan analog to mammalian p53, sets the stage for exploring potential antigenic similarities, aligning with the specific focus of our study.

While the observed agreement is modest, the discussion contextualizes the study within the realm of amoebiasis diagnosis, drawing on previous studies suggesting potential antigenic mimicry between E. histolytica and tumoral cells. The positive outcomes in western blotting and immunohistochemistry raise critical questions about the role of the detected antigen in colorectal adenocarcinoma that demand thorough investigation.

This study signals a need for further research. Exploring alternative monoclonal antibodies, such as the Macrophage Inhibitory Factor (MIF), holds promise for expanding our understanding. Additionally, investigating the clinical implications of the detected antigen as a potential diagnostic biomarker could influence prognosis and treatment strategies.

Our study contributes to the evolving understanding of E. histolytica and its potential connections to colorectal adenocarcinoma. The nuanced results highlight the complexity of the interaction between this protozoan and tumor cells. Further investigations are essential to unravel the intricacies of this relationship, offering potential insights for innovative diagnostic and therapeutic approaches. The call for continued research is crucial to comprehensively grasp E. histolytica's impact on colorectal cancer and advance our understanding of protozoan-cancer interactions in the scientific community.

-Ethics approval and consent to participate:

This study received approval from the ethics committee of Tarbiat Modares University, bearing the code number: IR.MODARES.REC.1402.004.

-Consent for publication

Not applicable.

-Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

-Competing interests

I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

-Funding

This research was financially supported by Tarbiat Modares University, Tehran, Iran. Grant No. MED33164270.

-Authors' contributions

L.H. conceptualization, data acquisition, data analysis, validation, writing—original draft.

A. D. Writing—review & editing, supervision, funding acquisition, conceptualization, methodology, validation, resources, data curation, formal analysis.

M. P. validation, data curation, review & editing.

F. Gh. methodology, validation, review & editing.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments:

The authors of this article would like to thank all the staff of the Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, as well as the colleagues of the Stem Cell Center of Tabriz University, for their assistance.

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No competing interests reported.

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Examining Antigenic Similarities: Comparative Analysis of the Light Chain Lectin of Entamoeba histolytica and p53 Protein on the Surface of Colon Adenocarcinoma Cells Biopsy

Status:

Version 1

Abstract

Background

Materials and Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Sample Collection:

Immunohistochemical staining

Western blotting technique:

Statistical analysis

Results

Discussion

Amoebiasis and Diagnostic Challenges:

Antigenic Mimicry and Previous Studies:

p53 and Its Significance in Cancer:

Evolutionary Perspective and Ehp53:

Results and Lack of Antigenic Agreement:

Implications and Future Directions:

Conclusion and Call for Further Research:

Declarations

References

Additional Declarations

Supplementary Files

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