Therapeutic effects, immunogenicity and cytotoxicity of a cell penetrating peptide-peptide nucleic acid conjugate against cagA of Helicobacter pylori in cell culture and animal model
-
Narges Nodeh Farahani
,
Behrooz Sadeghi Kalani
,
Seyed Hamidreza Monavari
,
Shiva Mirkalantari
,
Fatemeh Montazer
,
Mohammad Sholeh
,
Zahra Javanmard
,
Gholamreza Irajian
Abstract
Background and Objectives: Helicobacter pylori causes several gastrointestinal diseases, including asymptomatic gastritis, chronic peptic ulcer, duodenal ulcer, lymphoma of the mucosa-associated lymphoid tissue (MALT), and gastric adenocarcinoma. In recent years, failure to eradicate H. pylori infections has become an alarming problem for physicians. It is now clear that the current treatment strategies may become ineffective, necessitating the development of innovative antimicrobial compounds as alternative treatments.
Materials and Methods: In this experimental study, a cell-penetrating peptide-conjugated peptide nucleic acid (CPP-PNA) was used to target the cagA expression. cagA expression was evaluated using RT-qPCR assay after treatment by the CPP-PNA in cell culture and animal model. Additionally, immunogenicity and toxicity of the CPP-PNA were assessed in both cell culture and animal models.
Results: Our analysis showed that cagA mRNA levels reduced in H. pylori-infected HT29 cells after treatment with CPP-PNA in a dose-dependent manner. Also, cagA expression in bacterial RNA extracted from stomach tissue of mice treated with PNA was reduced compared to that of untreated mice. The expression of inflammatory cytokines also decreased in cells and tissue of H. pylori-infected mice after PNA treatment. The tested CPP-PNA showed no significant adverse effects on cell proliferation of cultured cells and no detectable toxicity and immunogenicity were observed in mice.
Conclusion: These results suggest the effectiveness of CPP-PNA in targeting CagA for various research and therapeutic purposes, offering a potential antisense therapy against H. pylori infections.
1. Kao CY, Sheu BS, Wu JJ. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis. Biomed J 2016;39:14-23.
2. Cover TL, Blaser MJ. Helicobacter pylori in health and disease. Gastroenterology 2009;136:1863-1873.
3. Plummer M, Franceschi S, Vignat J, Forman D, de Martel C. Global burden of gastric cancer attributable to Helicobacter pylori. Int J Cancer 2015;136:487-490.
4. Bravo D, Hoare A, Soto C, Valenzuela MA, Quest AF. Helicobacter pylori in human health and disease: Mechanisms for local gastric and systemic effects. World J Gastroenterol 2018;24:3071-3089.
5. den Hoed CM, Kuipers EJ. Gastric Cancer: How can we reduce the iIncidence of this disease? Curr Gastroenterol Rep 2016;18:34.
6. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449-490.
7. Stein M, Rappuoli R, Covacci A. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci U S A 2000;97:1263-1268.
8. Matos JI, de Sousa HA, Marcos-Pinto R, Dinis-Ribeiro M. Helicobacter pylori CagA and VacA genotypes and gastric phenotype: a meta-analysis. Eur J Gastroenterol Hepatol 2013;25:1431-1441.
9. Hatakeyama M. Helicobacter pylori CagA and gastric cancer: a paradigm for hit-and-run carcinogenesis. Cell Host Microbe 2014;15:306-316.
10. Boonyanugomol W, Chomvarin C, Baik S-C, Song J-Y, Hahnvajanawong C, Kim K-M, et al. Role of cagA-positive Helicobacter pylori on cell proliferation, apoptosis, and inflammation in biliary cells. Dig Dis Sci 2011;56:1682-1692.
11. Argent RH, Hale JL, El-Omar EM, Atherton JC. Differences in Helicobacter pylori CagA tyrosine phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8 secretion. J Med Microbiol 2008;57:1062-1067.
