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Laterosporulin10 (LS10) is a defensin like peptide from Brevibacillus sp. strain SKDU10 that inhibited microbial pathogens. However, in this study, anticancer activity of LS10 was examined against different cancer cell lines and compared with normal cells. LS10 displayed cytotoxicity against cancer cells like MCF-7, HEK293T, HT1080, HeLa and H1299 at below 10μM concentration, but not against prostate epithelium cells RWPE-1. Additionally, no hemolysis was observed at significantly higher concentration compared to IC50 values observed for different cancer cell lines. Release of lactate dehydrogenase from cancer cell lines at 15μM concentration upon 120min treatment indicated the lytic ability of LS10. Accordingly, electron microscopy experiments also confirmed the necrotic effect of LS10 at 15μM concentration against cancer cells. Furthermore, flow cytometry analysis of treated cancer cell lines revealed that LS10 induce apoptosis even at 2.5μM concentration. Nevertheless, RWPE-1 cells remained viable even at 20μM concentration. These results provide evidence that LS10 is an anticancer bacteriocin, which causes apoptotic and necrotic death of cancer cells at lower and higher concentrations, respectively. Taken all results together, the present study signifies that LS10 is an anticancer peptide that could be further developed for therapeutic applications.
Cancer is one of the leading cause of death worldwide (http://seer.cancer.gov/statfacts) and the frightening statistics underscore the need to examine the novel anticancer agent and modes of therapy1,2,3. The major goal of modern oncology program is to discover better anticancer entities with novel modes of action. Ideally, anticancer drugs should specifically target cancer cells without any toxic effect against normal cells but, unfortunately, most of the available anticancer drugs display severe side effects. Moreover, development of multidrug resistance by cancer cells4,5 makes the situation even more critical. Therefore, to tackle this grim situation, considerable efforts are being made throughout the world over past several years to discover novel and better therapeutic candidates for cancer therapy. In this context, recent utilization of small peptides in cancer treatment6,7 has been attracted a lot of scientific attention as cancer therapeutics.
A large number of naturally occurring antimicrobial peptides (AMPs) from various sources have been reported in the literature that displayed anticancer properties8,9,10. In fact, in the recent past, many live or attenuated bacteria were patented as potential anticancer agents11,12,13. Additionally, microbial products including toxins, enzymes, antibiotics, various proteins, peptides and other low molecular weight products have also been evaluated for their anticancer properties12,14. Various bacterial peptides with antimicrobial activity were also demonstrated activity against cancer cells15,16,17,18,19, however, only a few of these peptides characterized in detail for anticancer activity20,21,22. AMPs produced by bacteria are relatively amenable to bioengineering and demonstrated considerable therapeutic efficacy23,24, therefore, such peptides are considered as promising agents for anticancer therapies19,25,26,27. Most of these AMPs, also known as bacteriocins, are reported to be non-cytotoxic and non-hemolytic in nature28,29. Accordingly, AMPs produced by members of the genus Brevibacillus, particularly Brevibacillus laterosporus produces defensin like antimicrobial peptides30, antibiotics like laterosporamine31, acyl dipeptides like tupuselei amides, antifungal polyketides like basilisk amides32, lipopeptide antibiotic like tauramamide33, cyclodecapeptides like laterocidin and its analogues34 which inhibits growth of both Gram-positive and Gram-negative bacteria. Additionally, novel thrombin inhibitors like bacithrocins A, B and C35 and anticancer antibiotic like spergualin are also reported from strains of B. laterosporus36.
In a recent study we have characterized a defensin like AMP laterosporulin10 (LS10) that showed antibacterial activity against pathogens like M. tuberculosis strain H37Rv and Staphylococcus aureus37. In fact, it selectively inhibited the growth of M. tuberculosis H37Rv at significantly low LD50 values (0.5μM) when compared to M. smegmatis MC2 155 in in vitro and ex vivo assays. Further, insights into the mechanism of action using electron microscopy and flow cytometry experiments assigned it to the membrane permeabilizing bacteriocins. Since, LS10 efficiently killed M. tuberculosis H37Rv residing inside the macrophages without any antagonistic activity against macrophages37, we further evaluated its anticancer potential and compared with normal cells.
