Enterocin A- R type pyocin-Lactocin-Ligand Against Gastric

(1)

Evaluation of Physicochemical Activity of Anticancer Fusion Proteins;

Enterocin A- R type pyocin-Lactocin-Ligand Against Gastric Cancer Cell Line by Real-Time RT PCR Technique

Article in International Journal of Peptide Research and Therapeutics · June 2021

DOI: 10.1007/s10989-021-10158-3

CITATIONS

5

READS

136 5 authors, including:

Some of the authors of this publication are also working on these related projects:

Prevalence of extended-spectrum-β-lactamase-producing Escherichia coli isolates among uropathogensin a pediatrics hospitalView project

Investigate the Inhibitory Effects of Satureja khuzestanica Essential Oil against Housekeeping fabD and exoA Genes of Pseudomonas aeruginosa from Hospital Isolates using RT-PCR TechniqueView project

Davoud Esmaeili

Baqiyatallah University of Medical Sciences 162PUBLICATIONS 692CITATIONS

SEE PROFILE

(2)

Vol.:(0123456789)

1 3

International Journal of Peptide Research and Therapeutics https://doi.org/10.1007/s10989-021-10158-3

Evaluation of Physicochemical Activity of Anticancer Fusion Proteins;

Enterocin A‑ R type pyocin‑Lactocin‑Ligand Against Gastric Cancer Cell Line by Real‑Time RT PCR Technique

Neda Jalalvand¹ · Davoud Esmaeili^2,3 · Moha mmad Mehdi Moghani Bashi¹ · Mohammad Raiszadeh⁴ · Sirous Naeimi¹

Accepted: 5 January 2021

Abstract

Gastric cancer treatment remains a major challenge. There are many reports on the positive efficacy of Bacteriocins associated with anti-cancer sequences. The main purpose of this study was to determine and design a fusion gene contraceptive containing anticancer Enterocin A, R-type pyocin, Lactocin, ligand against AGS gastric cancer cell line using Real Time_

RT_ PCR technique. This study was designed EntA-PynR-Lac recombinant proteins containing ligand and anticancer using bioinformatics software. Escherichia coli BL-21 was used to express cloned genes in expression vectors that have a T7 bacteriophage promoter. The specific ligand of the AGS cell line was designed and added to the recombinant construct sequence, and its three-dimensional structures, conformity, and stability were evaluated. Synthesis of fusion gene concentrate was performed in the expression vector, the transformation of the recombinant plasmid containing anticancer fusion gene construct in the respective host. Confirmation and purification of the recombinant protein by Western blotting and nickel column chromatography through the His tag, cytotoxicity determination against AGS cell line via the MTT technique, and finally, Real-time PCR was performed. Protein fusion at a concentration of 80 μg/ml in 24 h kills 83% of the cells tested and treatment of the cells with this concentration leads to increased expression of caspase3 and bax genes and decreased expression of bcl2. The results show that the treatment of gastric cancer cells with recombinant protein at a concentration of 80 μg/ml induces apoptosis in this cell line.

Keywords Bacteriocins · Recombinant proteins · Enterocin A,pyocin R · Lactocin · Anticancer

Introduction

Antimicrobial peptides (AMPs) are generally, positive charge peptides found in a wide range of live organisms from microorganisms to mammalians. These peptides demonstrated capabilities to kill pathogenic and nonpathogenic bacteria, fungi, enveloped viruses, and even transformed or cancerous cells with different mechanisms such as destabi- lize host cell membrane, forming channels in host cell transmembrane, or enhancing host immunity by functioning as immune- modulators (Ibrahim 2019).

With the discovery of therapeutic peptides, scientists have turned their attention to the use of bacteriocins as a new therapeutic remedy against cancer. Accordingly, the cationic peptides of bacteriocins have a high affinity toward the negatively charged surface of the cancer cells rather than non-cancer cells. Secondly, the high fluidity of the tumor cell membrane results in destabilization, which can explain

* Davoud Esmaeili [email protected]

* Moha mmad Mehdi Moghani Bashi [email protected] Neda Jalalvand

[email protected] Sirous Naeimi

[email protected]

1 Faculty of Basic Sciences, Department of Genetics, Kazerun Branch, Islamic Azad University, Kazerun, Iran

2 Department of Microbiology and Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

4 School of Medicine, Trauma Research Center, Bqiyatallah Al-Azam Hospital, Baqiyatallah University of Medical Sciences, Tehran, Iran

(3)

the selective cytotoxicity toward cancer cells compared to non-cancer cells.

