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Journal of The American Society for Mass Spectrometry

, Volume 30, Issue 9, pp 1690–1699 | Cite as

Investigating the Proteomic Profile of HT-29 Colon Cancer Cells After Lactobacillus kefiri SGL 13 Exposure Using the SWATH Method

  • Jessica Brandi
  • Claudia Di Carlo
  • Marcello Manfredi
  • Federica Federici
  • Alda Bazaj
  • Eleonora Rizzi
  • Giuseppe Cornaglia
  • Laura Manna
  • Emilio Marengo
  • Daniela CecconiEmail author
Research Article

Abstract

Despite some studies revealed that kefir acts on different cancers, such as colorectal cancer, the proteomic changes that occur in the colon cancer cells remain to be explored. In this study, the proteomic analysis was combined with determination of kefir characteristics (e.g., adhesion capacity, gastrointestinal and antibiotic resistances), in order to confirm its use as a probiotic. Therefore, a label-free strategy based on SWATH-MS was applied to investigate the proteomic profile of HT-29 cells after exposure for 24 h to a specific strain of Lactobacillus kefiri named SGL 13. We identified a total of 60 differentially expressed proteins in HT-29 cells, among which most are located into the extracellular exosome, playing important/crucial roles in translation and cell adhesion, as indicated by the enrichment analysis. The eIF2 and retinoid X receptor activation pathways appeared to be correlated with the anti-tumoral effect of SGL 13. Immunoblot analysis showed an increase in Bax and a decrease in caspase 3 and mutant p53, and ELISA assay revealed inhibition of IL-8 secretion from HT-29 cells stimulated with LPS upon SGL 13 treatment, suggesting pro-apoptotic and anti-inflammatory properties of kefir. In conclusion, the results of this study, the first of its kind using co-culture of kefir and colon cancer cells, demonstrate that L. kefiri SGL 13 possesses probiotic potency and contribute to elucidate the molecular mechanisms involved in the L. kefiri–colon cancer cell interactions.

Keywords

SWATH-MS Label-free proteomics Colon cancer Lactobacillus kefiri 

Notes

Acknowledgements

This work was supported by FUR of the MIUR Ministry or Education, University and Research (Italy).

Compliance with Ethical Standards

Conflict of Interest

The authors declared the following potential conflicts of interest with respect of the research, authorship, and/or publication of this article: FF, ER, and LM are employees of Sintal Dietetics s.r.l., Italy, performing all research and development activities for Sintal Dietetics. Sintal Dietetics s.r.l. deposited Lactobacillus kefiri SGL 13 for purposes of European patent.

Supplementary material

13361_2019_2268_MOESM1_ESM.xlsx (116 kb)
Supplemental Table S1 Identified and quantified proteins (XLSX 116 kb)
13361_2019_2268_MOESM2_ESM.xlsx (50 kb)
Supplemental Table S2 Differentially expressed proteins (n = 60). (XLSX 49 kb)
13361_2019_2268_MOESM3_ESM.xlsx (18 kb)
Supplemental Table S3 IPA canonical-enriched pathways. (XLSX 17 kb)

