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The anti-adhesion effect of nisin as a robust lantibiotic on the colorectal cancer cells
Hesam Soleimanifar1, Hamideh Mahmoodzadeh Hosseini1, Sadra Samavarchi Tehrani2, Seyed Ali Mirhosseini1
1 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
2 Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| Date of Submission | 25-Aug-2021 |
| Date of Acceptance | 09-May-2022 |
| Date of Web Publication | 28-Apr-2023 |
Correspondence Address:
Dr. Seyed Ali Mirhosseini
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Vanak Sq. Molasadra St., Tehran
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_267_21
Background: Bacteriocins are a type of antimicrobial peptide that are produced by probiotics. They have been studied as possible therapeutic drugs and have been used to suppress bacterial development in foods. Nisin is a potent bacteriocin having the anti-microbial and anti-cancer characteristics produced by Lactococcus lactis. The aim of the present paper is to evaluate the influence of Nisin on cell adhesion and its two related genes, mmp-2 and mmp-9, in the colorectal cancer cell line.
Materials and Methods: For this purpose, HT-29 cells were treated with various concentrations of Nisin and the cell cytotoxicity, cell adhesion, and gene expression were evaluated using the MTT assay, cell adhesion assay, and real-time PCR.
Results: Our findings showed that 32 to 1024 μg/ml of Nisin resulted in a significant reduction in cell viability (P < 0.05). Furthermore, 128 and 256 μg/ml of Nisin significantly reduced the cell adhesion, and mmp-2 and mmp-9 gene expressions (P < 0.05).
Conclusion: Our findings suggested that Nisin could prevent metastasis and cancer progression.
Keywords: Bacteriocins, colorectal cancer, lactococcus lactis, metastasis, nisin
How to cite this article:
Soleimanifar H, Mahmoodzadeh Hosseini H, Samavarchi Tehrani S, Mirhosseini SA. The anti-adhesion effect of nisin as a robust lantibiotic on the colorectal cancer cells. Adv Biomed Res 2023;12:113 |
How to cite this URL:
Soleimanifar H, Mahmoodzadeh Hosseini H, Samavarchi Tehrani S, Mirhosseini SA. The anti-adhesion effect of nisin as a robust lantibiotic on the colorectal cancer cells. Adv Biomed Res [serial online] 2023 [cited 2023 Jun 7];12:113. Available from: https://www.advbiores.net/text.asp?2023/12/1/113/375313 |
| Introduction | |  |
Cancer is an intricate disease that encompasses more than 100 disorders, each with its own set of symptoms. Despite significant breakthroughs in cancer treatment, many cancer therapy strategies have negative side effects for patients.[1] Colorectal cancer (CRC) occurs when the cells that line the colon or rectum become aberrant and proliferate out of control. Regrettably, some CRCs may go undetected for years without causing any symptoms.[2],[3] Mortality rates for CRC vary dramatically around the world. With 1.65 million new cases and nearly 835,000 deaths in 2015, CRC is the third most frequent cancer in men and the second most prevalent disease in women.[4] CRC is caused by a combination of genetic and lifestyle factors. Genetic variables, on the other hand, are not particularly impressive.[5] There is accumulating evidence that mutations in genes that control cell proliferation are involved in CRC initiation and progression.[6] The tumor may create secondary tumors in other areas when cells detach from the tumor and invade adjacent tissues, a process known as metastasis. Preventing and combating metastases is the best strategy for CRC treatment. The spread of this type of cancer is still a severe issue, and many people die as a result of metastatic dissemination.[7] Recent studies have focused on the use of microbial metabolites, probiotics, and toxins in cancer treatment.[8],[9] Probiotics are considered live microbial species that are now being researched in the treatment of a variety of diseases, especially cancer.[10],[11] Bacteriocins are a type of antimicrobial peptide that are produced by probiotics. They have been studied as possible therapeutic drugs and have been used to suppress bacterial development in foods.[12],[13] Nisin is a potent bacteriocin having the anti-microbial and anti-cancer characteristics produced by Lactococcus lactis.[14] Nisin is not active against gram-negative bacteria, but liposomes of gram-negative bacteria. Nisin's anticancer effects on cancer cells have been studied in a limited number of research.[15],[16] Nisin has recently been studied for cancer cell growth prevention and has been shown to cause selective apoptosis, cell cycle arrest, and inhibit cell proliferation in HNSCC cells.[17] It is an anion carrier because it can generate pores in cells, causing the release of ions, amino acids, and ATP. Calcium plays a crucial role in apoptosis, and Nisin may be mediated by changes in intracellular calcium levels.[18] The inherent features of tumor cells and the reaction of tissue invasion are two processes of metastasis. In addition, it inhibited cell growth in part by preventing cell cycle arrest caused by cdc2 phosphorylation.[19] Matrix metalloproteinase (MMPs) is a group of proteins that are momentous in cancer progression and involved in different events such as metastases and cell viability. MMP2 and MMP9 are the two main members of this family.[20]
The goal of this study was to see how Nisin, a strong lantibiotic and metabolite generated from probiotics, affected the metastatic index, cell adhesion, and changes in the expression of some key genes in the metastatic, including mmp2 and mmp9, in CRC cells.
