Yeast probiotic Saccharomycopsis fibuligera alleviates the colorectal cancer by inhibiting the choline metabolism

doi:10.21203/rs.3.rs-4817265/v1

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Yeast probiotic Saccharomycopsis fibuligera alleviates the colorectal cancer by inhibiting the choline metabolism

https://doi.org/10.21203/rs.3.rs-4817265/v1

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The role of the microbiome in disease pathogenesis and anti-inflammatory effects of certain commensal microorganisms support the probiotic-based therapies for tumor treating. Saccharomycopsis fibuligera (S.fibuligera) is the major amylolytic yeast used in food fermentation. As a natural medicine, S.fibuligera has been found to protect the intestinal mucosa, while its role and related mechanisms in colorectal cancer (CRC) remain unexplored. In this study, we found that S.fibuligera could alleviate the development of CRC in mice, inhibit the proliferation of tumor cells and induce their apoptosis in vitro. Mechanistically, several metabolic pathways of tumor cells changed after S.fibuligera treatment, among which choline metabolism changed significantly. S.fibuligera inhibited choline metabolism of CRC cells by targeting and inhibiting the Ras/PI3K/AKT pathway, thereby reducing the expression of related metabolites, activating the AMPK pathway and inhibiting the mTOR pathway, which in turn induced apoptosis of CRC cells, ultimately alleviating CRC. Above all, our study has clarified anti-tumor effect of S.fibuligera and demonstrated that S.fibuligera alleviates CRC by inhibiting the choline metabolism, providing a promising new strategy for CRC treatment with high clinical translational value.

S.fibuligera

choline metabolism

colorectal cancer

metabolism reprogramming

Colorectal cancer (CRC) is the third most common malignancy worldwide and the second leading cause of cancer death[1]. Among CRCs, colitis-associated cancer (CAC) is caused by long-term exposure to intestinal inflammation[2]. Compared with sporadic CRC, CAC usually has more lesions, more severe pathological types, and worse prognosis[3]. Current treatments for CRC, such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, suffer from significant drawbacks, including high side effects and limited efficacy[4, 5]. Thus, novel strategies for the treatment of CRC are urgently needed.

Dysregulation of the gut microbiota affects the development of CRC[6], and probiotics are able to play a role in tumor immunotherapy by modulating the intestinal flora[7], including lactobacillus and bifidobacteria[8, 9]. Yeast, one of the most common fungi, is widely used in food production and fermentation[10]. It has a certain tolerance to acidic environment and can inhibit the growth of intestinal pathogens. Currently, Saccharomyces boulardii has been found to treat inflammatory colitis by altering the composition of intestinal microorganisms[11]. Meanwhile, yeast has also shown potential in tumor therapy due to its natural targeting capabilities[12], immunomodulatory properties, and high biosafety[13]. S.fibuligera is the major amylolytic yeast used in food fermentation for rice and cassava, capable of secreting amylase, acid protease, and β-glucosidase[14]. In an in vitro study assessing the therapeutic potential of five yeast probiotics from different sources, S.fibuligera exhibited stronger anti-cancer, anti-mutagenic, and anti-oxidant properties compared to other yeast probiotics[15]. Recent research has also found that S.fibuligera can protect the mouse intestine, reducing mortality and promoting intestinal barrier function [16]. However, its potential role in cancer therapy remains unexplored.

To meet the bioenergetic, biosynthetic and redox demands of malignant cells, tumors reprogram nutritional acquisition and metabolic pathways, a phenomenon known as metabolic reprogramming[17]. Cancer cells, for example, exhibit decreased mitochondrial oxidative phosphorylation and increased aerobic glycolysis, which contribute to tumor progression and chemoresistance[18]. Therefore, targeting cancer-specific metabolic pathways could lead to the development of novel cancer therapies. Metabolic abnormalities of glucose, amino acid, lipid, and nucleotide have been demonstrated in CRC, and drugs targeting enzymes involved in CRC metabolic reprogramming have been developed[19]. In addition, abnormal choline metabolism stands as a hallmark of cancer[20]. The proliferation of tumor cells requires an abundance of phosphatidylcholine (PC), a vital component of the phospholipid bilayer membrane[21]. Choline kinase alpha 1 (Chok-α1), as the key enzyme catalyzing PC synthesis, also positively regulates tumor cell proliferation and survival through the activation of signaling pathways such as PI3K/AKT [22].