References

  1. 1.
    Chauvin, A., Boisvert, F.M.: Clinical proteomics in colorectal cancer, a promising tool for improving personalised medicine. Proteomes. 6, (2018)Google Scholar
  2. 2.
    Hendler, R., Zhang, Y.: Probiotics in the treatment of colorectal cancer. Medicines (Basel). 5, (2018)Google Scholar
  3. 3.
    Guzel-Seydim, Z.B., Kok-Tas, T., Greene, A.K., Seydim, A.C.: Review: functional properties of kefir. Crit. Rev. Food Sci. Nutr. 51, 261–268 (2011)CrossRefGoogle Scholar
  4. 4.
    Sanders, M.E.: Probiotics: definition, sources, selection, and uses. Clin. Infect. Dis. 46(Suppl 2), S58–S61; discussion S144–151 (2008)CrossRefGoogle Scholar
  5. 5.
    Wells, J.M.: Immunomodulatory mechanisms of lactobacilli. Microb. Cell Factories. 10(Suppl 1), S17 (2011)CrossRefGoogle Scholar
  6. 6.
    Carey, C.M., Kostrzynska, M.: Lactic acid bacteria and bifidobacteria attenuate the proinflammatory response in intestinal epithelial cells induced by Salmonella enterica serovar Typhimurium. Can. J. Microbiol. 59, 9–17 (2013)CrossRefGoogle Scholar
  7. 7.
    Carasi, P., Ambrosis, N.M., De Antoni, G.L., Bressollier, P., Urdaci, M.C., Serradell Mde, L.: Adhesion properties of potentially probiotic Lactobacillus kefiri to gastrointestinal mucus. J Dairy Res. 81, 16–23 (2014)CrossRefGoogle Scholar
  8. 8.
    Sharifi, M., Moridnia, A., Mortazavi, D., Salehi, M., Bagheri, M., Sheikhi, A.: Kefir: a powerful probiotics with anticancer properties. Med. Oncol. 34, 183 (2017)CrossRefGoogle Scholar
  9. 9.
    Khoury, N., El-Hayek, S., Tarras, O., El-Sabban, M., El-Sibai, M., Rizk, S.: Kefir exhibits antiproliferative and proapoptotic effects on colon adenocarcinoma cells with no significant effects on cell migration and invasion. Int. J. Oncol. 45, 2117–2127 (2014)CrossRefGoogle Scholar
  10. 10.
    Federici, F., Manna, L., Rizzi, E., Galantini, E., Marini, U.: Draft genome sequence of Lactobacillus kefiri SGL 13, a potential probiotic strain isolated from kefir grains. Microbiol. Resour. Announc. 7, (2018)Google Scholar
  11. 11.
    Chenoll, E., Casinos, B., Bataller, E., Astals, P., Echevarria, J., Iglesias, J.R., Balbarie, P., Ramon, D., Genoves, S.: Novel probiotic Bifidobacterium bifidum CECT 7366 strain active against the pathogenic bacterium Helicobacter pylori. Appl. Environ. Microbiol. 77, 1335–1343 (2011)CrossRefGoogle Scholar
  12. 12.
    Candela, M., Seibold, G., Vitali, B., Lachenmaier, S., Eikmanns, B.J., Brigidi, P.: Real-time PCR quantification of bacterial adhesion to Caco-2 cells: competition between bifidobacteria and enteropathogens. Res. Microbiol. 156, 887–895 (2005)CrossRefGoogle Scholar
  13. 13.
    Manfredi, M., Brandi, J., Conte, E., Pidutti, P., Gosetti, F., Robotti, E., Marengo, E., Cecconi, D.: IEF peptide fractionation method combined to shotgun proteomics enhances the exploration of rice milk proteome. Anal. Biochem. 537, 72–77 (2017)CrossRefGoogle Scholar
  14. 14.
    Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A.: DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, P3 (2003)CrossRefGoogle Scholar
  15. 15.
    Brandi, J., Dando, I., Pozza, E.D., Biondani, G., Jenkins, R., Elliott, V., Park, K., Fanelli, G., Zolla, L., Costello, E., Scarpa, A., Cecconi, D., Palmieri, M.: Proteomic analysis of pancreatic cancer stem cells: functional role of fatty acid synthesis and mevalonate pathways. J. Proteome. 150, 310–322 (2017)CrossRefGoogle Scholar
  16. 16.
    Speziali, G., Liesinger, L., Gindlhuber, J., Leopold, C., Pucher, B., Brandi, J., Castagna, A., Tomin, T., Krenn, P., Thallinger, G.G., Olivieri, O., Martinelli, N., Kratky, D., Schittmayer, M., Birner-Gruenberger, R., Cecconi, D.: Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells. J. Proteome. 181, 118–130 (2018)CrossRefGoogle Scholar
