Evaluation of Apoptotic Activity of Protein Fusion Enterocin A- Pyocin R-Lactocin, Bacteriocins and Specific Ligand of Gastric Cancer Cell Line (AGS)

doi:10.21203/rs.3.rs-788144/v1

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Evaluation of Apoptotic Activity of Protein Fusion Enterocin A- Pyocin R-Lactocin, Bacteriocins and Specific Ligand of Gastric Cancer Cell Line (AGS)

https://doi.org/10.21203/rs.3.rs-788144/v1

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Conventional antimicrobial and anti-cancer treatments, including chemotherapy, surgery, radiation therapy, etc., have all been associated with antimicrobial resistance (AMR) and side effects on healthy cell damage. Recently, bacteriocins have emerged as a promising option in antimicrobial and anticancer therapies.In this study, EntA-PynR-Lac gene sequence was obtained using bioinformatics software. The designed fusion gene was synthesized and cloned into the pET22b expression vector and transferred to the engineered E. coli BL21 bacterium for expression of the recombinant protein. The three-dimensional structures and stability of the designed recombinant protein were evaluated. Confirmation and purification of the recombinant protein was performed by Western blotting and nickel column chromatography and determination of cell lethality of different concentrations of the recombinant protein against AGS cell line by MTT technique was conducted. Treatment of cells with different concentrations of protein to evaluate the apoptosis of AGS cells was performed by flow cytometry. The results showed that the percentage of cells treated with recombinant protein at a concentration of 80µg/ml with total apoptosis was 46.91% and increased by 36.25% compared to untreated cells. Due to the anti-apoptotic properties of fusion protein, it can be used to inhibit cancer cells as a therapeutic supplement and prevention.

Cancer Biology

Oncology

Apoptosis

Protein Fusion

EnterocinA-PyocinnR-Lactosin

Bacteriocins

Gastric Cancer Cell Line

Despite much progress in the diagnosis and treatment of cancer, they constitute as the main reasons of deaths worldwide. Side effects related to chemotherapy and drug resistance are also current treatment problems. Hence, the search for new anti-cancer agents is very important. Bacterial proteins and peptides are among the promising active compounds in the potential treatment of cancer.¹

However, many anticancer agents do not typically have specific properties for transformed cells. As a result, they also kill healthy cells that are rapidly proliferating resulting in toxic side effects. Another limitation of chemotherapy is the development of resistance by cancer cells.²

Therefore, a more effective option may be other drug groups that target cancer cells without toxicity to normal cells. Another necessity is to develop less resistance to such agents, compared to conventional chemotherapy protocols.³

Resistance to antimicrobial agents is also a problem that has recently caused concern around the world. Extensive and inappropriate use of antibiotics in the treatment of infectious diseases has led to the emergence of multidrug-resistant bacteria.⁴ To overcome this problem, innovative antimicrobial agents must be developed.⁵

In order to reduce therapeutic doses, a combination of antimicrobial agents may also reduce the emergence of resistant strains in nosocomial infections.⁶

Bacteria produce a wide range of bacteriocins and antimicrobial peptides (AMPs) that prevent other bacterial strains from invading their own site. The bacterial peptides can also prevent tumor cell growth. Due to cationic charge and amphiphilic nature, bacteriocins have an anti-cancer effect.This feature enables bacteriocin to act as an "active membrane" by interacting with a negatively charged cell membrane. The electrostatic interaction between cationic bacteriocins and cancer cells plays an important role in cytotoxicity. Cancer cells also have low cholesterol levels, and as the cancer progresses, the membrane fluidity increases, making the cancer cells more prone to AMPs.⁷

Bacteriocins can disrupt membrane integrity and cause apoptosis among cancer cells. Another mechanism involves inhibiting angiogenesis and then preventing cancer from progressing.⁸

Bacteriocins are substances ribosomally synthesized by bacteria. They exhibit antimicrobial activity towards other bacterial species.⁹ Majority of bacteriocins are extremely potent compared with most of their eukaryotic counterparts, exhibiting antimicrobial activity at nanomolar (nM) concentrations.¹⁰ The selectivity and safety profile of bacteriocins have been highlighted as superior advantages over traditional antibiotics.¹¹