12. Papadakos KS, Sougleri IS, Mentis AF, Hatziloukas E, Sgouras DN. Presence of terminal EPIYA phosphorylation motifs in Helicobacter pylori CagA contributes to IL-8 secretion, irrespective of the number of repeats. PLoS One 2013;8(2):e56291.
13. Di Mario F, Cavallaro LG, Scarpignato C. 'Rescue' therapies for the management of Helicobacter pylori infection. Dig Dis 2006;24:113-130.
14. Fleitas Martinez O, Cardoso MH, Ribeiro SM, Franco OL. Recent advances in anti-virulence therapeutic strategies with a focus on dismantling bacterial membrane microdomains, toxin neutralization, quorum-sensing interference and biofilm inhibition. Front Cell Infect Microbiol 2019;9:74.
15. Sully EK, Geller BL. Antisense antimicrobial therapeutics. Curr Opin Microbiol 2016;33:47-55.
16. Dias N, Stein CA. Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther 2002;1:347-355.
17. Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug
Discov 2010;9:117-128.
18. Javanmard Z, Kalani BS, Razavi S, Farahani NN, Mohammadzadeh R, Javanmard F, et al. Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori. Acta Microbiol Immunol Hung 2020;67:66-72.
19. Ramirez-Lazaro MJ, Lario S, Casalots A, Sanfeliu E, Boix L, Garcia-Iglesias P, et al. Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding. PLoS One 2011;6(5):e20009.
20. Kalani BS, Najafi M, Mohammadzadeh R, Razavi S, Ohadi E, Norkhoda S, et al. Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule. Microb Pathog 2019;136:103689.
21. Zhang M, Wang G, Tao Y, Zhang H. The proinflammatory effect and molecular mechanism of IL- 17 in the intestinal epithelial cell line HT-29. J BUON 2015;20:120-127.
22. Liu Y, Wang Y, Chen Q, Jiao F, Wang L, Gong Z. HDAC2 inhibitor CAY10683 reduces intestinal epithelial cell apoptosis by inhibiting mitochondrial apoptosis pathway in acute liver failure. Histol Histopathol 2019;34:1173-1184.
23. Shin HS, Lee HJ, Pyo MC, Ryu D, Lee KW. Ochratoxin a-induced Hepatotoxicity through phase I and phase II reactions regulated by AhR in liver cells. Toxins (Basel) 2019;11:377.
24. Graber DJ, Harris BT, Hickey WF. Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system. J Neuroinflammation 2011;8:122.
25. He X, Sun J, Huang X. Expression of caspase-3, Bax and Bcl-2 in hippocampus of rats with diabetes and subarachnoid hemorrhage. Exp Ther Med 2018;15:873-877.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
27. He Q, Wang JP, Osato M, Lachman LB. Real-time quantitative PCR for detection of Helicobacter pylori. J Clin Microbiol 2002;40:3720-3728.
28. Montazersaheb S, Hejazi MS, Nozad Charoudeh H. Potential of peptide nucleic acids in future therapeutic applications. Adv Pharm Bull 2018;8:551-563.
29. Oh E, Zhang Q, Jeon B. Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni. J Antimicrob Chemother 2014;69:375-380.
30. Mitev GM, Mellbye BL, Iversen PL, Geller BL. Inhibition of intracellular growth of Salmonella enterica serovar Typhimurium in tissue culture by antisense peptide-phosphorodiamidate morpholino oligomer. Antimicrob Agents Chemother 2009;53:3700-3704.
31. Patenge N, Pappesch R, Krawack F, Walda C, Mraheil MA, Jacob A, et al. Inhibition of growth and gene expression by PNA-peptide conjugates in Streptococcus pyogenes. Mol Ther Nucleic Acids 2013;2(11):e132.
32. Rasko DA, Moreira CG, Li de R, Reading NC, Ritchie JM, Waldor MK, et al. Targeting QseC signaling and virulence for antibiotic development. Science 2008;321:1078-1080.
