Probiotic Lactiplantibacillus plantarum WB4404 and WB4503 from Kimchi Induce Apoptosis in Human Colorectal Cancer Cells In Vitro

TL;DR

This study shows that two probiotic bacteria from kimchi can trigger cell death in human colorectal cancer cells in the lab.

Contribution

The novel contribution is identifying L. plantarum WB4404 and WB4503 as probiotics that induce apoptosis in colorectal cancer cells via intrinsic pathways.

Findings

L. plantarum WB4404 and WB4503 significantly inhibited HT-29 cell proliferation and activated apoptosis.

Apoptosis was confirmed through flow cytometry and DNA fragmentation observed via DAPI staining.

Exopolysaccharides from the bacteria reduced cell viability, suggesting a role in anticancer effects.

Abstract

Lactic acid bacteria isolated from kimchi include beneficial microorganisms with diverse health-promoting properties. Among these, Lactiplantibacillus plantarum has attracted attention owing to its potential anticancer effects. In this study, we investigated the anticancer activity and underlying mechanisms of L. plantarum WB4404 and L. plantarum WB4503, isolated from kimchi, in HT-29 human colorectal cancer cells in vitro. Both strains significantly inhibited HT-29 cell proliferation and activated apoptotic pathways. Flow cytometry analysis confirmed apoptosis, and DAPI staining revealed DNA fragmentation. The expression of apoptosis-related proteins, such as caspase-3, caspase-9, and the Bax/Bcl-2 ratio, was significantly altered, indicating activation of the intrinsic cell death pathway. Furthermore, crude exopolysaccharide reduced HT-29 cell viability, suggesting that bacterial metabolites may contribute to the overall anticancer effect rather than acting as independent determinants. These findings highlight that L. plantarum WB4404 and L. plantarum WB4503 induce intrinsic apoptosis in HT-29 human colorectal cancer cells in vitro and suggest their potential as probiotic candidates for functional food applications.

Figures

A](#F1)), reflecting impaired mitochondrial activity—a characteristic feature of early apoptosis [[30](#ref30), [31](#ref31)]. However, as MTT primarily assesses mitochondrial metabolism, it does not directly distinguish between apoptotic stages. LDH release increased modestly in response to the bacterial treatment ([Fig. 2](#F2)), suggesting that mitochondrial dysfunction occurred more prominently than late-stage membrane damage. Furthermore, all strains maintained over 80% viability in MRC-5 normal fibroblasts, indicating that the observed growth inhibition in HT-29 cells was unlikely to res

Full text

Introduction

Kimchi is a representative Korean fermented food, containing various beneficial microorganisms that proliferate during fermentation and exert positive effects on human health [1]. Lactic acid bacteria (LAB) isolated from kimchi have been reported to exhibit diverse physiological activities, including intestinal microbiota modulation, immune regulation, antioxidant, and anticancer effects [2, 3]. Owing to these biofunctional properties, increasing attention has been directed toward the isolation of novel probiotic strains from kimchi and elucidation of their underlying mechanisms of action [4].

Among kimchi-derived LAB, Lactiplantibacillus plantarum is a well-known probiotic with strong resistance to gastric and bile conditions and high adhesion ability to intestinal epithelial cells, allowing close host-microbe interactions [4, 5]. The consumption of probiotics has been shown to alter the intestinal microbial environment, and such modulation is particularly associated with the prevention and adjuvant treatment of colorectal cancer [6, 7]. Importantly, the beneficial effects of probiotics may result not only from live bacteria but also from heat-killed cells or bacterial metabolites [8]. Recent studies have demonstrated that L. plantarum exerts direct anticancer effects by modulating immune responses, inhibiting oxidative stress pathways, and suppressing cancer cell proliferation [9].

Exopolysaccharides (EPS), which are extracellular polysaccharides produced and secreted by LAB, have been reported to influence host physiological responses through immune modulation and antioxidant, anti-inflammatory, and cytoprotective effects [10]. Several studies have shown that probiotic-derived EPS can suppress the survival of cancer cell and promote apoptosis by inducing the accumulation of reactive oxygen species and inhibiting nuclear factor-κB signaling. Therefore, EPS may act as a crucial mediating factor contributing to the overall anticancer potential of probiotic strains [11].