| Materials and Methods | | |
Cell culture
The HT29, as a human colorectal adenocarcinoma cell line, was obtained from the Pasture Institute (Tehran, Iran). Cells were grew in RPMI 1640 comprising 10% FBS, penicillin/streptomycin (100 U/ml/100mg/ml), 40 μg/ml gentamicin, and L-glutamine (0.3 g/L) at 37°C and 5% CO2. All culture mediums and reagents were prepared by Ideh Zist Company (Tehran, Iran).
MTT assay
To survey the impact of Nisin on the proliferation of HT 29 cells, MTT assay was performed. In brief, 8 × 103 cells were transferred to each well of a 96-well plate containing 100 μl complete medium. After 24 h, the cells were treated with different concentrations of Nisin including 8, 16, 32, 64, 128, 256, 512, and 1024 μg/ml. After 24 h, the medium of each well was replaced with the 100 μl fresh medium without FBS, and then 10 μl of MTT (5 mg/ml) (Sigma-Aldrich, Germany) was added to each well and incubated at 37°C and 5% CO2 for 4 h. After that, the medium of each well was removed and 100 μl Dimethyl sulfoxide was added and the optical density of each well was recorded at 570 nm using ELISA reader. All tests were done three times. The PBS was tested as a negative control.
Adhesion assay
To assess the adhesion of HT29 cells, the protocol of Pereira et al.[21] was used. In brief, approximately 5 × 104 cells were exposed to four discrepant concentrations of Nisin including 32, 64, 128, and 256 μg/ml for 1 h and then transferred to a 96-well plate and incubated at 37°C and 5% CO2 for 3 h. In the next step, to remove un-adherent cells, the plate was washed twice with PBS. The attached cells were fixed using cold methanol for 5 min. The cells were stained with 1% Toluidine blue in sodium tetraborate 1% for 5 min. After washing with PBS, the colored cells were dissolved in SDS 1% for 20 min at 37°C and the optical density of each well was recorded at 540 nm using an Eliza reader. All tests were done three times. The PBS was tested as a negative control.
Gene expression analysis
To evaluate the effect of Nisin on the expression of mmp2 and mmp9 genes, a real-time PCR method was done. In brief, 5 × 106 HT29 cells were exposed to four discrepant concentrations including 32, 64, 128, and 256 μg/ml for 24 h. To isolate total mRNA, the RNX-Plus kit (Cinnagen, Iran) was utilized. After confirming the quality and quantity of mRNA by the agarose gel electrophoresis and spectrophotometer, respectively, 1 μg of each mRNA sample was used to synthesize cDNA by cDNA synthesis kit (Takara, Japan) containing two universal primers, random hexamer and oligo dT primers, and M-MLV reverse transcriptase in accordance with manufacture's guideline. In the next step, the relative expression of mmp2 and mmp9 genes was investigated by real-time PCR. [Table 1] showed the specific primers for mmp2 and mmp9 genes. β-actin gene was analyzed as a housekeeping gene. In brief, 1.5 μl of each cDNA was added to a mixture reaction containing 2X cyber green solution and 10 pmol of specific primer with a final volume of 20 μl. The thermal program was executed by Corbett Rotor-Gene6000 real-time PCR cycler (Qiagen Corbett, Hilden, Germany) as 3 min for the initial denaturation step at 94°C, 38 cycles of 15 s at 94°C, 30 s at annealing temperature, and 40 s at 72°C. The relative expression of mmp2 and mmp9 was computed by Rest 2009 based on 2-ΔΔCT (Qiagen, USA).
