Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the GastricCancer Cell

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Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the Gastric Cancer Cell

Article in Probiotics and Antimicrobial Proteins · October 2021

DOI: 10.1007/s12602-021-09770-y

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https://doi.org/10.1007/s12602-021-09770-y

Anticancer Effect of Enterocin A‑Colicin E1 Fusion Peptide on the Gastric Cancer Cell

Hadis Fathizadeh¹ · Mahmood Saffari¹ · Davoud Esmaeili^2,3 · Rezvan Moniri^1,4 · Javad Amini Mahabadi^5,6

Accepted: 5 March 2021

Abstract

Cancer is one of the most causes of death all over the world, although improvements in its treatment and recognition.

Due to the limitations of common anticancer methods, including surgery, chemotherapy, and radiotherapy, attention has been drawn to other anti-cancer compounds, especially natural peptides such as bacteriocins. In this study, we used a combination of two bacteriocins, colicin E1 and enterocin A, against AGS gastric cancer cell lines. In order to evaluate anticancer properties of fusion peptide, we applied MTT assay, real-time PCR, and flow cytometry tests.

This is the first report to show the cell growth inhibitory activity of the enterocin A in combination with colicin E1 against AGS human cancer cells. The results of this study showed that this fusion peptide at a concentration of 60.4 µg/mL and 24 h was able to kill half of the tested cancer cells, and treatment of the cells with this concentration increased the expression of bax and caspase 3 genes and reduced the expression of bacl-2 in 24 h. Flow cytometry analysis of annexin V-FITC/propidium iodide results also showed that our peptide was able to induce apoptosis in treated cells compared with control. Taken together, enterocin A-colicin E1 (ent A-col E1) can be considered as a good candidate for anticancer therapies.

Keywords Bacteriocins · Enterocin A · Colicin E1 · Anticancer activity · Apoptosis

Introduction

Gastric cancer is the third most common cause of death from cancer and the fourth most commonly diagnosed cancer in men [1–3]. The origin of this cancer is the glandular epi- thelium of gastric mucosa [4, 5]. Cancer routine treatments, such as chemotherapy, have many side effects due to their non-specific toxicity and killing normal cells in addition to cancer cells. Also, sometimes cancer cells become resistant to chemotherapy over time [6]. Despite many advances in other treatments, including surgery and radiation therapy, these treatments still have a limited response in gastric cancer, and survival rates are poor [7, 8]. Therefore, it is important to develop more effective strategies for treating patients [6].

With unprecedented advances in biotechnology, the use of peptides and proteins, especially natural peptides/proteins, have been suggested as new anticancer agents to kill cancer cells [9]. Bacteriocins, small peptides synthesized by ribosomes, have a diverse range of antibacterial and anticancer activity and are produced by a broad group of bacteria [10]. Being anticancer and therapeutic events is bacteriocins’ most exciting property [11]. There have been

* Mahmood Saffari

[email protected]

* Davoud Esmaeili [email protected]

1 Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, 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 Anatomical Science Research Center, Kashan University of Medical Sciences, Kashan, Iran

5 Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran

6 Department of Biology, School of Advanced Sciences in Regenerative Medicine, Tehran Medical Sciences Branch,

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Probiotics and Antimicrobial Proteins

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several reports of the anticancer effects of bacteriocins against cancer cells [12–15]. Characteristics of bacteriocins in cancer treatment: A) Bacteriocins are cationic by nature, so can be attached to the membrane of cancer cells, which have a higher negative charge compared to normal cells [16]. B) Cancer cell membrane, due to having a very large number of microvilli compared to a normal cell, allows binding of more anticancer peptides [17]. C) Easy destruction of cancer cell membranes due to having complex membrane flexibility as compared to normal cells [18]. Cancer cells show high levels of apoptosis, and the induction of apoptosis in cells greatly affects them [19].