Cancer is characterized by uncontrolled cell proliferation and tumor formation, and colorectal cancer is the third most diagnosed cancer and one of the leading causes of cancer-related deaths worldwide [12]. Its incidence is closely associated with lifestyle factors, such as westernized diets, obesity, alcohol consumption, and smoking, and has been increasing among younger populations [13]. Over the past decade, research has revealed that dysbiosis of the gut microbiota is closely associated with the onset and progression of colorectal cancer [7]. An imbalance in the gut microbiota induces inflammatory responses, enhances carcinogen production, and weakens intestinal barrier integrity, thereby promoting tumor development. Conversely, probiotic intake has been reported to restore microbial balance, alleviate inflammation, and act as a preventive or adjunctive therapy for colorectal cancer [14]. In this context, the anticancer mechanisms of L. plantarum are particularly relevant in colorectal cancer models, in which intestinal homeostasis and immune modulation play critical roles. Several previous studies have reported pro-apoptotic and anticancer effects of L. plantarum strains and their EPS on colorectal cancer cell lines [15]. However, these reports generally described apoptosis induction without distinguishing strain-level differences. In contrast, the present study explicitly compares the apoptosis profiles of two newly characterized strains, L. plantarum WB4404 and L. plantarum WB4503, thereby highlighting strain-specific variation within the same species. By doing so, this work extends prior findings beyond general cytotoxicity and defines distinct apoptotic responses associated with individual strains.

In this study, we investigated the anticancer activity and apoptosis-inducing mechanisms of kimchi-derived L. plantarum strains in HT-29 human colorectal cancer cells. HT-29 cells were treated with live L. plantarum to evaluate changes in cell proliferation and apoptosis-related protein expression, and EPS fractions isolated from the same strains were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the potential anticancer effects of bacterial metabolites.

Materials and Methods

Isolation and Identification of LAB

Traditionally prepared homemade kimchi samples (including cabbage kimchi and young radish kimchi) at 100 μl was incubated in 3 ml of pH 2.5-controlled MRS broth (Difco Laboratories, USA) for 30 min. After serial dilution, the samples were cultured on MRS agar at 37°C for 24 h. To isolate single colonies, the samples were double plated on MRS agar. The LAB were identified using 16S rRNA gene sequencing (Bionics Co., Ltd., Republic of Korea).

Bacteria Strains and Sample Preparation

The isolated strains, L. plantarum WB4404 and L. plantarum WB4503, were compared with Lacticaseibacillus rhamnosus GG (LGG), an industrially important probiotic strain that has been extensively studied. LGG, L. plantarum WB4404, and L. plantarum WB4503 were cultured in MRS broth at 37°C for 18 h, followed by washing twice with phosphate-buffered saline solution (PBS; Hyclone, USA). Cells were resuspended in the Roswell Park Memorial Institute 1640 medium (RPMI; Hyclone) at a final concentration of 1 × 10^8^ CFU/ml for subsequent experiments [16].

Extraction and Quantification of Ethanol-Precipitated EPS

Ethanol precipitation of EPS was performed as follows [17]. The cultures were grown in MRS broth at 37°C for 21 h. The cell-free supernatant was obtained through centrifugation at 14,000 ×g for 30 min at 4°C. To remove proteins, 4% (w/v) trichloroacetic acid was added to the supernatant, followed by stirring for 30 min, and the precipitate was removed through centrifugation. To precipitate EPS, three volumes of pre-chilled 99% ethanol were slowly added to the supernatant, and the mixture was incubated at 4°C for 24 h. The crude EPS was collected through centrifugation and analyzed using the phenol-sulfuric acid method.

Cell Culture

The MRC-5 (KCLB 10171) and HT-29 human colon adenocarcinoma (KCLB 30038) cells were obtained from the Korean Cell Line Bank (Republic of Korea). MRC-5 cells were maintained in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% streptomycin/penicillin (Hyclone) at 37°C in a humidified environment with 5% CO_2_. HT-29 cells were cultured in RPMI containing 10% fetal bovine serum and 1% streptomycin/penicillin under the same incubation conditions.