Statistical analysis
To calculate the discrepancy between test and control groups, a one-way ANOVA test was carried out. The analysis was done by SPSS Version 11 software (SPSS, Chicago, IL, USA). P values < 0.05 are regarded statistically significant.
| Results | | |
Cytotoxicity effects
To investigate the impacts of Nisin on the proliferation of HT29 cells, an MTT assay was performed. As shown in [Figure 1], 32, 64, 128, 256, 512, and 1024 μg/ml of Nisin resulted in significant reduction in the cell viability of HT29 cells (71.74, 68.72, 62.21, 49.59, 38.85, and 31.74%, respectively) compared to the negative control group (100%) (P < 0.05). | Figure 1: The cell viability percentage of HT29 cells after exposure to different concentrations of Nisin. 32, 64, 128, 256, 512, and 1024 μg/ml of Nisin caused to significant decrease in the cell viability (71.74, 68.72, 62.21, 49.59, 38.85, and 31.74%, respectively). (*) and (**) indicate significant results with P values less than 0.05 and 0.01, respectively. Neg Cont means the negative control group
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Cell adhesion analysis
To identify the interference properties of Nisin on the cell attachment, an adhesion assay was carried out. As illustrated in [Figure 2], treating HT29 cells with 32, 64, 128, and 256 μg/ml of Nisin significantly attenuated the percentage of attached cells (88.9, 80.7, 75.7, and 70.43%, respectively) in comparison with the negative control (100%). Only 128 and 256 μg/ml of Nisin significantly decreased the cell adhesion (P < 0.05). | Figure 2: The percentage of attached HT29 cells after being treated with different concentrations of Nisin. A decrease in cell attachment was significantly seen at 128 and 256 μg/ml of Nisin (75.7 and 70.43%, respectively). Star (*) indicates significant results with P values less than 0.05. Neg Cont means the negative control group
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Gene expression analysis
[Figure 3] illustrates the mRNA expression pattern of mmp2 and mmp9 genes (part (a) and (b), respectively) after treatment with Nisin. Findings from the real-time PCR assay revealed that the gene expression ratio of mmp2 was 0.87, 0.7, 0.559, and 0.36-fold after exposure to 32, 64, 128, and 256 μg/ml, correspondingly. The level of mmp2 expression was significantly attenuated in the cells treated with 64, 128, and 256 μg/ml Nisin relative to untreated cells (P < 0.05). Furthermore, the expression of mmp9 gene were remarkably reduced after exposure to 128 and 256 μg/ml Nisin (0.626 and 0.518-fold, respectively) compared to untreated cells (P < 0.05). | Figure 3: Relative expression of mmp-2 gene (a) and mmp-9 gene (b) in HT29 cells at 32, 64, 128, and 256 μg/ml Nisin. (a) Level of mmp2 expression was significantly reduced after treating with 64, 128, and 256 μg/ml Nisin (0.7, 0.559, and 0.36 fold, respectively) compared to untreated cells. (b) Level of mmp9 expression significantly reduced after exposing to 128 and 256 μg/ml Nisin (0.626 and 0.518 fold, respectively) compared to untreated cells. Star (*) indicates significant results with P values less than 0.05. Neg Cont means the negative control group
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| Discussion | | |
Metastatic cancer is an aggressive and lethal type of cancer that in spite of the great efforts of researchers, there is no proper treatment for it. Current therapeutic strategies and the synthetic chemical drug had no proper efficiency to prevent metastatic events.[22] For solving this problem, some studies offered compounds based on plants or bacteria that some of these compounds are useful to treat cancer.[23],[24] In addition, it is believed that probiotics and their metabolites directly or indirectly impact the signaling with the anti-cancer properties.[25] Nisin is the FDA-approved bacteriocins with anti-cancer properties.[16] In our previous study, we observed the apoptotic effect of Nisin on the SW480 as a colon cancer cell line.[26] We showed that Nisin induced apoptosis via the intrinsic pathway by increasing the BAX/BCL-2 ratio index. In addition, we found the anti-proliferative effect of Nisin.[27] Herein, we studied the impacts of Nisin on cell adhesion and the expression of mmp2 and mmp9 genes involved in the detachment of cells to the extracellular matrix.
Previous studies observed the cytotoxic effect of Nisin in the discrepant cancer cell lines. The IC50 of Nisin in the current study was about 76.8 μM which is lower than previous reports. MCF7 (breast cancer cell line) and HepG2 (hepatic cancer cell line) were examined by Paiva et al.,[28] with the IC50 of 105.46 and 112.25 mM, respectively, which is more than our study. Moreover, Begde et al.,[29] reported that 225 mM is the IC50 of Nisin for the studied cell lines, Jurkat cell line, and human lymphocyte without apoptotic effects. In another study performed on colorectal cancer cells, Maher and a co-worker reported that 89.9 μM and 115 μM of Nisin showed an anti-proliferative impact on HT29 and Caco-2 cell lines, respectively.[30] The effective concentration of Nisin in that study is approximately similar to our study.