Another special feature of cationic peptides, including bacteriocins, is their ability to induce apoptosis in tumor cells, which is why they are considered as potential agents in the treatment of cancer [19].

Colicins and enterocins are examples of these bacteriocins. Colicins are toxic peptides of high molecular mass that are made by colicinogenic strains of E. coli and other rel- evant species of Enterobacteriaceae [10]. Various studies have examined the anticancer role of a variety of colicins, such as colicin A, N, E1, E3, and U. It has been shown that these compounds can inhibit cell proliferation and induce apoptosis in different cancer cell lines [10, 20–22].

A large group of bacteria living as microflora in humans and animals’ intestinal tracts is called lactic acid bacteria (LAB) that are potent in producing bacteriocins [23].

Enterococci are members of the LAB family that can produce a bacteriocin called enterocin. Bacteriocins produced by Enterococcus include bacteriocin 35, enterocin A, B, L50A/B, and P, which belong to class II bacteriocins [24]. Several reports suggested that secreted metabolites of Enterococcuses such as enterocins have potential anticancer activity against HT-29, HeLa, Caco2, MCF-7, and AGS cancer cells [12, 13, 25–27]. To validate the potential development of an effective anticancer peptide, the cytotoxicity and apoptosis-inducing effects of enterocin A-colicin E1 on the AGS cells was elucidated in this study.

Methods

In our previous study, the initial designs of fusion peptide consisting of enterocin A and colicin E1 was performed using NCBI site and various software. After the synthesis of this peptide, in the next step, physiochemical characterization, secondary and tertiary structure prediction, and molecular dynamic simulation, and stability confirmation of fusion peptide was done by the in silico method. Cloning and expression in E. coli BL21, confirmation of expression by Western blot- ting, and purification of fusion peptide by Ni-NTA column was performed [28].

Cell Culturing Conditions

In this study, the AGS cell line (human gastric adenocarcinoma, IBRC C10071, Iran) was purchased from Iran National Genetic and Biological Resources Center. Cells were cultured under standard conditions in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-glutamine, and 1% non-essential amino acids. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂. In Vitro Cytotoxicity Assay of ent A‑col E1

AGS cell lines were grown in DMEM with 10% FBS. The growth inhibitory activity of ent A-col E1 fusion peptide on cancer cells was determined by using MTT assay [15].

Briefly, the AGS cells were seeded in a 96-well plate at a density of 5 ± 10³ cells/well and incubated in a CO₂ incubator at 37 °C. After cells reach near 80% confluence, the purified ent A-col E1 fusion peptide was tested at different concentrations (25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 µg/mL) for 24 h and 48 h (control was made with no ent A-col E1 fusion peptide and only cells were grown for all cell lines in this experiment). After incubation the ent A-col E1 peptide was removed from the cells and MTT stock solution (5 mg/mL in phosphate buffer saline or PBS, pH 7.5) was added to each well of 96-well plates and incubated for 4 h at 37 °C. MTT solution was discarded, and DMSO was added to each well;

then, the mixture was gently swirled at room temperature for 10 min to dissolve the blue formazan crystals. Imme- diately readings were collected by using a micro-ELISA plate reader at a test wavelength of 540 nm and a refer- ence wavelength of 595 nm. DMSO was used as a blank in this experiment. The percentage of cell viability was determined based on the formula: % Viability = (optical density of sample/optical density of control) × 100.

Statistical analysis and IC50 calculation performed by GraphPad Prism, version 8.4.3 (GraphPad Software).

Gene Expression Analysis by Quantitative Real‑Time PCR

To assess the impact of ent A-col E1 on the expression of bax, bcl-2, and caspase3, key genes involved in mitochondrial apoptosis, real-time PCR was carried out. Briefly, 5 × 10⁵ AGS cells were treated with 60.4 μg/mL ent A-col E1 for 24 h, and then, the total mRNA was extracted by RNA extraction kit (Favorgen Biotech Corporation, Ping-Tung, Taiwan) by the manufacturer’s instruction. The quality and quantity of extracted mRNA were evaluated by the Nanodrop spectrophotometer (Thermos,

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USA) and electrophoresis on 1.5% gel agarose, respectively.