Many bacteriocins substances that are synthesized ribosomally by bacteria, show antimicrobial activity against other bacterial species.⁹ Most of them are very strong compared to most of their eukaryotic counterparts and show antimicrobial activity even at the level of nanomolar (nM) concentrations.¹⁰ The selective and safe action of bacteriocins is one of their advantages over traditional antibiotics.¹¹

The mechanical action of bacteriocins includes bactericidal and bacteriostatic effects with or without cell lysis. Inhibition of cell growth and consequent lack of cell wall synthesis, changes in membrane permeability or pore formation cause the death of target cells.¹² Another potential effect of bacteriocins is their effect on cancer therapy by inhibiting the synthesis of DNA and membrane proteins that cause apoptosis or cytotoxicity in tumor cells.¹³

By increasing the amount of tumor necrosis factor alpha (TNFα) secreted by macrophages and T cells, it binds to cell surface receptors, and by trimer zing them, intracellular caspases are activated. With the relative change of pro-apoptotic mediators (such as Bax) and anti-apoptotic mediators (like Bcl-2), the permeability of the mitochondrial membrane to cytochrome C increases, and when released, the apoptosis forms and activates caspase.¹⁴

The results of a study by Yousefy et al. also showed that protein fusion caused the highest level of apoptosis in AGS cells. The AGS cell line treated with concentrations of nisin - enterocin-epidermicin protein fusion showed high levels of apoptosis.¹⁵

In order to design the recombinant fusion gene construct, we used a peptide sequence with anticancer activity and a specific ligand of AGS cell line. Because the anti-cancer activity of bacteriocins such as lactocin, R-type pyocin and enterocin has been studied separately in different cell lines in the past, induction of apoptosis was observed. In this study, we sought to induce apoptosis by designing a recombinant fusion gene construct against gastric cancer cell line (AGS) to be used to inhibit pathogens and cancer cells.

The aim of this study was to evaluate the apoptotic and antibacterial activity of anti-cancer protein fusion, enterocin A, R-type pyocin, lactocin containing specific ligand in gastric cancer cells.

The results of flow cytometry are reported as follows (Figure 1):

Annexin V- / PI- region: In this region, living cells were located.
Annexin V + / PI- region: Primary apoptotic cells were located in this region.
Annexin V + / PI + region: In this region, dead cells were located by secondary necrosis and apoptosis.
Annexin V- / PI + region: In this region, damaged cells were present during the preparation process (considered as necrosis and no apoptosis occurred in them).

The percentage of cells treated with recombinant protein with primary apoptosis (Q4 region), secondary apoptosis (Q3 region) and necrosis increased compared to untreated cells (Figure 2). The number of healthy cells (Q1 region) in the untreated cell group was significantly higher than the treated cells. These results indicate that treatment of gastric cancer cells with recombinant protein at a concentration of 80μg/ml induces apoptosis in this cell line.

In a study in 2014, the anti-cancer effects of bacterioicin isolated from Enterococcus Mundtii strain C4L10 was evaluated. It was showed that this bacteriocin has anti-proliferative activities against different human cancer cell lines.¹⁶

In a study conducted by Ankaiah et al. the bacteriocin enterocin-B produced by E.faecium exhibited anti-cancer activity against HeLa, HT-29, AGS cancer cells and it was observed that the anti-cancer activity was dramatically increased when cancer cells treated with the heterodimer of the bacteriocins enterocin-A+B.¹⁷

Nami et al. evaluated the therapeutic effects and probiotic properties of proteins secreted by Ent. faecalis. In this experiment, the anti-cancer effect of enterocin on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) as well as a normal cell line (HUVEC) was investigated. Their findings revealed that the metabolites produced by this vaginal Ent. faecalis strain can induce the apoptosis of cancer cells.¹⁸