33. Cegelski L, Marshall GR, Eldridge GR, Hultgren SJ. The biology and future prospects of antivirulence therapies. Nat Rev Microbiol 2008;6:17-27.
34. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 2005;310:670-674.
35. Hilleringmann M, Pansegrau W, Doyle M, Kaufman S, MacKichan ML, Gianfaldoni C, et al. Inhibitors of Helicobacter pylori ATPase Cagalpha block CagA transport and cag virulence.Microbiology (Reading) 2006;152:2919-2930.
36. Sayer JR, Wallden K, Pesnot T, Campbell F, Gane PJ, Simone M, et al. 2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion. Bioorg Med Chem 2014;22:6459-6470.
37. Patel RR, Sundin GW, Yang CH, Wang J, Huntley RB, Yuan X, et al. Exploration of using antisense peptide nucleic acid (PNA)-cell penetrating peptide (CPP) as a novel bactericide against fire blight pathogen Erwinia amylovora. Front Microbiol 2017;8:687.
38. McClorey G, Banerjee S. Cell-penetrating peptides to enhance delivery of oligonucleotide-based therapeutics. Biomedicines 2018;6:51.
39. Hegarty JP, Stewart Sr DB. Advances in therapeutic bacterial antisense biotechnology. Appl Microbiol Biotechnol 2018;102:1055-1065.
40. McMahon BM, Mays D, Lipsky J, Stewart JA, Fauq A, Richelson E. Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic Acid Drug Dev 2002;12:65-70.
41. Zeng Z, Han S, Hong W, Lang Y, Li F, Liu Y, et al. A tat-conjugated peptide nucleic acid tat-PNA-DR inhibits Hepatitis B virus replication in vitro and in vivo by targeting LTR direct repeats of HBV RNA. Mol Ther Nucleic Acids 2016;5(3):e295.
42. Pandey VN, Upadhyay A, Chaubey B. Prospects for antisense peptide nucleic acid (PNA) therapies for HIV. Expert Opin Biol Ther 2009;9:975-989.
43. Shen X, Corey DR. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res 2018;46:1584-1600.
44. Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol 2018;14:9-21.
45. Lan KH, Lee WP, Wang YS, Liao SX, Lan KH. Helicobacter pylori CagA protein activates Akt and attenuates chemotherapeutics-induced apoptosis in gastric cancer cells. Oncotarget 2017;8:113460-113471.
46. Chen Y, Sheppard D, Dong X, Hu X, Chen M, Chen R, et al. H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis. Cell Death Dis 2020;11:667.
2. Cover TL, Blaser MJ. Helicobacter pylori in health and disease. Gastroenterology 2009;136:1863-1873.
3. Plummer M, Franceschi S, Vignat J, Forman D, de Martel C. Global burden of gastric cancer attributable to Helicobacter pylori. Int J Cancer 2015;136:487-490.
4. Bravo D, Hoare A, Soto C, Valenzuela MA, Quest AF. Helicobacter pylori in human health and disease: Mechanisms for local gastric and systemic effects. World J Gastroenterol 2018;24:3071-3089.
5. den Hoed CM, Kuipers EJ. Gastric Cancer: How can we reduce the iIncidence of this disease? Curr Gastroenterol Rep 2016;18:34.
6. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449-490.
7. Stein M, Rappuoli R, Covacci A. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci U S A 2000;97:1263-1268.
8. Matos JI, de Sousa HA, Marcos-Pinto R, Dinis-Ribeiro M. Helicobacter pylori CagA and VacA genotypes and gastric phenotype: a meta-analysis. Eur J Gastroenterol Hepatol 2013;25:1431-1441.
9. Hatakeyama M. Helicobacter pylori CagA and gastric cancer: a paradigm for hit-and-run carcinogenesis. Cell Host Microbe 2014;15:306-316.