Eukaryotic defensins are multifunctional peptides known to exhibit anticancer properties. Therefore, in order to determine the similarity of defensin like bacteriocin LS10 with known eukaryotic defensins, we selected an earlier reported defensin like bacteriocin laterosporulin (LS), human β-defensins (HBD1, HBD2, HBD3) and human neutrophil defensins (HNP1, HNP2, HNP3) which are active against mammalian cells38,39,40. All the sequences were aligned to compare with LS10 (Fig. 1A). As observed for LS, LS10 also contained six cysteine residues which are involved in disulfide bond formation at conserved positions a characteristic feature of eukaryotic defensins30,37. Thus, to get more insight into structural aspects, we have performed circular dichroism (CD) for LS10. Interestingly, CD spectrum of the LS10 in water (Fig. 1B) was characterized by the presence of negative values of molar ellipticity (θ) for wavelengths shorter than 200nm and a minimum close to 200nm, a characterstic of random coil conformation41,42. Further, CD spectra obtained in membrane mimicking environments including 5% SDS and 100% TFE were also confirmed the randomic structure of LS1043,44. However, LS10 might acquire a secondary structure as a negative shift is found to be minimum in 5% SDS or 100% TFE when compared to water. The calculations of percentage or level of helicity (H) obtained from the deconvolution of LS10 concentrations and molar ellipticity (θ) confirm the predominance of randomic structures in 5% SDS and 100% TFE as well (Fig. 1B).
The anticancer activity of LS10 was examined using five different human cancer cells including HeLa, MCF-7, HT1080, H1299, and HEK293T. The cell lines were incubated with increasing concentration of LS10 (1–20μM) in their respective growth medium revealed LS10 to be cytotoxic in nature against all cancer cell lines tested (Fig. 2). A dose-dependent cytotoxicity was observed on all cancer cell lines with maximum activity being observed at 10μM and highest activity against MCF-7 cells. While 40% cytotoxicity was observed on MCF-7 at 5μM concentration of LS10, only 20% cytotoxicity was displayed by HT1080, HEK293T and H1299. However, no significant cytotoxicity was observed on HeLa cells at 5μM concentration, but 80% cytotoxicity was observed at 10μM for HeLa and other cell lines (Fig. 2).
To be a good anticancer agent, LS10 essentially should not be toxic towards normal cells. Therefore, we sought to examine the cytotoxic effect of LS10 against normal cells with increasing concentration (1–20μM) against normal prostate epithelium cells (RWPE-1). The results demonstrated that LS10 did not show any cytotoxicity against normal cells up to 15μM, while significant cytotoxicity was observed against cancer cell lines at this concentration. At 10μM concentration, more than 95% of normal cells remained viable while 80% of cancer cells lost viability. However, at 20μM concentration about 20% of normal cells were found to lose viability (Fig. 2). To examine the cytotoxicity of LS10 against red blood cells (RBCs), if any, we performed hemolysis assay in a time-dependent manner for 30min, 180 min and 24h intervals using various concentrations (1–100μM) of LS10. As shown in Fig. 3, no significant hemolysis was observed up to 40μM concentration of LS10. Therefore, LS10 was concluded as non-hemolytic bacteriocin as it did not cause hemolysis even at 20 times higher concentration of IC50 values against various cancer cell lines (Fig. 3).
To evaluate the activity of LS10 on membrane integrity of cancer and normal cells, we have carried out lactate dehydrogenase (LDH) release assay with HeLa, MCF-7 and RWPE-1 cells. For this, cells were incubated with increasing concentration of LS10 (2, 5, 10 and 15μM) and LDH release was monitored up to 24h at regular time intervals. As shown in Fig. 4, significant LDH release was not observed in HeLa and MCF-7 cells up to 60min of incubation with all tested concentrations of LS10. However, 50% and 75% increase in LDH release was observed for HeLa and MCF-7 cells respectively, after 120min of incubation with 15μM concentration of LS10 (Fig. 4). These results are in accordance with the cell viability assay that displayed significant cytotoxicity against HeLa and MCF-7 cells at 15μM concentrations of LS10. Interestingly, no LDH release was observed in RWPE-1 cells even after 24h of incubation at 15μM concentration of LS10 (Fig. 4).