Lastly, the presence of a large number of microvilli in the cancer cell membrane facilitates the binding and uptake of Bacteriocin molecules (Kaur and Kaur 2015).

Some bacteriocins have shown selectivity toward cancer cells with unknown mechanisms of action. Some factors may be attributed to the selectivity of bacteriocins toward cancer cells rather than normal cells. Firstly, cancer cells have negatively charged surface due to the presence of substantial amounts of anionic phosphatidyl serine, gangliosides, hepa- rin sulfates, and O-glycosylated mucins. The outer surface of non-cancer cells consists of neutral phospholipids including sphingomyelins and phosphatidyl choline whereas the inner surface contains amino phospholipids (Riedl et al. 2011).

Enterocins are a class of cationic bacteriocins produced by Enterococcus and are active against gram-negative and gram-positive bacteria; they are resistant to a great range of temperatures and pH levels, apart from being easily destroyed by digestive proteases. Moreover, they present a broad spectrum of activity against gram-positive and gram- negative microorganisms (Martínez-Bueno and Gálvez 2017).

Enterocins, like most bacteriocins, mainly target the cytoplasmic membrane of cells, forming pores which affect the transmembrane potential and/or the pH gradient and results in the leakage of intracellular molecules crucial for cellular maintenance (Cleveland 2001). As a result, they are used as probiotic strains as well as being developed for several emerging therapeutic areas including the treatment of antibiotic resistance and cancer (Aziz et al. 2019).

Otherwise, Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may syn- thesize several pyocins (Michel-Briand and Baysse 2002).

Three types of pyocins are known. R-type pyocins resem- ble non-flexible and contractile tails of bacteriophages. All R-type pyocins are nuclease- and protease-resistant. They cause depolarization of the cytoplasmic membrane with pore formation in target bacteria (Kaur and Kaur 2015).

Pyocins target cells through specific receptors. Based on their structure, pyocins are classified as R, F, or S-types.

R-type pyocins are nuclease-protease resistant and it is thought that they have evolved from phage tails because their structure resembles non-flexible and contractile tails of bacteriophages. Their mechanism of action is through depolarization of the cytoplasmic membrane by pore formation.

F-type pyocins are high molecular weight protease-resistant proteins which structure is similar to R-type pyocins, except for the flexible and non-contractile rod-like structure.

S-type pyocins are colicin-like, protease-sensitive, and their structure consists of two components: the more significant component executes the killing activity (DNase, RNase, or channel-forming activities) while the smaller component, by

showing sequence homology with colicin E2, is considered as an immunity protein. S-type pyocins cause cell death by DNA breakdown (pyocin AP41, S1, S2, S3) and pore formation (pyocin S5) (Michel-Briand and Baysse 2002; Parret et al. 2005).

Among the antimicrobials their biological variations can be mentioned with lactic acid bacteria (LAB) and the varieties of microbial metabolites produced from this group or associated groups, being some of them. LABs secrete a wide variety of Bacteriocins like nisin, pediocins, Lactocin, divergent Bacteriocin CDSs, diplocin, Lactostrepcins, etc.

Nisin is the only Bacteriocin allowed to be used in food products, and it is a GRAS (Generally Regarded as Safe) food preservative (FDA 1998).

The active chemicals Lactucin is capable of inducing pharmacological effects in humans. Lactucin and its deriva- tives possess analgesic activities in thermal models of nocip- tion similar to that observed for ibuprofen used as a standard drug (Wesolowska et al. 2006).

Meanwhile, the bcl2 family of proteins includes apoptosis-related molecules involved in normal physiology, as well as cancer pathology. Members of our team have discovered and cloned the novel gene BCL2L12, which codes for a protein member of the bcl2 family. The BCL2L12 expression has been studied extensively in various types of cancer and its important clinical value has been underlined.

