Bacillus mojavensis postbiotics: transcriptomic and anticancer effects in colon cancer cells

doi:10.21203/rs.3.rs-7574147/v1

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Bacillus mojavensis postbiotics: transcriptomic and anticancer effects in colon cancer cells

https://doi.org/10.21203/rs.3.rs-7574147/v1

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Probiotics have been shown to exert antiproliferative effects on colon cancer cells. While these effects are often attributed to microbiome regulation, they may also result from bioactive metabolites produced by probiotic bacteria. In the present study, we investigated the impact of a cell-free extract, hereafter referred to as a postbiotic, derived from Bacillus mojavensis, a strain isolated from aguamiel (a traditional Mexican beverage). The antiproliferative activity was evaluated in SW480 human colon cancer cells using MTT and crystal violet assays, while antimigratory effects were assessed through a wound-healing assay. In addition, the ability of the postbiotic to counteract inflammatory proliferation was evaluated in SW480 cells treated with lipopolysaccharide (LPS). Biosafety was tested using peripheral blood mononuclear cells (PBMCs) from healthy donors. The results demonstrated that treatment with 25 or 50 µg/mL of B. mojavensis postbiotic reduced the viability of more than 75% of SW480 cells and significantly inhibited cell migration after 24 h. Moreover, the postbiotic decreased LPS-induced proliferation without exerting any cytotoxic effect on PBMCs, underscoring its selectivity toward malignant cells. To elucidate the underlying mechanisms, transcriptomic profiling was performed, revealing extensive modulation of oncogenes and tumor suppressors, with enrichment of PI3K–Akt, MAPK, apoptosis, and cytokine receptor pathways. In conclusion, postbiotics from B. mojavensis isolated from aguamiel exhibit selective anticancer activity by inhibiting proliferation, migration, and inflammation-induced growth in colorectal cancer cells. Transcriptomic findings further support these effects.

Bacillus mojavensis

postbiotics

colon cancer cells

aguamiel

Bacillus mojavensis postbiotics selectively inhibit the proliferation and migration of SW480 colon cancer cells without harming PBMCs.

Postbiotics counteract LPS-induced inflammatory proliferation, supporting their anti-inflammatory and biosafety profile.

Transcriptomic profiling revealed reprogramming of cancer-related pathways, including PI3K–Akt, MAPK, apoptosis, and exosome-mediated communication.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality and the third most common type of cancer worldwide, with an expected increase particularly in countries with a high or very high Human Development Index (HDI) (Morgan et al. 2023). Survival prognosis strongly depends on the stage of the disease: patients in stage I present a survival rate close to 90%, whereas those in stage III show a 5-year survival of ~ 50%, and patients in stage IV have less than 10% survival expectancy (Chen et al. 2019; Van der Jeught et al. 2018). Current therapies include surgery, chemotherapy, radiotherapy, and targeted therapies; however, drug resistance and the high metastatic potential remain the main challenges (Van der Jeught et al. 2018).

An increasing body of evidence demonstrates a strong correlation between intestinal microbiome alterations (dysbiosis) and CRC progression (Ahn et al. 2013; Arthur et al. 2012; Kostic et al. 2013; Sobhani et al. 2011; Yang et al. 2021; Yang et al. 2019). Notably, metabolites derived from bacteria of healthy donors inhibit tumor growth and metastasis (Bell et al. 2022). These microbial products modulate cellular processes such as transcription, proliferation, and inflammation, directly or indirectly impacting oncogenic pathways (Ibragimova et al. 2021). Consequently, the intestinal microbiome and its metabolites are increasingly considered both for prevention and as adjuvant treatments in CRC (Van der Jeught et al. 2018; Saeed et al. 2022).

Probiotics have been widely studied for their beneficial effects, including improving intestinal barrier integrity, restoring microbiome composition, degrading carcinogenic compounds, and secreting molecules with antitumor activity (Tripathy et al. 2021). More recently, postbiotics, defined as “preparations of inanimate microorganisms and/or their components that confer a health benefit on the host” (Salminen et al. 2021), have emerged as promising alternatives. Compared with live probiotics, postbiotics offer advantages in terms of stability, safety, and reproducibility. Several metabolites, including secondary bile acids, short-chain fatty acids (SCFAs), and reuterin, have been linked to reduced intestinal inflammation, modulation of the redox balance, and decreased proliferation of colon cancer cells (Bell et al., 2022; Song et al., 2020).