To further confirm the results of LDH release and MTT assays, scanning electron microscopy was performed using HeLa and MCF-7 cancer cells along with RWPE-1 normal cells treated with LS10. We have primarily observed changes in cell morphology, microscopically upon treatment with LS10 (Figure S1), which were further confirmed by electron microscopy performed under the identical conditions against cancer and normal cells. As shown in Fig. 5, MCF-7 cells treated with 5μM concentration of LS10, showed significant alterations on the cell membrane. However, HeLa cells displayed profound effect at 15μM but not at 5μM concentration of LS10. As a result of LS10 effect, cell membrane of MCF-7 cells became smooth and microvilli were lost from the cell surface even at 5μM concentration but in case of HeLa cells, the microvilli were completely vanished at 15μM concentration of LS10 (Fig. 5). On the other hand, no such effect was observed on RWPE-1 cells even after 3h of treatment with LS10 (Fig. 5). These results were in agreement with the MTT and LDH release assay.
In order to examine the possibility of LS10 to exhibit any other mechanisms of action, we have evaluated its ability to induce apoptosis in cancer and normal cells. Thus, HeLa, MCF-7 and RWPE-1 cells were incubated with sub-lethal concentration (2.5μM) of LS10 for 2h and 24h. The induction of apoptosis was examined by Annexin V/PI staining followed by flow cytometry analysis. Results clearly demonstrated that LS10 induced apoptosis in cancer cells as approximately 90% of HeLa and MCF-7 cells were found to be Annexin V positive after 2h of treatment when compared to untreated cells (Fig. 6). However, only about 40% RWPE-1 cells were found to be Annexin V positive (Fig. 6). Results after 24h of treatment with LS10 (Figure S2) were also followed the same pattern as observed for 2h treatment. These results suggest that LS10 induces apoptosis in cancer cells even at very low concentration and less toxic towards normal cells such as RWPE-1.
LS10, a defensin like class IId bacteriocin, was isolated from Brevibacillus sp. strain SKDU10 and found to inhibit microbial pathogens but did not found cytotoxic towards macrophages37. Therefore, in the present study we have made an attempt to explore the anticancer potential of defensin like bacteriocin LS10. Sequence alignment of LS10 with other known anticancer defensins demonstrated that cysteine residues are conserved in position with LS that showed typical disulfide bonding pattern with human β-defensins, a key feature defensin like peptides.
So, in order to examine the anticancer activities of LS10, cell viability assays were performed with different cancer cell lines along with normal cell line. LS10 displayed significant cytotoxicity against cancer cell lines with comparable MIC values found against bacterial cells37. Though studies revealed LS10 to be effective against all tested cancer cell lines with maximum potency against MCF-7 and low cytotoxicity against normal cells, further to get more insights, hemolytic properties of LS10 was also tested at different time points (30min, 180min and 24h). Interestingly, LS10 did not show any significant differences in hemolysis at prolonged durations when compared to values obtained during the earlier study37. It is pertinent to mention that most of the AMPs, despite their high anticancer activities, could not move forward in drug development pipeline because of their high hemolytic nature8,45.
The surface of cancer cells is different from the normal cells in many ways46 and one of the major difference is found to be in surface charge. Cancer cells are relatively more negatively charged compared to the normal cells due to the high expression of negatively charged lipid molecules47,48. Therefore, most of the cationic AMPs display broad spectrum of anticancer activities while their selective activity against cancer cells is due to the net high positive charge49,50. AMPs show diverse mechanisms of action against cancer cells, while most of them act on cell membrane, a few others cause the disintegration of mitochondrial membranes also19,49,51. Nisin and its variants are commonly used as a food preservative, and recently they have been reported to induce apoptosis, cell cycle arrest, and inhibition of cell proliferation in HNSCC (Head and neck squamous cell carcinoma) cells19,25. The mitochondrial membrane is believed to originate from endosymbiotic prokaryotes and having high similarities with bacterial cell membranes, which is justified by mitochondrial membrane specific activity of AMPs19,51,52. In fact, eukaryotic defensins are also reported to have antimicrobial and anticancer activities9,53,54. Thus, in order to determine defensin like bacteriocin LS10 mediated cancer cell cytotoxicity by membrane disintegration, we have performed LDH release assay, but at 2 to 5μM concentrations it did not trigger any LDH release. However, at 10 and 15μM concentrations, LS10 caused significant LDH release after 120min of treatment in HeLa and MCF-7 cells, suggesting the loss of membrane integrity. These observations were also confirmed by electron microscopy. Interestingly, RWPE-1 did not show any LDH release or adverse effect at 15μM concentration of LS10 even after 24h of treatment. Taken all these results together, it can be concluded that LS10 mediated cancer cell cytotoxicity is mainly due to membrane disintegration.