The related cDNA of mRNA was synthesized using a Favor- gen kit (Favorgen Biotech Corporation, Ping-Tung, Taiwan) by random hexamer, oligo dt primers, and M-MLV reverse tran- scriptase. In the next step, the relative expression of bax, bcl-2, and caspase 3 genes was measured using real-time PCR by the specific primers (Table 1). The gapdh was tested as a house- keeping gene. One microliter of cDNA was amplified in 20-μL mixture reaction solution containing 2X cyber green solution and 10 pmol of described primers. PCR procedure was done by Corbett Rotor-Gene 6000 real-time PCR cycler (Qiagen Cor- bett, Hilden, Germany) with a temperature gradient program included an initial denaturation for 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60

°C for 60 s, and extension at 72 °C for 15 s.

Apoptotic Cells Detection by Flow Cytometry

For this study, AGS cancer cells (1.2 × 10⁵ cells per well) were seeded into a 6-well culture plate and treated with ent A-col E1 (60.4 µg/mL) for 24 h. After incubation, cells were collected by trypsinization and washed twice with PBS (pH 7.2). Apoptosis detection by flow cytometry “Phosphati- dyl Serine Detection” kit (IQ Products, Groningen, the Netherlands) was applied to assay induction of apoptosis in AGS cells according to instructions of the manufacturer.

The cells were resuspended in 1X binding buffer containing 1.4 M NaCl and 25 mM CaCl2 at a concentration of 1 × 10⁶ cells/mL. The treated and un-treated cell suspen- sion was transferred to 1.5 mL Eppendorf tubes. In the next step, 5-μL FITC labeled Annexin V was added to the tubes including 100 μL of cells and was incubated 15 min in dark at 4 °C. After incubation, 1-mL cold binding buffer was added and cells were washed and centrifuged for 5 min at 1500 g. Then, 3-μL propidium iodide was added and incubated 10 min at 4 °C and the cells were analyzed using BD FACS Calibur (BD Biosciences, San Jose, CA, USA).

The percentage of live, apoptotic, and dead cells were calculated by using FlowJo 7.6 software (Tree Star, Inc., Ashland, USA). The experiment was repeated twice, and using the kit, we detected ted viable cells (AnnexinV-PI-), early apoptotic (Annexin V+PI-), late apoptotic (Annexin V+PI+), and necrotic cells (Annexin V-PI+).

Statistical Analysis

Findings from all tests were computed by the one-way ANOVA test by GraphPad Prism, version 8.4.3 (GraphPad Software). A p ˂ 0.05 was statistically considered significant. The statistical analysis of gene expression was performed by Rest 2009 (Qiagen, Germantown, Maryland, USA).

Results

In Vitro Cytotoxicity Assay of ent A‑col E1

The quantitative colorimetric MTT assay was employed to investigate the effect of different concentrations of ent A-col E1 on the viability of AGS cells. The results indi- cate a concentration-dependent decline in oxidoreductase enzymatic activity in AGS cells exposed to ent A-col E1.

As illustrated in Fig. 1, a concentration of ≥ 60.4 μg/mL of ent A-col E1 led to a significant cytotoxic impact on cancer cells after 24-h treatment. Inverted photo-microscopic picture of ent A-col E1 effect on the AGS, after 24 h of incubation at 5% CO₂, is shown in Fig. 2. The non-viable cells are seen floating.

Gene Expression Analysis by Quantitative Real‑Time PCR

To evaluate the effect of ent A-col E1 on the AGS cell apoptosis, the mRNA expression of bcl-2, bax, and cas- pase3 genes was determined by real-time PCR. Bax is a pro-apoptotic gene, and bcl-2 is an anti-apoptotic gene.