Determination of Acid and Bile Salt Tolerance

To simulate the human gastrointestinal environment, MRS broth supplemented with 0.3% (w/v) pepsin (Sigma-Aldrich, USA) was adjusted to pH 2.5, and MRS broth containing 0.3% (w/v) bile salts (Difco Laboratories Inc., USA) was prepared. Pre-cultured Lactobacillus strains were inoculated into each medium to assess their survival under simulated gastric and intestinal conditions. The inoculated cultures were incubated for 2 or 24 h, after which appropriate dilutions were plated onto MRS agar to enumerate viable cells [18].

Adhesion Assay of Probiotic Strains to HT-29 Cells

To evaluate the adhesion ability of the probiotic strains, HT-29 cells were cultured in 6-well plates to obtain a confluent monolayer. One day before the adhesion assay, the culture medium was replaced with RPMI 1640 without penicillin and streptomycin and incubated for 24 h. Bacterial suspensions were then prepared and added to each well at a final concentration of 1 × 10^8^ CFU/ml. The plates were incubated at 37°C for 2 h to allow bacterial adhesion. After incubation, the wells were washed twice with PBS to remove non-adherent bacteria. Adherent bacteria were detached by treating the cells with 0.1% Triton X-100. The number of adherent bacteria was quantified and expressed as a percentage of the total bacterial count to determine the adhesion rate [19].

Cytotoxicity Evaluation of LAB Strains

To assess the inhibitory effect of LAB on cancer cell growth and the extent of cell death, a modified MTT assay was performed [20]. MRC-5 and HT-29 cells were seeded a density of 2 × 10^4^ cells/well in 96-well plates and incubated for 24 h under standard culture conditions (37°C, 5% CO_2_). The cells were treated with the LAB strains and EPS for 48 h in RPMI 1640 with penicillin and streptomycin. The cells were washed twice with PBS, and MTT solution (0.5 mg/ml) was added at a volume of 100 μl/well and incubated for 4 h. After incubation, 200 μl of dimethyl sulfoxide was added to dissolve the resulting formazan crystals, followed by shaking for 15 min. The absorbance was measured at 570 nm.

Lactate Dehydrogenase (LDH) Cytotoxicity Assay

The cytotoxic effect of LAB on HT-29 cells was assessed by quantifying LDH release using an EZ-LDH Cell Cytotoxicity Assay Kit (DG-LDH500) (DoGENBio Co., Ltd., Republic of Korea) [21]. HT-29 cells were seeded at 2 × 10^4^ cells/well in 96-well plates and incubated for 24 h at 37°C in 5% CO_2_. LAB was then added to reach a final concentration of 1 × 10^8^ CFU/ml, and the cells were further incubated for 48 and 72 h. Then, 10 μl of cell-free supernatants were transferred to new 96-well plates, mixed with 100 μl of LDH reaction mixture, and incubated for 30 min in the dark. The absorbance was measured at 450 nm.

Quantification of Relative mRNA Expression Using Quantitative Reverse Transcriptase Real-Time Polymerase Chain Reaction (qRT-PCR)

Apoptosis-related gene expression was assessed using qRT-PCR [22]. The β-actin gene was used as an internal reference for normalization. Total RNA was isolated using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA), and cDNA was synthesized with the SensiFAST cDNA Synthesis Kit. For the quantification of apoptosis-related genes (Table 1), SYBR Green-based qRT-PCR was conducted, and relative gene expression levels were analyzed using the 2^-ΔΔCt^ method.

Caspase-9 and Caspase-3 Colorimetric Assay

To investigate the apoptotic pathways of L. plantarum WB4404 and L. plantarum WB4503 in HT-29 cells, caspase activity was measured [23]. HT-29 cells were seeded at 1 × 10^6^ cells/well and incubated for 24 h, followed by treatment with LAB (1 × 10^8^ CFU/well) for 24, 48, and 72 h. After treatment, cells were washed twice with PBS, lysed with the provided lysis buffer on ice for 10 min, and centrifuged to obtain cytosolic extracts. The amount of protein extracted at each time point was quantified, and the Caspase-3 Colorimetric Assay Kit (Abcam, UK) and Caspase-9 Colorimetric Assay Kit (Abcam) were used according to the manufacturer’s instructions. Caspase activity was expressed as a fold change relative to the control group