Bcl2 and bax are the most important proteins in balanc- ing apoptosis. These proteins have anti-apoptotic and pro- apoptotic properties (Alshatwi 2011).

The expression of bax is upregulated by the tumor suppressor protein p53, and bax is involved in p53-mediated apoptosis. The p53 protein is a transcription factor that, when activated as part of the cell’s response to stress, regulates many downstream target genes, including bax.

Wild-type p53 has been demonstrated to upregulate the transcription of a chimeric reporter plasmid utilizing the consensus promoter sequence of bax approximately 50-fold over mutant p53. Thus it is likely that p53 promotes bax’s apoptotic faculties in vivo as a primary transcription factor.

However, p53 also has a transcription-independent role in apoptosis. In particular, p53 interacts with bax, promoting its activation as well as its insertion into the mitochondrial membrane (Miyashita and Reed January 1995).

The main aim of this study is to investigate the inhibitory effect of EntA-PynR-Lac recombinant proteins containing ligand and anticancer compounds against the AGS cell line using the Real-time RT-PCR technique. This study also included the expression of bcl2, bax, caspase3, and GAPDH reference genes.

(4)

International Journal of Peptide Research and Therapeutics

1 3

Materials and Methods

This strain of the Escherichia coli strain BL-21 is used to express cloned genes in expression vectors that have a T7 bacteriophage promoter. This strain of bacteria has also been obtained from the Pasteur Institute of Iran.

The pET system is the most powerful system developed for the cloning and expression of recombinant proteins in E. coli, and each of the pET family vectors is designed for a specific purpose. There is a wide variety of pET family vectors, all derived from the pBR322 plasmid. One of the fundamental differences between different types of pET systems is the sequence diversity upstream of the input fragment.

The identification of pET systems is a gene of resistance to an antibiotic. Fusion-tag sequences are a peptide sequence added by the vector itself that facilitate the detection and purification of recombinant proteins or may alter biological activity. The pET 22b ( +) vector series has a T7 bacteriophage promoter and has two replication ini- tiation regions, one derived from pBR322 and the other ori f1. The vector with a length of 5493 bp is controlled by the T7 promoter and the lac operator. This vector contains the ampicillin resistance gene in the position 4038–4895 and has two sequences encoding His-Tag in positions 140–157 and 207–239. The multiple cloning vector area is located in position158–203 of the map. (Fig. 1).

LB-Broth and LB-Agar cultures from Micro media Company were used in this research. To prepare Luria

Bertani (LB) liquid medium, one liter of LB broth, we weighed 20 g of the powder, dissolved it in some distilled water, and then increased its volume to one liter. It was then autoclaved at 15 ppm and 121 °C for 15 min and after cooling to room temperature, it was refrigerated at 4 °C.

To prepare one liter of solid LB agar medium, we weighed 40 g of powder and dissolved in some distilled water like the LB broth medium and then increased its volume to one liter.

It was then autoclaved for 15 min at 15 lb per square inch at 121 °C. After cooling to room temperature, we divided it into plates and kept it in the refrigerator at 4 °C before use.

0.1 mg of ampicillin antibiotic made by Sigma Company was dissolved in 1 ml of sterile distilled water to reach a final concentration of 100 μg/ml. After preparation and fil- tration in microtubes, it was divided into smaller volumes and stored in a freezer at − 20 °C. After autoclaving the LB broth and LB agar medium and reaching a temperature of 50–40 °C, we added 1 μl of antibiotic stock at a concentration of 100 μg/ml per 1 ml of LB medium.

To prepare a recombinant gene cassette containing anticancer-entA-PynR-Lac-Ligand gene sequence in the Pet22b vector, firstly we designed a recombinant gene cassette containing anti-cancer -entA-PynR-Lac-Ligand sequence in Pet22b vector. For this purpose, we extracted the sequences of entA-PynR-Lac genes from the NCBI site. Then, studies such as blasting and comparing genes with similar sequences and codon optimization (via Gene Script.com) for their expression in E. coli were performed.

We then used the optimized sequence as a candidate.

Using AGS cell line-specific ligand BDB software and

Fig. 1 The structure and components of the pET-22b vector

(5)

through AntiCP and IACP web-based software, we designed the anti-cancer sequence and added the recombinant contrast sequence.