  17. 17.
    Zheng, Q., Ye, J., Cao, J.: Translational regulator eIF2alpha in tumor. Tumour Biol. 35, 6255–6264 (2014)CrossRefGoogle Scholar
  18. 18.
    Modarai, S.R., Gupta, A., Opdenaker, L.M., Kowash, R., Masters, G., Viswanathan, V., Zhang, T., Fields, J.Z., Boman, B.M.: The anti-cancer effect of retinoic acid signaling in CRC occurs via decreased growth of ALDH+ colon cancer stem cells and increased differentiation of stem cells. Oncotarget. 9, 34658–34669 (2018)CrossRefGoogle Scholar
  19. 19.
    Su, Y., Zeng, Z., Chen, Z., Xu, D., Zhang, W., Zhang, X.K.: Recent progress in the design and discovery of RXR modulators targeting alternate binding sites of the receptor. Curr. Top. Med. Chem. 17, 663–675 (2017)CrossRefGoogle Scholar
  20. 20.
    Morris, J., Moseley, V.R., Cabang, A.B., Coleman, K., Wei, W., Garrett-Mayer, E., Wargovich, M.J.: Reduction in promotor methylation utilizing EGCG (epigallocatechin-3-gallate) restores RXRalpha expression in human colon cancer cells. Oncotarget. 7, 35313–35326 (2016)Google Scholar
  21. 21.
    Nagy, L., Thomazy, V.A., Shipley, G.L., Fesus, L., Lamph, W., Heyman, R.A., Chandraratna, R.A., Davies, P.J.: Activation of retinoid X receptors induces apoptosis in HL-60 cell lines. Mol. Cell. Biol. 15, 3540–3551 (1995)CrossRefGoogle Scholar
  22. 22.
    Tang, H., Mirshahidi, S., Senthil, M., Kazanjian, K., Chen, C.S., Zhang, K.: Down-regulation of LXR/RXR activation and negative acute phase response pathways in colon adenocarcinoma revealed by proteomics and bioinformatics analysis. Cancer Biomark. 14, 313–324 (2014)CrossRefGoogle Scholar
  23. 23.
    Savill, J., Dransfield, I., Hogg, N., Haslett, C.: Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature. 343, 170–173 (1990)CrossRefGoogle Scholar
  24. 24.
    Hashimoto, K., Ikeda, N., Nakashima, M., Ikeshima-Kataoka, H., Miyamoto, Y.: Vitronectin regulates the fibrinolytic system during the repair of cerebral cortex in stab-wounded mice. J. Neurotrauma. 34, 3183–3191 (2017)CrossRefGoogle Scholar
  25. 25.
    Canonici, A., Pellegrino, E., Siret, C., Terciolo, C., Czerucka, D., Bastonero, S., Marvaldi, J., Lombardo, D., Rigot, V., Andre, F.: Saccharomyces boulardii improves intestinal epithelial cell restitution by inhibiting alphavbeta5 integrin activation state. PLoS One. 7, e45047 (2012)CrossRefGoogle Scholar
  26. 26.
    Singh, B., Su, Y.C., Riesbeck, K.: Vitronectin in bacterial pathogenesis: a host protein used in complement escape and cellular invasion. Mol. Microbiol. 78, 545–560 (2010)CrossRefGoogle Scholar
  27. 27.
    Widom, R.L., Rhee, M., Karathanasis, S.K.: Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid. Mol. Cell. Biol. 12, 3380–3389 (1992)CrossRefGoogle Scholar
  28. 28.
    Zhu, C., Di, D., Zhang, X., Luo, G., Wang, Z., Wei, J., Shi, Y., Berggren-Soderlund, M., Nilsson-Ehle, P., Xu, N.: TO901317 regulating apolipoprotein M expression mediates via the farnesoid X receptor pathway in Caco-2 cells. Lipids Health Dis. 10, 199 (2011)CrossRefGoogle Scholar
  29. 29.
    Gkouskou, K.K., Ioannou, M., Pavlopoulos, G.A., Georgila, K., Siganou, A., Nikolaidis, G., Kanellis, D.C., Moore, S., Papadakis, K.A., Kardassis, D., Iliopoulos, I., McDyer, F.A., Drakos, E., Eliopoulos, A.G.: Apolipoprotein A-I inhibits experimental colitis and colitis-propelled carcinogenesis. Oncogene. 35, 2496–2505 (2016)CrossRefGoogle Scholar
  30. 30.
