Volume 26, Issue 4 (Autumn 2020)                   Intern Med Today 2020, 26(4): 364-381 | Back to browse issues page

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Yavari M, Ahmadizadeh C. Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells. Intern Med Today 2020; 26 (4) :364-381
URL: http://imtj.gmu.ac.ir/article-1-3414-en.html
1- Department of Microbiology, Ahar Branch, Islamic Azad University, Ahar, Iran.
2- Department of Microbiology, Ahar Branch, Islamic Azad University, Ahar, Iran. , ch_ahmadizadeh@iau_ahar.ac.ir
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1. Introduction
robiotics are dietary supplements consisting of living microorganisms that, after digestion, improve the health of the host and have many benefits [1, 2]. They have great therapeutic efficacy for humans, for instance, they are involved in the treatment of diarrhea caused by irritable bowel syndrome and colorectal cancer [3, 4]. The role of probiotics in modulating the immune response may be due to the effect of these beneficial microorganisms on the secretion of some cytokines and the expression of genes involved in innate immunity [56]. The type of probiotic effect depends on the production of their biochemically active metabolites, molecules present on the surface of these microorganisms, or their secreted components [7, 8]. In general, probiotics produce a variety of substances, including short-chain fatty acids such as acetate, lactate, succinate, butyrate, H2O2, and bacteriocin compounds that act on both Gram-positive and Gram-negative bacteria [9, 10]. Defensins are cysteine-rich antimicrobial cationic peptides that are part of innate and acquired immune systems because they can invoke monocytes and neutrophils, and are effective in inducing the production of various cytokines [11]. Defensins are produced by different groups of cells, including neutrophils and intestinal secretory cells, and show high antimicrobial activity in vitro against Gram-negative and Gram-positive bacteria, fungi, and viruses [12]. Among these compounds, human β-defensin 2 (hBD-2) has been widely studied because of its effects in strengthening the innate and acquired immune systems and on neutrophils which are the first line of defense in the immune system [12]. Probiotics, by modulating the intestinal microbial flora, exert beneficial effects on the health of the host, and are generally from human sources and considered non-pathogenic. The mechanism of action of probiotics is not fully understood, but some have been proposed to justify their preventive and therapeutic effects in human diseases [13]. Studies have shown that probiotics play an effective role in fighting against cancer by affecting the digestive enzymes of animals and humans, inhibiting carcinogens in the body and in vitro conditions, and suppressing mutations, cancer inducers, and tumors in laboratory animals [1415]. 
Cancer is a genetically heterogeneous disease and the second leading cause of death in the world after cardiovascular diseases. One of the most common types of gastrointestinal cancer in Iran is colorectal cancer which is the third most common cancer in men and the fourth in women [16]. In vitro experiments have shown that probiotics suppress primary neoplastic ulcers and colorectal cancer tumors in rat models [17]. One of the functional mechanisms of probiotics, including Lactobacilli, is the anti-proliferative property of cancer cells by the induction of apoptosis [18]. Therefore, probiotic therapy can treat cancer by two mechanisms: directly inhibiting the growth of pathogenic bacteria by the probiotics, and indirectly inhibiting the secretion of β-defensin and antimicrobial peptides by human cells [19]. BAX is a proapoptotic gene that causes cell death and BAX protein induces programmed cell death [20]. 