Western Blot Analysis

The expression levels of apoptosis-related proteins were analyzed using western blotting using a slightly modified version of a previously described method [24]. HT-29 cells were seeded in 6-well plates (1 × 10^6^ cells/well). They were then treated with LAB (1 × 10^8^ CFU/well), washed twice with PBS, and harvested for protein extraction. Proteins were extracted using Pro-Prep lysis buffer (iNtRON Biotechnology, Republic of Korea) supplemented with a protease inhibitor cocktail (Thermo Scientific Pierce, USA). After centrifugation at 15,000 ×g for 30 min at 4°C, the supernatant was collected and protein quantified. Equal amounts of protein (25 μg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with primary antibodies (Cell Signaling Technology, USA). Subsequently, the membranes were incubated with the appropriate secondary antibodies (Cell Signaling Technology) for 1 h. After washing with Tris-buffered saline containing Tween 20 (ChemBio, Republic of Korea), the protein bands were visualized using enhanced chemiluminescence (Advansta, USA).

Apoptosis Analysis Using Annexin V-Fluorescein Isothiocyanate / Propidium Iodide (PI) Staining

Flow cytometric analysis with Annexin V/PI staining was conducted to discriminate apoptotic from necrotic cell death [25]. HT-29 cells were seeded and incubated for 24 h, followed by treatment with LAB samples for 48 and 72 h. After treatment, cells were harvested by centrifugation at 800 ×g for 5 min at 4°C and washed twice with 3 ml of PBS. The cells were then stained using the Dead Cell Apoptosis Kit with Annexin V for Flow Cytometry (Invitrogen, USA), and apoptosis was analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, USA).

Cell Cycle Using Flow Cytometry

Cell cycle analysis was performed to investigate the mechanism of growth inhibition [26]. Both floating and adherent cells were collected and centrifuged at 800 ×g for 5 min at 4°C. The cells were washed twice with 3 ml of PBS, fixed in 70% ethanol at 4°C overnight, and centrifuged to remove ethanol. After washing with PBS, the cells were incubated with 300 μl of staining solution containing PI (50 μg/ml, Biosesang, Republic of Korea) and RNase A (10 μg/ml, Sigma-Aldrich, USA) at 37°C for 30 min. The cell cycle distribution was analyzed using a flow cytometer (CytoFLEX, Beckman Coulter).

DAPI Staining and Morphological Analysis

HT-29 cells were seeded in 6-well plates at a density of 1 × 10^6^ cells/well, treated with LAB for 48 h, washed three times with PBS, and observed for morphological changes using a DS-Ri2 digital camera (Nikon Co., Ltd., Japan).

DAPI staining was performed to observe nuclear condensation and fragmentation, indicative of apoptosis [27]. HT-29 cells were seeded in confocal dishes (1 × 10^5^ cells/well; SPL Life Sciences, Republic of Korea) and incubated for 24 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 15 min, followed by three additional washes with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 (200 μl) for 5 min and subsequently stained with DAPI (1 μg/ml; 4',6-diamidino-2-phenylindole) at 37°C for 15 min in the dark. After washing with PBS, the nuclei were visualized using a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany).

Statistical Analysis

All data are expressed as the mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed using IBM SPSS software version 25.0. Prior to analysis, the assumptions of normality and homogeneity of variance were assessed. Differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test as a post hoc analysis to identify statistically significant differences among multiple treatment groups (p < 0.05).

Results

Gastrointestinal Tolerance and Adhesion of LAB Strains

Acid and bile tolerance, as well as the adhesion ability to intestinal epithelial cells, of L. plantarum WB4404 and L. plantarum WB4503 isolated from kimchi were evaluated (Table 2).

Under simulated gastric conditions (pH 2.5), the survival rates of LGG, L. plantarum WB4404, and L. plantarum WB4503 was 100.40 ± 1.01, 99.80 ± 0.73, and 99.79 ± 0.43%, respectively. All strains demonstrated strong acid tolerance. In simulated bile conditions, the survival rates were 107.17 ± 1.06, 100.45 ± 0.92, and 99.94 ± 0.43%, respectively. The adhesion rates of LGG, L. plantarum WB4404, and L. plantarum WB4503 were 80.9 ± 0.52, 88.34 ± 0.16, and 81.08 ± 0.55%, respectively.