As shown in Fig. 3a, the ratio of bax expression was sig- nificantly raised after exposing to tested ent A-col E1 concentration. Moreover, the ratio of expression of bcl-2 was reduced after treating with 60.4 μg/mL ent A-col E1 compared to the negative control (Fig. 3b). The ratio of expression of caspase3 was enhanced after treating with 60.4 μg/mL ent A-col E1 compared to the negative control (Fig. 3c). while the gapdh gene expression had no change before and after treatment.

Table 1 The sequence of primer

pairs Gene Forward Reverse

Bax 5′-CAT CTT CTT CCA GAT GGT GA-3′ 5′-GTT TCA TCC AGG ATC GAG CAG-3′

Bcl-2 5′-GAG ACA GCC AGG AGA AAT CA-3′ 5′-CCT GTG GAT GAC TGA GTA CC-3′

Caspase 3 5′-CAA ACT TTT TCA GAG GGG ATCG-3′ 5′-GCA TAC TGT TTC AGC ATG GCAC-3′ Gapdh 5-GAA GGT GAA GGT CGG AGT CA-3 5-GAA GAT GGT GAT GGG ATT TC-3

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Determination of Apoptosis by Flow Cytometry Assay

Induction of apoptosis in AGS was observed by using annexin V-FITC/PI flow cytometric assay. The AGS cells were incubated with ent A-col E1 (60.4 µg/mL) for 24 h,

and results were observed that 20.1, 26.0, and 4.60% of AGS cells were present in dead, late apoptotic, early apoptotic phases, and 49.2% of AGS cells were present in live phase respectively compared to control cells (untreated). In control, the majority (68%) of cells were viable and non- apoptotic (Fig. 4).

Fig. 1 The effect of ent A-col E1 on cell viability of AGS cells. a, b After 24-h treatment.

c, d After 48-h treatment. Error bars represent the standard deviation of the mean

Fig. 2 Inverted photomicro- scopic picture of ent A-col E1 effect on the viability of the AGS cell line. a Untreated AGS cell line, b treated AGS cell line, after 24-h of incubation at 5% CO₂ and observed at × 20 magnification. The non-viable cells are seen floating (arrows)

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Fig. 3 The effect of ent A-col E1 on apoptosis-related genes. a bax gene amplification curve using RT-PCR technique: As outlined in the figure, the Ct levels of the bax gene were different before and after treatment (Ct mean before treatment = 22 and Ct mean after treatment = 18). b bcl-2 gene amplification curve using RT-PCR technique: As outlined in the figure, the Ct levels of the bcl-2 gene were different before and after treatment (Ct mean before treatment = 21.5 and Ct mean after treatment = 18). c Caspase 3 gene amplification

curve using RT-PCR technique: As outlined in the figure, the Ct levels of the caspase3 gene were different before and after treatment (Ct mean before treatment = 21.5 and Ct mean after treatment = 21).

However, there was no change in the Ct levels of the gapdh gene before and after treatment (Ct mean before treatment = 20.5 and Ct mean after treatment = 20.5). The gapdh gene expression had no change before and after treatment. All graphs represent mean ± SEM.

*P < 0.05; **P < 0.01; ***P < 0.001 compared to untreated group

Fig. 4 Evaluation of apoptosis induction effect of ent A-col E1 on AGS cells by Flow cytometry analysis. a Treated AGS cells with ent A-col E1. b) Control, non-treated AGS cells; Q₄: live cells (Annexin

V- /PI-); Q₃: early apoptosis (Annexin V+/PI-); Q₂: late apoptosis (Annexin V+/PI+); Q₁: necrotic cells (Annexin V-/PI+)