Despite the membrane disintegration many AMPs have been reported to induce apoptosis in cancer cells52,55,56. Similarly, LS10 also induce apoptosis in cancer cells. In fact, LS10 was found to be highly apoptotic for cancer cells at low concentration (2.5μM) and a multi-action bacteriocin, which induces both apoptosis and necrotic effects when treated with lower and higher concentrations, respectively. It is interesting to note that at 2.5μM concentration, despite the induction of high apoptosis (90%) in HeLa cells no significant cell death was observed in MTT assay. As synthetic anticancer drugs are very expensive and considering the production of bacteriocin LS10 produced by bacteria with novel antibacterial and anticancer activities against human cancer cells, indicates its potential for applications when compared to eukaryotic/human defensins. In fact, production of small AMPs naturally from bacteria is highly desirable and beneficial to test against cancer cells23,57. In summary, the bacteriocin LS10 is a potential novel anticancer molecule that displayed no adverse effect against normal cells like RWPE-1 and did not cause hemolysis.
Human cervical cancer cell line, HeLa and human normal prostate epithelium cell line RWPE-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Other cell lines like human embryonic kidney cancer cell line (HEK293T), human fibro-sarcoma cell line (HT1080), human lung carcinoma (H1299) and human breast cancer cell line (MCF-7) were gifted by Prof. R.N.K. Bamezai, National Centre of Applied Human Genetics, New Delhi, India. HeLa, HEK293T, HT1080 and MCF-7 cells were grown in DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin cocktail (Sigma, USA). RWPE-1 cells were grown in keratinocyte serum-free medium supplemented with recombinant human epidermal growth factor and bovine pituitary extract (Invitrogen, USA). All cells were maintained in CO2 incubator (ThermoScientific, USA) at 37°C and 5% CO2 environment.
The peptide was extracted from Brevibacillus sp. strain SKDU10 and purified as mentioned previously37. Amino acid sequence of LS10 was compared and aligned with laterosporulin and other human defensins with anticancer properties using Bioedit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
CD experiments were performed using a Jasco 815spectropolarimeter (JASCO, USA), coupled to a Peltier Jasco PTC-423L system for temperature control. HPLC purified LS10 (0.05mg/ml) in water was used for the experiments carried out in the presence of SDS micelles (5% SDS) (BioRad, USA) and 2,2,2-trifluoroethanol (100% TFE) (Sigma, USA). LS10 samples were also prepared to a final concentration of 0.05mg/ml in 10mM Tris-HCl (pH 7.0) for both SDS and TFE experiments. Spectra were collected and averaged over six scans in the spectral range of 195–250nm, with 0.2cm path length quartz cells (Starna Scientific, USA) at 37°C. Parameters used in experiments were 0.2nm step resolution, 50nm/min speed, 1s response time and 1nm bandwidth. Following baseline correction, the observed ellipticity, θ (m degree) was converted to the molar ellipticity [θ] (degree.cm2.dmol−1)43.
Cell viability was determined by using MTT assay. Briefly, HeLa, HEK293T, HT1080, H1299, MCF-7 and RWPE-1 cells (5×103 cells/well) were seeded in 96 well plates. Upon 24h of incubation, the growth medium was replaced with fresh medium containing increasing concentrations of LS10 (1–20μM). While 1% Triton X-100 (G Biosciences, USA) was used as positive control, blank growth medium as negative control. The plates were incubated for additional 24h and each well was added with 20μl of MTT solution (5mg/ml in PBS) and incubated for 3h at 37°C. Subsequently, the MTT containing medium was removed and 50μl of Dimethyl sulfoxide (Sigma, USA) was added to each well. To assess the percentage of live cells in samples, absorbance was read at 590nm on ELISA plate reader (ThermoScientific, USA).
