29 May 2019

Characterizing Cancer-Fighting Lipopeptides | Sample Preparation

Lipopeptides are cyclic structures produced by Bacillus and Pseudomonas species. They are hydrophilic peptides, usually between 7 and 10 amino acids long, linked to a hydrophobic fatty acid structure. They are part of a class of compounds that have been shown to have medicinal properties. This was shown by the ability of bacillus to successfully compete against plant and fungal pathogens in an antibiotic fashion. And also, by their ability to stimulate the defensive capabilities of athe plant. 

a lipopeptide

As the research work continued the antibiotic compounds produced from various Bacillus microbes were identified as polymeric biosurfactants (Stein, 2005). These biosurfactants include compounds such as lipopeptides, glycolipids, phospholipids, and fatty acids. In particular, it is believed lipopeptides play a key role in disease suppression, because of their high surface activity and antibiotic properties. However, this has also moved a stage further as Bacillus vallismortis lipopeptide metabolites have shown cytotoxic activity against colon cancer carcinoma cells.

And the Bacillus amyloliquefaciens BACY1 (BLE) lipopeptide extract is able to induce cell death in human oral squamous cell carcinoma (OSCC) cells SCC4 and SCC25. The results showed that BLE had both anti-proliferative, and apoptotic effects making it an ideal therapeutic candidate.

According to the World Health Organization (WHO), OSCC accounts for the majority of oral cancer.

According to the World Health Organization (WHO), OSCC accounts for the majority of oral cancer. And late stage malignant OSCC cells, are well-known to develop drug resistance to standard chemotherapeutic agents in existing therapy. 

The Mechanism of Action in Chronic Myeloid Leukemia (CML)

There are a huge number of different Bacillus species in the environment and all of them produce a range of biosurfactant lipopeptides. This chemical and physical diversity of lipopeptide antibiotics gives a huge potential for therapeutic anticancer and antibiotic applications. Chronic myeloid leukemia is a cancer of bone marrow hematopoietic stem cells and is responsible for around 15% of adult leukemia.

Chronic myeloid leukemia is a cancer of bone marrow hematopoietic stem cells and is responsible for around 15%25 of adult leukemia.

Bacillus subtilis lipopeptides are known to have anti-tumor effects. These include inhibitory effects on human breast cancer by disruption of the Akt pathway, apoptosis of melanoma A 375 cells by plasma membrane disruption and action against human leukemia K562 cells. The lipopeptides in this series have a peptide ring and fatty acid chain structure and have shown a range of anti-bacterial, anti-inflammatory, anti-viral, and anti-tumour function. Bacillus cyclic lipopeptides consist of three major groups known as the surfactin, iturin, and fengycin families and all have potential as anti-tumour compounds.

  • Surfactin slows the proliferation of the human colon carcinoma cell line LoVo. It is also active in TPA-induced breast cancer cell invasion through the inhibition of MMP-9 expression and can kill the MCF-7 human breast carcinoma cell line through a ROS/JNK-mediated mitochondrial/caspase pathway. It has also been valuable in preventing platelet aggregation and enhancing fibrinolysis
  • Iturin inhibits the proliferation of breast cancer cells MDA-MB-231 and MCF-7, alveolar adenocarcinoma A549, renal carcinoma A498, K562 chronic myelogenous leukemia cells, and colon adenocarcinoma HCT-15
  • Fengycin is able to block non-small cell lung cancer cell 95D and inhibit the growth of xenografted 95D cells in denuded mice

Isolating and Analysing 

Lipopeptide biosurfactants are cyclic peptides with varying lengths of attached lipid chains. Their extraction and isolation from bacillus cells are generally accomplished by a combination of maceration, acidification of cell material with ammonium sulphate and ethanol, chloroform or acetone extraction. Many of these lipopeptides are still not fully characterised and have enormous potential in future medicine. The extract obtained will still be a mixture requiring separation.

This can be accomplished by High-Pressure Liquid Chromatography (HPLC) followed by semi-preparative chromatography using C18 reverse phase Luna type columns. An alternative for separating lipopeptide mixtures is the use of one-dimensional sodium dodecyl sulphate polyacrylamine gel electrophoresis (1D SDS-PAGE). This is generally used for proteomic experiments to separate protein or lipopeptide mixtures and to estimate the molecular mass of these high molecular weight biosurfactants. 

Analysis and Characterisation

Analysis and characterisation of lipopeptides is a two-stage process. The first is obtaining a molecular weight and structure of the molecule and this is usually accomplished by Fourier Transform Infrared spectrophotometer (FTIR) and matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis. This will give an accurate molecular weight of the whole molecular ion and also fragments if required. MALDI is a soft ionisation mass spectrometry technique allowing identification of whole compounds.

Samples are mixed with a glycerol matrix and dried on a target holder. A laser is fired with various energies to form gaseous ions, which are then separated in a time of flight analyser (TOF) and detected. MALDI can be used to determine the full molecular mass of high molecular weight biosurfactants and lipopeptide biosurfactants that have been purified. FTIR can then identify chemical groups on the main molecular chain that could modify the properties. Following this, if sufficient quantities are obtained, the purified lipopeptide can be used in assays to determine its biological activity. 

Preparing Samples for FTIR

FTIR is an excellent technique for characterising the chemical groups and structure of unidentified compounds. The preparation of samples is important to achieve consistency in results. Some researchers of lipopeptides have used Calcium fluoride plates with a sample of the lipopeptide sandwiched in between to achieve high-resolution IR spectra (400-4000cm-1).

However, one of the most consistent sample preparation methods is the potassium bromide disk. The sample is fully recoverable and by using a hydraulic press from Specac such as the Autotouch hydraulic press the disk compression can be fully controlled to achieve consistent results.

The KBr disk thickness and transparency is crucial in the comparison of different spectra and transferability between different FT-IR instrumentation. The Automatic hydraulic press from Specac is fully automatic and programmable to produce a consistent pressing for the KBr disk and also has controlled pressing weights of 8T, 15T, 25T, and 40T for consistent thickness. 

References and Further Reading

  1. Zhao H, Yan L, Xu X, et al. Potential of Bacillus subtilis lipopeptides in anti-cancer I: induction of apoptosis and paraptosis and inhibition of autophagy in K562 cells. AMB Express. 2018;8(1):78. Published 2018 May 9.
  2. Chen-Hui Kuo, Yun-Wei Lin, Ruey-Shyang Chen, Lipopeptides Extract from Bacillus Amyloliquefaciens Induce Human Oral Squamous Cancer Cell Death, Asian Pac J Cancer Prev, 16 (1), 91-96. 
  3. Romero D, de Vicente A, Rakotoaly RH, et al (2007). The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe Int, 20, 430-40
  4. T. J. P. Smyth, Isolation and Analysis of Lipopeptides and High Molecular Weight Biosurfactants, K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_290, Springer-Verlag Berlin Heidelberg, 2010
  5. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, Fracchia L, Smyth TJ, Marchant R (2010) Microbial biosurfactants production, applications and future potential. Appl Microbiol Biotechnol 87:427–444
  6. Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra, SS (2002) Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68: 6210–6219

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