Under normal conditions, BAX is cytosolic in the body, mainly through retransferring from mitochondria to the cytosol by BCL2L1/Bcl-xL, and prevents the accumulation of toxic BAX levels in the mitochondrial outer membrane. Under stress, a conformation change occurs which transfers it to the mitochondrial membrane, leading to the release of cytochrome c, which in turn causes apoptosis [21]. Ogushi et al. showed that several pathogenic bacteria of probiotics, including Lactobacilli, would cause innate immunity by inducing defensin. Pathogenic strains of Salmonella spp. and Helicobacter pylori were also reported to stimulate hBD-2 expression [22]. Bacterial components of probiotic Lactobacilli induce the expression of defensins and thus affect the antibacterial activity of the intestine. Therefore, the induction of defensins by probiotics, including Lactobacilli may be a new therapeutic strategy to strengthen innate defense mechanisms [23]. This study aimed to investigate the effect of Lactobacillus casei on the expression of hBD-2 and BAX genes in HT29 cancer cells and to determine the possibility of hBD-2 secretion from HT29 cells.
2. Materials and Methods
The present study was conducted at the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in Iran in 2017. Cell line HT29 was obtained from the Pasteur Institute of Iran and bacteria were obtained from the Persian Type Culture Collection Center. To accurately identify the used Lactobacillus species, they were first identified using the molecular method. The MRS broth medium containing the bacteria was transferred to the tubes and centrifuged at 10000 rpm for 5 minutes. Then, 800 μL of lysis buffer was added to the tubes. The tubes were then placed in a water bath at 85°C for 30 minutes. Next, the tubes were placed in a freezer at -70°C for 10 minutes and then placed back in the water bath, after adding 700 μL of chloroform-isoamyl alcohol solution to each tube. The tubes were then centrifuged and the supernatant of the tubes containing the DNA was transferred to new tubes. Cold isopropanol, twice the sample amount, was added to the new tubes, which were placed in a -70°C freezer overnight. The samples were then centrifuged at 12000 rpm for 5 minutes and the supernatant was poured out. Then, 50 mL of DNase-free distilled water was added to each tube. After the DNAs were extracted, the primers were first evaluated and ordered for synthesis.
To investigate the lethal effect of Lactobacillus casei cell extract on cell line HT29, a colorimetric assay (MTT kit, Sigma Aldrich, Germany) was used. Around 0.025, 0.05, 0.1, 0.25, 0.5, and 1 mL/µg of killed bacterial cell extract were treated on HT29 cell line at 24-hour intervals. After that, the contents of 96-well plates were carefully removed and methylthiazolyldiphenyl-tetrazolium bromide (MTT) dye was added and kept for 4 hours in 5% CO2 at 37°C. Then, MTT dye was isolated and formazan crystals were produced and dissolved in isopropanol by living cells.
DAPI staining
To directly investigate the effects of killed Lactobacillus casei extract and living bacteria on HT29 cells, after cell culture and cell proliferation in a 6-well plate, different concentrations of killed bacteria, as well as live bacteria, were poured 20 times the cell size into each 6-well plate along with mesh wells. The bacteria were then treated for 24 hours, fixed by adding 60 mL of 4% paraformaldehyde to each well, and then the cell supernatant was poured out. The cells inside each well were washed with PBS 3 times, and the cells inside each well were permeabilized with 0.1% Triton X-100 for 10 minutes.
RNA extraction
All treated and untreated RNAs were extracted using an RNA extraction kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions and its concentration was measured by a spectrophotometer (NanoDrop ND 1000, Thermo Fisher Scientific, USA).
cDNA synthesis
The complementary DNA molecules were fabricated with a kit (Fermentas, Germany). The used primers were designed by oligo 5 software and entered into Primer-BLAST online tool at the NCBI website (www.ncbi.nlm.nih.gov) whose information is presented in Table 1.