Comparison of Crude EPS Productivity among LAB Strains

The productivity and total carbohydrate contents of the crude EPS are presented in Table 3. The EPS yield, expressed as a percentage, varied among the strains. The crude EPS production of LGG, L. plantarum WB4404, and L. plantarum WB4503 were 2.13 ± 0.08, 5.57 ± 0.18, and 2.69 ± 0.04 μg EPS/8 log CFU for the respective strains, with L. plantarum WB4404 showing the highest level.

Growth Inhibition of HT-29 Cells by LAB and Their Crude EPS Fractions

The inhibitory effects of LAB and their crude EPS on HT-29 cells were evaluated based on the reduction in metabolic activity, as measured using the MTT assay (Fig. 1). In MRC-5 normal fibroblasts, all LAB strains maintained high cell viability, exceeding 80% across treatments. In contrast, the viability of HT-29 human colorectal cancer cells treated with LGG, L. plantarum WB4404, and L. plantarum WB4503 was 66.67 ± 2.34, 8.41 ± 0.25, and 43.53 ± 3.98%, respectively. Both L. plantarum strains exhibited stronger antiproliferative effects than the reference strain LGG, with L. plantarum WB4404 inducing the most pronounced reduction in viability, indicating a high cytotoxic potential toward HT-29 cells in vitro. To further investigate the contribution of EPS, HT-29 cells were treated with the crude EPS extracted from each strain at concentrations ranging from 12.5 to 500 μg/ml. Metabolic inhibition increased in a concentration-dependent manner, and a similar trend was observed for their EPS fractions, although the magnitude differed from that of the whole cells, suggesting that EPS may contribute in part, but does not fully account for, the effects observed with whole cells.

LDH Cytotoxicity Assay by LAB Strains

To confirm the cytotoxic potential of the LAB observed in the MTT assay, the release of LDH from HT-29 cells was measured after 48 and 72 h of treatment (Fig. 2). After 48 h of treatment, the cytotoxicity of LGG, L. plantarum WB4404, and L. plantarum WB4503 groups was -1.29 ± 0.56, 16.77 ± 0.52, and 1.31 ± 0.52%, respectively. After 72 h, LDH release significantly increased in all LAB-treated groups, each strains reached 41.58 ± 0.58, 30.32 ± 0.15%, and 12.52 ± 0.66%, respectively. L. plantarum WB4404 exhibited the highest cytotoxicity at both time points.

Expression of Apoptosis-Related Genes Using qRT-PCR by LAB Strains

The expression levels of pro-apoptotic (caspase-9, caspase-3, and Bax) and anti-apoptotic (Bcl-2) genes were determined using quantitative RT-PCR to assess the apoptotic response in HT-29 cells treated with LAB (Fig. 3). Compared with the control group, LGG moderately upregulated caspase-9 and caspase-3 expression by 1.65 and 1.13-fold, respectively. In contrast, L. plantarum WB4404 and L. plantarum WB4503 treatments significantly increased caspase-9 (2.59 and 2.42-fold, respectively) and caspase-3 (1.85 and 1.49-fold, respectively) mRNA expression levels, indicating stronger activation of the caspase cascade than LGG. Consistently, the Bax/Bcl-2 ratio, an indicator of the balance between pro- and anti-apoptotic signaling, was substantially higher in both treatment groups, with the most pronounced increase observed in cells treated with L. plantarum WB4404 (approximately 3.40-fold relative to the control).

Caspase-9 and Caspase-3 Activity by LAB Strains

The enzymatic activities of caspase-9 and caspase-3 in LAB-treated HT-29 cells were measured using colorimetric assay kits (Fig. 4). L. plantarum WB4404 increased caspase-9 activity in a time-dependent manner, with the highest activity observed at 48 h, followed by a decrease at 72 h. In contrast, L. plantarum WB4503 maintained higher activity levels than the control at 24 and 48 h and reached its maximum activity at 72 h. For caspase-3, both L. plantarum WB4404 and L. plantarum WB4503 exhibited significantly higher activity than the control at 24 and 48 h, with L. plantarum WB4404 maintaining the highest activity at 72 h.