For gene optimization and prediction of mRNA structure, initially, wild-type genes related to Pseudomonas aeruginosa, Enterococcus faecium, and Lactobacillus sakei were analyzed by Gene script online software. Then, because the codons of these genes were not preferred for expression in the E. coli expression host, the original sequence codons were replaced by the more preferred codons in E. coli by the Jcat software. Finally, the optimized gene was re-examined by Gene script online software. The second structure of the mRNA gene was analyzed by bioinformatics tools such as the mfold program at http://www.bioin fo.rpi.edu/appli catio ns/mfold ).

The overall properties of the protein were extracted using https ://web.expas y.org/protp aram (ProtParam Expasy) to determine the characteristic of the recombinant protein.

Garnier-Osgothorp-Robson version IV (GOR4) software was used to evaluate and analyze the secondary structural features of the recombinant protein.

Finally, the specific sequence of anticancer- entA-PynR- Lac -ligand fusion gene was ordered from Biomatik(Gene Synthesis Service) for synthesis and cloning in the pET22b expression vector, and the plasmid containing the recombinant gene was lyophilized by the company.

Escherichia coli BL21 strain is used to express genes in expression vectors containing the T7 bacteriophage promoter. Then the prepared recombinant plasmid was pre- sented to the BL-21 bacterial cell suspension.

Competence cells were prepared from E. coli BL-21 (DE3). Among the available methods for transformation, the heat shock method was used. Transformation or transfer of the generated recombinant vector into susceptible cells was performed by the heat shock method.

After the colonies grew, the plates were removed from the incubator and examined. In the case of the recombinant vector, the correct colony was selected by colony screen- ing. Thus, several colonies were grown on LB agar medium containing the ampicillin antibiotic.

In this study, due to the use of the alkaline lysis method, the company’s plasmid extraction kit (Bioneer, Korea) was used. To obtain small amounts of plasmids or clones during different stages of research, recombinant plasmid DNA was extracted.

Qualitative and quantitative evaluation of the extracted DNA was performed using two methods of spectrophotom- etry and agarose gel electrophoresis. Primers were designed using Primer 3 and Gene Runner software. Using the BLAST program, the suitability of the designed primer was confirmed. Primers were prepared and PCR was performed.

Induction and expression of recombinant protein were performed. Recombinant protein expression was assessed by

SDS-PAGE. Polyacrylamide gel staining was performed and the expression of recombinant protein was also confirmed by Western blotting.

Gene expression was induced by adding IPTG so that 5 μl (1 μl of IPTG per milliliter of culture medium) was added to the rest of the culture medium prepared from one-molar IPTG stock. Incubation was performed in a shaker incubator at 37 °C for 1, 2, 3, 4, and 5 h 24 h a day.

After optimizing the production conditions and confirm- ing the expression of proteins and to prepare a high volume of bacterial and recombinant protein samples, the steps of culture, induction, and expression were performed in 1-L volumes.

The recombinant proteins were then purified using Nickel column chromatography using his-tag. Sonication is one of the physical methods of cytolysis and ultrasound waves are used to destroy cells. In this method, the cells in the suspension are lysed.The prepared precipitate was sonicated in bulk cytolysis buffer (20 mM Tris–HCl, 0.5 M NaCl, 0.3%

TritonX100, 0.005% PMSF, 10 mM Imidazole, pH 8.0). It was then broken by a sonicator in 10 40-s cycles under 4°

C.The sonicated sample was then centrifuged at 12,000 rpm for 20 min and the supernatant was used for purification on a column.

The protein supernatant was passed through a column containing nickel resin and after collecting the extracted liquid; the column was washed with a wash buffer (20 mM Tris, 0.5 M Nacl, 20 mM Imidazole, pH 8.0). In order to remove the recombinant proteins with histidine sequence from the resin, the separation buffer (20 mM Tris, 0.5 M Nacl, 0.6 M Imidazole, pH 8.0) was passed through the column.After collecting the protein fractions, SDS-PAGE electrophoresis was performed to evaluate their purity.

Protein assays were also performed by the Bradford method,the company’s bradford protein assay kit (Biotech, Iran) was used.