Although Lactobacillus and Bifidobacterium have been the most studied, the anticancer properties of postbiotics derived from Bacillus have been less explored, despite the clear advantages of this genus, such as spore formation, high resistance to adverse environmental conditions, and survival in the gastrointestinal tract (Mingmongkolchai and Panbangred 2018; Song et al. 2023). In this context, in the present study, we analyzed the effect of the postbiotic derived from a strain of Bacillus mojavensis isolated from aguamiel (the sap used to prepare pulque, a traditional Mexican beverage). This strain (B-F2A1) has been characterized through microscopy, 16S rRNA sequencing, mass spectrometry, and biochemical profiling. It has demonstrated antagonistic effects against multidrug-resistant pathogens, supporting its potential as both probiotic and postbiotic (Martínez-Ortiz et al. 2024).

Given the urgent need for novel, selective, and safe therapeutic strategies against CRC, in this study, we evaluated the effect of B. mojavensis postbiotics on SW480 colon adenocarcinoma cells. We analyzed their impact on cell viability, migration, and inflammatory proliferation induced by lipopolysaccharide (LPS), while simultaneously determining their safety profile in human peripheral blood mononuclear cells (PBMCs). We also performed a transcriptomic profile using microarrays to identify molecular mechanisms associated with cancer.

Bacterial culture and Postbiotic (CFS).

Bacillus mojavensis (strain B-F2A1 NCBI OQ469319, CDBB B-2078) was cultured in MRS broth (De Man, Rogosa and Sharpe; Sigma-Aldrich, St. Louis, MO, USA) for 24 h at 37°C to obtain a pre-inoculum. Optical density was adjusted to 0.5 McFarland (~ 10⁸ CFU/mL) using a densitometer (Thermo Fisher Scientific). To collect postbiotics from bacterial cultures grown in MRS and DMEM/F12 medium, the pre-inoculum culture was inoculated at 2% into fresh MRS broth or DMEM/F12 and incubated for 48 h at 37°C. After incubation, the cultures were centrifuged at 4,500 rpm for 10 min at 4°C (Eppendorf 5810R, Hamburg, Germany), and the pH was adjusted to 7.4. The cell-free supernatants (CFS, hereafter referred to as postbiotic) were recovered, filtered through a 0.22 µm PES membrane (Millipore, Burlington, MA, USA) to ensure sterility, and supplemented with 10% of fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific). The postbiotic preparations were stored at 4°C until use.

Protein quantification of postbiotic preparations

The protein concentration of the Bacillus mojavensis postbiotic was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. The postbiotic concentration was expressed in micrograms of protein (Bradford assay) as a practical and reproducible standardization criterion. This strategy allowed for the standardization of treatments across experiments. We recognize that the biological effects are not exclusively due to proteins, but rather to the complex mixture of peptides, metabolites, short-chain fatty acids, and other bioactive molecules.

Cell culture

SW480 human colon adenocarcinoma cells (ATCC CCL-228) were cultured in DMEM/F12 medium (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific), 2 mM L-glutamine (Sigma-Aldrich), and antibiotics (50 U/mL penicillin and 50 µg/mL streptomycin; Gibco, Thermo Fisher Scientific). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. The protein concentration of the postbiotic was quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA).

MTT assay

Cell viability was determined using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich). SW480 cells were seeded at a density of 40,000 cells/well in 48-well plates (Corning, NY, USA) and treated with 25 or 50 µg/mL of B. mojavensis postbiotic for 24 h. Following treatment, cells were incubated with MTT solution (0.5 mg/mL) for two hours at 37°C in the dark. The supernatant was removed, and the formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich). Absorbance was measured at 540 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific). Cells treated with the DMEM F12 free cell supernatant were cultured with 7.5%, 15% and 30% (V/V) of the postbiotic. All experiments were conducted in triplicate, and data were normalized against untreated control cells.

