Skip to main content
PMC home page
Protein & Cell logoLink to Protein & Cell
. 2010 Jul 7;1(6):584–594. doi: 10.1007/s13238-010-0072-4

Effect of themicrobial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

Xiangyang Liu 1,2, Xinyi Tao 1, Aihua Zou 1, Shizhong Yang 1, Lixin Zhang 2,✉, Bozhong Mu 1,✉
PMCID: PMC4875320  PMID: 21204010

Abstract

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C13-lipopeptide < C14-lipopepitde < C15-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC50 values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.

Keywords: Bacillus subtilis, lipopeptide, antitumor activity, membrane fatty acid, apoptosis

Contributor Information

Lixin Zhang, Email: zhanglixin@im.ac.cn.

Bozhong Mu, Email: bzmu@ecust.edu.cn.

References

  1. Ando H., Wen Z.M., Kim H.Y., Valencia J.C., Costin G.E., Watabe H., Yasumoto K., Niki Y., Kondoh H., Ichihashi M., et al. Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin-proteasome pathway. Biochem J. 2006;394:43–50. doi: 10.1042/BJ20051419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arima K., Kakinuma A., Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun. 1968;31:488–494. doi: 10.1016/0006-291X(68)90503-2. [DOI] [PubMed] [Google Scholar]
  3. Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Bonmatin J.M., Laprévote O., Peypoux F. Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activitystructure relationships to design new bioactive agents. Comb Chem High Throughput Screen. 2003;6:541–556. doi: 10.2174/138620703106298716. [DOI] [PubMed] [Google Scholar]
  5. Bortolato M., Besson F., Roux B. Inhibition of alkaline phosphatase by surfactin, a natural chelating lipopeptide from Bacillus subtilis. Biotechnol Lett. 1997;19:433–435. doi: 10.1023/A:1018387909310. [DOI] [Google Scholar]
  6. Cao X., Wang A.H., Jiao R.Z., Wang C.L., Mao D.Z., Yan L., Zeng B. Surfactin induces apoptosis and G(2)/M arrest in human breast cancer MCF-7 cells through cell cycle factor regulation. Cell Biochem Biophys. 2009;55:163–171. doi: 10.1007/s12013-009-9065-4. [DOI] [PubMed] [Google Scholar]
  7. Cao X.H., Liao Z.Y., Wang C.L., Cai P., Yang W.Y., Lu M.F., Huang G.W. Purification and antitumour activity of a lipopeptide biosurfactant produced by Bacillus natto TK-1. Biotechnol Appl Biochem. 2009;52:97–106. doi: 10.1042/BA20070227. [DOI] [PubMed] [Google Scholar]
  8. Cao X.H., Liao Z.Y., Wang C.L., Yang W.Y., Lu M.F. Evaluation of a lipopeptide biosurfactant from Bacillus natto Tk-1 as a potential source of anti-adhesive. Antimicrobial and Antitumor Activities. Braz J Microbiol. 2009;40:373–379. doi: 10.1590/S1517-83822009000200030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cao X.H., Wang A.H., Wang C.L., Mao D.Z., Lu M.F., Cui Y.Q., Jiao R.Z. Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. Chem Biol Interact. 2010;183:357–362. doi: 10.1016/j.cbi.2009.11.027. [DOI] [PubMed] [Google Scholar]
  10. Carrillo C., Teruel J.A., Aranda F.J., Ortiz A. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim Biophys Acta. 2003;1611:91–97. doi: 10.1016/S0005-2736(03)00029-4. [DOI] [PubMed] [Google Scholar]
  11. Dai R.J., Phillips R.A., Zhang Y., Khan D., Crasta O., Ahmed S.A. Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation. Blood. 2008;112:4591–4597. doi: 10.1182/blood-2008-04-152488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Deleu M., Bouffioux O., Razafindralambo H., Paquot M., Hbid C., Thonart P., Jacques P., Brasseur R. Interaction of surfactin with membranes: a computational approach. Langmuir. 2003;19:3377–3385. doi: 10.1021/la026543z. [DOI] [Google Scholar]
  13. Dufour S., Deleu M., Nott K., Wathelet B., Thonart P., Paquot M. Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties. Biochim Biophys Acta. 2005;1726:87–95. doi: 10.1016/j.bbagen.2005.06.015. [DOI] [PubMed] [Google Scholar]