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Discussion

Among non-communicable diseases in the world, cancer is the most important cause of death [29]. Although many studies have been conducted on the antibacterial properties of bacteriocins, our knowledge of their anticancer effects on the types of cancer cells is not sufficient. After design- ing and analyzing the stability of ent A-col E1 peptide and its expression in the host bacterium [28], we examined its anticancer properties using MTT, real-time PCR, and flow cytometry methods. This is the first time that research has been done on the anticancer activity of a combination of enterocin A and colicin E1 against one of the cancer cell lines. Cell proliferation inhibition by various bacteriocins, including pyocin, pediocin, microcin, and colicins, has been proven in different cancer cells [30–33]. The ability of NAD(P)H-dependent cellular oxidoreductase enzymes to decrease MTT to formazan can be considered a reflec- tion of the rate of viable cells present [34]. The results of this study showed that ent A-col E1 can kill 50% of cells at a concentration of 60.4 µg/mL after 24 h. In line with our results, Ankaiah et al. [13] demonstrated that Ent A produced by strain Enterococcus faecium had anticancer activity against human colon, gastric, and cervical cancer cells. They stated that Ent A was able to kill 50% of cells at a concentration of 120 µg/mL after 48 h. In a study, the effects of a variety of colicins, including A, U, and E1 on 11 cancer cell lines, were examined. In the case of colicin E1, the results showed that this bacteriocin had an inhibitory effect on most cell lines and only the colon carcinoma cells were insensitive to this bacteriocin [20].

One study reported that Ent P had selective toxicity against colorectal cancer cells (IC50: 320 µg/mL) [26]. In another investigation conducted by Paiva et al. [35], the IC50 of nisin against breast cancer and hepatic cancer cell line was reported 105.46 and 112.25 μM, respectively, while our results show that ent A-col E1 at a lower concentration can kill cancer cells. Also, Fuska et al. [36] found that the proliferative activity of mouse P388 leukemia cells could be inhibited by colicin E3 in a dose- and time- dependent assay. This cell line was tested by colicins D, A, and E2, and the inhibitory effects of these bacteriocins on the viability of this cell line were determined by reducing the number of cells. The process that leads to the synthesis of functional products using data stored on DNA is called gene expression [37]. Apoptotic and anti- apoptotic genes produce compounds and proteins which are capable of apoptosis regulation. Any disturbance in the expression of these genes may be involved in the car- cinogenic pathway [38]. The susceptibility rate of cancer and normal cells to the apoptosis induction depends on the balance of pro-apoptotic and anti-apoptotic proteins [39].

In this study, the real-time PCR technique was used to the investigation of ent A-col E1 effect on the expression of bcl-2, bax, caspase 3, and in the AGS cell line. Bcl-2 is an anti-apoptotic protein that prevents programmed cell death without affecting cell proliferation. Bax is a pro-apoptotic compound that induces programmed cell death via its heterodimerization and homodimerization with bcl-2 and other members of the bcl-2 protein family [40]. Caspase-3, a family of cysteine proteases, works downstream of bax/

bcl-2 control and has an important role in the apoptosis process. Caspases are activated in two ways: the intrinsic path (dependent on mitochondria) and the extrinsic pathway (dependent on death receptors). In the intrinsic pathway, with the relative change of pro-apoptotic and anti-apoptotic mediators, mitochondrial permeability to cytochrome C increases, and with its release, apoptosome is formed and activates Caspase 3 and 9 [41]. Cytotoxicity and apoptotic attributes of bacteriocins have been veri- fied previously; this study assessed the effect of ent A-col E1 on the mitochondrial apoptosis pathway through the expression analysis of three key genes, bax, bcl-2, and caspase 3, in the intrinsic apoptosis pathway. Herein, we observed that ent A-col E1 can increase bax expression rate and reduce bcl-2 expression rate in the mRNA levels.