In a study by Abdi-Ali et al., the cytotoxic effect of pyocin S2, derived from Pseudomonas aeruginosa, on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF was surveyed. Indeed the results indicated that pyocin S2 exhibit an inhibitory effect on tumor cell lines.¹⁹ In another similar study, the cytotoxicity of pyocin S2 was evaluated against human malignant cells and normal cells in the same species. According to the results, the inhibitory effects on the cells increased with an increase of pyocin S2 activity.²⁰

Kim and colleagues examined the anticancer effect of cytoplasmic fraction from Lactococcus lactis ssp. lactis. The results of these researchers indicated the proliferation of human colon cancer cell line was inhibited by the treatment with cytoplasmic fraction of Lc. lactis.²¹

In another study in 2020, PI staining was performed and revealed apoptosis in Colicin N-treated cells, but no detectable fluorescent red was associated with necrosis.²²

In a study by Chumchalova et al., considered colicin E1, A and U stopped the cell cycle in five tumor cell lines. Colicin E1 and A, induced apoptosis in three cancer cell lines by 7 to 58%.²³

Ankaiah et.al studied the anti-cancer activity of enterocin-A against human colon, gastric (HT-29, Caco-2 and AGS) and cervical (HeLa) cancer cell lines and they found out that ent-A was able to induce apoptosis in HeLa cell line at a concentration of 120μg/ml, so that 53.93% of the cells had delayed apoptosis and 5.8% showed a primary apoptosis.²⁴

Fathizadeh et al. in a review study investigated the role of bacteriocins as new potential therapeutic candidates in cancer therapy. They find out that Evidence suggests that bacteriocins such as colcins, nisins, pediocins, pyocins, and bovocins are able to inhibit tumor growth at various stages by activating or inhibiting various cellular and molecular signaling pathways.²⁵They also showed that enterocin A-colicin E1 fusion peptide can be used as an effective antibacterial compound to treat or prevent bacterial infections.²⁶

Jalalvand et al. were investigated cloning and expression of the fusion gene construct of enterosin A, pyocin R, and lactocin, along with the anticancer peptide and specific ligand of the AGS cell line in a bacterial host. When this recombinant protein was exposed to AGS cell line, it resulted in a significant increase in the expression of Bax and caspase 3 genes and a significant decrease in Bcl2 gene expression compared to the GAPDH reference gene.²⁷

In our study, the anticancer effect of peptide fusion, bacteriocins and specific ligand of AGS cell line were investigated. In general, the recombinant protein induced apoptosis in the AGS cell line, and it can be suggest that this recombinant protein activated apoptosis internally, leading to cell death.

Flow cytometry results showed that the frequency of cancer cells treated with recombinant protein that had apoptosis was significantly higher than untreated cells. This indicates that treatment of AGS cell line with recombinant protein at a concentration of 80μg/ml induces apoptosis.

Under certain conditions, this recombinant protein may be used as adjuvants in chemotherapeutic drug combinations or as a tool to manipulate the expression of genes induced in apoptosis and ultimately control the growth and proliferation of cancer cells.

Through the present study, the following findings were specifically obtained:

Tests to measure the ability to inhibit cell proliferation and induce apoptosis in the cancer cell line showed that recombinant protein at a concentration of 80μg/ml can induce apoptosis and death in the gastric cancer cell line.
In general, recombinant protein may be introduced as a candidate to control microbial infections as well as cancer, and further attention and research.

These results can be the basis for more extensive studies to comprehensively study the effects of this recombinant protein for use in the treatment of cancer. In vitro results show that this protein has the necessary properties for in vivo studies.

Initially, a recombinant gene cassette containing anticancer sequences, lactocin-enterocin, pyocin and specific ligand genes was designed and their sequences were prepared for synthesis and cloning in pET22b expression vector from Biomatik Corporation.²⁷ Transformation of the generated vector into susceptible cells was performed by heat shock method.

