The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase

Abstract

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics.

Results: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains.

Conclusion: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

Keywords: Lactococcus lactis; Anti-cancer; Arginine deiminase; Cell-free supernatant; Cytostatic; Lactic acid bacteria; Postbiotics; Probiotics; Protein release; Therapeutic vector.

Fig. 1

Both CFS and > 50 kDa fraction from L. lactis Lc4 strain demonstrate a protein-dependent reduction in cell proliferation. a, c) Confluence of HT-29 (left) and HCT116 (right) in time after treatment with 33% (v/v) CFS (a) or F > 50 kDa (c). GM17 or GM17 > 50 kDa was used as additional control. Graphs represent one biological repetition, performed in 3 technical replicates, error bars represent SD. b, d) Cell growth rate constant after treatment with CFS (b) or F > 50 kDa (d) (n = 3 independent experiments). Data are presented as mean with min to max range. e) Effect of protein digestion on the cytostatic activity of F > 50 kDa fraction. Results represent mean +/- SD for n = 2 independent experiments (*p < 0.05, **p < 0.01, by one-sided t-test)

Fig. 2

Identification of proteins in fractions responsible for the observed cytostatic effect. (a) Diagram depicting the experimental set-up of identifying potentially active proteins in the F > 50 kDa fraction. (b) SDS-PAGE analysis of active fractions separated via different chromatography techniques. Bands excised for MS analysis are marked with red rectangles, with their corresponding molecular weight provided. (c) Proteins identified by MS within the excised SDS-PAGE bands, along with their respective emPAI index. (d) Gene Ontology biological process enrichment analysis performed with the ShinyGO package [59]. Visualized are the top 20 significantly enriched pathways. Pathways associated with glucose degradation and its metabolic processes are marked in red rectangles. Details of the analysis are summarized in the Materials and Methods section, and full results of GO enrichment analysis are presented in Supplementary Table 2. Analysis parameters: FDR cut-off: 0.05, pathway size: Min. 10, Max. 2000; removed redundancy, and abbreviated pathways. Fold Enrichment was defined as the percentage of genes above the FDR cut-off, belonging to a pathway, divided by the corresponding percentage in the background (MS hits in F > 50 kDa).

Fig. 3

F-IEX fraction induces cell cycle arrest in the G0/1 phase, decreases c-Myc expression, and reduces phosphorylation of the p70 S6 kinase. (a) Confluence changes in HT-29 and HCT116 cells over time (0-72 h) after F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10 mM HEPES buffer – 5% v/v) or no treatment (control). Scans were performed every 2 h, each time point represents mean +/- SD (n = 3 independent experiments). (b) Growth rate constant of F-IEX-treated HT-29 and HCT116 cells. (c) Microscopic images from the IncuCyte S3 Live-Cell imaging system of HT-29 and HCT116 cells treated for 72 h with F-IEX (5% v/v) or vehicle (same as in a) ) as a control. (d) SYTOX Green-exclusion experiments. Green area-to-phase area ratio as quantification of changes in cells with disrupted membrane integrity during 72 h treatment with F > 50 kDa and GM17 > 50 kDa (5% v/v) and 5 µM of SYTOX Green (left graph) with corresponding confluence (right graph). Puromycin (0.5 µg/ml) was used as a cell death-inducing positive control. (e) Cell cycle analysis of HT-29 cells after 24 h treatment with 5% (v/v) F-IEX and vehicle (same as in a). (f) Bar-chart quantifying result from panel e. Data presented as mean +/- SD from n = 3 independent experiments. (g) Western blots showing c-Myc and phosphorylated p70-S6 kinase (P-p70-S6K) levels. HT-29 and HCT116 cells were treated with 5% vehicle (same as in a) or 5% (v/v) of F-IEX for 24 h. Total protein was isolated from the cells, resolved in SDS-PAGE gels, and analyzed using antibodies against indicated proteins. Actin was used as a loading control. n = 3 independent experiments. (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test)