MMP family plays a momentous role in cancer cell migration, invasion, and metastasis via an effect on the extracellular matrix (ECM).[31] MMPs are involved in cancer development, cancer cell growth, angiogenesis, and cancer progression.[32] Furthermore, high expression of MMP-2 and MMP-9 is related to metastasis to lymph nodes.[33] MMP-9 are able to secrete the ECM factors such as TGF-β, VEGF, and FGF-2 causing to proliferate and migrant endothelial cells and finally angiogenesis.[34]
In this study, we observed that Nisin is able to upregulate the expression of both mmp-2 and mmp-9 genes at 128 and 256 μg/ml. Decrease expression of these genes could be protective against tumor cell migration and metastasis. But, our finding showed that despite declining the expression of mmp-2 and mmp-9 genes, the cell adhesions decreased with increasing the Nisin concentration. The reason for this observation can be due to the increased cell death at higher concentrations and attenuated cell number during the time of treatment. Moreover, numerous proteins, factors, and molecular signaling are involved in cell detachment and metastasis, which has not been studied here; therefore, the conclusion about the anti-metastatic effect of Nisin is difficult.
| Conclusion | | |
Overall, Nisin is able to prevent metastasis via decreased expression of mmp2 and mmp9 genes in addition to cytotoxicity impacts. However, to confirm this data, further assessment of other mechanisms involved in metastatic events is essential.
Ethics approval
The study protocol was approved by the Research Ethics Committees of Baqiyatallah University of Medical Sciences. (Approval ID: IR.BMSU.REC.1396.028).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Pucci C, Martinelli C, Ciofani G. Innovative approaches for cancer treatment: Current perspectives and new challenges. Ecancermedicalscience 2019;13.  |
| 2. | Aghabozorgi AS, Ebrahimi R, Bahiraee A, Tehrani SS, Nabizadeh F, Setayesh L, et al. The genetic factors associated with Wnt signaling pathway in colorectal cancer. Life Sci 2020;256:118006. |
| 3. | Mir SM, Nezhadi A, Tehrani SS, Jamalpoor Z. The clinical significance of VDR and WIFI downregulation in colorectal cancer tissue. Gene Rep 2020;20:100762. |
| 4. | Global Burden of Disease Cancer C, Fitzmaurice C, Akinyemiju TF, Al Lami FH, Alam T, Alizadeh-Navaei R, et al. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 1990 to 2016: A systematic analysis for the global burden of disease study. JAMA Oncol 2018;4:1553-68. |
| 5. | Cho YA, Lee J, Oh JH, Chang HJ, Sohn DK, Shin A, et al. Genetic risk score, combined lifestyle factors and risk of colorectal cancer. Cancer Res Treat 2019;51:1033-40. |
| 6. | Chen W-Z, Jiang J-X, Yu X-Y, Xia W-J, Yu P-X, Wang K, et al. Endothelial cells in colorectal cancer. World J Gastrointest Oncol 2019;11:946-56. |
| 7. | Biller LH, Schrag D. Diagnosis and treatment of metastatic colorectal cancer: A review. JAMA 2021;325:669-85. |
| 8. | Elinav E, Garrett WS, Trinchieri G, Wargo J. The cancer microbiome. Nat Rev Cancer 2019;19:371-6. |
| 9. | Fang Y, Yan C, Zhao Q, Xu J, Liu Z, Gao J, et al. The roles of microbial products in the development of colorectal cancer: A review. Bioengineered 2021;12:720-35. |
| 10. | Drago L. Probiotics and colon cancer. Microorganisms 2019;7:66. |
| 11. | Lu K, Dong S, Wu X, Jin R, Chen H. Probiotics in cancer. Front Oncol 2021;11:638148. |
| 12. | Huang F, Teng K, Liu Y, Cao Y, Wang T, Ma C, et al. Bacteriocins: Potential for human health. Oxid Med Cell Longev 2021;2021:5518825. |
| 13. | Kumariya R, Garsa AK, Rajput YS, Sood SK, Akhtar N, Patel S. Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria. Microb Pathog 2019;128:171-7. |
| 14. | Małaczewska J, Kaczorek-Łukowska E. Nisin-A lantibiotic with immunomodulatory properties: A review. Peptides 2021;137:170479. |