    Inaba, T., Matsuda, M., Shimamura, M., Takei, N., Terasaka, N., Ando, Y., Yasumo, H., Koishi, R., Makishima, M., Shimomura, I.: Angiopoietin-like protein 3 mediates hypertriglyceridemia induced by the liver X receptor. J. Biol. Chem. 278, 21344–21351 (2003)CrossRefGoogle Scholar
  31. 31.
    Morikawa, K., Hanada, H., Hirota, K., Nonaka, M., Ikeda, C.: All-trans retinoic acid displays multiple effects on the growth, lipogenesis and adipokine gene expression of AML-I preadipocyte cell line. Cell Biol. Int. 37, 36–46 (2013)CrossRefGoogle Scholar
  32. 32.
    Meador, J.A., Ghandhi, S.A., Amundson, S.A.: p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation. Radiat. Res. 175, 689–699 (2011)CrossRefGoogle Scholar
  33. 33.
    Sijts, E.J., Kloetzel, P.M.: The role of the proteasome in the generation of MHC class I ligands and immune responses. Cell. Mol. Life Sci. 68, 1491–1502 (2011)CrossRefGoogle Scholar
  34. 34.
    Han, N.Y., Choi, W., Park, J.M., Kim, E.H., Lee, H., Hahm, K.B.: Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease. J. Dig. Dis. 14, 166–174 (2013)CrossRefGoogle Scholar
  35. 35.
    Kolukula, V.K., Sahu, G., Wellstein, A., Rodriguez, O.C., Preet, A., Iacobazzi, V., D'Orazi, G., Albanese, C., Palmieri, F., Avantaggiati, M.L.: SLC25A1, or CIC, is a novel transcriptional target of mutant p53 and a negative tumor prognostic marker. Oncotarget. 5, 1212–1225 (2014)CrossRefGoogle Scholar
  36. 36.
    Catalina-Rodriguez, O., Kolukula, V.K., Tomita, Y., Preet, A., Palmieri, F., Wellstein, A., Byers, S., Giaccia, A.J., Glasgow, E., Albanese, C., Avantaggiati, M.L.: The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis. Oncotarget. 3, 1220–1235 (2012)CrossRefGoogle Scholar
  37. 37.
    Infantino, V., Iacobazzi, V., Menga, A., Avantaggiati, M.L., Palmieri, F.: A key role of the mitochondrial citrate carrier (SLC25A1) in TNFalpha- and IFNgamma-triggered inflammation. Biochim. Biophys. Acta. 1839, 1217–1225 (2014)CrossRefGoogle Scholar
  38. 38.
    Gosslau, A., Jao, D.L., Butler, R., Liu, A.Y., Chen, K.Y.: Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A. J. Cell. Physiol. 219, 485–493 (2009)CrossRefGoogle Scholar
  39. 39.
    de Almeida Jr., O.P., Toledo, T.R., Rossi, D., Rossetto Dde, B., Watanabe, T.F., Galvao, F.C., Medeiros, A.I., Zanelli, C.F., Valentini, S.R.: Hypusine modification of the ribosome-binding protein eIF5A, a target for new anti-inflammatory drugs: understanding the action of the inhibitor GC7 on a murine macrophage cell line. Curr. Pharm. Des. 20, 284–292 (2014)CrossRefGoogle Scholar
  40. 40.
    Tan, B.S., Tiong, K.H., Choo, H.L., Chung, F.F., Hii, L.W., Tan, S.H., Yap, I.K., Pani, S., Khor, N.T., Wong, S.F., Rosli, R., Cheong, S.K., Leong, C.O.: Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF). Cell Death Dis. (6), e1826 (2015)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  • Jessica Brandi
    • 1
  • Claudia Di Carlo
    • 1
  • Marcello Manfredi
    • 2
    • 3
    • 4
  • Federica Federici
    • 5
  • Alda Bazaj
    • 6
  • Eleonora Rizzi
    • 5
  • Giuseppe Cornaglia
    • 6
  • Laura Manna
    • 5
  • Emilio Marengo
    • 3
    • 7
  • Daniela Cecconi
    • 1
    Email author
  1. 1.Department of Biotechnology, Proteomics and Mass Spectrometry LaboratoryUniversity of VeronaVeronaItaly
  2. 2.ISALIT s.r.l.NovaraItaly
  3. 3.Center for Translational Research on Autoimmune & Allergic Diseases—CAADNovaraItaly
  4. 4.Department of Translational MedicineUniversity of Eastern PiedmontNovaraItaly
  5. 5.Sintal Dietetics s.r.l.TeramoItaly
  6. 6.Department of Diagnostics and Public HealthUniversity of VeronaVeronaItaly
  7. 7.Department of Sciences and Technological InnovationUniversity of Eastern PiedmontAlessandriaItaly

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