In this study, we demonstrated that S.fibuligera significantly alleviated the development of CRC in mice, inhibited the proliferation of tumor cells and induced their apoptosis in vitro. Mechanistically, S.fibuligera inhibited choline metabolism of CRC cells by regulating the Ras/PI3K/AKT pathway. It also promoted the apoptosis of tumor cells by activating the AMPK pathway and inhibiting the mTOR pathway, thereby alleviating the development of CRC. Overall, our study demonstrates that S.fibuligera exerts an inhibitory effect on CRC development both in vivo and in vitro, providing a promising new strategy for CRC treatment.

Animals

Female C57L/6J mice aged 6–8 weeks were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were fed for one week in accordance with SPF feeding standards, with the temperature controlled at 18–22 ℃, humidity controlled at 40%-70%, and full nutrition feed. The experimental method followed the relevant regulations of Jiangsu Experimental Animal Management Committee.

All animal procedures were carried out in compliance with guidelines set by the Animal Care Committee at Nanjing University(Nanjing, China) under Grant Number IACUC-D2202134.

Strain

SHBCC D13478 Saccharomycopsis fibuligera was purchased from China General Microbiological Culture Collection Center (Beijing, China).

Cell line

Human colon cancer cell line HCT116 and mouse colon cancer cell line MC38 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China).

Methods

Cultivation of S.fibuligera

The freeze-dried powder was dissolved in the prepared YM medium and placed in a shaker for culturing at 28℃, 140r/min.

Construction of the colon cancer models

After a week of adaptive feeding, the mice were randomly divided into 3 groups: control group, CAC group and S.fibuligera group. On the first day of the experiment, mice in CAC group and S.fibuligera group were intraperitoneally injected with azoxymethane (12mg/kg, Sigma, USA), and after a week, the drinking water of mice was changed into 2.5%DSS, which was freely consumed for a week. At the third week of the experiment, 2.5%DSS was replaced with normal drinking water for two weeks. After repeating the above three-week cycle three times, the mice continued to drink normal water until the 100th day. The mice in the S. fibuligera group underwent 5 days of intragastric administration of S. fibuligera (1×10⁷CFU/mL) one week before the AOM injection, and were administered with S. fibuligera (1×10⁷CFU/mL) intragastrically every two days during the modeling period. The mice in the CAC group were administered with the same volume of PBS by intragastric gavage as control. On the 100th day of the experiment, the mice were euthanized, and anatomical dissection and organ collection were performed.

In the experiment of mitigating CAC development through inhibiting choline metabolism by S. fibuligera, two additional groups of mice were included: the Cho group and the Cho + S. fibuligera group. Both of the groups were fed a diet containing excess choline (choline chloride 5g/kg) from the first day of the experiment, while the other groups were fed a regular diet (choline chloride 1.5g/kg).

H&E staining

The cut tissues were fixed in 4% paraformaldehyde, then dehydrated, embedded, sectioned, dewaxed, hydrated, stained, dehydrated and sealed sequentially.

RNA extraction, reverse transcription and QPCR

10-30mg tissue was lysed in 1ml Trizol (Vazyme, Nanjing, China) using a homogenizer. Chloroform was added, shaken vigorously, and centrifuged. The aqueous phase was mixed with isopropanol, centrifuged, and the RNA precipitate was washed with 75% ethanol. The precipitate was dissolved in RNase-free water and heated at 56°C for 10 minutes. After determining the concentration, cDNA Synthesis Super Mix(Vazyme, Nanjing, China) was used to perform reverse transcription. Then the SYBR Green super mix (Vazyme, Nanjing, China) was used for processing qPCR experiment according to the manufacturer’s instructions. Ultimately, we calculated the relative expression level of mRNA using the 2^−ΔΔCt method.