Protein Expression of Caspase-9, Caspase-3, Bax, and Bcl-2 by LAB Strains

The protein expression levels of caspase-9, caspase-3, Bax, and Bcl-2 in HT-29 cells were examined using western blotting (Fig. 5). L. plantarum WB4404 and L. plantarum WB4503 increased the cleaved caspase-9/procaspase-9 ratio, which was accompanied by elevated cleaved caspase-3 expression, particularly in L. plantarum WB4404-treated cells. Moreover, L. plantarum WB4404 and L. plantarum WB4503 treatments upregulated Bax expression and downregulated Bcl-2 expression compared with the control.

Apoptosis Analysis by LAB Strains Using Annexin V/PI-Staining

Flow cytometry revealed changes in early and late apoptosis in Annexin V/PI-stained HT-29 cells following LAB treatment (Fig. 6). After 48 h of incubation, all LAB groups displayed a significant increase in early apoptosis compared with the control. L. plantarum WB4404 showed the highest level of early apoptosis, with an approximately 8.9-fold increase relative to the control. When the incubation time was extended to 72 h, L. plantarum WB4404 progressed to late apoptosis, whereas LGG and L. plantarum WB4503 induced further increases in early apoptotic populations.

Cell Cycle by LAB Strains

The effect of LAB treatment on cell cycle progression in HT-29 cells was analyzed using flow cytometry with PI staining (Fig. 7). LAB treatment resulted in an increased proportion of cells in the sub-G1 phase, representing DNA-fragmented apoptotic cells. After 48 h of treatment, both L. plantarum WB4404 and L. plantarum WB4503 showed a significant increase in the sub-G1 population relative to the control. At 72 h, further time-dependent accumulation of sub-G1 cells was observed, with the highest proportion detected in L. plantarum WB4404-treated cells.

Morphological Change of HT-29 Cells by LAB Strains

Optical microscopy revealed clear differences in cell density following LAB treatment, with L. plantarum WB4404 and L. plantarum WB4503 showing a marked reduction in cell density and adhesion compared with the control. Nuclear morphology was examined using DAPI staining. The control HT-29 cells displayed regular oval-shaped nuclei with homogeneous chromatin, whereas the cells treated with L. plantarum WB4404 or L. plantarum WB4503 displayed irregular nuclear margins, chromatin condensation, and nuclear fragmentation (Fig. 8).

Discussion

Previous studies have mainly evaluated the anticancer activity of LAB using cell-free supernatants or crude extracts, largely due to safety concerns regarding the administration of live bacteria in immunocompromised patients [15]. While these approaches have contributed to identification of LAB-derived anticancer effects, they are limited in addressing strain-specific mechanistic differences. In the present study, we employed live L. plantarum strains isolated from kimchi to investigate apoptosis induction in colorectal cancer cells, which enabled us to identify strain-dependent signaling differences involving Bax/Bcl-2 modulation and caspase activation. In addition, our results indicate that EPS contribute as components of the overall anticancer phenotype rather than acting as independent inducers of apoptosis, thereby refining the current understanding of LAB-mediated anticancer activity.

Kimchi contains a diverse community of LAB, and strains isolated from this environment withstand a wide range of environmental stressors [1, 28]. In this study, L. plantarum WB4404 and L. plantarum WB4503, isolated from kimchi, exhibited strong acid tolerance, bile resistance, and robust adhesion to intestinal epithelial cells under simulated gastrointestinal conditions (Table 2). These findings indicate that these strains are viable probiotic candidates with the potential to exert functional effects in vivo. Establishing these physiological traits prior to anticancer evaluation is essential to predict the likelihood of biological activity in the gut environment.

Apoptosis is an essential process for maintaining the balance between cell death and regeneration, and its induction is well recognized as a common mechanism through which various anticancer agents suppress tumor growth [29]. To investigate the antiproliferative and apoptosis-inducing effects of the strains on HT-29 human colorectal cancer cells, MTT and LDH assays were conducted. Both strains (8 log CFU/ml) significantly reduced formazan formation in the MTT assay (Fig. 1A), reflecting impaired mitochondrial activity—a characteristic feature of early apoptosis [30, 31]. However, as MTT primarily assesses mitochondrial metabolism, it does not directly distinguish between apoptotic stages. LDH release increased modestly in response to the bacterial treatment (Fig. 2), suggesting that mitochondrial dysfunction occurred more prominently than late-stage membrane damage. Furthermore, all strains maintained over 80% viability in MRC-5 normal fibroblasts, indicating that the observed growth inhibition in HT-29 cells was unlikely to result from nonspecific cytotoxicity (Fig. 1B). Based on these observations, further analyses were conducted to elucidate the specific cell death pathways involved.