  14. Eeman M., Berquand A., Dufrêne Y.F., Paquot M., Dufour S., Deleu M. Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization. Langmuir. 2006;22:11337–11345. doi: 10.1021/la061969p. [DOI] [PubMed] [Google Scholar]
  15. Elstein K.H., Thomas D.J., Zucker R.M. Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis. Cytometry. 1995;21:170–176. doi: 10.1002/cyto.990210209. [DOI] [PubMed] [Google Scholar]
  16. Geiser F., McAllan B.M., Kenagy G.J. The degree of dietary fatty acid unsaturation affects torpor patterns and lipid composition of a hibernator. J Comp Physiol B. 1994;164:299–305. doi: 10.1007/BF00346446. [DOI] [PubMed] [Google Scholar]
  17. Geyer R.P., Bennett A., Rohr A. Fatty acids of the triglycerides and phospholipids of HeLa cells and strain L fibroblasts. J Lipid Res. 1962;3:80–83. [Google Scholar]
  18. Gong J., Traganos F., Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem. 1994;218:314–319. doi: 10.1006/abio.1994.1184. [DOI] [PubMed] [Google Scholar]
  19. Gorczyca W., Gong J., Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 1993;53:1945–1951. [PubMed] [Google Scholar]
  20. Grau A., Gómez Fernández J.C., Peypoux F., Ortiz A. A study on the interactions of surfactin with phospholipid vesicles. Biochim Biophys Acta. 1999;1418:307–319. doi: 10.1016/S0005-2736(99)00039-5. [DOI] [PubMed] [Google Scholar]
  21. Haddad N.I.A., Liu X.Y., Yang S.Z., Mu B.Z. Surfactin isoforms from Bacillus subtilis HSO121: separation and characterization. Protein Pept Lett. 2008;15:265–269. doi: 10.2174/092986608783744225. [DOI] [PubMed] [Google Scholar]
  22. Hagve T.A. Effects of unsaturated fatty acids on cell membrane functions. Scand J Clin Lab Invest. 1988;48:381–388. doi: 10.3109/00365518809085746. [DOI] [PubMed] [Google Scholar]
  23. Heerklotz H., Seelig J. Detergent-like action of the antibiotic peptide surfactin on lipid membranes. Biophys J. 2001;81:1547–1554. doi: 10.1016/S0006-3495(01)75808-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Heerklotz H., Seelig J. Leakage and lysis of lipid membranes induced by the lipopeptide surfactin. Eur Biophys J. 2007;36:305–314. doi: 10.1007/s00249-006-0091-5. [DOI] [PubMed] [Google Scholar]
  25. Howlett N.G., Avery S.V. Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 1997;48:539–545. doi: 10.1007/s002530051093. [DOI] [PubMed] [Google Scholar]
  26. Huang X.Q., Lu Z.X., Zhao H.Z., Bie X.M., Xia L.F., Yang S.J. Antiviral activity of antimicrobial lipopeptide from Bacillus subtilis fmbj against Pseudorabies Virus, Porcine Parvovirus, Newcastle Disease Virus and Infectious Bursal Disease Virus in Vitro. Int J Pept Res Ther. 2006;12:373–377. doi: 10.1007/s10989-006-9041-4. [DOI] [Google Scholar]
  27. Kameda Y., Oira S., Matsui K., Kanatomo S., Hase T. Antitumor activity of bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2311. Chem Pharm Bull (Tokyo) 1974;22:938–944. doi: 10.1248/cpb.22.938. [DOI] [PubMed] [Google Scholar]
  28. Kikuchi T., Hasumi K. Enhancement of plasminogen activation by surfactin C: augmentation of fibrinolysis in vitro and in vivo. Biochim Biophys Acta. 2002;1596:234–245. doi: 10.1016/S0167-4838(02)00221-2. [DOI] [PubMed] [Google Scholar]
  29. Kim S.Y., Kim J.Y., Kim S.H., Bae H.J., Yi H., Yoon S.H., Koo B. S., Kwon M., Cho J.Y., Lee C.E., et al. Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression. FEBS Lett. 2007;581:865–871. doi: 10.1016/j.febslet.2007.01.059. [DOI] [PubMed] [Google Scholar]
  30. Kuo M.T. Redox regulation of multidrug resistance in cancer chemotherapy: molecular mechanisms and therapeutic opportunities. Antioxid Redox Signal. 2009;11:99–133. doi: 10.1089/ars.2008.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu X.Y., Haddad N.I.A., Yang S.Z., Mu B.Z. Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121. Protein Pept Lett. 2007;14:766–773. doi: 10.2174/092986607781483642. [DOI] [PubMed] [Google Scholar]