| 15. | Goudarzi F, Asadi A, Afsharpour M, Jamadi RH. In vitro characterization and evaluation of the cytotoxicity effects of nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2) and blood (K562) cancer cell lines. AAPS PharmSciTech 2018;19:1554-66. |
| 16. | Norouzi Z, Salimi A, Halabian R, Fahimi H. Nisin, a potent bacteriocin and anti-bacterial peptide, attenuates expression of metastatic genes in colorectal cancer cell lines. Microb Pathog 2018;123:183-9. |
| 17. | Patil SM, Kunda NK. Nisin ZP, an antimicrobial peptide, induces cell death and inhibits non-small cell lung cancer (NSCLC) progression in vitro in 2D and 3D cell culture. Pharm Res 2022. |
| 18. | Joo NE, Ritchie K, Kamarajan P, Miao D, Kapila YL. Nisin, an apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigenesis via CHAC1. Cancer Med 2012;1:295-305. |
| 19. | Kamarajan P, Hayami T, Matte B, Liu Y, Danciu T, Ramamoorthy A, et al. Nisin ZP, a bacteriocin and food preservative, inhibits head and neck cancer tumorigenesis and prolongs survival. PLoS One 2015;10:e0131008. |
| 20. | Yao Z, Yuan T, Wang H, Yao S, Zhao Y, Liu Y, et al. MMP-2 together with MMP-9 overexpression correlated with lymph node metastasis and poor prognosis in early gastric carcinoma. Tumour Biol 2017;39:1010428317700411. |
| 21. | Pereira FV, Ferreira-Guimaraes CA, Paschoalin T, Scutti JA, Melo FM, Silva LS, et al. A natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies. PLoS One 2014;9:e96141. |
| 22. | Ganesh K, Massagué J. Targeting metastatic cancer. Nat Med 2021;27:34-44. |
| 23. | Buyel JF. Plants as sources of natural and recombinant anti-cancer agents. Biotechnol Adv 2018;36:506-20. |
| 24. | Sedighi M, Zahedi Bialvaei A, Hamblin MR, Ohadi E, Asadi A, Halajzadeh M, et al. Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities. Cancer Med 2019;8:3167-81. |
| 25. | Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA. The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy. Cancer Cell 2018;33:570-80. |
| 26. | Hosseini SS, Goudarzi H, Ghalavand Z, Hajikhani B, Rafeieiatani Z, Hakemi-Vala M. Anti-proliferative effects of cell wall, cytoplasmic extract of lactococcus lactis and nisin through down-regulation of cyclin D1 on SW480 colorectal cancer cell line. Iran J Microbiol 2020;12:424-30. |
| 27. | Ahmadi S, Ghollasi M, Hosseini HM. The apoptotic impact of nisin as a potent bacteriocin on the colon cancer cells. Microb Pathog 2017;111:193-7. |
| 28. | Paiva AD, de Oliveira MD, de Paula SO, Baracat-Pereira MC, Breukink E, Mantovani HC. Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity. Microbiology 2012;158:2851-8. |
| 29. | Begde D, Bundale S, Mashitha P, Rudra J, Nashikkar N, Upadhyay A. Immunomodulatory efficacy of nisin--a bacterial lantibiotic peptide. J Pept Sci 2011;17:438-44. |
| 30. | Maher S, McClean S. Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem Pharmacol 2006;71:1289-98. |
| 31. | Najafi M, Farhood B, Mortezaee K. Extracellular matrix (ECM) stiffness and degradation as cancer drivers. J Cell Biochem 2019;120:2782-90. |
| 32. | Cabral-Pacheco GA, Garza-Veloz I, Castruita-De la Rosa C, Ramirez-Acuña JM, Perez-Romero BA, Guerrero-Rodriguez JF, et al. The roles of matrix metalloproteinases and their inhibitors in human diseases. Int J Mol Sci 2020;21:9739. |
| 33. | Saleh M, Khalil M, Abdellateif MS, Ebeid E, Madney Y, Kandeel EZ. Role of matrix metalloproteinase MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) in the clinical progression of pediatric acute lymphoblastic leukemia. Hematology (Amsterdam, Netherlands) 2021;26:758-68. |
| 34. | Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: Causes, consequences, challenges and opportunities. Cell Mol Life Sci 2020;77:1745-70. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1]
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