Annexin V-fluorescein isothiocyanate/PI flow cytometry was performed to determine cell death pathways. Both strains showed markedly increased early apoptotic cells while maintaining low necrosis levels, and L. plantarum WB4404 induced significantly higher early apoptosis compared with LGG (Fig. 6). Sub-G1 accumulation was also detected in the treated cells (Fig. 7), indicating DNA damage and fragmentation [32]. Because sub-G1 analysis is sensitive to sample preparation artifacts, these findings were interpreted along with DAPI staining. DAPI staining confirmed nuclear fragmentation (Fig. 8), providing morphological evidence of apoptosis.

At the molecular level, L. plantarum WB4404 and L. plantarum WB4503 activated key components of the intrinsic apoptotic pathway. Treatment increased the expression of caspase-9 and caspase-3 at both mRNA and protein levels, elevated the pro-apoptotic protein Bax, and decreased the anti-apoptotic protein Bcl-2, leading to a substantial increase in the Bax/Bcl-2 ratio (Fig. 5). This shift enhances mitochondrial outer membrane permeability [33], promotes cytochrome c release, and activates caspase-9, which subsequently promotes cleavage of executioner caspases (caspase-3/7) and poly (ADP-ribose) polymerase, ultimately leading to apoptosis [34, 35]. Caspase activation was particularly pronounced in the L. plantarum WB4404-treated group, indicating stronger engagement of the mitochondria-dependent apoptotic cascade by this strain.

Strain-specific differences in anticancer activity may be attributed to variations in cell-surface structures or EPS. Previous studies have reported that EPS from certain Lactobacillus spp. and L. plantarum strains can exert anticancer effects on colorectal cancer cells, suggesting that the chemical composition and structural features of EPS are associated with this activity [36]. Consistent with these findings, crude EPS extracted from L. plantarum WB4404 reduced HT-29 cell viability more effectively than EPS extracted from other strains in the MTT assay (Fig. 1C). This observation suggests that EPS derived from L. plantarum WB4404 may contribute to the overall anticancer effect observed in this study, rather than acting as independent determinants. However, the anticancer effects of whole bacterial cells cannot be explained by EPS alone, and EPS likely represents only one part of the overall mechanism. Moreover, because the EPS used in this study was an unpurified crude extract, further research using purified EPS is required to determine its specific role independent of the co-extracted metabolites.

Taken together, these results demonstrates that kimchi-derived L. plantarum WB4404 and L. plantarum WB4503 activate mitochondria-dependent intrinsic apoptosis in HT-29 human colorectal cancer cells through activation of the caspase cascade and modulation of Bax/Bcl-2 expression. Nonetheless, these findings are based on in vitro assays, and the evaluation of toxicity toward normal cells is limited to general screening. The selective anticancer activity in intestinal tissues, interactions with the immune system, and ecological stability of the gut microbiota cannot be fully addressed in vitro. To clearly delineate strain-specific anticancer mechanisms, future studies should include EPS structural analysis, receptor-binding studies, and detailed profiling of membrane-associated signaling pathways, followed by in vivo validation.

Despite these limitations, this study provides substantial in vitro evidence that kimchi-derived L. plantarum strains possess functional properties beyond conventional probiotic activity and may represent potential probiotic candidates for functional food development targeting colorectal cancer-related pathways.

Conclusion

L. plantarum WB4404 and L. plantarum WB4503, isolated from kimchi, were identified as potential probiotic strains with strong acid and bile tolerance and high adhesion capacity under simulated gastrointestinal conditions. Both strains inhibited the proliferation of HT-29 human colorectal cancer cells by activating the mitochondria-dependent intrinsic apoptotic pathway, L. plantarum WB4404 exhibited stronger anticancer activity through enhanced Bax/Bcl-2 modulation and increased activation of caspase-9 and caspase-3. These findings suggest that kimchi-derived L. plantarum strains possess functional properties beyond conventional probiotic traits and may serve as promising candidates capable of influencing intrinsic apoptotic responses in colorectal cancer cells, potentially through strain-specific extracellular metabolites such as EPS. Further in vivo and mechanistic studies are required to validate and extend these findings.