The concentration of recombinant protein was deter- mined and then the cells used for this study were purchased from AGS gastric cancer cell line from the cell bank of the Genetic and Biological Resources Center of Iran. Cells were cultured under standard conditions in Dulbecco’s Modified Essential Medium/Nutrient Mixture F-12 (DMEM/F-12) containing 10% fetal bovine serum (FBS), 1% penicillin/

streptomycin. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂. Due to the large proliferation of cells, we prepared a subculture to prevent them from dying.

For this purpose, trypsin / EDTA 0.25% was used to separate the cells from the bottom of the flask. Using a Neobar slide and a conventional microscope, we calculated the number of cells required for the new flask by volume and transferred it to the new flask containing the complete culture medium.

To treat the cells with different concentrations of recombinant protein, 100,000 cells were poured into each cavity of

(6)

1 3

the 96-well plate and incubated for 24 h in a CO₂-containing incubator at 37 °C to allow the cells to adhere to the bottom of the plate. After 24 h, we examined the proliferation and adhesion of cells to the bottom of the cavities under a microscope. Then 19 different concentrations of recombinant protein were prepared as follows:

10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 µg/ml.

The incubation time was 24 h. After this time, MTT test was performed and the company’s MTT assay kit (BIO- IDEA, Iran) was used. The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt to purple formazan crystals by metabolically active cells. The viable cells contain NAD (P) H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and The test result was read as Optical Density (OD) using ELIZA reader. Holding the OD values obtained, the control OD (C) and the OD of each concentration (T) were calculated. In the next step, by placing OD of each concentration (T) and control OD (C) in the formula of percentage of lethality (mentioned below), the amount (percentage) of lethality of each concentration was calculated:

To study gene expression by Real-time RT- PCR technique, cells were treated at 80 μg/ml concentrations for 24 h to study gene expression. Then, expression of bcl2, bax, Caspase3 genes was performed by the Real-time RT-PCR technique.

RNA extraction using nanodrops from AGS cancer cells, the company’s RNA extraction kit (Jena Bioscience,Germany) was used. Removal of genomic DNA contamination, and determination of RNA concentrations were the next steps in the research. Then, the extracted RNAs were electrophoresed and their quality was evaluated,

%Cytotoxicity=1−T∕C×100

cDNA synthesis, RNA-primer mixture preparation, and cDNA mixture preparation were performed.

An RT-PCR kit made by Sinagen Company (Vivantis,Iran) was used to prepare cDNA. This is a 2-step kit and includes cDNA fabrication and PCR of cDNA samples. cDNA synthesis was performed according to the kit instructions (Vivantis Sinagen kit).

Sequences of bcl2, bax, Caspase3, and GAPDH (Glyc- eraldehyde-3-phosphate dehydrogenase) genes were extracted from the NCBI database and then the respective primers were designed by GENE Runner software and then BLASTed with the entire human genome. (Table 1).

Real-time RT-PCR technique was used to determine the expression of bcl2, bax, Caspase3, and GAPDH genes,the q-PCR kit made by Jena Bioscience Company(Germany) was used.Quantitative determination of bcl2, bax, Caspase3, and GAPDH genes before and after exposure to the recombinant protein was also performed using the Real-time RT- PCR technique and then, Livak method was used to evaluate the expression changes of the studied genes. Table 2 showes the temperature program of quantitive real time PCR.

This study is a cross-sectional and laboratory type and its results are described descriptively. Statistical analysis of ΔCt results obtained from real-time PCR of genes was performed by SPSS version22 software using the ANOVA method. In this study, relative quantification was used to analyze gene expression.

To determine the specificity of the reaction product, after real-time PCR, the reaction product was electrophoresed on 2% agarose gel to confirm this reaction and the relativity of the product.