Western blotting

After lysing the tissue in RIPA lysis buffer (Solarbio, Beijing, China), the liquid was centrifuged to obtain the supernatant, and the protein concentration was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China). We heated the protein in a 99°C metal bath for 10 minutes to fully denature it. Then, the protein was separated and transferred to polyvinylidene difluoride membranes. After incubation overnight at 4°C with the primary antibody and incubating with the secondary antibody for 2h, the protein bands were detected and visualized on Chemiluminescence System(BIO-RAD, USA).

TUNEL fluorescence staining

The cell smears were washed once with PBS. Cells were fixed with 4% paraformaldehyde for 30 minutes and washed once more with PBS. PBS of 0.3% Triton X-100 was added and incubated at room temperature for 5 minutes. Subsequently, 50µL of TUNEL detection reagent (Beyotime, Shanghai, China) was applied to the samples, which were then incubated in the dark at 37ºC for 60 minutes. We observed the samples under a fluorescence microscope, utilizing an excitation wavelength range of 450-500nm and an emission wavelength range of 515-565nm for clear visualization.

Cytokine assays

Mouse peripheral blood was centrifuged at 3000rpm for 20min, and the supernatant was used for ELISA (Dakewe, Shenzhen, China) to measure cytokine levels.

Cell viability assays

In a 96-well plate, 1×104 cells were seeded into each well and incubated for a period of time under conditions of 37°C and 5% CO₂. After the cells were attached to the wall, they were exposed to fungal stimulation at different multiplicity of infection (MOI) ratios: 1, 2, and 4. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (DOJINDO, Japan) following the provided procedure. The cells were incubated at 37℃ for 2 hours. Then, the absorbance at 450nm was measured using a microplate reader (BIO-RAD, USA).

Apoptosis assays

The Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China) was used to detect apoptosis. In a 12-well plate, 5×10⁵ cells were seeded into each well and incubated for a period of time under conditions of 37°C and 5% CO₂. After the cells were attached to the wall, they were exposed to fungal stimulation at different multiplicity of infection (MOI) ratios: 1, 2, and 4. Cells were harvested after stimulation and washed in cold PBS twice. Then, the cells were resuspended in 200µl of 1X binding buffer, and 5µl of Annexin V-FITC and 5µl of PI dye were added, followed by incubation in the dark for 10 minutes. Finally, we used a flow cytometer (Becton, Dickinson and Company, USA) to detect the samples and FlowJo10 to analyze the data and create graphs.

Untargeted metabolomics

The medium was quickly poured off, and the culture dish was rinsed 2–3 times with pre-cooled PBS to thoroughly remove residuals. Subsequently, the bottom of the culture dish was briefly exposed to liquid nitrogen for about a minute to facilitate cell detachment. A pre-cooled methanol-water mixture (with a ratio of 4:1 methanol to water) was added, and cells were gently scraped off using a cell scraper. The culture dish was then rinsed with the same methanol-water mixture to ensure all cells were collected. The collected cell samples were transferred to small glass vials in two portions, each with 500 µL of the methanol-water mixture. After adding 200 µL of chloroform, the mixture was gently agitated and subjected to sonication on ice to disrupt cell walls and release contents. An internal standard was added, followed by further sonication in an ice-water bath for extraction. Following centrifugation, the supernatant was transferred to LC-MS sample vials and the solvent was allowed to evaporate. The sample was re-dissolved in fresh methanol-water mixture, and centrifuged again. The supernatant was then aspirated, filtered through a filter, and transferred to an LC sample vial, which was stored at -80°C until LC-MS (Liquid Chromatography-Mass Spectrometry) analysis. The analytical instrument used in this experiment was a liquid chromatography-mass spectrometry system consisting of an ACQUITY UPLC I-Class plus Ultra-Performance Liquid Chromatography coupled with a QE High-Resolution Mass Spectrometer.

Metabolite content assay

The ELISA kit (AiFang, Hunan, China) was used to detect the contents of PC, PCho, and GPC in the cells.

Statistical analysis

Graphs and statistical analyses presented in this article were produced using GraphPad Prism version 8.00, with the results expressed in mean ± SEM format. One-way ANOVA was employed to analyze the differences between the control and treatment groups. P-value style: *p < 0.05; **p < 0.01; ***p < 0.001. All results were replicated three times or more.