Crystal violet assay

SW480 cells were seeded at 40,000 cells/well in 48-well plates and treated with MRS medium (control) or postbiotic (25 or 50 µg/mL) for 24 h. After treatment, cells were washed three times with phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific), fixed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min, and stained with 0.05% crystal violet solution (Sigma-Aldrich) in 20% ethanol for 1 min. Excess stain was removed by washing with PBS, and plates were visualized under an inverted microscope (Nikon Eclipse Ts2, Tokyo, Japan).

Wound-healing assay

SW480 cells were seeded at 20,000 cells/well in 24-well plates (Corning) and grown to confluence. A linear scratch was made across the monolayer with a sterile 200 µL pipette tip. Detached cells were removed by washing with PBS, and cells were then treated with MRS (control) or postbiotic (25 or 50 µg/mL) in serum-free DMEM/F12 medium. Images were captured at 0 and 24 h using a phase-contrast inverted microscope (Nikon Eclipse Ts2). After 24 h, cells were fixed and stained with crystal violet as described above, and wound closure was quantified.

LPS treatment

SW480 cells were seeded at 40,000 cells/well in 48-well plates and treated with 10 µg/mL lipopolysaccharide (LPS from Escherichia coli O111:B4; Sigma-Aldrich, Cat. No. L2630-25) for 24 h. To evaluate the effect of the postbiotic, cells were co-treated with 25 or 50 µg/mL of B. mojavensis postbiotic. Cell viability was determined by using the MTT assay as described previously.

PBMC isolation and postbiotic treatment

PBMCs were isolated from 5 mL of venous blood obtained from healthy donors using density gradient centrifugation with Lymphoprep™ (STEMCELL Technologies, Cat. No. 07801). PBMCs were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM L-glutamine (Sigma-Aldrich), 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells (40,000/well) were treated with 30% or 50% (v/v) postbiotic for 24 h, and cell viability was evaluated using the MTT assay. Human PBMCs samples were obtained and processed in compliance with the Helsinki Declaration and approved by the local ethics committee (Comité de Bioética en Investigación del Decanato de Ciencias Médicas UPAEP, CEIEUPAEP06/2023).

Statistical analysis

All experiments were performed at least in triplicate. Data are expressed as mean ± SEM. Before statistical testing, data were assessed for normality and equality of variances. Statistical differences among groups were evaluated using one-way ANOVA followed by Dunnett’s post hoc test. Analyses were performed using GraphPad Prism software (v8.0; GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

Microarray experiments

SW480 cells were seeded at 40,000 cells/well in 48-well plates and treated with 15% (v/v) MRS or postbiotic for 10 h. Longer incubations or higher concentrations did not yield intact RNA. Total RNA was extracted using TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA purity and concentration were determined with a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific). Four datasets were generated using the Human Cancer Oligonucleotide Array Hcan_06 (Microarray Unit, Cellular Physiology Institute, UNAM, Mexico City), containing 50-mer probe sets representing 1,920 transcripts. For probe preparation, 5 µg of total RNA were reverse-transcribed into cDNA and labeled with Alexa Fluor 555 and Alexa Fluor 647 dyes (Thermo Fisher Scientific). Hybridizations were performed in duplicate, scanned using the ScanArray 4000 system (Packard BioChips, Billerica, MA, USA), and analyzed with the corresponding software.

Data Analysis

Microarray fluorescence data were normalized using the Z-score transformation method (Cheadle et al. 2003). Genes with a z-ratio > ± 1.5 were considered significantly differentially expressed. The fluorescence intensity matrix was curated and deposited in the Gene Expression Omnibus (GEO/OMNIBUS) under accession GSE307410.

Pathway and Enrichment Analysis

Pathway and enrichment analyses were conducted in R software (v4.2.2) using RStudio, with the Bioconductor package clusterProfiler v4.9. Visualization and pathway mapping were performed using Pathview and Heatmapper. Analyses were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases, including Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories. Additional functional enrichment of the top up- and down-regulated genes was performed using g:Profiler (Kolberg et al. 2023) under default parameters.