Table 1 The specification to the

studied primers Oligo sequences 5′ → 3’

Number Primer Name Sequence 5′ → 3′ (10–50 bp)* Length TM

1 cas3-F CAA ACT TTT TCA GAG GGG ATCG 22 62.33

2 cas3-R GCA TAC TGT TTC AGC ATG GCAC 22 65.47

3 GAP-F GAA GGT GAA GGT CGG AGT CA 20 63.28

4 GAP-R GAA GAT GGT GAT GGG ATT TC 20 57.91

5 bax-F CAT CTT CTT CCA GAT GGT GA 20 58.70

6 bax-R GTT TCA TCC AGG ATC GAG CAG 21 63.27

7 bcl2-F GAG ACA GCC AGG AGA AAT CA 20 61.19

8 bcl2-R CCT GTG GAT GAC TGA GTA CC 20 60.65

Table 2 Temperature program for Real- time PCR

Cycle no Time Temp (°C) Step

1 2 min 50 UNG treatment

35 2 min 95 Initial denaturation

35 15 s 95 Denaturation

35 1 min 60 Annealing and elongation

(7)

Results

Characteristic of Recombinant Protein

It is necessary to have information about the desired protein to continue working more accurately. This information was extracted using the server https ://web.expas y.org/protp aram (ProtParam Expasy) and the results are as follows:

The recombinant protein contains 236 amino acids with a molecular weight of 25,823.48Daltons and an isoelectric point of 8.65. In recombinant protein, the total number of negatively charged amino acid stores is (Asp + Glu) = 25, and the total number of positively charged amino acids is (Arg + Lys) = 29. The predicted half-life of the protein in reticulocytes and mammals is approximately 30 h in vitro, more than 20 h in yeast (in vivo), and more than 10 h in E. coli (in vivo).

The instability index is 28.92 and is classified as a stable protein. The aliphatic index is 80.25 and the average hydro- phobicity (GRAVY) is -0.31.

Results of Secondary Structure and Tertiary Structure of the Recombinant Protein

The results showed that the structural content of the protein includes alpha helix, random coil, and extended strand. The results of the secondary structure prediction for the recombinant protein include 34.75% alpha-helix, 47.88% random coil, and 17.37% extended strand. Also, the results obtained about the three-dimensional structure

of this protein by I-TASSER software can be seen in Fig. 2.

Among these images predicted from the third structure for the recombinant protein, the image with the highest C-Score is selected as the best option, which is the first image (Fig. 3).

Results of Expression of bcl2, bax, Caspase3 Genes and GAPDH Reference Gene by Real‑Time RT‑PCR Technique

Results of the bcl2 gene expression and GAPDH reference gene by Real-time RT-PCR technique show that the Ct level of the bcl2 gene increased after treatment, which indicates a decrease in gene expression. The expression of the bcl2 gene, which is an anti-apoptotic protein, decreased by 4 times compared to the reference gene, which was reported as a decrease in bcl2 gene expression.

The Ct level of the bax gene decreased after treatment, indicating increased gene expression. Therefore, The expression of the bax gene, which belongs to the family of pro-apoptotic proteins, was 16 times higher than the expression of the reference gene, which indicates an increase in the expression of the bax gene.

The amount of Ct level of caspase3 decreased after treatment, the expression of the caspase3 gene was 11.31 times higher than the expression of the reference gene which indicates an increase in gene expression. The ct level of the reference gene did not change significantly before and after treatment.

Fig. 2 The structure alignment between the I-TASSER model

(8)

1 3

Results of Expression of the Recombinant Protein by SDS‑PAGE Method

To evaluate the expression of recombinant protein, the SDS-PAGE method with Coomassie Brilliant Blue – SDS- PAGE staining in the presence of a protein marker was used (Fig. 4).

Results of Confirmation of Recombinant Protein Expression by Western Blotting

In order to confirm the specificity of the recombinant protein with histidine sequence and molecular weight of 25.8 KD, Western blotting method with the presence of anti-his tag monoclonal antibody and also labeled secondary antibody (Goat anti-mouse—Peroxidase conjugate) was used. The results are shown in Fig. 5.

MTT Test Results

The results show that the recombinant protein at a concentration of 80 μg/ml is able to reduce the enzymatic activity of oxidoreductase in treated AGS cells. According to Fig. 6, after 24 h of incubation of AGS cells exposed to the recombinant protein at the desired concentration, more than 80% of the cells were destroyed. Also, by observing these cells under inverted microscopy, it was found that

Fig. 3 Comparison of relative changes in mRNA expression of bcl2, bax, caspase3, and GAPDH reference genes in AGS cells treated and untreated with recombinant protein

Fig. 4 Wells T1, T2, T3, T4, T5, T6 are IPTG-induced bacterial deposits at times 1, 2, 3, 4, 5, and 24 h after induction. well T0 is a bacterial deposit before induction

Fig. 5 Western blotting of recombinant protein. No. 1: Pre-induction deposition (B.I), No. 2: Post-induction deposition 2 h

(9)

the recombinant protein with a concentration of 80 μg/ml has a significant cytotoxic effect on these cells.