The LDH release assay was performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, USA). Briefly, HeLa, MCF-7 and RWPE-1 cells (about 5×103 cells/well) were seeded in 96 well plates (BD Falcon, USA). Upon 24h incubation, culture medium was replaced with fresh medium containing LS10 (2, 5, 10 and 15μM). Blank growth medium was used as negative control and 1% Triton X-100 as positive control. After, 30min, 60min, 120min and 24h of treatment with different concentrations of LS10, culture medium was collected and centrifuged. Aliquots of 50μl from each reaction were incubated with 50μl of reaction buffer supplied with CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, USA). After 30min incubation in dark at room temperature, 50μl stop solution was added to each reaction well and the release of LDH was measured by reading absorbance at 490nm using ELISA plate reader (ThermoScientific, USA). Cytotoxicity of LS10 was determined by comparing LDH release with the positive control (1% Triton X-100 treated cells).
HeLa, MCF-7 and RWPE-1 cells (about 106 cells/well) were seeded on poly-lysine (Sigma, USA) coated hemocytometer cover slips, placed in 24 well plates (BD Falcon) and incubated in 5% CO2 environment at 37°C temperature. After 24h, growth medium was replaced with medium containing LS10 (5 and 15μM, in triplicate). Cells without peptide treatment served as negative control. After 3h treatment with LS10, cells were washed thrice with ice cold PBS to remove all unattached cells and fixed in modified karnovsky’s fixative for 2h. Subsequently, cells were treated with OsO4 (Sigma, USA) for 1h. Further, cells were dehydrated in graded ethanol sequentially with 30min incubation for each step at 4°C (30–100%). Ethanol dehydrated samples were freeze-dried and processed further as mentioned earlier58. Cover slips were placed on aluminium stubs using silver paint and sputter coated with gold. Cells were then observed and photographed using S-260, Leica Cambridge, scanning electron microscope (BRUKER, USA).
Apoptosis assay was performed using apoptosis assay kit (Invitrogen, USA). Briefly, HeLa, MCF-7 and RWPE-1 cells were seeded in 24 well tissue culture plates (about 2×105 cells/well) and incubated for 24h. Subsequently, culture medium was replaced with fresh medium containing 2.5μM of LS10 along with a negative control (cells without peptide). Apoptosis induced by the peptide was examined by Annexin V/PI staining followed by flow cytometry analysis of stained cells. Briefly, after 2h and 24h of incubation with LS10, cells were harvested, washed twice with cold PBS and resuspended in 1X Annexin binding buffer. Cells were then transferred to fresh 1.5ml tube containing 100μl of binding buffer and 5μl of FITC-conjugated Annexin V and 1μl of PI were added. The cells were vortexed gently and incubated for 15min at room temperature in dark. After incubation, 200μl of 1X Annexin binding buffer was added to each tube and stained cells were analyzed by Accuri flow cytometry (BD Biosciences, USA) with CellQuest software (BD Biosciences, USA) used for analysis of the results.
All comparisons were based on the mean +/− standard deviation of the mean (SD). Parametric data were analyzed using two-way analysis of variance (ANOVA) with the Bonferroni posttest method for comparison between groups. Column statistics for nonparametric data were analyzed by using the D’Agostino and Pearson omnibus normality test along with a one-sample t test. Results were considered significant when P values were <0.05. All experiments were performed independently three times in triplicate.
How to cite this article: Baindara, P. et al. Anticancer properties of a defensin like class IId bacteriocin Laterosporulin10. Sci. Rep. 7, 46541; doi: 10.1038/srep46541 (2017).
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Financial assistance from the Department of Biotechnology (grant no. DBT/In-Bz/2013-16/16/SO-R1) is duly acknowledged and also the Council of Scientific and Industrial Research (CSIR network project BSC-119, Human microbiome; Man as superorganism). We thank Dr. Mohammad Askandar Iqbal, Department of Biotechnology, Jamia Millia Islamia, New Delhi for useful discussions and National Center of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Delhi, for sharing the cancer cell lines. We thank Ms. Sumeeta Kumari for help in maintainance of the cell lines. We also thank Mr. Anil Theophilus and Randeep Sharma for their help in electron microscopy.
The authors declare no competing financial interests.
Author Contributions P.B., A.G., G.P.S.R. and S.K. involved in experiment design. P.B. and A.G. performed the experiments. P.B., A.G., G.P.S.R. and S.K. involved in analysis of results and wrote the paper.