Bacillus mojavensis postbiotic selectively reduces the viability and migration of SW480 colon cancer cells without affecting PBMCs.

To determine the antiproliferative potential of B. mojavensis postbiotic, we first assessed its effect on SW480 colorectal cancer cells. Exposure to 25 or 50 µg/mL of the postbiotic for 24 h significantly reduced cell viability as measured by MTT assays (****p < 0.0001 vs. control), with both concentrations showing comparable effects. These findings were confirmed by crystal violet staining, which revealed a marked decrease in adherent cells and monolayer disruption, indicating loss of proliferative capacity and viability (Fig. 1A–B).

We next evaluated the impact on cell migration, a critical step in metastatic dissemination. Wound-healing assays showed that postbiotic treatment at both concentrations inhibited SW480 migration after 24 h compared to MRS control (Fig. 1C). This antimigratory activity may involve interference with cytoskeletal dynamics, extracellular matrix remodeling, or adhesion signaling pathways that sustain tumor invasiveness. Moreover, the effects on cell viability may be attributed to the metabolites secreted by B. mojavensis, regardless of the culture medium in which the postbiotic is generated. This is supported by the fact that when the postbiotic is produced in the DMEM F12 cell culture medium, it still decreases the viability of SW480 cells (Supplementary Fig. 1).

Given the therapeutic relevance, it was essential to determine whether these effects were selective for malignant cells or also detrimental to healthy immune cells. Therefore, PBMCs from healthy donors were exposed to postbiotic (30% v/v) or MRS control for 24 h. Interestingly, no cytotoxicity was observed in PBMCs. In fact, postbiotic-treated PBMCs displayed significantly higher viability compared to MRS (****p < 0.0001) (Fig. 1D). This result supports the selectivity of the postbiotic towards cancer cells, supporting its potential as a safe therapeutic adjuvant and ruling out general cytotoxicity. The inclusion of PBMCs was explicitly designed to evaluate biosafety, a critical requirement in developing biotherapeutic candidates.

Bacillus mojavensis postbiotic attenuates LPS-induced proliferative signaling in SW480 cells.

The tumor microenvironment of colorectal cancer is characterized by chronic inflammation, often exacerbated by bacterial products such as lipopolysaccharide (LPS), which engages Toll-like receptor 4 (TLR4) and activates NF-κB/MAPK cascades that promote tumor growth and survival. To model this inflammatory stimulus, SW480 cells were incubated with 10 µg/mL LPS for 24 h. As expected, LPS induced a strong proliferative response, increasing cell viability more than fourfold relative to control. Remarkably, co-incubation with B. mojavensis postbiotic (25 or 50 µg/mL) abrogated this effect, reducing proliferation by up to 375.68% compared to LPS alone (****p < 0.0001) (Fig. 1E). This finding indicates that the postbiotic may interfere with LPS-driven pro-inflammatory and proliferative cascades, although the precise molecular mediators remain to be identified.

Bacillus mojavensis postbiotic modulates oncogenic gene expression in SW480 cells.

Microarray analysis of SW480 cells exposed to 15% v/v postbiotic for 10 h revealed extensive transcriptional reprogramming. A total of 1,920 transcripts were interrogated, and the 20 genes with the highest positive (up-regulated) and negative (down-regulated) z-ratio values were represented in the heatmap (Fig. 2A). The heatmap illustrates the differential expression of these genes, with hierarchical clustering clearly separating the samples into two groups: control (MRS) and B. mojavensis postbiotic (BM). Red and blue colors indicate up- and down-regulated genes, respectively. The up-regulated genes included PLOD2, MMP14, RPS19, CD80, DCT, PRDX2, RAB10, ABCC4, SSTR2, and RCV1, while the down-regulated genes comprised PBOV1, SERPINB1, FLJ38507, OGN, RFXAP, MEST, SLC12A2, ENO1, HLA-DQA1, and RAD1.

