A Combination of Two Probiotics, Lactobacillus sporogenes and Clostridium butyricum, Inhibits Colon Cancer Development: An In Vitro Study

Abstract

Cancer remains a leading cause of death worldwide and, even though several advances have been made in terms of specific treatment, the late-stage detection and the associated side effects of the conventional drugs sustain the search for better treatment alternatives. Probiotics are live microorganisms that have been proven to possess numerous health benefits for human hosts, including anticancer effects. In the present study, the in vitro effect of the association of two probiotic strains (PBT), Lactobacillus sporogenes and Clostridium butyricum, were tested against colon (HT-29 and HCT 116), lung (A549), and liver (HepG2) cancer cell lines, alone or in combination with 5-fluorouracil (5FU). Moreover, the underlying mechanism of PBT and PBT-5FU against the HT-29 cell line was evaluated using the Hoechst 33342 staining, revealing characteristic apoptotic modifications, such as chromatin condensation, nuclear fragmentation, and membrane blebbing. Furthermore, the increase in the expression of pro-apoptotic Bax, Bid, Bad, and Bak proteins and the inhibition of the anti-apoptotic Bcl-2 and Bcl-XL proteins were recorded. Collectively, these findings suggest that the two strains of probiotic bacteria, alone or in association with 5FU, induce apoptosis in colon cancer cells and may serve as a potential anticancer treatment.

Keywords: 5-fluorouracil; Clostridium butyricum; Lactobacillus sporogenes; apoptosis; colon cancer; liver cancer; lung cancer; probiotics.

Figure 1

In vitro evaluation of the effect exerted by PBT (Lactobacillus sporogenes:Clostridium butyricum TO-A = 25:1, 10⁷ million CFU), 5FU (5-fluorouracil 25 μM), and PBT-5FU after 24, 48, and 72 h of treatment on HT-29 and HCT 116 cells’ viability by performing the MTT assay. Data are presented as viability percentages (%) normalized to a control (untreated cells) and expressed as mean values ± SD of three independent experiments performed in triplicate. The statistical differences between untreated and the treated cells were analyzed by applying the one-way ANOVA method followed by Dunnett’s multiple comparisons post-test (** p < 0.01; **** p < 0.0001).

Figure 2

In vitro evaluation of the effect exerted by PBT (Lactobacillus sporogenes:Clostridium butyricum TO-A = 25:1, 10⁷ million CFU), 5FU (5-fluorouracil 25 μM), and PBT-5FU after 24, 48, and 72 h of treatment on A549 and HepG2 cells’ viability by performing the MTT assay. Data are presented as viability percentages (%) normalized to a control (untreated cells) and expressed as mean values ± SD of three independent experiments performed in triplicate. The statistical differences between untreated and the treated cells were analyzed by applying the one-way ANOVA method followed by Dunnett’s multiple comparisons post-test (** p < 0.01; *** p < 0.001 **** and p < 0.0001).

Figure 3

Morphological and shape changes produced by 5FU, PBT and PBT-5FU in HT-29 cells after 24, 48, and 72 h of treatment. The scale bars indicate 50 µm.

Figure 4

Hoechst 33342 staining of HT-29 cells’ nuclei following the 24 h treatment with: PBT, 5FU, and PBT-5FU. The orange and green arrows indicate nuclei expressing abnormal features. The scale bars represent 100 µm.

Figure 5

Relative fold change expression of mRNA of pro-apoptotic (Bax, Bid, Bad, and Bak) and anti-apoptotic (Bcl-2 and Bcl-XL) markers in human adenocarcinoma colorectal cells (HT-29)—72 h after exposure to PBT, 5FU, and PBT-5FU. mRNA expression levels normalized to beta-actin expression; mean values ± SD of three independent experiments presented; one-way ANOVA with Tukey’s post-test used to identify the statistical differences (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).