  32. Liu X.Y., Yang S.Z., Mu B.Z. Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121. J Pept Sci. 2008;14:864–875. doi: 10.1002/psc.1017. [DOI] [PubMed] [Google Scholar]
  33. Marsh S., McLeod H.L. Pharmacogenetics and oncology treatment for breast cancer. Expert Opin Pharmacother. 2007;8:119–127. doi: 10.1517/14656566.8.2.119. [DOI] [PubMed] [Google Scholar]
  34. Menendez J.A., Vellon L., Lupu R. Targeting fatty acid synthase-driven lipid rafts: a novel strategy to overcome trastuzumab resistance in breast cancer cells. Med Hypotheses. 2005;64:997–1001. doi: 10.1016/j.mehy.2004.09.027. [DOI] [PubMed] [Google Scholar]
  35. Michel V., Bakovic M. Lipid rafts in health and disease. Biol Cell. 2007;99:129–140. doi: 10.1042/BC20060051. [DOI] [PubMed] [Google Scholar]
  36. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  37. Pelz K., Hopfener K., Wiedmann-Al-Ahmad M., Jahnke H., Wittmer A., Otten J.E. Differences in the fatty acid composition of KB-cells and gingival keratinocytes is culture medium additive dependent. Biomed Chromatogr. 2006;20:870–880. doi: 10.1002/bmc.610. [DOI] [PubMed] [Google Scholar]
  38. Qi L.F., Xu Z.R., Chen M.L. In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles. Eur J Cancer. 2007;43:184–193. doi: 10.1016/j.ejca.2006.08.029. [DOI] [PubMed] [Google Scholar]
  39. Rakheja D., Kapur P., Hoang M.P., Roy L.C., Bennett M.J. Increased ratio of saturated to unsaturated C18 fatty acids in colonic adenocarcinoma: implications for cryotherapy and lipid raft function. Med Hypotheses. 2005;65:1120–1123. doi: 10.1016/j.mehy.2005.05.045. [DOI] [PubMed] [Google Scholar]
  40. Savage P.B., Li C., Taotafa U., Ding B., Guan Q. Antibacterial properties of cationic steroid antibiotics. FEMS Microbiol Lett. 2002;217:1–7. doi: 10.1111/j.1574-6968.2002.tb11448.x. [DOI] [PubMed] [Google Scholar]
  41. Schlager S.I., Ohanian S.H., Borsos T. Correlation between the ability of tumor cells to incorporate specific fatty acids and their sensitivity to killing by a specific antibody plus guinea pig complement. J Natl Cancer Inst. 1978;61:931–934. [PubMed] [Google Scholar]
  42. Sheppard J.D., Jumarie C., Cooper D.G., Laprade R. Ionic channels induced by surfactin in planar lipid bilayer membranes. Biochim Biophys Acta. 1991;1064:13–23. doi: 10.1016/0005-2736(91)90406-X. [DOI] [PubMed] [Google Scholar]
  43. Singletary S.E. Breast cancer management: the road to today. Cancer. 2008;113:1844–1849. doi: 10.1002/cncr.23649. [DOI] [PubMed] [Google Scholar]
  44. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev. 1992;56:395–411. doi: 10.1128/mr.56.3.395-411.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vollenbroich D., Pauli G., Özel M., Vater J. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl Environ Microbiol. 1997;63:44–49. doi: 10.1128/aem.63.1.44-49.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Walker P.R., Kwast-Welfeld J., Gourdeau H., Leblanc J., Neugebauer W., Sikorska M. Relationship between apoptosis and the cell cycle in lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1. Exp Cell Res. 1993;207:142–151. doi: 10.1006/excr.1993.1173. [DOI] [PubMed] [Google Scholar]
  47. Wang C.L., Ng T.B., Yuan F., Liu Z.K., Liu F. Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2. Peptides. 2007;28:1344–1350. doi: 10.1016/j.peptides.2007.06.014. [DOI] [PubMed] [Google Scholar]
  48. Ye C.L., Liu J.W., Wei D.Z., Lu Y.H., Qian F. In vitro anti-tumor activity of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone against six established human cancer cell lines. Pharmacol Res. 2004;50:505–510. doi: 10.1016/j.phrs.2004.05.004. [DOI] [PubMed] [Google Scholar]

Articles from Protein & Cell are provided here courtesy of Oxford University Press

RESOURCES