In Fig. 2B, functional enrichment analysis of the 20 most representative DEGs is shown as a bar plot integrating multiple databases (GO, KEGG, Reactome, WikiPathways, miRTarBase, Transcription Factor, Human Protein Atlas, Corum, and Human Phenotype Ontology). Enriched categories are displayed on the X-axis and adjusted significance values (− log10 Padj) on the Y-axis. Only GO:CC (Cellular Component), GO:BP (Biological Process), and Transcription Factors (TF) showed significant enrichment. Among the most relevant terms were extracellular exosome, endocytic vesicles, transcription regulator complexes, and cell adhesion, highlighting the role of the postbiotic in modulating intercellular communication, transcriptional regulation, and vesicle organization. These findings suggest that treatment with B. mojavensis postbiotics alters both the dynamics of exosome secretion and gene regulation mediated by transcription factors that are critical in cancer, paving the way for future studies to identify metabolites and their effects.

In Fig. 2C, enrichment in GO Cellular Component (CC) is represented as a circular plot linking modulated genes to the cellular structures in which they act. Among the significantly enriched components were extracellular exosomes and endocytic vesicles, where RAB10 and ABCC4 were up-regulated (genes implicated in vesicular trafficking and metabolite export), while SLC12A2 and RCV1 were repressed. These changes indicate that the postbiotic actively regulates vesicle and exosome release, modulating intercellular communication and the transfer of pro-oncogenic signals. In the mitochondrial category, PRDX2 (peroxiredoxin-2, with antioxidant activity) was up-regulated. At the same time, ENO1 (a glycolytic enzyme essential for tumor metabolism) was repressed, pointing to a rebalancing of oxidative metabolism and a potential reduction of the glycolytic phenotype typical of cancer cells (Warburg effect). Within DNA repair complexes, RAD1 (a DNA damage sensor) was down-regulated, suggesting that the postbiotic interferes with the tumor cells’ ability to respond to genomic instability. Finally, in transcriptional regulator complexes, RFXAP and MEST were both repressed, indicating modulation of epigenetic regulation and cell differentiation programs.

Finally, Fig. 2D shows enrichment in GO Molecular Function (MF), which revealed an equally relevant profile with key functions modulated by specific genes. For transcription factor binding, CD80 (up-regulated) was associated with immune modulation through T cell activation via CTLA4/CD28 interactions. In contrast, CREB (down-regulated) is a transcription factor known to promote cell proliferation and survival, suggesting that the postbiotic exerts an inhibitory effect on proliferative programs. Regarding kinase activity and nucleotide binding, PIK3CG was strongly repressed, a central mediator of the PI3K–Akt pathway that supports tumor growth and resistance to apoptosis. Its suppression points to direct inhibition of this signaling axis. In ligand and receptor binding, VEGF and PDGFA were up-regulated, both linked to tyrosine kinase receptors. Although these are typically pro-oncogenic, their upregulation in a context of simultaneous repression of major metabolic and proliferative drivers (e.g., PIK3CG and ENO1) may reflect a compensatory feedback mechanism induced by the postbiotic. For adaptor molecular functions, GNB2 was up-regulated, while GNG11 was repressed, indicating a reorganization of G-protein–coupled receptor (GPCR) signaling cascades.

Pathway and Gene Ontology (GO) enrichment analysis of DEGs in SW480 cells treated with Bacillus mojavensis postbiotic.

Enrichment analysis revealed that the transcriptional reprogramming induced by the postbiotic involved key oncogenic and immune-related pathways (Fig. 3). KEGG pathway enrichment (Panel A) highlighted significant modulation of PI3K–Akt signaling, MAPK signaling, cytokine–cytokine receptor interaction, apoptosis, and general pathways in cancer, consistent with the repression of PIK3CG and CREB and the up-regulation of IL2, VEGF, and PDGFA. GO Biological Process enrichment (Fig. 3B) identified terms such as regulation of MAPK cascade, apoptotic signaling, cell adhesion, lymphocyte differentiation, and cellular stress response, linking postbiotic treatment to reduced proliferative capacity and enhanced immune regulation. GO Molecular Function enrichment (Fig. 3C) indicated enrichment in kinase activity, transcription factor binding, cytokine receptor binding, and growth factor binding, reflecting broad modulation of signaling and transcriptional control. Finally, GO Cellular Component enrichment (Fig. 3D) revealed significant associations with focal adhesions, exosomes, vesicles, mitochondria, and transcription regulator complexes, supported by the up-regulation of RAB10 and ABCC4 and the down-regulation of SLC12A2. Collectively, these results support the idea that the postbiotic disrupts proliferative and survival pathways while enhancing immune signaling and vesicle-mediated communication, although further studies are warranted to confirm these mechanistic interpretations.