The increase of the ratio of bax to bcl-2 is the marker of apoptosis activation in the cells. Similar to the results of our study, a study by Ahmadi et al. [42] on colon cancer cells showed that the tested bacteriocin was able to induce apoptosis by increasing bax expression and decreasing bcl-2 expression. In one study, nisin ZP was reported to have activated caspase 8 but had no efficacy on caspase 3 in cancer cells [43]. Our research has shown that ent A-col E1 has a favorable effect on caspase 3 and increases its expression and subsequently activates apoptosis from the intrinsic path. Ankaia et al. [15] showed in a study that enterocin B produced by Enterococcus faecium could have anti-cancer activity against Hela, gastric cancer, and HT-29 cell line and fluorescent microscopic observations also provided the apoptotic morphological changes such as membrane blebbing, nuclear fragmentation, and apoptotic body formation. Cellular changes involved in the process of cell death include loss of symmetry of membrane phospholipids during the early stages of apoptosis.

In most living cells, phosphatidylserine is located in the cytoplasmic side of the cell membranes by the aminophos- pholipid translocase activity. With the onset of apoptosis, the transfer of phosphatidylserine from the inside to the outside of the membrane is a common occurrence [44, 45]. This event causes phosphatidylserine to be bound to Annexin V-FITC (in the presence of calcium). However, in the necrosis process, phosphatidylserine is also available due to the destruction of membrane integrity, except that

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in necrosis, PI binds to cellular DNA in cells whose membranes are destructed due to death. Therefore, apoptotic cells and necrotic cells can be distinguished [44, 46]. In a study by Arunmanee et al. [47], the assay of the induction of apoptosis by colicin N showed acceptable results from the effect of colicin N in inducing programmed cell death in treated human lung cancer cells compared with the control group. Also, a report of the effect of enterocin A in the induction of apoptosis in Hela and HT29 cells showed a higher percentage of apoptotic cells in the test group than control group cells [13]. In one investigation, flow cytometry showed that the pore-forming colicins A, U, and E1 affected the 5 cell lines. Colicin E1 was able to inhibit 50%

of fibrosarcoma HS913T cells, while standard fibroblast MRC5 cell line was less sensitive (11%) than the s tumor cells. In three of the lines, colicins A and E1 enhanced the number of cells in apoptosis (by 7–58%). Colicin A increases the number of cells in the G1 phase in the MRC5 cell line and tumor HS913T line having the mutagenesis in p53 gene (12-22 %). This increase in MDA-MB-231 cell line was equivalent to 5%. Subsequently, in cell treatment with colicin E1, their cycle was changed just in the MCF7 cell line with p53, while the ratio of cells in the G1 phase enhanced by 26%. After treatment of cells with colicin A, the rate of apoptosis increased by 7–28%, except for the HS913T cell line, which increased by 16%. Colicin E1 was also able to increase apoptosis by 58% in the MCF7 cell line and by 14% in the HS913T cell line [20].

In our study, the concentration of ent A-col E1, equivalent to the IC₅₀ obtained in the MTT test, was considered and the cells were exposed to peptide for 24 h. The results showed that apoptosis was induced in about 30% of treated cells, of which 26% were late apoptotic and 4% were early apoptotic cells. Compared to control group cells that did not receive any treatment, the number of living cells was lower and the percentage of apoptotic cells was higher. These favorable findings suggest that ent A-col E1 can induce apoptosis in cancer cells and destroy these cells.

In conclusion, our outcomes revealed that ent A-col E1 treatment has cytotoxic effects on the AGS cell lines and induces apoptosis by increasing the bax/bcl-2 ratio in mRNA levels. Flow cytometry results also show that this peptide can kill cancer cells by inducing apoptosis. These findings suggested that ent A-col E1 can be a good candidate for cancer therapy. However, to clarify the precise mechanisms of ent A-col E1 function, further researches are required.

Author Contribution HF and DE designed and performed experiments and analyzed data. HF and MS wrote the manuscript with support from RM, MS, and JM.

Funding This research was supported by the Kashan University of Medical Sciences (Grant no. 97149).

Data Availability All data analyzed during this study are included in this published article.

Declarations

Disclaimer The funding agency had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Competing Interests The authors declare that they have no competing interests.

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