10. Boonyanugomol W, Chomvarin C, Baik S-C, Song J-Y, Hahnvajanawong C, Kim K-M, et al. Role of cagA-positive Helicobacter pylori on cell proliferation, apoptosis, and inflammation in biliary cells. Dig Dis Sci 2011;56:1682-1692.
11. Argent RH, Hale JL, El-Omar EM, Atherton JC. Differences in Helicobacter pylori CagA tyrosine phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8 secretion. J Med Microbiol 2008;57:1062-1067.
12. Papadakos KS, Sougleri IS, Mentis AF, Hatziloukas E, Sgouras DN. Presence of terminal EPIYA phosphorylation motifs in Helicobacter pylori CagA contributes to IL-8 secretion, irrespective of the number of repeats. PLoS One 2013;8(2):e56291.
13. Di Mario F, Cavallaro LG, Scarpignato C. 'Rescue' therapies for the management of Helicobacter pylori infection. Dig Dis 2006;24:113-130.
14. Fleitas Martinez O, Cardoso MH, Ribeiro SM, Franco OL. Recent advances in anti-virulence therapeutic strategies with a focus on dismantling bacterial membrane microdomains, toxin neutralization, quorum-sensing interference and biofilm inhibition. Front Cell Infect Microbiol 2019;9:74.
15. Sully EK, Geller BL. Antisense antimicrobial therapeutics. Curr Opin Microbiol 2016;33:47-55.
16. Dias N, Stein CA. Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther 2002;1:347-355.
17. Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug
Discov 2010;9:117-128.
18. Javanmard Z, Kalani BS, Razavi S, Farahani NN, Mohammadzadeh R, Javanmard F, et al. Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori. Acta Microbiol Immunol Hung 2020;67:66-72.
19. Ramirez-Lazaro MJ, Lario S, Casalots A, Sanfeliu E, Boix L, Garcia-Iglesias P, et al. Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding. PLoS One 2011;6(5):e20009.
20. Kalani BS, Najafi M, Mohammadzadeh R, Razavi S, Ohadi E, Norkhoda S, et al. Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule. Microb Pathog 2019;136:103689.
21. Zhang M, Wang G, Tao Y, Zhang H. The proinflammatory effect and molecular mechanism of IL- 17 in the intestinal epithelial cell line HT-29. J BUON 2015;20:120-127.
22. Liu Y, Wang Y, Chen Q, Jiao F, Wang L, Gong Z. HDAC2 inhibitor CAY10683 reduces intestinal epithelial cell apoptosis by inhibiting mitochondrial apoptosis pathway in acute liver failure. Histol Histopathol 2019;34:1173-1184.
23. Shin HS, Lee HJ, Pyo MC, Ryu D, Lee KW. Ochratoxin a-induced Hepatotoxicity through phase I and phase II reactions regulated by AhR in liver cells. Toxins (Basel) 2019;11:377.
24. Graber DJ, Harris BT, Hickey WF. Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system. J Neuroinflammation 2011;8:122.
25. He X, Sun J, Huang X. Expression of caspase-3, Bax and Bcl-2 in hippocampus of rats with diabetes and subarachnoid hemorrhage. Exp Ther Med 2018;15:873-877.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
27. He Q, Wang JP, Osato M, Lachman LB. Real-time quantitative PCR for detection of Helicobacter pylori. J Clin Microbiol 2002;40:3720-3728.
28. Montazersaheb S, Hejazi MS, Nozad Charoudeh H. Potential of peptide nucleic acids in future therapeutic applications. Adv Pharm Bull 2018;8:551-563.
29. Oh E, Zhang Q, Jeon B. Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni. J Antimicrob Chemother 2014;69:375-380.
30. Mitev GM, Mellbye BL, Iversen PL, Geller BL. Inhibition of intracellular growth of Salmonella enterica serovar Typhimurium in tissue culture by antisense peptide-phosphorodiamidate morpholino oligomer. Antimicrob Agents Chemother 2009;53:3700-3704.