S. fibuligera alleviated the development of CAC in vivo

To figure out whether S.fibuligera has a protective effect against CAC, we established a mouse CAC model. The CAC model combined the use of a chemical carcinogen, azoxymethane (AOM), and a pro-inflammatory agent, dextran sulfate sodium (DSS), to induce CAC in mice through a sequence of inflammation and tumorigenesis. During this period, S.fibuligera was administered via gavaging every two days, repeated for three cycles (Fig. 1B). By dissecting and measuring the lengths of the colons, the results demonstrated that the colons length of mice treated with S.fibuligera became longer compared to the control group(Fig. 1C-D), which suggested that S.fibuligera reduced the intestinal inflammation. Additionally, the spleen weight in the S.fibuligera-treated group was significantly lower than that in the control group (Fig. 1E-F). We then evaluated the number, size and burden of tumor, as shown in the Fig. 1G, S.fibuligera treatment markedly reduced the tumor number, tumor size, and tumor burden in CAC mice.

Histological examination with H&E staining showed that S.fibuligera treatment reduced the proportion of adenocarcinoma in mice and decreased the histological scores of tumor tissues in the S.fibuligera-treated group(Fig. 1H). Immunohistochemical analysis of tumor tissues for Ki-67 and PCNA expression revealed that S.fibuligera treatment reduced the expression of these markers, indicating that S.fibuligera can inhibit tumor cell proliferation (Fig. 1I). Moreover, we quantitatively analyzed the expression levels of inflammatory cytokines in the tumor tissues. The S.fibuligera treatment group showed a reduction in the serum levels of IL-6 and increasing IL-10 levels compared to the control group (Fig. 1J). As expected, it also suppressed IL-6 mRNA expression and enhanced IL-10 mRNA expression in tumor tissues (Fig. 1K). These results suggested that S.fibuligera may play a key role in inhibiting AOM/DSS-induced CAC in vivo.

S.fibuligera inhibited the cell proliferation and promoted the apoptosis of CRC cells in vitro

We further verified the the impact of S.fibuligera on the function of CRC cells in vitro. We stimulated the colorectal cancer cell lines MC38 and HCT116 with different concentrations of S.fibuligera. Employing the CCK-8 assay,we assessed the proliferation and viability of tumor cells. The CCK-8 results demonstrated that S.fibuligera effectively inhibited the viability of both MC38 and HCT116 tumor cells, with a dose-dependent effect observed in HCT116 cells(Fig. 2A). Western blot analysis showed that treatment with S.fibuligera promoted the expression of apoptosis-related proteins cleaved-caspase3 and BAX, while inhibiting the expression of survivin(Fig. 2B). Besides, to further investigate the influence of S.fibuligera on cell apoptosis, we utilized Annexin V/PI double staining. Similarly, compared to the control group, treatment with S.fibuligera significantly induced apoptosis in both MC38 and HCT116 cells(Fig. 2C). Interestingly, it also demonstrated a dose-dependent response, with higher concentrations of S.fibuligera correlating with increased rates of tumor apoptosis.The subsequent TUNEL fluorescence staining also indicated that the number of apoptotic cells increased with higher concentrations of S.fibuligera(Fig. 2D). These results collectively suggested that S.fibuligera can inhibit the proliferation of CRC cells and induce their apoptosis.

S.fibuligera played a key role in regulating metabolic reprogramming of CRC cells

S.fibuligera promoted metabolic reprogramming of CRC cells

Metabolic abnormalities in tumor cells are crucial for tumorigenesis. Li et al. found that β-Galactosidase secreted by S.thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft[23], demonstrating the probiotics can play an important role in regulating tumor cell metabolism.

To determine whether S. fibuligera exerts its effects by influencing the metabolism of CRC cells, we performed untargeted metabolomics on S.fibuligera-stimulated MC38 cells based on LC-MS/MS technology. This approach showed changes in various metabolic pathways following S.fibuligera treatment.

Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed for multivariate statistical analysis of the samples to distinguish overall metabolic profile differences between groups and identify differential metabolites. As shown in Fig. 3A-C, significant differences in metabolites between the S.fibuligera-treated group (S.F) and the control group (CON) were observed. Subsequently, we used T-tests and fold change analysis to compare the differential metabolites between the two groups. Volcano plots were utilized to visualize p-values and fold changes(Fig. 3D). In the volcano plot, red dots represent significantly upregulated metabolites in the S.F group, blue dots represent downregulated metabolites, and gray dots represent those not significantly different. S.fibuligera treatment can induce significant changes in the metabolites of MC38 cells.

By querying the KEGG database, we further identified associated metabolic pathways and performed pathway enrichment analysis on the differential metabolites. A bubble plot was created for the top 20 downregulated pathways(Fig. 3E). Following S.fibuligera treatment, significant changes were observed in various metabolic pathways, including choline metabolism, central carbon metabolism, pentose and glucuronate interconversions, and primary bile acid biosynthesis. Among these, the alterations in the choline metabolism pathway were the most notable. The Kennedy pathway, also known as the CDP-choline pathway, is crucial for the synthesis of phosphatidylcholine (PC), in which metabolites can regulate the tumor cells growth and provide a promising approach for creating anticancer treatments[24, 25]. We found that the abundance of Kennedy pathway-related metabolites (Phosphocholine(PCho), Phosphatidylcholine(PC), and Glycerophosphate choline(GPC)) was significantly lower in the S. fibuligera-treated group (Fig. 3F). As expected, we obtained similar conclusions by measuring the levels of choline metabolism-related metabolites in HCT116 cells using ELISA kits. Treating with the different concentrations of S.fibuligera, the levels of phosphatidylcholine(PC), phosphocholine(PCho), and glycerophosphate choline(GPC) all decreased compared with the control(Fig. 3G). Further investigation of the relevant mRNA in this metabolic pathway revealed that treatment with S.fibuligera significantly suppressed the gene expression associated with the Kennedy pathway(Fig. 3I). Among these genes, CHKA showed the most pronounced inhibitory effect, and Chok-α is a key enzyme in choline metabolism. Moreover, we conducted Western blot analysis to assess its downstream expression levels. The results demonstrated a significant decrease in Chok-α protein expression in CRC cells treating with S.fibuligera (Fig. 3H). These findings collectively indicated that S.fibuligera can inhibit choline metabolism in CRC cells in vitro and significantly suppress the expression of the key enzyme Chok-α in the Kennedy pathway.

S.fibuligera promoted apoptosis in CRC cells by inhibiting choline metabolism

In order to investigate whether choline metabolism can affect the function of CRC cells, we treated CRC cells with the Chok-α inhibitor MN58b to inhibit choline metabolism. Western Blot results indicated that MN58b significantly inhibited the expression of Chok-α at the protein level (Fig. 4A). Moreover, we demonstrated that both MN58b alone and the combination treatment of S.fibuligera and MN58b promoted the expression of apoptotic proteins, including BAX and survivin in the MC38 and HCT116 cells(Fig. 4B). Subsequently, we used the CCK-8 assay to detect cell proliferation, and MN58b alone can significantly inhibit the proliferation of MC38 cells. Furthermore, the combination of S.fibuligera and MN58b showed a more pronounced inhibition of CRC proliferation compared with MN58b or S.fibuligera alone (Fig. 4C). Annexin V/PI double staining and TUNEL results also indicated that MN58b could induce apoptosis in CRC cells, and when used in combination with S.fibuligera, the induction of apoptosis was more obvious (Fig. 4D-E).

S.fibuligera relieved the development of CAC by inhibiting choline metabolism in vivo

In order to confirm the role of choline metabolism in S.fibuligera's mitigation of CAC, C57BL/6J mice were fed a diet with excessive choline (choline chloride 5g/kg) during changing with AOM/DSS (Fig. 5A). Compared to the CAC group mice fed a regular diet, the choline chloride(Cho) group mice exhibited worsened CRC, characterized by shorter colons and higher tumor burden. However, after oral administration of S.fibuligera, the Cho + S.fibuligera group showed alleviation of the exacerbated effects of excessive choline. In comparison to the Cho group, the Cho + S.fibuligera group displayed longer colons(Fig. 5B-C), increased spleen weight (Fig. 5D-E), and reduced tumor number, size, and burden (Fig. 5F).