Discussion and Conclusion

In this study, the effect of recombinant protein on the expression of bax,bcl2, and caspase3 genes was investigated. After the production of recombinant protein, it was exposed to the AGS cell line, and the results were analyzed and compared by other studies.

In a study conducted by Yusuf et al. The anti-cancer effects of Enterococcus mundtii strain c4l10 on the catego- ries of oral cancer cells, breast cancer cell, and colon cancer were investigated. It was observed that this Bacteriocin exerts its anti-cancer effects on the studied cell lines by its anti-proliferative mechanism (Yusuf 2014). In another study in 2018, the anti-cancer effects of Enterosin B on cell lines including HeLa, HT-29, and human AGS cancer cells were measured and the effects of induction of apoptosis were also observed in these cell lines. Likewise, it was found that the anticancer effect of Enterosin A and B heterodimer on cancer cells is increasing significantly. (Ankaiah 2018).

Another study in 2014 evaluated the therapeutic effects of a metabolite produced by the Enterococcus faecalis (derived from the human vagina) probiotic agent. Throughout the study, the anticancer effect of Enterosin on AGS, HeLa, MCF-7, and HT-29 cancer cell lines as well as a normal cell line (HUVEC) was evaluated and the induced effect of apoptosis was observed on cancer cell lines. (Nami 2014).

In a study conducted by Abdi-Ali et al. The cytotoxic effect of Pseudomonas aeruginosa-derived Pyocin S2 on HepG2 and Im9 tumor cell lines and a normal HFFF cell line was investigated. According to the results, the inhibitory effect of Pyocin S on tumor cell lines was observed (Abdi- Ali et al. 2004).

In another similar study, the cytotoxicity of Pyocin S2 was evaluated against cancer cell lines and normal mouse cells. (Watanabe et al. 1980) Kim and colleagues examined

the effect of cellular components of ten different probiotics on eleven types of cancer cell lines. The results of these researchers indicated the effect of probiotics on the inhibi- tion of colon cancer cell lines. (Kim 2003).

Another study examined the cloning and expression of Enterococcus faecium T136-derived Enterocin A in a yeast host. The results show an increase in antimicrobial activity against various bacterial pathogens. (Uteng et al. 2003).

In our study, cloning and expression of Bacteriocin fusion gene constructs of entA-PynR-Lac along with anticancer peptide and specific ligand of AGS cell line in the bacterial host were investigated. When this recombinant protein was exposed to the AGS cell line, it resulted in a significant increase in the expression of bax and caspase3 genes and a significant decrease in bcl2 gene expression compared to the GAPDH reference gene.

In general, in the present study, 24-h treatment of human gastric cancer cells (AGS) with a concentration of 80 μg/ml of recombinant protein-induced apoptosis in the studied cell line. This recombinant protein activates apoptosis internally, leading to cell death.

In this study, we investigated the effect of recombinant protein on the internal pathway of apoptosis and evaluated the apoptotic induction of this recombinant protein. The results showed that treatment with recombinant protein has cytotoxic effects on gastric cancer cells. The findings also show that the recombinant protein can activate apoptosis through the internal pathway.

This recombinant protein can be introduced as an effec- tive inhibitor of the proliferation and growth of gastric cancer cell line (AGS) cells, which in certain conditions, may be used as adjuvants in chemotherapeutic agents combinations.

This recombinant protein is a tool to manipulate the expression of genes induced in apoptosis and ultimately control the growth and proliferation of cancer cells. The above results can be the basis for more extensive studies to comprehen- sively 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.