The development of colorectal cancer (CRC) involves a complex interaction between intrinsic and extrinsic factors, including gut dysbiosis. Probiotics, mainly Lactobacillus, have been proposed as a strategy to restore microbial balance, either by modulating bacterial populations or through their metabolic products, known as postbiotics. Based on this evidence, postbiotics have gained attention as safer and more stable alternatives to probiotics, since they may exert anticancer effects without requiring the survival of live microorganisms. In our study, treatment with 25 and 50 µg/mL of B. mojavensis postbiotics reduced the viability and migration of SW480 cells. These include bacteriocins, biosurfactants, exopolysaccharides, siderophores, immunomodulatory components and other bioactive metabolites released during microbial growth or lysis (Kanmani et al. 2013; Uesugi et al. 2023). Given these diverse mechanisms, members of the genus Bacillus have emerged as promising sources of bioactive postbiotics. The genus Bacillus has also demonstrated anticancer properties. For example, the supernatant of B. coagulans upregulates pro-apoptotic genes such as bax, casp3, and casp9 in SKBR3 breast cancer cells (Dolati et al. 2022), while an B. subtilis extracellular polymeric substance exerts antioxidant and antitumoral activities (Zhang and Yi 2022).

The antimigratory effect observed in SW480 cells (Fig. 1C) is consistent with reports of probiotic-derived molecules with antimetastatic activity. Mixtures of B. longum, B. bifidum, L. acidophilus, and L. plantarum reduce invasion and tumor growth (Shang et al. 2020). Likewise, the supernatant of L. acidophilus and L. rhamnosus GG has been reported to decrease MMP2/MMP9 expression and increase TIMP-1/TIMP-2 in HeLa and HT29 cells (Nouri et al. 2016). Our transcriptomic analyses support this observation, showing down-regulation of genes associated with extracellular matrix and cell adhesion, suggesting that migration inhibition is not solely dependent on loss of viability.

In this context, bacterial products such as lipopolysaccharide (LPS) play a central role by activating inflammatory pathways that promote tumor progression. In our model, LPS increased the viability of SW480 cells, supporting its ability to induce proliferation through TLR4–NF-κB/MAPK signaling, as previously described (ZHU et al. 2019). Importantly, we showed that B. mojavensis postbiotics counteracted the proliferative effect of LPS, suggesting that they interfere with pro-inflammatory cascades in colorectal cancer.

On the other hand, biosafety is an indispensable requirement for the use of probiotic-derived products. In our work, PBMCs treated with 30% v/v postbiotic showed no cytotoxicity and, in fact, presented greater viability than cells cultured with MRS, evidencing the selectivity of the effect toward malignant cells. Similar findings have been described with parasporins from B. thuringiensis, which exert selective cytotoxicity on tumor cells without affecting PBMCs (Borin et al. 2021). However, it is essential to consider that the increased viability of PBMCs exposed to postbiotics should be further studied to determine whether it reflects a phenotypic change and ultimately to ensure their safety. Therefore, additional in vivo and mechanistic studies are needed to fully establish the safety profile of B. mojavensis postbiotics.

Microarray analysis revealed differentially expressed genes that collectively enriched PI3K–Akt, MAPK, apoptosis, and cytokine-cytokine receptor interaction pathways. Up-regulation of TP53 supports the activation of tumor suppressor mechanisms, while down-regulation of PIK3CG and CREB indicates inhibition of proliferative and survival signaling. In addition, enrichment in exosome- and vesicle-related processes suggests modulation of intercellular communication, potentially limiting the spread of pro-oncogenic signals. These molecular results reinforce the proliferation and migration assays and open an entire line of research aimed at identifying the active components of the postbiotic responsible for its biological effects on cancer cells.