In IPTG-induced pET systems, T7 RNA polymerase was produced, and finally this RNA polymerase was transcribed from the target gene located on this vector. Then, the transcribed RNA was translated into the target protein. Induction of recombinant plasmid and production of recombinant fusion protein in E.coli BL21 was performed as follows:

At a time interval of one hour after induction (1-5 hours), 1ml of the sample was collected from the culture medium and collected in a clean microtube. An induced sample was also prepared around the clock. After centrifugation, the precipitates were placed at -20°C until the start of SDS-PAGE process. Then, the expression of recombinant protein was assessed by SDS-PAGE.

Western blot (immunoblot) also was used to specifically detect the produced fusion protein and evaluate its antigenicity. The cleaved protein bands in SDS-PAGE were first electrically transferred to a membrane of nitrocellulose or polyvinylidene difluoride (PVDF) with porosity (0.45μm). The presence of recombinant protein was then evaluated using polyclonal antibodies.²⁷

Cell treatment and study of apoptosis

In each cavity of the 96-well plate, 100,000 AGS cells from the cell bank of the Genetic and Biological Resources Center of Iran (IBRC C10071) were poured and incubated for 24 hours in a CO2-containing incubator at 37°C to allow the cells to adhere to the bottom of the plate. Then, the proliferation and adhesion of cells to the bottom of the cavities were examined, and cases where the cell size was much lower or much higher were removed. Different concentrations of recombinant protein were prepared at intervals of 10 (10-100 µg/ml). The incubation period was 24 hours and after MTT test, the results were determined as Optical Density (OD) using ELISA plate reader. By detecting the values of OD, control OD (C) and OD of each concentration (T) were also calculated and the lethal percentage of each concentration was determined.

With the onset of apoptosis, phosphatidylserine, which is normally present on the inner surface of the cell membrane, was transferred to the outside. Annexin V can bind specifically to phosphatidylserine in the presence of calcium. Under these conditions, annexin V was conjugated to the Fluorescein isothiocyanate (FITC).

Propidium iodide solution (PI) was used for staining. The compound only penetrates cells whose plasma membrane structure has been destroyed and then attaches to DNA. Thus, cells that are in the early stages of apoptosis are identified from those that are in the final stages, and the frequency of cancer cell death is determined in terms of necrosis, primary and secondary apoptosis.

To evaluate apoptosis, a cell culture plate of 24 cells was used and after 24 hours, the cells adhered to the bottom of the plates. To all three wells of the plates (as triplicates) was added 80μg/ml of freshly prepared recombinant protein, and a few wells were used as controls that were not treated with recombinant protein.

Then, the final volume was increased to one cc by culture medium. To treat the cells, the plate was placed in a CO2 incubator at 37°C for 24 hours.

After removing the supernatant fluid and washing with PBS, trypsin was added to remove the cells from the bottom of the plates. An inverted microscope was used to examine the cell separation. Then, the treated and untreated cells with recombinant protein at a concentration of 80μg/ml were poured into a centrifuge tube and centrifuged at 1500 rpm for 5 minutes. Then cell sediments were isolated for flow cytometry.

Ethics approval and Consent to Participate:

This study was approved by Islamic azad university- Kazerun branch ethics committee under IR.IAU.KAU.REC.1398.153. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication: Not applicable

Availability of data and materials: Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study

Competing Interest: The authors declare that they have no competing interests

Authors' contributions:

D.E., N. J., M. R., M, M., and S.N. designed the study. N.J. performed research. D.E. performed data analysis. D. E. and N.G. wrote the article. M.M., S. N., and M.R. read and approved the final manuscript.

Acknowledgements:

This article was extracted from the thesis prepared by Neda Jalalvand to fulfill the requirements required for earing the PhD degree. The authors would like to thank the Department of Microbiology and Applied Virology Research Center of Baqiyatallah University of Medical Sciences, Tehran, Iran for their support, cooperation and assistance throughout the period of study.

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No competing interests reported.

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Evaluation of Apoptotic Activity of Protein Fusion Enterocin A- Pyocin R-Lactocin, Bacteriocins and Specific Ligand of Gastric Cancer Cell Line (AGS)

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