Additionally, the Cho group mice showed severe structural abnormalities in the colon by HE staining, which showed a significant alleviation after treating with S.fibuligera (Fig. 5G). Immunohistochemical staining of tumor tissues for Ki-67 and proliferating cell nuclear antigen (PCNA) also revealed similar results (Fig. 5H-I).

TUNEL staining indicated that S.fibuligera treatment induced apoptosis in tumor cells (Fig. 5J). Although choline metabolites in tumor tissues increased after feeding excessive choline, oral administration of S.fibuligera reduced the production of Kennedy pathway metabolites, inhibiting the tumor growth. Additionally, we examined the expression of genes related to the Kennedy pathway and choline transporters in tumor tissues. Interestingly, the Cho group did not show a significant increase in the expression of these genes. However, S.fibuligera treatment in mice fed excessive choline significantly reduced the expression of CHKA, CHKB, and CHPT1 (Fig. 5K), affecting the expression of choline kinase and choline phosphotrasferase. These findings suggested that S.fibuligera may inhibit CRC cell proliferation and induce apoptosis by downregulating choline metabolism.

The mechanisms of S.fibuligera to inhibit choline metabolism in CRC cells

S.fibuligera inhibited choline metabolism in CRC cells by regulating RAS / PI3K / AKT signaling pathway

Previous studies have shown that Chok-α is mainly activated by genes such as RAS, with lipid kinase PI3K, one of the most extensively studied Ras effectors, likely participating in this process. Ras mutations in NIH3T3 fibroblasts increase ChoK activity through Ral-GDS and PI3K pathways[26]. In human prostate and colon cancer cells, treatment with the PI3K inhibitor PI-103 reduces Chok-α and PC levels[27].

To explore whether S.fibuligera affects Chok-α expression via the RAS/PI3K/AKT pathway, we first examined changes in RAS expression and PI3K/AKT phosphorylation levels treating with S.fibuligera. Western Blot results indicated that, compared to the control group, S.fibuligera led to reduced Ras expression and inhibited AKT phosphorylation (Fig. 6A). Subsequently, we used the PI3K inhibitor 3-MA to treat CRC cells and found that 3-MA alone could inhibit PI3K and Chok-α expression. The inhibitory effect was enhanced when 3-MA and S.fibuligera were combined (Fig. 6B). Similarly, the levels of choline metabolism-related metabolites decreased with 3-MA treatment, and the combination of S.fibuligera and 3-MA suppressed the production of choline metabolism-related metabolites more effectively(Fig. 6C). These results suggested that S.fibuligera can inhibit Chok-α expression by suppressing the Ras/PI3K/AKT pathway, thereby inhibiting choline metabolism in CRC cells.

S.fibuligera induced CRC cells apoptosis by activating AMPK pathway and inhibiting mTOR pathway

In the metabolic pathway enrichment analysis, significant changes were observed in the expression of the mTOR and AMPK pathways in MC38 cells treating with S.fibuligera (Fig. 6D). AMPK has been demonstrated to directly phosphorylate p53 at serine15 to activate tumor apoptosis, regulating p53-dependent apoptosis[28]. Interestingly, inhibition of ChoK-α can lead to rapid activation of the AMPK metabolic stress sensor through the phosphorylation of its catalytic subunit α at residue T172. AMPK activation also inhibits the mTORC1 pathway by dephosphorylating mTOR at S2448. In turn, mTOR inhibition affects the activity of several known downstream targets which can regulate the growth, metabolism and proliferation and affect the tumorgenesis, such as 4E-BP1, p70S6K, S6 ribosomal protein, and GSK3[29]. Thus, changes in choline metabolism can play a crucial role in the regulation of AMPK activation.

Consequently, we examined the phosphorylation status of the AMPK/mTOR pathway in CRC cells. We found that S.fibuligera treatment promotes AMPK phosphorylation while inhibiting mTOR phosphorylation in CRC cells(Fig. 6E). Subsequently, we treated CRC cells with MN58b to inhibit choline metabolism and observed similar results (Fig. 6G).