31. Patenge N, Pappesch R, Krawack F, Walda C, Mraheil MA, Jacob A, et al. Inhibition of growth and gene expression by PNA-peptide conjugates in Streptococcus pyogenes. Mol Ther Nucleic Acids 2013;2(11):e132.
32. Rasko DA, Moreira CG, Li de R, Reading NC, Ritchie JM, Waldor MK, et al. Targeting QseC signaling and virulence for antibiotic development. Science 2008;321:1078-1080.
33. Cegelski L, Marshall GR, Eldridge GR, Hultgren SJ. The biology and future prospects of antivirulence therapies. Nat Rev Microbiol 2008;6:17-27.
34. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ. Small-molecule inhibitor of Vibrio cholerae virulence and intestinal colonization. Science 2005;310:670-674.
35. Hilleringmann M, Pansegrau W, Doyle M, Kaufman S, MacKichan ML, Gianfaldoni C, et al. Inhibitors of Helicobacter pylori ATPase Cagalpha block CagA transport and cag virulence.Microbiology (Reading) 2006;152:2919-2930.
36. Sayer JR, Wallden K, Pesnot T, Campbell F, Gane PJ, Simone M, et al. 2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion. Bioorg Med Chem 2014;22:6459-6470.
37. Patel RR, Sundin GW, Yang CH, Wang J, Huntley RB, Yuan X, et al. Exploration of using antisense peptide nucleic acid (PNA)-cell penetrating peptide (CPP) as a novel bactericide against fire blight pathogen Erwinia amylovora. Front Microbiol 2017;8:687.
38. McClorey G, Banerjee S. Cell-penetrating peptides to enhance delivery of oligonucleotide-based therapeutics. Biomedicines 2018;6:51.
39. Hegarty JP, Stewart Sr DB. Advances in therapeutic bacterial antisense biotechnology. Appl Microbiol Biotechnol 2018;102:1055-1065.
40. McMahon BM, Mays D, Lipsky J, Stewart JA, Fauq A, Richelson E. Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic Acid Drug Dev 2002;12:65-70.
41. Zeng Z, Han S, Hong W, Lang Y, Li F, Liu Y, et al. A tat-conjugated peptide nucleic acid tat-PNA-DR inhibits Hepatitis B virus replication in vitro and in vivo by targeting LTR direct repeats of HBV RNA. Mol Ther Nucleic Acids 2016;5(3):e295.
42. Pandey VN, Upadhyay A, Chaubey B. Prospects for antisense peptide nucleic acid (PNA) therapies for HIV. Expert Opin Biol Ther 2009;9:975-989.
43. Shen X, Corey DR. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res 2018;46:1584-1600.
44. Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol 2018;14:9-21.
45. Lan KH, Lee WP, Wang YS, Liao SX, Lan KH. Helicobacter pylori CagA protein activates Akt and attenuates chemotherapeutics-induced apoptosis in gastric cancer cells. Oncotarget 2017;8:113460-113471.
46. Chen Y, Sheppard D, Dong X, Hu X, Chen M, Chen R, et al. H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis. Cell Death Dis 2020;11:667.
| Files | ||
| Issue | Vol 13 No 3 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i3.6399 | |
| Keywords | ||
| Helicobacter pylori; cag A; Peptide nucleic acid; Immunogenicity; Cytotoxicity | ||
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How to Cite
1.
Nodeh Farahani N, Sadeghi Kalani B, Monavari SH, Mirkalantari S, Montazer F, Sholeh M, Javanmard Z, Irajian G. Therapeutic effects, immunogenicity and cytotoxicity of a cell penetrating peptide-peptide nucleic acid conjugate against cagA of Helicobacter pylori in cell culture and animal model. Iran J Microbiol. 2021;13(3):360-371.