The primers were synthesized by Takapouzist Company.
Real-time polymerase chain reaction
Real-time Polymerase Chain Reaction (PCR) in the iQ5 detection system (Bio-Rad, USA) was performed in triplicate using a thermal cycler, the information of which is available in Table 2

In real-time PCR tubes, 1 μL of cDNA and 19 μL of SYBR green master mix containing 1 μL of forward primer (0.2 micromolar), 1 μL of reverse primer (0.2 micromolar), 7 μl of DEPC, and 10 μL of master mix 1x real-time were poured. Then, the tubes were inserted in the real-time PCR device.
Gene expression data analysis
The Cycle Threshold (CT) was determined for each sample. The expression level of each sample was calculated for the relevant genes and GAPDH gene using CT values. The expression ratio of the treated samples to that of control samples normalized by the expression of the GAPDH gene was calculated using the formula provided by Pfaffl (Formula 1): 

3. Results
As shown in Figure 1, the sharp band of Lactobacillus casei DNA is observed in agarose electrophoresis.

The quantity and quality of DNA were evaluated using the NanoDrop device. All extracted DNA samples had high quality and quantity, with the highest absorption peak at 260 nm. The highest absorption wavelengths of RNA and protein were 230 and 280 nm, respectively. Cancer cells were treated in two ways. In the first method, the cells were treated for 4 hours by co-culturing in plates containing 0.1-μm pore-size transfer wells and then the transfer wells containing bacteria were removed. The second method was to kill the bacteria by heating and cooling them continually and treating the cells. In both methods, 12 and 24 hours were considered. After treating the cells with Lactobacillus casei, the cells were observed to move towards apoptosis. The probability of hBD-2 gene expression in cells was zero, although the cells were dying. Co-cultured colon cancer cells with nuclear staining indicate apoptosis of these cells. However, an increase in the expression was observed in the molecular study.
After cell treatment, DAPI staining was performed to evaluate cell apoptosis. Figure 2 shows stained and unstained cells imaged by light and fluorescence microscopy. 

Qualitative evaluation of the extracted RNA
To evaluate the changes in gene expression, after HT29 treatment with Lactobacillus casei and its cell extract, RNA was extracted from the treated HT29 cells. Then, the RNA concentration of the samples was measured in the NanoDrop device. To evaluate the quality of the extracted RNAs, the samples underwent electrophoresis on agarose gel (Figure 3). 

Real-time PCR results
To perform real-time PCR, cells in 6-well plates with 50% inhibitory concentration (IC50) of culture medium were treated with Lactobacillus casei bacteria and its cell extract (Figure 4).

The expression levels of BAX and hBD-2 genes in the co-culturing of HT29 colon cancer cells with Lactobacillus casei are shown in Figures 5 and 6, which were treated for 12 and 24 hours, and the gene expression changes were measured by real-time PCR. 