Known as an energy sensor, AMPK can regulate cell growth. To investigate whether enhanced AMPK phosphorylation is associated with inducing apoptosis in CRC cells, we assessed cell apoptosis after adding the AMPK inhibitor Dorsomorphin. We found a significant reduction in apoptosis compared to treatment with S.fibuligera alone when Dorsomorphin was added (Fig. 6F). As expected, the combined treatment of Dorsomorphin and S.fibuligera resulted in increased apoptosis rates compared to the Dorsomorphin group (Fig. 6H-I). These findings demonstrated that S.fibuligera inhibits choline metabolism in CRC cells, leading to AMPK pathway activation and mTOR pathway inhibition, thereby mediating cell apoptosis.

In this study, we focused on the S.fibuligera and demonstrated its in vivo and in vitro anti-tumor effects and explored the deeply anti-tumor molecular mechanism, demonstrating S.fibuligera plays a key role in regulating metabolic reprogramming of CRC cells. The results showed that S.fibuligera is able to promote the apoptosis of CRC cells and relieve the development of CAC by inhibiting the choline metabolism. Mechanically, S.fibuligera inhibited choline metabolism in CRC cells by suppressing the RAS/PI3K/AKT signaling pathway and induced CRC cells apoptosis by activating AMPK pathway and inhibiting mTOR pathway. The study demonstrated the potential anti-tumor effects of S.fibuligera and its mechanism in regulating the metabolism reprogramming.

Probiotic plays an important role in preventing and treating different types of cancer[30, 31]. However, previous researches have primarily focused on the bacteria. Lactobacillus, Lactococcus, Bifidobacterium and Enterococcus are common bacterial probiotics in anti-tumor treatment[32–34]. S Karimi Ardestani et al. demonstrated the heat-killed probiotics have the ability to inhibit the tumor growth and induce apoptosis of HT-29 cells in human colon cancer[35]. Suguru Saito et al. also showed that the heat-killed Lactococcus lactis subsp. Cremoris C60 can enhance the major histocompatibility complex class I-restricted antigen presentation, increase the number of CD8⁺T and attenuate tumor growth[36]. Given the safety and acid-resisting characterization of yeasts, more and more yeasts such as S.boulardii and S.fibuligera are also accepted as potential probiotics mediating the intestinal microenvironment[37–39]. A study showed that S.fibuligera can provide protective effects on the mouse intestinal barrier, reduce intestinal barrier permeability, and induce CD4 + T cells produce IL-22[40]. This study mainly focused on the potential tumor treatment ability of S.fibuligera. S.fibuligera could alleviate CAC by reducing tumor burden, mitigating histopathological damage, and inhibiting the production of inflammatory cytokines in vivo. It also played a role in inhibiting cell proliferation and inducing apoptosis in different CRC cell lines in vitro.

To find out the reason how S.fibuligera alleviated the tumor burden, we performed untargeted metabolomics on S.fibuligera-stimulated MC38 cells based on LC-MS/MS technology. After stimulation with S.fibuligera, a significant decrease in choline metabolism was observed in CRC cells. Additionally, the gene levels of enzymes and transport proteins and metabolites involved in the Kennedy pathway were also found to be reduced. These results suggested that S.fibuligera can inhibit choline metabolism in CRC cells. CHOK-α overexpression is associated with the malignant phenotype, metastatic capability and drug resistance in human cancers, and thus has been recognized as a robust biomarker and therapeutic target of cancer[41–43]. In our results, it is shown that the expression of Chok-α in CRC cells significantly decreased at both the gene and protein levels after stimulation by S.fibuligera. When we treated the cells with the Chok-α inhibitor MN58b, we observed that expression of Chok-α was suppressed, inhibiting the proliferation of CRC cells and inducing their apoptosis. By feeding the mice with choline-rich diet, the Kennedy pathway was activated. We found that the colon cancer treating with choline-rich food was more severe compared to the CAC group. However, after the treatment with S.fibuligera, there was a certain degree of alleviation. S.fibuligera played a key role in mediating the choline metabolism, the results also indicated that S.fibuligera might inhibit the Kennedy pathway by suppressing Chok-α.