Fig. 4

Arginine supplementation rescues the cytostatic effect of F-IEX and L. lactis Lc4 CFS. (a) Confluence changes of HT-29 (top) and HCT116 (bottom) cells over time (0-72 h) after F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10mM HEPES buffer – 5% v/v) treatment, with and without the addition of 5 mM L-arginine (ARG). Cell proliferation was monitored with the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point represents mean +/- SD, n = 3. (b) Growth rate constant of data presented in panel a. (c) Cell cycle analysis of HT-29 cells after 24 h of treatment with 5% (v/v) F-IEX and vehicle (same as in a) with the addition of 5mM L-arginine (ARG). Measured DNA content corresponds to the percentage of cells in each cell cycle phase (G0/1, S, G2/M). (d) Bar chart quantifying results from panel c. Data presented as mean +/- SD from n = 3 independent experiments. (e) Western blots showing c-Myc and phosphorylated p70 S6 kinase (P-p70-S6K) levels. HT-29 and HCT116 cells were treated with 5% vehicle (same as in a) or 5% of F-IEX for 24 h, with and without the addition of 5mM L-arginine. Total protein was isolated from the cells, resolved in SDS-PAGE gels, and analyzed using antibodies against indicated proteins. Actin was used as a loading control. n = 3. (f) The effect of 5 mM L-arginine addition on the cytostatic activity of Lc4 CFS evaluated on HT-29 cells with the MTS assay (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test (b, d) or one-way ANOVA with Bonferroni correction (f)

Fig. 5

Heterologously expressed recombinant wild-type ADI protein exhibited the same cytostatic phenotype as F-IEX. (a) SDS-PAGE gels analysis of purified proteins. Left gel: ADI WT protein after two steps of purification on Superdex75 (GF75) and Resource Q (IEX) columns (three consecutive fractions collected after IEX separation are shown). Middle gel: Catalytically inert mutant of ADI – C400A. Right gel: Direct comparison of F-IEX and heterologously expressed recombinant ADI WT protein. The red rectangles show the bands corresponding to the calculated size of respective ADI variants (ADI WT or C400A). (b) Representative graphs of confluence changes of HT-29 (top) and HCT-116 (bottom) cells over time (0-72 h) after ADI (0.66% v/v), F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10mM HEPES buffer) treatment with or without the addition of 5 mM L-arginine (ARG). The proliferation of cells was monitored using the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point presents a mean +/- SD. (c) Growth rate constant of data presented in b) (n = 3 independent experiments). (d) Confluence changes of HT-29 (left) and HCT116 (right) cells over time (0-72 h) after ADI (0.66% v/v), vehicle (same as in b) and C400A catalytic mutant (0.66% v/v) treatment. The proliferation of cells was monitored using the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point presents a mean +/- SD. (n = 3 independent experiments). (e) Table with enzymatic activity values of F-IEX, heterologously expressed recombinant ADI WT protein, and ADI C400A catalytic mutant (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test)

Fig. 6

The enzymatic and cytostatic activity of the F > 50 kDa fraction is a unique feature of the Lc4 strain. (a) SDS-PAGE gel stained with PageBlue (Thermo Fisher) with protein profiles of F > 50 kDa fractions from Lc4, Lc2, and Lc3 L. lactis strains. (b) Growth rate constant of HT-29 (left) and HCT116 (right) cells treated with F > 50 kDa fractions from Lc4, Lc2, Lc3, and GM17 > 50 kDa (as a control) (n = 3 independent biological replicates). Data presented as a mean with min to max range. (c) Enzymatic activity of F > 50 kDa fractions from Lc4, Lc2, and Lc3 strains. Data presented as mean +/- SD from n = 3. (d) Stacked bar chart presenting the percentage of injured, dead, and live bacterial cells (cultivation condition described in the Materials and Methods section). Data are presented as a mean from n = 3 biological replicates (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-way ANOVA with Dunnet’s correction)

Fig. 7

ADI release is the consequence of bacterial cell damage and protein leakage. (a) Changes in the viability of L. lactis Lc4 cells during cultivation with respect to ADI enzymatic activity. The enzymatic activity, as an amount of L-citrulline produced by ADI, was measured for F > 50 kDa obtained from 25 ml of CFS. Data points are represented as mean +/- SD (n = 3 independent biological replicates). (b) SDS-PAGE gel analysis of protein profiles of Lc4 F > 50 kDa obtained from different bacterial cultivation time points. Protein concentration was measured with the Bradford method and equal protein amounts of each sample were resolved in SDS-PAGE gel. (c) SEM image of Lc4 strain cells after 24 h of cultivation. Blue rectangles indicate cells undergoing the lysis process. Red ovals mark possible cytoplasmic blebs and/or leakage events. (d) TEM image of cytoplasm leakage from Lc4 cells. The red oval indicates altered membrane integrity