References

Abdi-Ali A, Worobec EA, Deezagi A, Malekzadeh F (2004) Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines. Can J Microbiol 50:375–381 Alshatwi A (2011) Anti-proliferative effects of organic extracts from

root bark of Juglans Regia L. (RBJR) on MDA-MB-231 human breast cancer cells: role of Bcl-2/Bax, caspases, and Tp53. Asian Pac J Cancer Prev 12:525–530

Ankaiah D et al (2018) Cloning, overexpression, purification of bacteriocin enterocin-B and structural analysis, interaction determination of enterocin-A B against pathogenic bacteria, and human cancer cells. Int J Biol Macromol 116:502–512

Fig. 6 MTT test results

(10)

1 3

Aziz F, Khan MN, Ahmed S, Andrews SC (2019) Draft genome sequence of Enterococcus faecium SP15, a potential probiotic strain isolated from spring water. BMC Res Notes 12(1):99 Cleveland J et al (2001) Bacteriocins: safe, natural antimicrobials for

food preservation. Int J Food Microbiol 71:1–20

Osama O Ibrahim (2019) Classification of antimicrobial peptides bacteriocins, and the nature of some bacteriocins with potential applications in food safety and bio-pharmaceuticals. EC Microbiol 7:591–608.

Kaur S, Kaur S (2015) Bacteriocins as potential anticancer agents.

Front Pharmacol 6:272. https ://doi.org/10.3389/fphar .2015.00272 Kim JY et al (2003) Cell cycle dysregulation induced by the cytoplasm

of Lactococcus lactis ssp. lactis in SNUC2A, a colon cancer cell line. Nutr Cancer 46:197–201

Martínez-Bueno M, Gálvez A (2017) Effect of combined physicochemical treatments based on Enterocin AS-48 on the control of Listeria. Food Control 21(4):478–486

Michel-Briand Y, Baysse C (2002) The pyocins of Pseudomonas aeruginosa. Biochimie 84(5–6):499–510. https ://doi.org/10.1016/

S0300 -9084(02)01422 -0

Miyashita T, Reed JC (1995) Tumor suppressor p53 is a direct tran- scriptional activator of the human bax gene. Cell 80(2):293–299.

https ://doi.org/10.1016/0092-8674(95)90412 -3

Nami Y et al (2014) A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances the apoptosis of human cancer cells. J Appl Microbiol 117:498–508

Parret AHA, Temmerman K, De Mot R (2005) Novel lectin-like bacteriocins of biocontrol strain Pseudomonas fluorescens Pf-5. Appl Environ Microbiol 71(9):5197–5207. https ://doi.org/10.1128/

AEM.71.9.5197-5207.2005

Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A et al (2011) In search of a novel target-Phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim Biophys Acta 1808:2638–2645.

https ://doi.org/10.1016/j.bbame m.2011.07.026

Uteng M, Hauge HH, Markwick PRL, Fimland G, Mantzilas D, Nis- sen-Meyer J, Muhle-Goll C (2003) Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridge. Biochemistry 42(39):11417–11426 Watanabe T, Saito H (1980) Cytotoxicity of pyocin S2 to tumor

and normal cells and its interaction with cell surfaces. Bio- chim Biophys Acta 633:77–86. https ://doi.org/10.1016/0304- 4165(80)90039 -2

Wesolowska A, Nikiforoka A, Michalka K (2006) Analgesic and seda- tive activities of lactucin and some lavctucin like guainolides in mice. J Ethanopharmacolgy 107:254–258

Yusuf MA, Ichwan SJ, Haziyamin T, Hamid THA (2014) Anti-proliferative activities of purified bacteriocin from Enterococcus mundtii strain C4L10 isolated from the caecum of Malaysian non-broiler chicken on cancer cell lines. Int J Pharm Pharm Sci 7:334–337 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

(11)

1.

2.

3.

4.

5.

6.

Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).

Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small- scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.

These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription (to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will apply.

We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as detailed in the Privacy Policy.

While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may not:

use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access control;

use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is otherwise unlawful;

falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in writing;

use bots or other automated methods to access the content or redirect messages override any security feature or exclusionary protocol; or

share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal content.

In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue, royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any other, institutional repository.

These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.

To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law, including merchantability or fitness for any particular purpose.

Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed from third parties.

If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not expressly permitted by these Terms, please contact Springer Nature at

[email protected]