When we treated CRC cells with the PI3K inhibitor 3-MA, we observed that the stimulation of CRC cells with S.fibuligera inhibited the expression of RAS and PI3K and the inhibition of PI3K also reduced the expression of Chok-α, demonstrating S.fibuligera can inhibit choline metabolism by suppressing the RAS/PI3K pathway. In the metabolic pathway enrichment analysis of MC38 cells stimulated by S.fibuligera, significant changes were found in the expression of the mTOR and AMPK pathways. AMPK and mTOR pathways are closely related to cell growth and development, particularly in terms of apoptosis and autophagy [44, 45]. We conducted Western Blot analysis to detect the phosphorylation levels of AMPK and mTOR proteins and found that S.fibuligera indeed activated the phosphorylation of AMPK and inhibitd the phosphorylation of mTOR in CRC cells, thereby inducing apoptosis. This phenomenon is mediated by Chok-α, as the inhibition of Chok-α resulted in reduced phosphorylation of AMPK. Subsequently, using an AMPK inhibitor, we demonstrated that activation of AMPK can induce apoptosis in CRC cells. These results indicated that S.fibuligera inhibited choline metabolism through the Ras/PI3K signaling pathway, thereby activating the AMPK pathway, promoting AMPK phosphorylation, inhibiting mTOR phosphorylation, and subsequently promoting apoptosis in CRC cells. However, the role of the S.fibuligera and its effect on the gut microbiota in CAC remains incompletely understood. Further research is necessary to comprehensively assess the changes in S.fibuligera expression profiles throughout disease progression and the gut microbiome changes in response to treatment, allowing a deeper understanding of S.fibuligera treatment impact on CAC.

This work provides a new sight in the therapeutic roles of S.fibuligera in managing CRC. Given S.fibuligera's ability to treat tumors and regulate cancer cell metabolic reprogramming to promote apoptosis and inhibit proliferation, it shows promise as a potential therapeutic tool in oncology. Further study about the engineering modifications of S.fibuligera such as whole recombinant yeast vaccine carriers, yeast surface display[37], may improve the efficacy and expand the application prospects of yeast in the field of tumor treatment. Collectively, this work presents a natural medicine for modulating the intestinal microenvironment, influencing tumor cell reprogramming, and inhibiting tumor initiation and progression with the ultimate goal of advancing the development of patient-friendly and yeast therapeutics for CRC.

AOM	Azoxymethane	CTL	Choline transporter like protein
CAC	Colitis-associated cancer	DSS	Dextransodium sulfate
Cho	Choline	GPC	Glycerophosphate choline
ChoK	Choline kinase	PC	Phosphatidylcholine
Chok-α	Choline kinase alpha	PCho	Phosphocholine
CRC	Colorectal cancer	S. fibuligera	Saccharomycopsis fibuligera

Acknowledgements

The authors are grateful to Professor Yue Zhao and Yuxin Yin from Nanjing University for their technical assistance.

Author contributions

TW and XW conceived and devised the study. XW, XL and CW designed the experiments and analysis. XL and CWformed the experiments, performed statistical analysis and wrote the paper. TW and XW provided technical support. All authors read and approved the submitted paper.

Funding Information

This work was supported by the China Postdoctoral Science Foundation under Grant Number 2023M741629 and 2024T170397; Jiangsu Funding Program for Excellent Postdoctoral Talent under Grant Number 2024ZB062.

Conflict of interest

The authors declare that they have no conflict of interest.

Data Availability

All data supporting the fndings of this study are available within the paper.

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Yeast probiotic Saccharomycopsis fibuligera alleviates the colorectal cancer by inhibiting the choline metabolism

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Abstract

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Introduction

Materials and methods

Animals

Strain

Cell line

Methods

Construction of the colon cancer models

H&E staining

RNA extraction, reverse transcription and QPCR

Western blotting

TUNEL fluorescence staining

Cytokine assays

Cell viability assays

Apoptosis assays

Untargeted metabolomics

Metabolite content assay

Statistical analysis

Results

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Version 1