The hBD-2 gene expression showed a significant increase in 24 hours compared to the control group (P= 0.023), while BAX gene expression did not show significant changes in 12 hours compared to the control group (P= 0.37).
4. Discussion
The results of this study showed that, by increasing the expression of the hBD-2 gene under the influence of live and killed extracts of Lactobacillus casei, the bacteria showed the ability to stimulate the production of hBD-2 from colon cells, and the live bacteria induced apoptosis in cancer cells; however, the expression of BAX gene did not show a significant change in the first 12 hours. The mechanism of action of probiotics is not fully understood, but some have been proposed to justify their preventive and therapeutic effects in human diseases, including the production of bacterial inhibitory compounds, strengthening the immune system, and stimulating the production of antimicrobial peptides from human cells [13]. Therefore, probiotic therapy can treat cancer by two mechanisms; first by directly inhibiting the growth of pathogenic bacteria by the probiotics, and second by indirectly inhibiting the secretion of hBD-2 and antimicrobial peptides by human cells. Antimicrobial peptides contain 10 to 150 amino acids whose antimicrobial function depends on various factors such as pH, peptide abundance, and salt concentration [19]. They are normally produced from different parts of the body such as skin, intestinal mucosa, oral mucosa, respiratory tract, eyes, and mesenchymal stem cells in bone marrow, but the expression of these proteins is inductively increased in infectious conditions such as wounds or inflammation. These peptides mainly target microbial membranes; hence, because of their applicability, they are new generation antibiotics [24]. A group of these peptides is defensins, which are divided into three groups: α, β, and θ. Beta defensins are one of the largest members of this family, and their copies are found in many vertebrates, invertebrates, and plants. Most defensins have six cysteine residues, which are not essential for antimicrobial activity but are highly resistant to bacterial proteolysis. Defensins are abundant in cells and tissues that are involved in host defense against microbial infections [25]. The BAX gene is a unique point for the intrinsic apoptosis signaling pathway. This pathway is initiated by various stimuli such as cytokine deprivation and cytotoxic stress and eventually induces programmed cell death [26]. Soltan Dallal et al. [27] examined the effect of two probiotic bacteria Lactobacillus acidophilus and Lactobacillus casei on the TLR2 and TLR4 genes expression in HT29 cells infected with Salmonella Enteritidis. Their results showed that after the treatment of healthy and non-infected HT29 cells with each of the two probiotic bacteria, the expression of TLR2 and TLR4 genes increased significantly. In contrast, the expression of these two genes in infected cells before and after treatment with each of the probiotic bacteria was significantly reduced. Karimi Ardestani et al. [28]. In molecular detection of heat-killed probiotic bacteria and the study of apoptosis induction on colon cancer HT-29 cell line, showed that Lactobacillus brevis reduced survival and proliferation of HT-29 colon cancer cells in a time- and dose-dependent pattern. The highest cytotoxic effect was related to dilution of 1000 μg/mL at 72 hours. The bioavailability depending on dose and time in HT-29 and HEK-293 cells was estimated to be 23% and 50%, respectively. DNA fragmentation test also confirmed the induction of apoptosis by heat-killed bacteria. 
Sattari and Ahmadizadeh [29] studied the expression of PTEN and AKT1 genes in the co-culturing of the HT29 colon cancer cell line with Streptococcus thermophilus. Their results showed that Streptococcus thermophilus decreased the expression of the AKT1 gene and increased the expression of PTEN and led the cancer cells to apoptosis. MTT test showed that the concentration of OD (0.05) had the highest lethality in 4 hours [29]. According to Taverniti and Guglielmetti [30], bacterial strains of Lactobacillus bulgaricus and Lactobacillus casei probiotics reduce the viability of HT29 and CACO-2 cancer cells. Their results showed that the cellular components of heat-killed bacteria, such as the peptidoglycan cell wall underlying cytoplasmic membrane had cytotoxic effects on cancer cells, which is consistent with our study. Yan et al. [31] showed that solution compounds secreted by Lactobacillus casei and Lactobacillus rhamnosus induce apoptosis in monocytic leukemia cells. As a result, probiotics can be considered as a safe agent for the fi ght against cancer with no side effects which is consistent with our study. Baldwin et al. [32] showed that Lactobacillus acidophilus and Lactobacillus casei can increase apoptosis induction in the LS315 carcinoma cell line and can be used as adjuvants with chemotherapy. This finding is in agreement with our results. 
Ali Asgari et al. [33] reported the effects of Lactobacillus casei cell extract on increasing the expression of the BAX gene and decreasing the expression of the BCL2 gene in the colon cancer cell and induction of programmed cell death. This finding is also consistent with our Findings. Abdalan et al. showed that aqueous extract of Quercus infectoria leaf has an anticancer effect on colon cancer cell line HT29, and can induce apoptosis by increasing the expression of BAX gene in HT29 cells which is consistent with our results [34].
5. Conclusion
Despite the stimulation of hBD-2 production by killed extract of Lactobacillus bacteria, the extract of these bacteria can be used to stimulate cancer cells to produce hBD-2, inhibit pathogens, prevent the stimulation of cellular proliferation signaling, and fight antibiotic-resistant bacteria.
Ethical Considerations
Compliance with ethical guidelines

This study obtained its ethical approval from the Research Ethics Committee of the Islamic Azad University of Ahar Branch (Code: 5984).
This study was extracted from the Master thesis of the first author approved by the Islamic Azad University of Ahar Branch. 
Authors' contributions
Both authors were equally contributed in preparing this article. 
Conflicts of interest
The authors declared no conflict of interest.

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Type of Study: Original | Subject: Basic Medical Science
Received: 2019/10/24 | Accepted: 2020/04/25 | Published: 2020/10/1

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