Although the obtained results demonstrate an apparent antiproliferative, antimigratory, and anti-inflammatory effect of B. mojavensis postbiotics on colorectal cancer cells, this study presents some limitations. First, only two fixed concentrations (25 and 50 µg/mL) were evaluated; therefore, future research should include a dose–response curve to calculate the IC₅₀, which would be relevant to compare effects among postbiotics. Second, functional analysis was performed in a single cell line (SW480); therefore, extrapolation to other CRC types or in vivo models should be made with caution. Third, although the microarray allowed identification of key molecular pathways, the specific metabolites or proteins responsible for the biological effect have not yet been characterized; thus, metabolomic and proteomic studies will be required to confirm these findings. Finally, biosafety evaluation was limited to PBMCs from healthy donors, so it will be essential to expand these studies to other cell types and animal models before considering clinical applications.

Despite these limitations, our results demonstrate that B. mojavensis postbiotics selectively inhibit proliferation, migration, and inflammation-induced growth in colorectal cancer cells. Integrating functional and transcriptomic assays provides new insights into the mechanism of action and therapeutic potential of B. mojavensis metabolites. Future studies should include metabolomic and proteomic analyses to identify the specific bioactive compounds responsible and to determine dose–response relationships in preclinical models.

Acknowledgements

We appreciate the technical and administrative support of the laboratories, especially Héctor Galvan Ramos and María Isabel Galvez Soto.

Author contribution

Conceptualization: MA T-L, E B-R; Methodology: MA T-L, MA G-V, E B-R, C M-O, JM S-L; Investigation (cell culture, viability and migration assays, RNA extraction): MA T-L, MA G-V, C M-O, VM M-O; Bioinformatics and microarray analysis: JM S-L, C M-O; Formal analysis: MA T-L, MA G-V, E B-R, JM S-L; Literature search: MA T-L, E B-R, C M-O, VM M-O; Resources: MA T-L, E B-R, VM M-O; Data curation: MA T-L, MA G-V, E B-R; Writing - original draft: MA T-L, E B-R; Writing-review and editing: MA T-L, MA G-V, E B-R, C M-O, VM M-O, JM S-L, EG E-K; Supervision: E B-R, EG E-K; Project administration: E B-R, B P-A, EG E-K; Funding acquisition: E B-R. All authors have read and approved the final manuscript.

Funding: This work was partially funded by the Mexican Academy of Sciences (AMC), L'Oréal-Mexico, the UNESCO Office in Mexico, the Mexican Commission for Cooperation with UNESCO (CONALMEX), and the UPAEP research fund.

Availability of data and materials: The datasets generated and analyzed during the current study are available in the genomics data repository GEO with accession GSE307410 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE307410)

Ethics approval and consent to participate. PBMCs were obtained from healthy adult donors under informed consent, following approval by the Institutional Research Ethics Committee of UPAEP (Under review: CEIUPAEP-006/2023). This study did not involve animal experimentation.

Consent for publication. All authors agree to be published.

Competing interests: The authors declare that they are clear of any financial or personal conflicts that might affect the research reported in this study.

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No competing interests reported.

Supplementaryfigure1.jpg
Supplementary fig 1. Viability of SW480 cells treated with postbiotic obtained in MRS and DMEM F12 cell media. Viability of SW480 cells treated with the postbiotic at concentrations of 30% (a), 15% (b) and 7.5% (c) grown in MRS media and 30% (d), 15% (e) and 7.5% (f) of postbiotic of B. mojavensisobtained in DMEM F12 eukaryotic cell media. Also DMSO at 5% (g) was used.

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Bacillus mojavensis postbiotics: transcriptomic and anticancer effects in colon cancer cells

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Abstract

Figures

Key Points

Introduction

Material and methods

Protein quantification of postbiotic preparations

Cell culture

MTT assay

Crystal violet assay

Wound-healing assay

LPS treatment

PBMC isolation and postbiotic treatment

Statistical analysis

Microarray experiments

Data Analysis

Pathway and Enrichment Analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1